Please note that many of the page functionalities won't work as expected without javascript enabled. The, The human fibrinogen Aα R35C mutation prevents FpA cleavage by thrombin. Statistical analysis and graphical representations were made using Prism (GraphPad Software, San Diego, CA, USA). Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia. Laser injuries were used to induce venous thrombosis and the time … ; project administration, M.N.-A. How Animal Research Helps Humans. Many use over-the-counter medications to manage gastroesophageal reflux disease, but there are different types of anti-reflux surgeries that can be a viable option for treatment and symptom control. Missense mutations (P59Q; D342E): Milder disease Point mutations → Stop codons: Severe disease G240X mutation common in Palestinian Arabs Functional effects of mutations differ N-terminal (P59Q) or PRAD region (107del215) Reduced Binding of ColQ to catalytic subunit of AChE; More distal mutations Nevertheless, patients with very high scores on any of the prediction models (i.e., MDF > 90 or MELD > 30) have very severe disease, which necessitates careful assessment for occult infection and other contraindications to corticosteroid treatment. HSC Cores. 2021. ; Lavik, K.I. MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Sci. ; Mackman, N. Animal Models of Thrombosis From Zebrafish to Nonhuman Primates: Use in the Elucidation of New Pathologic Pathways and the Development of Antithrombotic Drugs. those of the individual authors and contributors and not of the publisher and the editor(s). Zebrafish as a model system for the study of hemostasis and thrombosis. Lozier, J.N. We attempted to detect the mutated Aα protein chain in plasma samples using mass spectrometry, searching for peptides corresponding to the fusion of exon 1- and exon 3-encoded residues. Zones of delayed neurogenesis, or non-neurogenic zones, are found around the proneural clusters (areas marked 1–4 in Fig. RNA was isolated from embryos with the mutation, or from adult liver samples, with Trizol (Thermo Fisher Scientific, Walthum, MA, USA), reverse transcribed (Superscript II, Thermo Fisher Scientific, Walthum, MA, USA), DNAse treated (Turbo DNAse, Thermo Fisher Scientific, Walthum, MA, USA) and amplified by PCR targeting the fga cDNA. ; Tsao, P.; Vo, A.H.; Huarng, M.C. ; Rost, M.S. The statements, opinions and data contained in the journal, © 1996-2021 MDPI (Basel, Switzerland) unless otherwise stated. and M.N.-A. Click here if you forgot your password. However, l-tyrosine supplementation was not able to attenuate the skeletal muscle dysfunction in zebrafish and the dominant skeletal muscle α-actin nemaline myopathy in mouse models . Orthotopic Tumor Model Studies. Iyer, N.; Tcheuyap, V.T. and M.N.-A. ; Sander, J.D. ; Menegatti, M.; Reyon, D.; Rost, M.S. ; Parant, J.M. ; Campbell, D.S. J. Mol. Babaei, F.; Ramalingam, R.; Tavendale, A.; Liang, Y.; Yan, L.S. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. ; Ruiz-Saez, A.; Echenagucia, M.; Rodriguez-Lemoin, A. Inhibition of thrombin generation in plasma by fibrin formation (Antithrombin I). Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia. ; Kim, S. Laser-induced thrombosis in zebrafish. "Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders" Int. ; supervision, R.J.F. Find support for a specific problem on the support section of our website. In vivo models (right side) include patient-derived xenografts (PDX) in mouse, zebrafish, and chicken egg chorioallantoic membrane (CAM) hosts. https://doi.org/10.3390/ijms22020655, Fish, Richard J.; Freire, Cristina; Di Sanza, Corinne; Neerman-Arbez, Marguerite. Evaluating for Contraindications to Corticosteroids Health Science Center. International Journal of Molecular Sciences. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Plasma fibrinogen inhibits platelets adhesion in flowing blood to immobilized fibrinogen. Endenburg, S.C.; Lindeboom-Blokzijl, L.; Zwaginga, J.J.; Sixma, J.J.; de Groot, P.G. Sci. Analysis of factor V in zebrafish demonstrates minimal levels needed for early hemostasis. Congenital heart defects are the most common type of birth defect, affecting nearly 1% of births in the United States each year, according to the Centers for Disease Control. Venous TTO at 3 dpf is expected to be slightly prolonged in models of both fibrinogen disorder classes in the heterozygous state. Our dedicated information section provides allows you to learn more about MDPI. CRISPR-Cas9 based genome edits of. Freire, C.; Fish, R.J.; Vilar, R.; Di Sanza, C.; Grzegorski, S.J. ; Tsai, S.Q. Peyvandi, F. Epidemiology and treatment of congenital fibrinogen deficiency. ; Yost, H.J. A nontransfected control sample was also prepared. The mutated fibrinogen alpha chains produced in our proposed models of dysfibrinogenemia (R28C) and hypodysfibrinogenemia (Δ19–56) lack the Aα amino terminal sequence for effective thrombin-mediated cleavage and FpA release. For a patient with a neurological disease, the neurons of knock-out embryos can be fluorescently labeled to see if they form incorrectly. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. De Bosch, N.B. Find support for a specific problem on the support section of our website. The authors declare no conflict of interest. Please let us know what you think of our products and services. and M.N.-A. 2021, 22, 655. This strategy allows us to assess tumor development in a relevant environment and evaluate efficacy in a preclinical tumor model that mimics the disease … ; Nichols, T.C. ; Liu, Y.; Vo, A.H.; Richter, C.E. You seem to have javascript disabled. ; Richter, C.E. Mosimann, C.; Kaufman, C.K. See further details. ; Shavit, J.A. Neerman-Arbez, M. Molecular basis of fibrinogen deficiency. ; investigation, R.J.F., C.F. The zebrafish heart ECG is similar to that of humans and thus zebrafish are considered as an ideal model for cardiovascular research (Fu et al., 2010; Zhang et al., 2015). For the present study an obvious limitation is that the disorders we model are diagnosed in part by the concordance of plasma fibrinogen levels and activity. The mutation was confirmed by DNA sequencing. ; Nagaswami, C.; Chernysh, I.N. Int. Casini, A.; de Moerloose, P. Can the phenotype of inherited fibrinogen disorders be predicted? J. Mol. At 24 h posttransfection the culture medium was removed, cells washed with PBS and then cultured for a further 24 h in OptiMEM (Thermo Fisher Scientific, Walthum, MA, USA) without serum. In several of these non-neurogenic zones, members of the hairy/enhancer of split family genes have been shown to prevent neural progenitors from entering … This study describes a novel mechanism: Seizures reduce connexin 36 levels in zebrafish models, and may contribute to the onset of subsequent seizures. D Zones of Delayed Differentiation. ; Fu, Y.; Reyon, D.; Maeder, M.L. To mimic this mutation in zebrafish fibrinogen (Aα R28C), and test whether it is also resistant to FpA cleavage, we first mutated a zebrafish Aα chain expression plasmid, pcDNA3.1-ZF-Aα, using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) and the oligonucleotides fgaR28C-F (5′GGACACAGTGGTGAACCCTTGCGGTGCTCGTCCTATTGAGC3′) and fgaR28C-R (5′GCTCAATAGGACGAGCACCGCAAGGGTTCACCACTGTGTCC3′). Khandekar, G.; Kim, S.; Jagadeeswaran, P. Zebrafish thrombocytes: Functions and origins. While clots are still likely to occur via B:b knob-hole interactions [, Thus far, the differences between the distinct model phenotypes we report are subtle. Subscribe to receive issue release notifications and newsletters from MDPI journals, You can make submissions to other journals. The zebrafish has the key attribute of accessible larval blood vessels which can be readily targeted with a laser to induce clotting and thrombosis. Animal models serving in research may have an existing, inbred or induced disease or injury that is similar to a human condition. ; Asselta, R.; Duga, S.; Peyvandi, F.; et al. Weyand, A.C.; Grzegorski, S.J. Syngeneic mouse models can be a powerful tool for testing immunotherapies, but they are only as good as their background data. ; Li, P.; Pugach, E.K. Embryos from natural matings were raised at 28.5 °C. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent, Mutations in the three fibrinogen genes give rise to congenital fibrinogen disorders [, Afibrinogenemia shows recessive inheritance, two mutated alleles of a fibrinogen gene are required for its appearance, and hypofibrinogenemia can be detected in heterozygous carriers of alleles that would cause afibrinogenemia in homozygosity [, While diagnosis can usually be achieved by laboratory tests and genetic studies of the fibrinogen genes, prediction of the clinical phenotype of fibrinogen disorders beyond a bleeding tendency is challenging [, Bleeding events linked to low plasma fibrinogen, or a dysfunctional fibrinogen molecule, can be explained by a deficiency in the quantity or quality of the major physiological substrate for coagulation-based clotting. Ariens, R.A. Fibrin(ogen) and thrombotic disease. Fish, R.J.; Di Sanza, C.; Neerman-Arbez, M. Targeted mutation of zebrafish. At Charles River, you’ll get syngeneic models that have been fully characterized with known checkpoint inhibitors, whole exome sequencing, and immunologic profiling data, backed by experienced scientists who can ensure your model runs smoothly. ; Stapleton, A.N. Prisca Chapouton, Leanne Godinho, in Methods in Cell Biology, 2010. ; Farrell, D.H. Incorporation of fibrin molecules containing fibrinopeptide A alters clot ultrastructure and decreases permeability. F1 offspring were raised and genomic DNA from F1 embryos extracted and assayed for the R28C mutation by PCR genotyping and HpaI digestion, and DNA sequencing to confirm the mutation. ... We are improving computer models and cell culture research, but animal research is still vital to save lives across species. B:b interactions are essential for polymerization of variant fibrinogens with impaired holes ‘a’. This corresponds to R28C in the zebrafish Aα (, On the TU zebrafish genetic background, we used CRISPR-Cas9-based genome editing to induce double-stranded genomic DNA breaks near the R28 codon in exon 2 of the zebrafish, To confirm the expected mutation at the RNA level, we generated cDNA from liver RNA extracted from adult, To understand why the genome edited sequence led to exon 2 skipping in fga mRNA, we tested the hypothesis that it was affecting an exon splicing enhancer sequence (ESES), using prediction software (RESCUE-ESE Web Server—genes.mit.edu). Zebrafish or zebra danio (danio rerio) are seen as one of the latest "models" for vertebrate embryological development studies.These embryos have the great advantage that they develop as "see through" embryos, that is, all internal development can be … The Tol2kit: A multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. In the future we aim to use this preliminary guide to assess the phenotype of newly uncovered mutations linked to congenital fibrinogen disorders and take steps towards correlating the larval zebrafish model phenotypes with clinical indicators in patients. Our dedicated information section provides allows you to learn more about MDPI. Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. ; Hynes, R.O. ; Hu, Z.; Liu, Y.; Yu, X.; Ferguson, A.C.; Madarati, H.; Friedmann, A.P. ; Ajuh, P.; Cheng, S.H. Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland. Menu. ; Al-Mondhiry, H.A. What to know about the COVID-19 vaccine. De Moerloose, P.; Casini, A.; Neerman-Arbez, M. Congenital fibrinogen disorders: An update. fibrinogen; fibrin; thrombocytes; thrombosis; zebrafish, Help us to further improve by taking part in this short 5 minute survey, Therapies for the Treatment of Cardiovascular Disease Associated with Type 2 Diabetes and Dyslipidemia, NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure–Activity Relationships, Ex Vivo Live Full-Thickness Porcine Skin Model as a Versatile In Vitro Testing Method for Skin Barrier Research, Accelerated Spatial Fibrin Growth and Impaired Contraction of Blood Clots in Patients with Rheumatoid Arthritis, Fibrinogen/Fibrin, Factor XIII and Fibrinolysis in Diseases, https://www.mdpi.com/1422-0067/22/2/655/s1, http://creativecommons.org/licenses/by/4.0/. ... Zebrafish and more; See more important resources on ethical animal research. But here in a Cincinnati Children's laboratory, the freshwater variant plays a vital role in scientific discovery. All this makes them suitable for studies on human disease and embryological development. Liu, Y.; Kretz, C.A. Hu, Z.; Lavik, K.I. ; Chi, N.C.; et al. those of the individual authors and contributors and not of the publisher and the editor(s). Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. Hu, Z.; Liu, Y.; Huarng, M.C. Grzegorski, S.J. This research was funded by the Swiss National Science Foundation, grant number #31003A_172864 to M.N.-A. ; DiOrio, J.P.; Siebenlist, K.S. and M.N.-A. ; Farrell, D.H. Our data suggest that laser-induced TTO values are affected by fibrinogen quantity or quality. ; Morgan, F.J. 22, no. ; Richter, C.E. ; Rosenstingl, S.; Laurian, C.; Bruneval, P.; Tobelem, G. Embolized ischemic lesions of toes in an afibrinogenemic patient: Possible relevance to in vivo circulating thrombin. Fish, R.J.; Freire, C.; Di Sanza, C.; Neerman-Arbez, M. Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders. ; methodology, R.J.F., C.F. These authors contributed equally to this work. Berget, S.M. We thank the University of Geneva, Faculty of Medicine facilities for animal care, proteomics and bioimaging. Author to whom correspondence should be addressed. ; Gross, P.L. Casini, A.; Neerman-Arbez, M.; Ariens, R.A.; de Moerloose, P. Dysfibrinogenemia: From molecular anomalies to clinical manifestations and management. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. ... in a mid- to high-throughput manner. Kwan, K.M. PCR products were cloned in pCRII TOPO (Thermo Fisher Scientific, Walthum, MA, USA) and sequenced. The statements, opinions and data contained in the journals are solely Ni, H.; Denis, C.V.; Subbarao, S.; Degen, J.L. This helps scientists learn new details in much less time. De Marco, L.; Girolami, A.; Zimmerman, T.S. Cores Administration; Amaxa Nucleofector – Resource Login; Cell Imaging – Training and consultation on use of Imaging Resources; CZAR Zebrafish – Housing, breeding and doing experiments with Zebrafish; DNA/Peptide Synthesis – a wide range of routine and specialty oligonucleotides as well as chemically synthesized peptides Please note that many of the page functionalities won't work as expected without javascript enabled. Flood, V.H. Koopman, J.; Haverkate, F.; Grimbergen, J.; Lord, S.T. 1 F). This demonstrated that fga exon 2 skipping occurred in transcripts where the Aα R28C codon was introduced, and encoded Aα, A plasmid for expression of the zebrafish fibrinogen AαE chain, under the control of a ubiquitin (, Early zebrafish embryos were microinjected with approximately 1 nL of injection mixes. The statements, opinions and data contained in the journal, © 1996-2021 MDPI (Basel, Switzerland) unless otherwise stated. These authors contributed equally to this work. Exon recognition in vertebrate splicing. ; funding acquisition, M.N.-A. Update on antithrombin I (fibrin). ; Zon, L.I. ; Norris, Z.G. Cells were cultured and transfected in DMEM supplemented with 10% FCS and antibiotics. Zebrafish serve as a powerful animal model, allowing researchers to evaluate the effects of connexin 36 on the whole brain in an intact living system during neural hyperactivity. This can be seen by the minor prolongation of mean TTO in, This leads us to propose an expected zebrafish phenotype profile for models of quantitative versus qualitative disorders in our two laser injury assays. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. ; writing—original draft preparation, R.J.F. The species or classification of animals used in testing largely depends on the goal of the experiment. ; Fujimoto, E.; Grabher, C.; Mangum, B.D. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish. The statements, opinions and data contained in the journals are solely ; Legrand, C.; Soria, J.; Soria, C.; et al. Liu, C.Y. The fibrinogen Aalpha R16C mutation results in fibrinolytic resistance. Zebrafish are good animal models for behavioral, biochemical, and physiological measurements (Huang et al., 2018; Poopal et al., 2020; Ramesh et al., 2020). A mathematical model is an abstract model that uses mathematical language to describe the behaviour of a system. Novel blood collection method allows plasma proteome analysis from single zebrafish. and M.N.-A. ; Maeder, M.L. ; Wagner, D.D. ; Sato, T.N. ; Koehn, J.A. Gregory, M.; Hanumanthaiah, R.; Jagadeeswaran, P. Genetic analysis of hemostasis and thrombosis using vascular occlusion. Register if you don't have an account. A hemophilia model in zebrafish: Analysis of hemostasis. Author to whom correspondence should be addressed. ; et al. ; Kanki, J.P.; Chien, C.B. However, they cannot be used to project a precise correlation between fibrinogen quantity, quality and a clinical phenotype. Hwang, W.Y. Tajdar, M.; Orlando, C.; Casini, A.; Herpol, M.; De Bisschop, B.; Govaert, P.; Neerman-Arbez, M.; Jochmans, K. Heterozygous. The model we used for afibrinogenemia has been reported previously [, Despite the defect in laser-induced thrombocyte binding we describe, we have no evidence for spontaneous bleeding in, We initially encountered difficulties in modelling the R35C dysfibrinogenemia mutation. A CRISPR-Cas9 strategy was used. At present, to our knowledge, plasma fibrinogen cannot be measured accurately in larval zebrafish blood due to low blood volumes and a lack of methodology. Mosesson, M.W. The time-to-occlusion (TTO) was measured in seconds after laser injury laser of the posterior cardinal vein (PCV) in 3-day postfertilization (3 dpf) larvae [. The larval injury models can therefore suggest the phenotypic effects of a disorder’s mutation, the disease inheritance mode, and detect detrimental functional effects of a mutation. ; Neerman-Arbez, M. A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AalphaE in early hemostasis. All authors have read and agreed to the published version of the manuscript. Okumura, N.; Terasawa, F.; Haneishi, A.; Fujihara, N.; Hirota-Kawadobora, M.; Yamauchi, K.; Ota, H.; Lord, S.T. Adult zebrafish were maintained at 26 °C, pH 7.5, and 500 µS conductivity. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. Jagadeeswaran, P.; Cooley, B.C. Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function. Data are available on request from the corresponding author. Analysis of blood coagulation in the zebrafish. Laser injury of the posterior cardinal vein in 3-day postfertilization (3 dpf) zebrafish larvae can lead to occlusive venous thrombosis and when monitored gives the time-to-occlusion (TTO, The adhesion and aggregation of fluorescent thrombocytes was assessed after laser injury of the PCV in 5 dpf, A frequently detected missense mutation found in dysfibrinogenemia patients is the, We aimed to produce zebrafish with a mutation equivalent to human Aα R35C. and C.D.S. and M.N.-A. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. ; Garieri, M.; Di Sanza, C.; Neerman-Arbez, M.; Fish, R.J. Chemical Modulators of Fibrinogen Production and Their Impact on Venous Thrombosis. Conditioned media were recovered, and cell lysates prepared in RIPA buffer. Early embryos of the zebrafish TU background were microinjected with a 1–2 nL mixture containing 0.5 ng/nL recombinant Cas9 nuclease (PNABio, Newbury Park, CA, USA), 250 pg/nL of a single guide RNA (sgRNA) with complementarity to zebrafish, 5′CCATACCCAGTCATCATCGGTACACCCTGGCCATTCTTTTGTCTGGCAGGTGTCTTGTGCCTTGAAGCCGTGCTCAATAGGACGAGCGCC, Microinjected F0 embryos were raised to adulthood and crossed with wild-type fish to identify a founder animal. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. ; Di Paola, J.; Shavit, J.A. ; Schneider, S.; Marshall, V.; Jagadeeswaran, P. Knockout of von Willebrand factor in Zebrafish by CRISPR/Cas9 mutagenesis. Animal experimentation was authorized by the Geneva cantonal authority (authorization GE/161/19, 07.11.2019). Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent, This is an open access article distributed under the, Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. For example, zebrafish are quick to breed, easy to house, and transparent as embryos - but they also carry 70 percent of the genes found in humans. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. ; Boulot, P.; Reyftmann, L.; de Moerloose, P.; Neerman-Arbez, M. Hypodysfibrinogenaemia due to production of mutant fibrinogen alpha-chains lacking fibrinopeptide A and polymerisation knob ‘A’. Jagadeeswaran, P.; Sheehan, J.P.; Craig, F.E. Research organisms are also useful because a disease’s natural course in humans can take dozens of years, whereas a research organism can quickly develop a version of that disease or some of its symptoms. M. thrombosis in zebrafish using a CRISPR-Cas system ; Duga, S. ; Zhou, ;. Radhakrishnan, U.P please enter your username and password models fail to support venous occlusion, whereas models quantitative. Classes in the journal, © 1996-2021 MDPI ( Basel, Switzerland ) unless otherwise stated P.!, J.W agreed to the published version of the page functionalities wo n't work as expected without javascript enabled a! Epidemiology zebrafish disease models treatment of Congenital fibrinogen deficiency specific problem on the support section our! Representations were made using Prism ( GraphPad Software, San Diego, CA, USA ) Fu... 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