Objective:To obtain a polyvalent single-chain variable fragment（scFv）against West Nile virus through PEGylation in order to improve its antigen-binding ability and neutralizing activity.Methods:A scFv carrying a C-terminal cysteine residue（scFvC）was constructed by introducing Cys into the C-terminal of scFv against West Nile virus. Then the multimerization of scFvC was achieved by targeting the thiol group of Cys with maleimide-activated polyethylene glycol. ELISA was used to detect the antigen-binding activity of the multivalent scFvC. Pseudovirus-based neutralization assay was used to evaluate the neutralizing activity of the multivalent scFvC in vitro. One-way analysis of variance was used for statistical analysis. Results:The PEGylated scFvC multimers showed higher antigen-binding ability than the monomeric scFvC. In the pseudovirus-based neutralization assay，both monomeric scFvC and PEGylated scFvC multimers showed good neutralizing activity compared with the control group（ P<0.000 1）. Moreover，the PEGylated scFvC multimers showed a more effective ability to block the pseudovirus infection in target cells（ P<0.05），suggesting that the PEGylated scFvC multimers could enhance their function in vitro through avidity effect. Conclusion:In this study，a scFvC targeting West Nile virus is successfully constructed and its polyvalent form is generated through PEGylation，which improves the antigen-binding and neutralizing activity of the parental scFv.

Objective:To obtain fucose-free anti-West Nile virus nonstructural protein 1 (NS1) antibody and evaluate its antibody-dependent cell-mediated cytotoxicity (ADCC).Methods:The guanosine diphosphate-fucose transporter SLC35C1 in CHO cells was knocked out using CRISPR/Cas9 gene editing technology to obtain the fucose-free cell line CHO SLC35C1 -/-. CHO SLC35C1 -/- cells were used to produce fucose-free anti-West Nile virus NS1 antibodies. The binding abilities of the antibodies to the target antigen of West Nile virus NS1 protein and the human high-affinity IgG Fc receptor hFcγRⅠ (hCD64) were detected by ELISA and flow cytometry, respectively. The ADCC activity of the antibodies was detected by ADCC reporter gene assay. One-way analysis of variance was used for statistical analysis. Results:CHO SLC35C1 -/- cells expressed green fluorescent protein but not Lens culinaris agglutinin. The anti-West Nile virus NS1 antibodies produced by CHO SLC35C1 -/- cells with a fucose content of 0.22% could bind to West Nile virus NS1 protein in a concentration-dependent manner. Compared with the wild-type antibodies, the fucose-free anti-West Nile virus NS1 antibodies showed a stronger binding ability to hFcγRⅠ(hCD64), as indicated by a significant increase in fluorescence intensity. The ADCC reporter gene assay showed that the fucose-free anti-West Nile virus NS1 antibodies had increased activity as compared with the wild-type antibodies ( P<0.001). Conclusion:The fucose-free anti-West Nile virus NS1 antibodies may be used to protect against West Nile virus infection.

Objective:To obtain fucose-free anti-West Nile virus nonstructural protein 1 (NS1) antibody and evaluate its antibody-dependent cell-mediated cytotoxicity (ADCC).Methods:The guanosine diphosphate-fucose transporter SLC35C1 in CHO cells was knocked out using CRISPR/Cas9 gene editing technology to obtain the fucose-free cell line CHO SLC35C1 -/-. CHO SLC35C1 -/- cells were used to produce fucose-free anti-West Nile virus NS1 antibodies. The binding abilities of the antibodies to the target antigen of West Nile virus NS1 protein and the human high-affinity IgG Fc receptor hFcγRⅠ (hCD64) were detected by ELISA and flow cytometry, respectively. The ADCC activity of the antibodies was detected by ADCC reporter gene assay. One-way analysis of variance was used for statistical analysis. Results:CHO SLC35C1 -/- cells expressed green fluorescent protein but not Lens culinaris agglutinin. The anti-West Nile virus NS1 antibodies produced by CHO SLC35C1 -/- cells with a fucose content of 0.22% could bind to West Nile virus NS1 protein in a concentration-dependent manner. Compared with the wild-type antibodies, the fucose-free anti-West Nile virus NS1 antibodies showed a stronger binding ability to hFcγRⅠ(hCD64), as indicated by a significant increase in fluorescence intensity. The ADCC reporter gene assay showed that the fucose-free anti-West Nile virus NS1 antibodies had increased activity as compared with the wild-type antibodies ( P<0.001). Conclusion:The fucose-free anti-West Nile virus NS1 antibodies may be used to protect against West Nile virus infection.

Objective:To obtain a polyvalent single-chain variable fragment（scFv）against West Nile virus through PEGylation in order to improve its antigen-binding ability and neutralizing activity.Methods:A scFv carrying a C-terminal cysteine residue（scFvC）was constructed by introducing Cys into the C-terminal of scFv against West Nile virus. Then the multimerization of scFvC was achieved by targeting the thiol group of Cys with maleimide-activated polyethylene glycol. ELISA was used to detect the antigen-binding activity of the multivalent scFvC. Pseudovirus-based neutralization assay was used to evaluate the neutralizing activity of the multivalent scFvC in vitro. One-way analysis of variance was used for statistical analysis. Results:The PEGylated scFvC multimers showed higher antigen-binding ability than the monomeric scFvC. In the pseudovirus-based neutralization assay，both monomeric scFvC and PEGylated scFvC multimers showed good neutralizing activity compared with the control group（ P<0.000 1）. Moreover，the PEGylated scFvC multimers showed a more effective ability to block the pseudovirus infection in target cells（ P<0.05），suggesting that the PEGylated scFvC multimers could enhance their function in vitro through avidity effect. Conclusion:In this study，a scFvC targeting West Nile virus is successfully constructed and its polyvalent form is generated through PEGylation，which improves the antigen-binding and neutralizing activity of the parental scFv.

OBJECTIVE To optimize the management model of drugs used in investigator-initiated trial(IIT).METHODS With benchmarking analysis,based on the practical work experience of a tertiary specialized hospital in the field of IIT drug management in Shanghai,a thorough review was conducted,involving relevant laws,regulations,and academic literature to establish benchmark criteria and the evaluation standards.Starting from the initiation of IIT projects,a detailed comparative analysis of key processes was carried out,such as the receipt,storage,distribution,use and recycling of drugs for trial.The deficiencies in the current management of IIT drugs were reviewed in detail and a series of optimization suggestions were put forward.RESULTS It was found that the authorized records of drug management were missing,the training before project implementation was insufficient,and the records of receipt and acceptance of IIT drugs were incomplete.In light of these existing problems,improvement measures were put forward,including strengthening the training of drug administrators and stipulating that only drug administrators with pharmacist qualifications be eligible to inspect and accept drugs,etc.The related systems were improved,and 17 key points of quality control for the management of IIT drugs were developed.CONCLUSIONS A preliminary IIT drug management system for medical institutions has been established,which helps to improve the institutional framework of medical institutions in this field.

Objective:To establish an in vivo infection model of West Nile virus (WNV) pseudovirus and evaluate the neutralizing activity of antibody WNV-XH1.Methods:A stable cell line that can package the WNV pseudovirus was established in the early stage to prepare the pseudovirus supernatant. The supernatant was concentrated and infected BHK21 cells to detect the titer of the pseudovirus. After intraperitoneal injection of the pseudovirus into C57BL/J mice, bioluminescence imaging was performed to observe the infection status of the pseudovirus in the mice. After simultaneous infection, blood was collected and ELISA was used to detect NS1 levels in mouse serum. The in vivo functional activity of antibody WNV-XH1 was evaluated using the established mouse infection model.Results:Fluorescence was detected in C57BL/J mice infected with WNV pseudovirus, and the NS1 levels in the peripheral blood serum of mice infected with pseudovirus were significantly higher than those of non infected mice (1.453±0.09vs0.305±0.018). After intravenous administration of WNV-XH1 antibody before the attack, the fluorescence signal in the mice decreased and the serum NS1 level decreased (0.384±0.015).Conclusions:A successful in vivo infection model of WNV pseudovirus was established, and it was confirmed that the antibody WNV-XH1 had a protective effect against WNV pseudovirus infection in vivo.

Objective:To establish an antibody expression system to reduce the antibody-dependent enhancement (ADE) effect of target antibody.Methods:Site-directed mutagenesis was used to mutate the 234 and 235 sites of the Fc region of the mammalian cell antibody expression vector-L234A and L235A to establish the antibody expression vector pFRT-IgG1κ-FcM. An antibody Wt-WNV with significant ADE effect obtained in previous work was selected and expressed by the pFRT-IgG1κ-FcM system to obtain mutant antibody FcM-WNV. The binding ability of FcM-WNV to target antigen West Nile virus envelope protein-DⅢ (WNV E-DⅢ) was detected by ELISA, and the its binding ability to human high-affinity IgG Fc receptor hFcγRⅠ (hCD64 ) was analyzed by flow cytometry. The neutralizing activity of FcM-WNV in vitro was detected by pseudovirus infection of host cells (BHK21 and K562). Results:The expression levels of FcM-WNV and Wt-WNV were comparable, and FcM-WNV could recognize and bind to WNVE-DIII in a concentration-dependent manner. Compared with Wt-WNV, the binding ability of FcM-WNV to hCD64 was significantly weakened, showing a significant decrease in fluorescence intensity. Consistent with the previous experimental results, Wt-WNV at a concentration of 5 μg/ml significantly enhanced the infection of K562 by WNV pseudovirus, while FcM-WNV at a concentration of 5 μg/ml could effectively block pseudovirus infection in both K562 and BHK21 cells.Conclusions:The established antibody expression system can effectively reduce the ADE effect of the target antibody.

Objective:To establish an in vivo infection model of H5N1 pseudovirus and to detect the neutralizing activity of FHA3 antibody using this model. Methods:Based on the sequence information of hemagglutinin (HA) and neuraminidase (NA) of A/Anhui/1/2005/H5N1 strain, two recombinant plasmids of pcDNA3.1-HA5 and pcDNA3.1-NA1 were constructed. The two plasmids and plasmid pNL4-3.Luc.R-E- were co-transfected into 293T cells to prepare H5N1 pseudovirus supernatant. The morphology of pseudovirus particles in the supernatant was observed by electron microscopy. MDCK cells were infected with the pseudovirus supernatant and the virus titer was detected. BALB/c mice were injected with the pseudovirus supernatant by intraperitoneal injection and subjected to bioluminescence imaging at 2, 5, 8, and 12 d after infection to detect the pseudovirus infection in vivo. The functional activity of FHA3 antibody in vivo was evaluated using the established mouse infection model. Results:The recombinant plasmids pcDNA3.1-HA5 and pcDNA3.1-NA1 were correctly constructed and could be used to prepare pseudovirus supernatants of high titer by co-transfecting 293T cells with the plasmid pNL4-3.Luc.R-E-. The virus particles were round under electron microscope. H5N1 pseudovirus-infected mice exhibits strong fluorescence signals, which were attenuated by FHA3 treatment before challenge.Conclusions:The in vivo infection model of H5N1 pseudovirus was successfully constructed and FHA3 antibody was proved to be protective against the pseudovirus infection.

Isochrysis galbana is considered an ideal bait for functional foods and nutraceuticals of humans because of its high fucoxanthin(Fx)content.However,multi-omics analysis of the regula-tory networks for Fx biosynthesis in I.galbana has not been reported.In this study,we report a high-quality genome assembly of I.galbana LG007,which has a genome size of 92.73 Mb,with a contig N50 of 6.99 Mb and 14,900 protein-coding genes.Phylogenetic analysis confirmed the monophyly of Haptophyta,with I.galbana sister to Emiliania huxleyi and Chrysochromulina tobinii.Evolutionary analysis revealed an estimated divergence time between I.galbana and E.huxleyi of～133 million years ago.Gene family analysis indicated that lipid metabolism-related genes exhibited significant expansion,including IgPLMT,IgOAR1,and IgDEGS1.Metabolome analysis showed that the content of carotenoids in I.galbana cultured under green light for 7 days was higher than that under white light,and β-carotene was the main carotenoid,accounting for 79.09％of the total carotenoids.Comprehensive multi-omics analysis revealed that the content of β-carotene,antheraxanthin,zeaxanthin,and Fx was increased by green light induction,which was significantly correlated with the expression of IgMYB98,IgZDS,IgPDS,IgLHCX2,IgZEP,IgLCYb,and IgNSY.These findings contribute to the understanding of Fx biosynthesis and its regulation,pro-viding a valuable reference for food and pharmaceutical applications.

Abstract Low level light therapy utilizes the photochemical effect of red light to induce reactions in the irradiated body tissue to achieve the therapeutic effect. Myopia and amblyopia are diseases which threaten the eye health of children and adolescents. Red light has been used to treat amblyopia for decades. Recently, its application in myopia prevention and control has become a new hotspot. This review summarizes the application of low level red light in children and adolescents with myopia and amblyopia from the aspects of intervention mode, effect, factors, mechanism and safety to provide reference for future researches.