On the basis of the origin comparison of known endothelial genesis inhibitors, a 417-bp cDNA fragment was amplified from umbilical cord by RT-PCR and cloned into the expression vector pPIC9, followed by transformation into Pichia pastoris GS115. The resulted yeast was induced with methanol to express recombinant protein. The resulted protein was purified from culture broth and designated as EDI-8t. The in vitro study showed that EDI-8t, originated from collagen VIII, could specifically inhibit the growth and migration of bovine aortic endothelial cells (BAEC) stimulated by basic fibroblast growth factor (bFGF). The protein also exhibited the activity to cause cell apoptosis. In vivo EDI-8t showed the identical activity comparing with endostatin to inhibit the growth of liver tumor transplanted into nude mice. Interestingly, EDI-8t showed higher activity than endostatin to inhibit tumor growth in metastatic model of melanoma mice.
Amino Acid Sequence ; Angiogenesis Inhibitors ; biosynthesis ; genetics ; isolation & purification ; Animals ; Antineoplastic Agents ; pharmacology ; Base Sequence ; Cattle ; Cells, Cultured ; Collagen Type VIII ; chemistry ; genetics ; Endothelium, Vascular ; metabolism ; Genetic Vectors ; genetics ; Human Umbilical Vein Endothelial Cells ; chemistry ; Humans ; Mice ; Mice, Nude ; Molecular Sequence Data ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana.

METHODSSeven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement.

RESULTSThe 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the p19 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus P1 protein produced no obvious enhancement GFP expression.

CONCLUSIONTransient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.

Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Plant Viruses ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Potexvirus ; genetics ; RNA Interference ; Tobacco ; genetics ; metabolism

OBJECTIVETo explore the mechanisms by which HFBI fusions increase recombinant fusion protein accumulation in plants.

METHODSThe HFBI sequence from Trichoderma reesei was synthesized and two plant expression vectors for expression of green fluorescence protein (GFP) and GFP-HFBI were constructed. The vectors were inoculated in Nicotiana benthamiana plants through agroinfiltration, and the expression levels and mRNA accumulation levels of GFP in Nicotiana leaves were examined by Western blotting, ELISA and RT-PCR.

RESULTSThe HFBI fusion tag significantly enhanced the accumulation of GFP in the leaves of N. benthamiana without causing toxic effects. Endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of spherical protein particles in the plant cells.

CONCLUSIONHFBI fusions can increase the accumulation of its fusion partner in plants by forming stable protein particles, which probably shields the target protein from endogenous protease-induced degadation. HFBI fusion technology provides an alternative to improving recombinant protein expression in plants from agroinfection-compatible expression vectors.

Endoplasmic Reticulum ; Genetic Engineering ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; Imidazoles ; chemistry ; Plant Leaves ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Tobacco ; genetics ; metabolism

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.
Adaptation, Physiological ; Butanols ; metabolism ; Environment ; Fermentation ; Pichia ; cytology ; metabolism

Objective: To study the effect of low-to-moderate dose glucocorticoid therapy on viral clearance time in patients with COVID-19. Methods: A total of 72 patients diagnosed with COVID-19 from January 19 to February 17, 2020 at the First Affiliated Hospital, School of Medicine, Zhejiang University were recruited. All patients received oral abidol and/or combined lopinavir/ritonavir, darunavir antiviral, and symptomatic supportive care. Among them, 51 patients received methylprednisolone (0.75-1.50 mg·kg^-1·d^-1) (glucocorticoid treatment group), and 21 patients who did not use glucocorticoid were the control group. The time of stable virologic conversion insputumand the time of radiologic recovery in lungsince onset were compared between the two groups and among the normal patients.The Kruskal-Wallis test or Fisher exact test was used to compare the difference between groups. Results: The median ages of the glucocorticoid group and the control group were 52 [interquartile range (IQR):45, 62] years and 46 (IQR: 32, 56)years, and the differences were significant (P<0.05). The clinical conditions at hospital admission were different between the two groups (P<0.01). There were 52.0% critical ill patients in the glucocorticoid treatment group, compared to that of 71.4% normal patients in the control group. The median times from the onset tostable virologic conversion to negative in the two groups were 15 (IQR:13,20) days and 14 (IQR:12,20) days (P>0.05), and the difference was no statistically significant. The median times from onset to radiologic recovery were 13 (IQR: 11,15) days and 13 (IQR:12,17) days in the two groups, and there was no difference (P>0.05). In ordinary patients, the median timesfrom the onset tostable virologic conversion insputum were no difference (P>0.05), with 13 (IQR:11,18) days in the glucocorticoid group and 13 (IQR:12,15) days in the control group; The median times from onset to radiologic recovery in lungwere also no difference (P>0.05), with 12 (IQR: 10,15)days in the glucocorticoid group and 13 (IQR: 12,17) days inthe control group. Conclusions Low-to-moderate glucocorticoid treatment has no effect on the time of virus clearance in patients with different clinical types of COVID-19. The glucocorticoid is not recommended since no effectiveness on accelerating the improvement of radiologic recovery in lung has been observed.

Objective:To study the effect of low-to-moderate dose glucocorticoid therapy on viral clearance in patients with COVID-19.Methods:A total of 72 patients diagnosed with COVID-19 from January 19 to February 17, 2020 at the First Affiliated Hospital, Zhejiang University School of Medicine were recruited. All patients received oral arbidol and combination of lopinavir/ritonavir or darunavir/cobistitat for antiviral therapy, and symptomatic supportive care. Among them, 51 patients received methylprednisolone (0.75-1.50 mg·kg -1·d -1) (glucocorticoid treatment group), and 21 patients did not use glucocorticoid (control group). The time of virologic negative conversion in sputum and the time of radiologic recovery in lung since onset were compared between the two groups. The Kruskal-Wallis test or Fisher exact test was used to compare the difference between groups. Results:The median ages of the glucocorticoid group and the control group were 52 (45, 62) and 46 (32, 56) years ( χ2=4.365, P<0.05). The clinical conditions at hospital admission were different between the two groups ( P<0.01). The severe cases accounted for 52.0%, while moderate cases in the control group accounted for 71.4%. The median times from the onset to virologic negative conversion in the two groups were 15 (13, 20) and 14 (12, 20) days ( P>0.05). The median times from onset to radiologic recovery were 13 (11, 15) and 13 (12, 17) days in the two groups ( P>0.05). In moderate cases, the median times from the onset to virologic conversion in sputum were 13 (11, 18) days in the glucocorticoid group and 13 (12, 15) days in the control group ( P>0.05). The median times from onset to radiologic recovery in lung were 12 (10, 15) and 13 (12, 17) days, respectively ( P>0.05). Conclusions:Low-to-moderate glucocorticoid treatment has no effect on the time of virus clearance in patients with different clinical types of COVID-19, and also no effect on accelerating radiologic recovery in lung, so it is not recommended.