To establish the mouse model of Gly374Arg mutation in fibroblast growth factor receptor 3(Fgfr3) and to analyze the phenotype of the mutant mice.The double PCR was used to introduce Gly374Arg point mutation into mouse Fgfr3. The electroporation of embryonic stem(ES) cells was carried out with targeting vector. The targeted ES cells were screened by Positive-Negative Selection of G418 and Ganciclovir, and Southern blot. The correct targeted ES cells were microinjected into blastula. Finally, mutant mice were obtained by crossing between EIIa-Cre transgenic mice and mice carrying recombined mutant Fgfr3 allele. The mice were genotyped by PCR, and phenotype was observed by skeleton staining, histology, etc.Fgfr3-Gly374Arg mutant mice exhibited small size, short tail, macrocephaly and had dome-shaped heads, the epiphyseal growth plates of mutant mice were narrower, and the hypertrophic chondrocyte zone was also obviously decreased. Meanwhile, the majority of female mice were infertile, and the uterus, ovary and mammal gland in mutant female mice were also smaller and underdeveloped.The model of Fgfr3-Gly374Arg mutation causing achondroplasia in mice has been established successfully.
Bronchial asthma (referred to as “asthma” hereinafter in this consensus document) is a chronic inflammatory airway disease with well-accepted heterogeneity and complex pathophysiological processes (1). Severe asthma is recognized as a poorly controlled condition that seriously affects quality of life, accounts for massive use of medical resources, and imposes huge socioeconomic burdens, representing a primary cause of asthma-related disability and death in the patients (2-5). In this regard, improving the diagnosis and treatment of severe asthma should be of paramount importance towards better outcomes in overall control and prognosis, as well as lower medical costs (5).
Abstract Heparinases, including heparinases I–III (HepI, HepII, and HepIII, respectively), are important tools for producing low‐molecular‐weight heparin, an improved anticoagulant. The poor thermostability of heparinases significantly hinders their industrial and laboratory applications. To improve the thermostability of heparinases, we applied a rigid linker (EAAAK) 5 (R) and a flexible linker (GGGGS) 5 (F) to fuse maltose‐binding protein (MBP) and HepI, HepII, and HepIII from Pedobacter heparinus , replacing the original linker from the plasmid pMAL‐c2X. Compared with their parental fusion protein, MBP‐fused HepIs, HepIIs, and HepIIIs with linkers (EAAAK) 5 or (GGGGS) 5 all displayed enhanced thermostability (half‐lives at 30°C: 242%–464%). MBP‐fused HepIs and HepIIs exhibited higher specific activity (127%–324%), whereas MBP‐fused HepIIIs displayed activity similar to that of their parental fusion protein. Kinetics analysis revealed that MBP‐fused HepIIs showed a significantly decreased affinity toward heparin with increased K m values (397%–480%) after the linker replacement, whereas the substrate affinity did not change significantly for MBP‐fused HepIs and HepIIIs. Furthermore, it preliminarily appeared that the depolymerization mechanism of these fusion proteins may not change after linker replacement. These findings suggest the superior enzymatic properties of MBP‐fused heparinases with suitable linker designs and their potential for the bioproduction of low‐molecular‐weight heparin.
To analyze the airway inflammatory phenotypes and clinical features of severe asthma compared to mild-moderate ("common") asthma.A total of 946 cases of asthma were retrospectively analyzed in our hospital from January 2013 to December 2014. Sixty-one patients were classified to the severe asthma group, and 885 patients to the common asthma group. Severe asthma was diagnosed based on the protocol from ATS/ERS guidelines. All patients received induced sputum cell counts and pulmonary function tests, and 543 of them received fractional exhaled nitric oxide (FeNO) tests. The airway inflammatory phenotypes were defined and the clinical features of patients of severe asthma were studied.The distribution of airway inflammatory phenotypes of asthma was as follows: eosinophilic subtype (46.6%, 441/946), mixed granulocytic subtype (27.5%, 260/946), neutrophilic subtype (21.5%, 203/946), and paucigranulocytic subtype (4.4%, 42/946). There were no differences between the severe asthma group and the common asthma group in the distributions. Compared with common asthma patients, severe asthma patients had higher sputum eosinophil percentages (29.1 % ± 28.5% vs 22.2% ± 25.2%, t = 1.98, P < 0.05), higher FeNO values [(66.4 ± 64.1) ppb vs (48.0 ± 43.7) ppb, P < 0.01], lower percentages of FEV1% pred [(63.7 ± 24.1) % vs (84.7 ± 23.7)%, P < 0.01], and lower ratios of FEV1/FVC [(56.4 ± 15.1) % vs (69.1 ± 14.5)% P < 0.01]. In severe asthma patients, FeNO values were higher in the eosinophilic subtype and mixed granulocytic subtype (P < 0.05). Neutrophilic subtype patients had the lowest sputum eosinophil percentage [(1.8 ± 0.8)%, P < 0.01], the lowest percentage of FEV1% pred [(46.6 ± 16.1)%, P < 0.01], and the lowest ratios of FEV1/FVC [(45.2 ± 16.1)%, P < 0.01].The common airway inflammatory phenotypes included eosinophilic subtype, mixed granulocytic subtype and neutrophilic subtype, in both severe and common asthma patients. Severe asthma patients had more severe eosinophilic airway inflammation and poorer lung function. Neutrophilic subtype might be the most intractable subtype with severely damaged pulmonary function in severe asthma.
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