Generation and utilization of endostatin-sensitive cell lines for assessing the biological activity of endostatin.

Recombinant proteins are gaining increasing popularity for treating human diseases. The clinical effectiveness of recombinant proteins is directly related to their biological activity, which is an important indicator in drug development and quality control. However, certain recombinant proteins have unclear or complex signal pathways, making detecting their activity in vitro difficult. For instance, recombinant human endostatin (endostatin), a new antitumor drug developed in China, lacks a sensitive and stable assay for its biological activity since being market approval. To address this issue, we performed a genome-wide screening of immortalized human umbilical vein endothelial cells (HUVECs) using a CRISPR/Cas9 knockout library containing 20,000 targeted genes. We identified two potential endostatin-resistant genes, NEPSPP and UTS2, and successfully constructed a highly sensitive cell line, HUVEC-UTS2-3#, by knocking down the UTS2 gene. Based on the optimized parameters of HUVEC-UTS2-3# cells, we established a new method for detecting the biological activity of endostatin. The method was validated, and it produced results consistent with primary HUVEC cells but with higher sensitivity and more stable data. The use of gene-editing technology provides a novel solution for detecting the biological activity of recombinant proteins that other methods cannot detect.

Development of an Adeno-Associated Virus-Vectored SARS-CoV-2 Vaccine and Its Immunogenicity in Mice.

Owing to the outbreak of the novel coronavirus (SARS-CoV-2) worldwide at the end of 2019, the development of a SARS-CoV-2 vaccine became an urgent need. In this study, we developed a type 9 adeno-associated virus vectored vaccine candidate expressing a dimeric receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) and evaluated its immunogenicity in a murine model. The vaccine candidate, named AAV9-RBD virus, was constructed by inserting a signal peptide to the N-terminus of two copies of RBD, spaced by a linker, into the genome of a type 9 adeno-associated virus. In vitro assays showed that HeLa cells infected by the recombinant AAV virus expressed high levels of the recombinant RBD protein, mostly found in the cell culture supernatant. The recombinant AAV9-RBD virus was cultured and purified. The genome titer of the purified recombinant AAV9-RBD virus was determined to be 2.4 × 1013 genome copies/mL (GC/mL) by Q-PCR. Balb/c mice were immunized with the virus by intramuscular injection or nasal drip administration. Eight weeks after immunization, neutralizing antibodies against the new coronavirus pseudovirus were detected in the sera of all mice; the mean neutralizing antibody EC50 values were 517.7 ± 292.1 (n=10) and 682.8 ± 454.0 (n=10) in the intramuscular injection group and nasal drip group, respectively. The results of this study showed that the recombinant AAV9-RBD virus may be used for the development of a SARS-CoV-2 vaccine.

The Impact of Haze Pollution on Regional Eco-Economic Treatment Efficiency in China: An Environmental Regulation Perspective.

"The Gray Great Wall" formed by haze pollution is an increasingly serious issue in China, and the resulting air pollution has brought severe challenges to human health, the socio-economy and the world ecosystem. Based on the facts above, this paper uses China's province-level panel data from 2009 to 2016, systematically measures the heterogeneous structure of regional ecological economic (eco-economic) treatment efficiency through a Super Slacks-Based Measure (SBM) model and dynamic threshold models, and analyzes the forcing mechanism of haze pollution pressure on regional eco-economic treatment efficiency from an environmental regulation perspective. Results indicated that China's eco-economic treatment has been vigorously promoted, which is significantly conducive to green growth upgrading. However, the process has a large developmental scope due to regional heterogeneity. Interestingly, the forcing impact of haze pollution on regional eco-economic treatment efficiency is limited by the "critical mass" of environmental regulations: a weak degree of regulation will facilitate an increase in regional eco-economic treatment efficiency through the forcing effect of haze pollution pressure. Once environmental regulation reaches a critical level, a stronger degree of regulation will suppress the forcing effect of haze pollution in turn and it will decrease the regional eco-economic treatment efficiency. This paper endeavors to clarify the differences, suitability and dependency in the process of ecological transformation for Chinese local governments in different regions and provide policy references for a regional ecological transformation matching system.