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Wienholds E van Eeden F Kosters M Mudde J Plasterk RH Cuppen E 《Genome research》2003,13(12):2700-2707
One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently,we described the first target-selected knockout in zebrafish. Here,we report on the further improvements of this procedure,resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total,255 mutations were identified,of which 14 resulted in a premature stop codon,7 in a splice donor/acceptor site mutation,and 119 in an amino acid change. By this method,we potentially knocked out 13 different genes in a few months time. Furthermore,we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms. 相似文献
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Dientamoeba fragilis is a globally occurring parasite that has been recognized as a causative agent of gastrointestinal symptoms. A single-round PCR was developed to detect D. fragilis DNA directly from human stool samples. The genetic diversity of D. fragilis from 93 patients and 6 asymptomatic carriers was examined by PCR followed by restriction fragment length polymorphism and sequencing of part of the small-subunit rRNA gene. The data show that D. fragilis sequences can be studied directly from fecal specimens despite the absence of a cyst stage and without the need for prior culturing. In addition, the results suggest strongly that D. fragilis shows remarkably little variation in its small-subunit rRNA gene. 相似文献
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Nilius B Weidema F Prenen J Hoenderop JG Vennekens R Hoefs S Droogmans G Bindels RJ 《Pflügers Archiv : European journal of physiology》2003,445(5):584-588
The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent inactivation. Interestingly, the number of green fluorescent cells, which is an index of the number of transfected cells, was significantly smaller for cells transfected with truncations shorter than E649 than for cells transfected with wild-type ECaC. However, the expression level of GFP was restored in the presence of the ECaC blocker ruthenium red, suggesting that these truncations resulted in deleterious Ca(2+) influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca(2+)-dependent inactivation. 相似文献
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Improved diagnostic stratification of digitised Barrett's oesophagus biopsies by p53 immunohistochemical staining 下载免费PDF全文
Myrtle J van der Wel Lucas C Duits Roos E Pouw Cornelis A Seldenrijk G J A Offerhaus Mike Visser Fiebo J ten Kate Katharina Biermann Lodewijk A A Brosens Michael Doukas Clement Huysentruyt Arend Karrenbeld Gursah Kats‐Ugurlu Jaap S van der Laan G van Lijnschoten Freek C P Moll Ariadne H A G Ooms Hans van der Valk Jan G Tijssen Jacques J Bergman Sybren L Meijer 《Histopathology》2018,72(6):1015-1023
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Curtis JP Alexander JH Huang Y Wallentin L Verheugt FW Armstrong PW Krumholz HM Van de Werf F Danays T Cheeks M Granger CB;ASSENT- ASSENT- Investigators 《The American journal of cardiology》2004,94(3):279-283
We investigated the effect of smaller dose, weight-adjusted heparin with earlier monitoring of activated partial thromboplastin time on the incidence of ischemic and hemorrhagic complications in patients with ST-elevation myocardial infarction treated with full-dose tenecteplase. We compared the outcomes of patients enrolled in the Second Assessment of the Safety and Efficacy of a New Thrombolytic Regimen (ASSENT-2; n = 8,461) who received heparin stratified by weight (patients weighing >67 kg received a 5,000-U bolus plus infusion at 1,000 U/hour; those weighing < or =67 kg received a 4,000-U bolus plus infusion at 800 U/hour) with patients in ASSENT-3 who received weight-adjusted heparin (60-U/kg bolus, maximum 4,000 U/hour, followed by a 12-U/kg/hour infusion, maximum 1,000 U/hour). Compared with patients in ASSENT-2, those in ASSENT-3 had similar rates of 30-day mortality, recurrent infarction, and intracranial hemorrhage, less major bleeding (2.2% vs 4.7%, p <0.001), and less refractory ischemia (6.5% vs 8.6%, p <0.001). After adjustment for baseline characteristics, patients in ASSENT-3 had similar rates of 30-day mortality (odds ratio [OR] 0.96, 95% confidence interval [CI] 0.77 to 1.19) and intracranial hemorrhage (OR 1.02, 95% CI 0.61 to 1.69) but less major bleeding (OR 0.49, 95% CI 0.35 to 0.67) than did patients in ASSENT-2. These findings support the use of smaller dose, weight-adjusted heparin in patients with ST-elevation myocardial infarction treated with tenecteplase. 相似文献
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John S. Van Dyke Freek Massee Milan P. Allan J. C. Séamus Davis Cedomir Petrovic Dirk K. Morr 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(32):11663-11667
To identify the microscopic mechanism of heavy-fermion Cooper pairing is an unresolved challenge in quantum matter studies; it may also relate closely to finding the pairing mechanism of high-temperature superconductivity. Magnetically mediated Cooper pairing has long been the conjectured basis of heavy-fermion superconductivity but no direct verification of this hypothesis was achievable. Here, we use a novel approach based on precision measurements of the heavy-fermion band structure using quasiparticle interference imaging to reveal quantitatively the momentum space (k-space) structure of the f-electron magnetic interactions of CeCoIn5. Then, by solving the superconducting gap equations on the two heavy-fermion bands with these magnetic interactions as mediators of the Cooper pairing, we derive a series of quantitative predictions about the superconductive state. The agreement found between these diverse predictions and the measured characteristics of superconducting CeCoIn5 then provides direct evidence that the heavy-fermion Cooper pairing is indeed mediated by f-electron magnetism.Superconductivity of heavy fermions is of abiding interest, both in its own right (1–7) and because it could exemplify the unconventional Cooper pairing mechanism of high-temperature superconductors (8–11). Heavy-fermion compounds are intermetallics containing magnetic ions in the 4f- or 5f-electronic state within each unit cell. At high temperatures, each f-electron is localized at a magnetic ion (Fig. 1A). At low temperatures, interactions between f-electron spins (red arrows Fig. 1A) lead to the formation of a narrow but the subtly curved f-electron band near the chemical potential (red curve, Fig. 1B), and Kondo screening hybridizes this band with the conventional c-electron band of the metal (black curve, Fig. 1B). As a result, two new heavy-fermion bands (Fig. 1C) appear within a few millielectron volts of the Fermi energy. Their electronic structure is controlled by the hybridization matrix element sk for interconversion of conduction c-electrons to f-electrons and vice-versa, such that[1]The momentum structure of the narrow bands of hybridized electronic states (Eq. 1 and Fig. 1C, blue curves at left) near the Fermi surface then directly reflects the form of magnetic interactions encoded within the parent f-electron band . It is these interactions that are conjectured to drive the Cooper pairing (1–5) and thus the opening up of a superconducting energy gap (Fig. 1C, yellow curves at right).Open in a separate windowFig. 1.Effects of f-electron magnetism in a heavy-fermion material. (A) The magnetic subsystem of CeCoIn5 consists of almost localized magnetic f-electrons (red arrows) with a weak hopping matrix element yielding a very narrow band with strong magnetic interactions between the f-electron spins. (B) The heavy f-electron band is shown schematically in red and the light c-electron band in black. (C) On the left, schematic of the result of hybridizing the c- and f-electrons in B into new composite electronic states referred to as heavy fermions (blue). On the right, the opening of a superconducting energy gap is schematically shown by back-bending bands near the chemical potential. The microscopic interactions driving Cooper pairing of these states, and thus of heavy-fermion superconductivity, have not been identified unambiguously for any heavy-fermion compound. 相似文献