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 investigate the safety and efficacy of laparoscopic fundoplication in the treatment of gastroesophageal reflux related cough (GERC).Methods:Clinical data of 138 GERC patients undergoing laparoscopic fundoplication at our center from Jan 2013 to Dec 2017 were analyzed and patients were followed up until Dec 2022.Results:A total of 138 patients underwent surgery successfully.The median follow-up time was 51 (7-130) months.There were no mortality,nor major complications. In 138 GERC patients, GERC was cured in 85 patients,the cure rate of fundoplication was 61.6%, and altogether 112 patients reported effective, the effective rate was 81.2%. Multivariate regression analysis showed that normal esophageal peristalsis was independent predictors of good efficacy of fundoplication[ OR=3.810(1.373,10.573), P=0.01]. Conclusions:Laparoscopic fundoplication has long term efficacy for refractory GERC. Esophageal motility is an important dynamic characteristic that affects the postoperative efficacy of GERC.

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 investigate the safety and efficacy of laparoscopic fundoplication in the treatment of gastroesophageal reflux related cough (GERC).Methods:Clinical data of 138 GERC patients undergoing laparoscopic fundoplication at our center from Jan 2013 to Dec 2017 were analyzed and patients were followed up until Dec 2022.Results:A total of 138 patients underwent surgery successfully.The median follow-up time was 51 (7-130) months.There were no mortality,nor major complications. In 138 GERC patients, GERC was cured in 85 patients,the cure rate of fundoplication was 61.6%, and altogether 112 patients reported effective, the effective rate was 81.2%. Multivariate regression analysis showed that normal esophageal peristalsis was independent predictors of good efficacy of fundoplication[ OR=3.810(1.373,10.573), P=0.01]. Conclusions:Laparoscopic fundoplication has long term efficacy for refractory GERC. Esophageal motility is an important dynamic characteristic that affects the postoperative efficacy of GERC.

ObjectiveTo investigate the effect of kinesin family member 15 （KIF15） on the proliferation of hepatocellular carcinoma （HCC） cells and its mechanism of action. MethodsTCGA and GEPIA datasets were analyzed to determine the expression of KIF15 in HCC and its effect on tumor stage and survival. Quantitative real-time PCR and Western blot were used to measure the expression level of KIF15 in human-derived HCC cell lines （HepG2， Hep3B， MHCC-97H， and LM3） and human normal liver cell line L02 cultured in vitro， and Hep3B and HepG2 were selected for subsequent studies. CCK-8 assay， plate colony formation assay， and EdU staining were performed for Hep3B cells transfected with shRNA-NC or shRNA-KIF15 and HepG2 cells transfected with LV-vector or LV-KIF15 to evaluate the viability and proliferative capacity of these cells. GSEA was used to analyze the potential signaling pathways associated with KIF15 in HCC， and Western blot was used for detection. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsThe analysis of TCGA and GEPIA datasets showed that in HCC patients， the expression of KIF15 in HCC tissue was significantly higher than that in normal tissue， and the HCC patients with high KIF15 expression tended to have a poorer prognosis. Compared with sh-NC-Hep3B， sh3-Hep3B showed significant reductions in the mRNA and protein levels of KIF15 （P<0.05）， cell viability， clone formation number， and EdU positive rate （all P<0.05）. Compared with vector-HepG2， LV-KIF15-HepG2 showed significant increases in the mRNA and protein levels of KIF15 （P<0.05）， cell viability， clone formation number， and EdU positive rate （all P<0.05）. Subcutaneous tumor assay showed that compared with sh-NC-Hep3B， sh3-Hep3B showed reductions in tumor volume and tumor weight， as well as a significant reduction in the immunohistochemical score of Ki67 and a significant increase in the immunohistochemical score of TUNEL （P<0.05）. GSEA analysis showed that the PI3K/AKT/mTOR pathway was positively correlated with KIF15 in HCC （NES=1.59， P<0.001）. Western blot showed that LY294002 could inhibit the PI3K/AKT/mTOR pathway upregulated in LV-KIF15-HepG2， and compared with LV-KIF15-HepG2， LY294002+LV-KIF15-HepG2 showed significant reductions in cell viability， clone formation number， and EdU positive rate （all P<0.05）. ConclusionKIF15 enhances the viability and proliferative capacity of HCC cells by upregulating the PI3K/AKT/mTOR signaling pathway.

Objective:To explore the long-term efficacy of laparoscopic fundoplication for proton pump inhibitor dependent gastroesophageal reflux disease (GERD).Method:Clinical data of proton pump inhibitor dependent GERD patients who underwent fundoplication at the Rocket Force Characteristic Medical Center from Jan to Jun 2012 were analyzed, including GERD symptom score, subjective symptom relief rate, PPI discontinuation rate and surgical satisfaction, as well as recurrence and complications.Result:A total of 160 GERD patients were included in this study, with 64% of patients having respiratory symptoms. Nissen and Toupet fundoplication were performed in 43 and 117 cases, respectively, with a follow-up time of (127±3) months. The postoperative GERD symptom scores of the patients were significantly lower than before treatment (all P<0.001); The subjective relief of overall symptoms in the digestive tract and airway problem was 90% (80%, 100%) and 100%, respectively. The PPI discontinuation rate was 86%, and the overall satisfaction rate of the treatment was 92%, and the satisfaction rate of patients with respiratory symptoms was 89%. 7% of patients experienced varying degrees of symptomatic recurrence, 4% of patients re-underwent endoscopic treatment and/or laparoscopic fundoplication due to symptom recurrence. The incidence of long-term postoperative dysphagia, bloating, belching, increased exhaust, abdominal pain, diarrhea, and constipation were 11.3%, 16.9%, 0, 1.3%, 0, 2.5%, and 5.6%, respectively. Conclusions:Laparoscopic fundoplication has good long-term efficacy in the treatment of GERD. A small number of patients may experience postoperative recurrence, as well as complications such as dysphagia and gas-bloat syndrome. Most recurrent patients can achieve good therapeutic effect by redoing endoscopic treatment or redoing surgery.

Since there is a lack of obvious clinical symptoms in the early stage of hepatocellular carcinoma (HCC), most patients have progressed to the advanced stage at the time of confirmed diagnosis. There are limited treatment options for HCC patients who miss the opportunity for surgery, so it is of great importance to find new therapeutic targets. Tumor-associated macrophages (TAMs) are a group of macrophages existing in the tumor immune microenvironment and affect the malignant behaviors of HCC cells and the state of immune escape within the tumor. This article introduces the origin and classification of TAM, summarizes the role and mechanism of TAMs in vascular proliferation, invasion and metastasis, formation and maintenance of stemness, and anti-tumor immunity in HCC, and briefly describes the current research advances in therapeutic targets for TAM, and it is pointed out that targeting TAM may be a promising direction for clinical treatment.

Objective:To investigate the safety and efficacy of remimazolam combined with afentanyl for fiberoptic bronchoscopy.Methods:Sixty patients admitted to Chifeng Hospital for fiberbronchoscopy from January to April 2022 were selected and divided into two groups by random number table method: remimazolam group (group R) and propofol group (group P), 30cases in each group. After intravenous injection of alfentanil for anesthesia induction, group R was sedated by intravenous injection of remidazolam besylate, and group P was sedated by intravenous injection of propofol emulsion. When sufficient sedation was achieved, fiberoptic bronchoscopy was performed. The patients were scored with the Mini-Mental State Examination (MMSE) before examination and before leaving the room. The recovery rate of sedation and the recovery rate of drugs during operation were compared. Blood pressure, heart rate (HR), bispectral index (BIS), SpO 2 value and Modified Observer′s Assessment of Alertness/Sedation (MOAA/S) score were compared before induction (T 0), at the beginning of examination (T 1), immediately when fiber bronchoscope reached juga (T 2), at the end of surgery (T 3), immediately, when patients regained consciousness (T 4). Drug onset and recovery time (time out of hospital) as well as the incidence of intraoperative and postoperative adverse reactions were recorded in both groups. Results:There was no statistically significant difference in general condition, MMSE score and examination time between the two groups (all P>0.05). There was no statistically significant difference between the two groups in the success rate of sedation and the number of sedative remedy times (all P>0.05). The number of additional drugs in group R was significantly higher than that in group P ( P<0.05). The systolic blood pressure, diastolic blood pressure and BIS values of patients in group P at T 1 and T 2 were significantly lower than those in group R (all P<0.05). After administration, the MOAA/S score of the two groups began to decrease, and the decrease of the P group was significantly greater than that of the R group, and the MOAA/S value of the patients was the lowest at the 3rd and 4th minutes after administration, respectively. The time from the beginning of administration to the MOAA/S score ≤3 in group P was significantly shorter than that in group R (all P<0.05). The incidence of pain and respiratory depression after injection in group P was significantly higher than that in group R ( P<0.05). Conclusions:The application of afentanil combined with remimazolam in the patients undergoing fiberoptic bronchoscopy has good sedative effect and high anesthesia quality, and has no obvious effect on cognitive function and few adverse reactions, so it is safe and effective.