Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.     

<Emphasis Type="Italic">Narcissus tazetta</Emphasis> lectin shows strong inhibitory effects against respiratory syncytial virus,influenza A (H1N1, H3N2, H5N1) and B viruses

A mannose-binding lectin (Narcissus tazetta lectin [NTL]) with potent antiviral activity was isolated and purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis, using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on mannose-agarose and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12. The purified lectin was shown to have an apparent molecular mass of 26 kDa by gel filtration and 13 kDa by SDS-PAGE, indicating that it is probably a dimer with two identical subunits. The cDNA-derived amino acid sequence of NTL as determined by molecular cloning also reveals that NTL protein contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension. Three-dimensional modelling study demonstrated that the NTL primary polypeptide contains three subdomains, each with a conserved mannose-binding site. It shows a high homology of about 60%–80% similarity with the existing monocot mannose-binding lectins. NTL could significantly inhibit plaque formation by the human respiratory syncytial virus (RSV) with an IC₅₀ of 2.30 μg/ml and exhibit strong antiviral properties against influenza A (H1N1, H3N2, H5N1) and influenza B viruses with IC₅₀ values ranging from 0.20 μg/ml to 1.33 μg/ml in a dose-dependent manner. It is worth noting that the modes of antiviral action of NTL against RSV and influenza A virus are significantly different. NTL is effective in the inhibition of RSV during the whole viral infection cycle, but the antiviral activity of NTL is mainly expressed at the early stage of the viral cycle of influenza A (H1N1) virus. NTL with a high selective index (SI=CC₅₀/IC₅₀≥141) resulting from its potent antiviral activity and low cytotoxicity demonstrates a potential for biotechnological development as an antiviral agent.     

Development of a fluorescent probe hydrolysis-insulated isothermal PCR for rapid and sensitive on-site detection of African swine fever virus

Highlights
1. A probe-based insulated isothermal PCR (iiPCR) assay was developed for rapid and onsite detection of ASFV.
2. The developed iiPCR showed similar sensitivity and specificity with OIE recommended real-time PCR.
3. Blood samples could be directly applied as PCR template in iiPCR without DNA extraction.     

MYH9: structure,functions and role of non-muscle myosin ⅡA in human disease

MYH9-related diseases (MYH9-RD) are a group of autosomal dominant diseases caused by mutations in the MYH9 gene, which are featured by thrombocytopenia, giant platelets and granulocyte cytoplasmic inclusion bodies. MYH9-RD patients generally suffer from bleeding syndromes, progressive kidney disease, deafness, or cataracts. Here, we reported on a case of MYH9-RD. A novel heterozygous mutation of MYH9 (c.2344-2345delGTinsTA, p.T782Y) was discovered by targeted sequencing technology. Immunofluorescence analysis of neutrophils confirmed abnormal aggregation of MYH9 protein. The results of this study should expand the MYH9 gene mutation spectrum and provide reference for subsequent researchers and genetic counseling.     

Anti-IL-33 antibody treatment inhibits airway inflammation in a murine model of allergic asthma

Interleukin (IL)-33 is a recently described member of the IL-1 family and has been shown to induce production of T helper type 2 cytokines. In this study, an anti-IL-33 antibody was evaluated against pulmonary inflammation in mice sensitized and challenged with ovalbumin. The anti-IL-33 or a control antibody (150 μg/mouse) was given intraperitoneally as five doses before the sensitization and antigen challenge. Treatment with anti-IL-33 significantly reduced serum IgE secretion, the numbers of eosinophils and lymphocytes, and concentrations of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid compared with administration of a control antibody. Histological examination of lung tissue demonstrated that anti-IL-33 significantly inhibited allergen-induced lung eosinophilic inflammation and mucus hypersecretion. Our data demonstrate for the first time that anti-IL-33 antibody can prevent the development of asthma in a mouse model and indicate that blockade of IL-33 may be a new therapeutic strategy for allergic asthma.     

Genome-Wide Identification, Classification, and Expression Analysis of Autophagy-Associated Gene Homologues in Rice (Oryza sativa L.)

Autophagy is an intracellular degradation process for recycling macromolecules and organelles. It plays important roles in plant development and in response to nutritional demand, stress, and senescence. Organisms from yeast to plants contain many autophagy-associated genes (ATG). In this study, we found that a total of 33 ATG homologues exist in the rice [Oryza sativa L. (Os)] genome, which were classified into 13 ATG subfamilies. Six of them are alternatively spliced genes. Evolutional analysis showed that expansion of 10 OsATG homologues occurred via segmental duplication events and that the occurrence of these OsATG homologues within each subfamily was asynchronous. The Ka/Ks ratios suggested purifying selection for four duplicated OsATG homologues and positive selection for two. Calculating the dates of the duplication events indicated that all duplication events might have occurred after the origin of the grasses, from 21.43 to 66.77 million years ago. Semi-quantitative RT–PCR analysis and mining the digital expression database of rice showed that all 33 OsATG homologues could be detected in at least one cell type of the various tissues under normal or stress growth conditions, but their expression was tightly regulated. The 10 duplicated genes showed expression divergence. The expression of most OsATG homologues was regulated by at least one treatment, including hormones, abiotic and biotic stresses, and nutrient limitation. The identification of OsATG homologues showing constitutive expression or responses to environmental stimuli provides new insights for in-depth characterization of selected genes of importance in rice.     

Cytological and morphology characteristics of natural microsporogenesis within Camellia oleifera

Camellia oleifera is believed to exhibit a complex intraspecific polyploidy phenomenon. Abnormal microsporogenesis can promote the formation of unreduced gametes in plants and lead to sexual polyploidy, so it is hypothesized that improper meiosis probably results in the formation of natural polyploidy in Camellia oleifera. In this study, based on the cytological observation of meiosis in pollen mother cells (PMCs), we found natural 2n pollen for the first time in Camellia oleifera, which may lead to the formation of natural polyploids by sexual polyploidization. Additionally, abnormal cytological behaviour during meiosis, including univalent chromosomes, extraequatorial chromosomes, early segregation, laggard chromosomes, chromosome stickiness, asynchronous meiosis and deviant cytokinesis (monad, dyads, triads), was observed, which could be the cause of 2n pollen formation. Moreover, we confirmed a relationship among the length–width ratio of flower buds, stylet length and microsporogenesis. This result suggested that we can immediately determine the microsporogenesis stages by phenotypic characteristics, which may be applicable to breeding advanced germplasm in Camellia oleifera.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01002-5.     

Cloning and expression of a new rat procarboxypeptidase B gene in Escherichia coli and purification of recombination carboxypeptidase B

A new coding sequence of the procarboxypeptidase B gene was obtained from SD rat fresh pancreas by RT-PCR and highly expressed in Escherichia coli in inclusion bodies. The folded procarboxypeptidase B was subjected to trypsin enzymatic cleavage to produce active carboxypeptidase B, subsequently, carboxypeptidase B was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Octyl FF, as a result, 40 mg carboxypeptidase B per litre cell culture with specific activity 7.42 u/mg was achieved. Further research showed that the obtained recombinant carboxypeptidase B could substitute carboxypeptidase B isolated from pancreas.