Objective:To construct three photosensitive fusion proteins between protein G C3 domain and Avitag for site-spe-cific biotinylation of goat IgG.Methods:DNA sequences of protein G C3 domain with amber codon mutation and C-terminal containing 1,2,3 Avitag were obtained by chemical synthesis,respectively,and sub-cloned into pTXB1 plasmid to construct three fusion pro-tein expression vectors.Three plasmids and pEVOL-pBpF plasmid were co-transformed into competent bacterium E.coli BL21,respec-tively.Target fusion proteins CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 were prepared using genetic codon extension technology,and were purified by His Trap Chelating HP column,then biotinylated by Avitag/BirA system.Photo-conjugation efficiency,crosslink site and antigen-binding activity of photo-conjugates were investigated by SDS-PAGE,control experiment,and competitive ELISA.Results:Three expression vectors were successfully constructed,and three designed photoactivatable fusion proteins CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 were successfully expressed with molecular weights consistent with their theoretical values.Heavy-chain conjugation efficiencies of biotinylated fusion protein with goat IgG were 85%with Fc-specific conjugation,and conjugates maintained its antigen-binding activity after photo-conjugation.Conclusion:CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 fusion proteins are successfully prepared,and mediates by those photosensitive fusion proteins,biotins molecules are conjugated to Fc fragment of goat IgG,results site-specific biotinylation of goat IgG.

Objective:To construct three photosensitive fusion proteins between protein G C3 domain and Avitag for site-spe-cific biotinylation of goat IgG.Methods:DNA sequences of protein G C3 domain with amber codon mutation and C-terminal containing 1,2,3 Avitag were obtained by chemical synthesis,respectively,and sub-cloned into pTXB1 plasmid to construct three fusion pro-tein expression vectors.Three plasmids and pEVOL-pBpF plasmid were co-transformed into competent bacterium E.coli BL21,respec-tively.Target fusion proteins CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 were prepared using genetic codon extension technology,and were purified by His Trap Chelating HP column,then biotinylated by Avitag/BirA system.Photo-conjugation efficiency,crosslink site and antigen-binding activity of photo-conjugates were investigated by SDS-PAGE,control experiment,and competitive ELISA.Results:Three expression vectors were successfully constructed,and three designed photoactivatable fusion proteins CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 were successfully expressed with molecular weights consistent with their theoretical values.Heavy-chain conjugation efficiencies of biotinylated fusion protein with goat IgG were 85%with Fc-specific conjugation,and conjugates maintained its antigen-binding activity after photo-conjugation.Conclusion:CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 fusion proteins are successfully prepared,and mediates by those photosensitive fusion proteins,biotins molecules are conjugated to Fc fragment of goat IgG,results site-specific biotinylation of goat IgG.

This study aimed to observe the protective effect of momordicine I, a triterpenoid compound extracted from momordica charantia L., on isoproterenol (ISO)-induced hypertrophy in rat H9c2 cardiomyocytes and investigate its potential mechanism. Treatment with 10 μM ISO induced cardiomyocyte hypertrophy as evidenced by increased cell surface area and protein content as well as pronounced upregulation of fetal genes including atrial natriuretic peptide, β-myosin heavy chain, and α-skeletal actin; however, those responses were markedly attenuated by treatment with 12.5 μg/ml momordicine I. Transcriptome experiment results showed that there were 381 and 447 differentially expressed genes expressed in comparisons of model/control and momordicine I intervention/model, respectively. GO enrichment analysis suggested that the anti-cardiomyocyte hypertrophic effect of momordicine I may be mainly associated with the regulation of metabolic processes. Based on our transcriptome experiment results as well as literature reports, we selected glycerophospholipid metabolizing enzymes group VI phospholipase A 2 (PLA2G6) and diacylglycerol kinase ζ (DGK-ζ) as targets to further explore the potential mechanism through which momordicine I inhibited ISO-induced cardiomyocyte hypertrophy.Our results demonstrated that momordicine I inhibited ISO-induced upregulations of mRNA levels and protein expressions of PLA2G6 and DGK-ζ. Collectively, momordicine I alleviated ISO-induced cardiomyocyte hypertrophy, which may be related to its inhibition of the expression of glycerophospholipid metabolizing enzymes PLA2G6 and DGK-ζ.

Objective:To explore the feasibility and value of performing a three-sided encapsulation procedure based on fascia anatomy in laparoscopic lateral lymph node dissection (LLND) for middle and low rectal cancer.Methods:This was a retrospective review. The study cohort comprised patients who met the diagnostic criteria for rectal cancer according to the Chinese Guidelines for the Diagnosis and Treatment of Colorectal Cancer, had a short lymph node diameter of >5 mm on the lateral side within the 15 days before surgery, were evaluated as feasible candidates for laparoscopic total mesorectal excision+LLND surgery, had been diagnosed with low or intermediate level rectal cancer, and whose tumor was less than 8 cm away from the anal verge according to pathological examination of the operative specimen. Patients with a history of other malignant tumors of the abdomen or with incomplete follow-up data were excluded. Forty-two patients with middle and low rectal cancer who had undergone lateral lymph node dissection in diagnosis and treatment center of Gastrointestinal Cancer of Guangdong Hospital of Chinese Medicine from Jan.2018 to Dec.2022 were enrolled. There were 24 men (57.1%) and 18 women (42.9%) aged 58.4±11.8 years and the median BMI was 22.5 (19.3–24.1) kg/m 2. The main point of the three-sided encapsulation procedure is to expand the external side medial to the external iliac artery and vein, narrowing the range of exterior side dissection. The anterior-medial side is designed to expand the vesical fascia to define the range of anterior-medial side extension. The internal side is fully extended to the ureterohypogastric nerve fascia; the distal point of the caudal extension reaches the level of the Alcock canal and the bottom reaches the piriformis, enabling dissection of the obturator nerve and No.283 lymph nodes. No.263D lymph nodes are dissected by exposing the internal iliac artery and its branches, dissecting the group No.263P lymph nodes, and severing the inferior vesical artery. Finally, the lateral lymphatic tissue is completely resected. Relevant variables were recorded, including the number of lateral lymph nodes detected, the rate of lymph node metastasis, operation duration, intraoperative blood loss, postoperative complications, postoperative hospital stay, and 3-year overall survival rate. Results:Laparoscopic surgery was successfully completed in all patients with no conversions to open surgery and no intraoperative complications. Twenty-seven (64.3%) of the study patients underwent left-sided LLND, 10 (23.8%) right-sided LLND, and five (11.9%) bilateral LLND, with lymph nodes cleared on both sides. All patients' lymph nodes were examined pathologically. A median of 17.0 (11.7, 26.0) lymph nodes was detected, the median of lateral lymph nodes being 5.0 (2.0, 10.2). The median operation time was 254.5 (199.0, 325.2) minutes. The median intra-operative blood loss was 50.0 (30.0, 100.0) mL. All patients were diagnosed with adenocarcinoma by pathological examination of the operative specimen. Two patients developed postoperative intestinal obstruction, one lymphatic leakage, and one a perineal incision infection. There were no cases of anastomotic leakage. The median postoperative hospital stay was 6.0 (5.0, 7.0) days and the median follow-up time 23.5 (9.0, 36.7) months. During follow-up, three patients (7.1%) died of tumor recurrence and metastasis. Two (4.8%) experienced mild urinary dysfunction, and one (2.4%) had moderate postoperative erectile dysfunction. One patient (2.4%) was found to have prostate and lung metastases 3 month after surgery. The 3-year overall survival rate was 74.4%.Conclusions:Three sided encapsulation is a safe and feasible procedure for LLND, achieving accurate and complete clearance of lateral lymphatic tissue.

Objective:To explore the ethical issues arising from stem cell research in China and propose countermeasures to regulate stem cell research.Methods:This article analyzed the ethical issues arising from stem cells in terms of regulatory systems, source channels, production processes, storage industries, and translational applications.Results:Combining the current ethical situation of stem cell research and the practical exploration of the authors' unit, we proposed countermeasures to address the ethical issues of stem cell research in five aspects, including forming a scientific and effective stem cell ethical regulatory system, building an open and transparent stem cell traceability mechanism, creating a GMP-compliant stem cell production platform, selecting a legal and compliant stem cell storage institution, and conducting ethical stem cell clinical research.Conclusions:It is of great practical significance to strengthen the ethical construction of stem cell research and regulate stem cell transformation research to promote the healthy development of the stem cell industry.

In recent years，with the change in lifestyle and social environment and the increase in pressure in both life and work，male fertility has decreased significantly in China，and the incidence of male infertility has increased year by year，which has brought great challenges to andrologists. Traditional Chinese medicine （TCM） has a definite curative effect in the treatment of male infertility and is widely applied in clinical practice. In order to clarify the role of TCM in different types and each stage of male infertility，the China Association of Chinese Medicine （CACM） invited outstanding young andrologists in the clinic of TCM and western medicine to discuss topics such as idiopathic oligospermia and teratospermia，abnormal semen liquefaction，varicocele，immune infertility，improving success ratio of assisted reproductive technology，and ameliorating depression or anxiety. They conducted in-depth discussions on the advantages，characteristics，disadvantages，diseases responding specifically，and advantageous aspects of TCM treatment. The causes of male infertility and related links of treatment were summarized. Due to the unclear etiology and complex pathogenesis of male infertility，western medicine cannot achieve a good curative effect，while TCM，taking the holistic view as the core，specializes in improving functional diseases and can correspond to multiple targets and factors，with comprehensive treatments such as internal treatment and external treatment. This study summarized the advantageous diseases and advantageous stages of TCM treatment alone and integrated TCM and western medicine treatment and put forward suggestions for the treatment of the diseases by TCM and western medicine in order to promote the therapeutic effects and advantages of TCM among andrologists，increase mutual learning and communication between TCM and western physicians，provide patients with excellent and personalized treatment plans in clinical practice，and improve the curative effect of male infertility and fertility of males in China.

Objective:To investigate the efficacy and safety of retroperitoneal soft tissue sarcoma resection combined with nephrectomy.Methods:The clinical data of 27 cases undergoing retroperitoneal soft tissue sarcoma resection combined with nephrectomy at the Gastrointestinal Tumor Center , Guangdong Provincial Hospital , Traditional Chinese Medicine from Jun 2017 to Aug 2022 were retrospectively analyzed for the indication of nephrectomy, postoperative progression of renal insufficiency and survival rate.Results:Twenty-six cases (96%) achieved R 0/R 1 resection and 1 case nderwent R 2 resection. Six cases underwent combined unilateral nephrectomy and 21 patients underwent combined multi-organ resection with a median number of resections of 4 (2,5). Postoperative pathology suggested that the combined resected kidney was positive for tumor infiltration in 17 cases. Five cases had Clavien-Dindo grade 3 or higher complications and no deaths occurred within 30 days after surgery. At the 90th day after surgery, 19 cases (70%) had decreased renal function ( Z=2.88, P=0.04), with a median decrease of -3.96 (-30.36, 0.31)ml·(min·1.73 m 2) -1, including 8 cases of preoperative Chronic Kidney Disease(CKD)1 stage progression (6 cases of CKD 2 stage, 2 cases of CKD 3 stage); 2 cases of CKD 2 stage progressed to CKD 3 stage; 1 case of preoperative CKD 3 stage progressed to CKD 4 stage. During the follow-up period of 3-38 months, no patient progressed to CKD 5 stage and no patient required dialysis treatment. Conclusion:Retroperitoneal soft tissue sarcoma resection combined with nephrectomy is safe and feasible while improving tumor radicality.

Objective:To explore the feasibility and value of performing a three-sided encapsulation procedure based on fascia anatomy in laparoscopic lateral lymph node dissection (LLND) for middle and low rectal cancer.Methods:This was a retrospective review. The study cohort comprised patients who met the diagnostic criteria for rectal cancer according to the Chinese Guidelines for the Diagnosis and Treatment of Colorectal Cancer, had a short lymph node diameter of >5 mm on the lateral side within the 15 days before surgery, were evaluated as feasible candidates for laparoscopic total mesorectal excision+LLND surgery, had been diagnosed with low or intermediate level rectal cancer, and whose tumor was less than 8 cm away from the anal verge according to pathological examination of the operative specimen. Patients with a history of other malignant tumors of the abdomen or with incomplete follow-up data were excluded. Forty-two patients with middle and low rectal cancer who had undergone lateral lymph node dissection in diagnosis and treatment center of Gastrointestinal Cancer of Guangdong Hospital of Chinese Medicine from Jan.2018 to Dec.2022 were enrolled. There were 24 men (57.1%) and 18 women (42.9%) aged 58.4±11.8 years and the median BMI was 22.5 (19.3–24.1) kg/m 2. The main point of the three-sided encapsulation procedure is to expand the external side medial to the external iliac artery and vein, narrowing the range of exterior side dissection. The anterior-medial side is designed to expand the vesical fascia to define the range of anterior-medial side extension. The internal side is fully extended to the ureterohypogastric nerve fascia; the distal point of the caudal extension reaches the level of the Alcock canal and the bottom reaches the piriformis, enabling dissection of the obturator nerve and No.283 lymph nodes. No.263D lymph nodes are dissected by exposing the internal iliac artery and its branches, dissecting the group No.263P lymph nodes, and severing the inferior vesical artery. Finally, the lateral lymphatic tissue is completely resected. Relevant variables were recorded, including the number of lateral lymph nodes detected, the rate of lymph node metastasis, operation duration, intraoperative blood loss, postoperative complications, postoperative hospital stay, and 3-year overall survival rate. Results:Laparoscopic surgery was successfully completed in all patients with no conversions to open surgery and no intraoperative complications. Twenty-seven (64.3%) of the study patients underwent left-sided LLND, 10 (23.8%) right-sided LLND, and five (11.9%) bilateral LLND, with lymph nodes cleared on both sides. All patients' lymph nodes were examined pathologically. A median of 17.0 (11.7, 26.0) lymph nodes was detected, the median of lateral lymph nodes being 5.0 (2.0, 10.2). The median operation time was 254.5 (199.0, 325.2) minutes. The median intra-operative blood loss was 50.0 (30.0, 100.0) mL. All patients were diagnosed with adenocarcinoma by pathological examination of the operative specimen. Two patients developed postoperative intestinal obstruction, one lymphatic leakage, and one a perineal incision infection. There were no cases of anastomotic leakage. The median postoperative hospital stay was 6.0 (5.0, 7.0) days and the median follow-up time 23.5 (9.0, 36.7) months. During follow-up, three patients (7.1%) died of tumor recurrence and metastasis. Two (4.8%) experienced mild urinary dysfunction, and one (2.4%) had moderate postoperative erectile dysfunction. One patient (2.4%) was found to have prostate and lung metastases 3 month after surgery. The 3-year overall survival rate was 74.4%.Conclusions:Three sided encapsulation is a safe and feasible procedure for LLND, achieving accurate and complete clearance of lateral lymphatic tissue.

To explore the potential mechanism of frankincense volatile oil in the prevention and treatment of cardiac hypertrophy based on in vitro cell experiment and network pharmacology. METHODS: The anti-hypertrophic effect of frankincense volatile oil was investigated by isoproterenol induced H9c2 cardiomyocytes hypertrophy model. The active chemical components and targets of frankincense volatile oil and targets associated with cardiac hypertrophy were obtained by CNKI, Pubmed, Pubchem databases, etc. String database and Cytoscape 3.8.0 software were used to construct protein-protein interaction network (PPI) and a network of "drug-active component-key target-disease" of frankincense volatile oil in order to screen the key targets of frankincense volatile oil against cardiac hypertrophy. The fluorescent quantitative PCR experiments were performed to verify those key targets. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis of key target genes were performed using David online analysis tool. RESULTS: In vitro cell experiments showed that frankincense volatile oil significantly inhibited the isoproterenol induced increases in cardiomyocytes surface area and protein synthesis, and upregulations of ANP and β-MHC mRNA. A total of 87 active components and 36 ingredient-disease targets of frankincense volatile oil were screened. Network analysis showed that ESR1, NOS3, PTGS2, TNF, MAPK14, and PPARG were key targets. Fluorescence quantitative PCR experiments results indicated that frankincense volatile oil inhibited isoproterenol induced upregulations of ESR1, PTGS2, TNF, and MAPK14 mRNA levels, and downregulations of NOS3, PPARG mRNA levels, respectively. In addition, the GO functional enrichment analysis showed that its biological pathways mainly included lipopolysaccharide-mediated signaling pathway, positive regulation of nitric oxide biosynthetic process, caveola, enzyme binding, etc. The KEGG pathway enrichment analysis included 22 KEGG pathways, which were closely related to VEGF signaling pathway, TNF signaling pathway, sphingolipid signaling pathway and others. CONCLUSION: The active components of frankincense volatile oil may regulate VEGF signaling pathway, TNF signaling pathway, Sphingolipid signaling pathway by acting on ESR1, NOS3, PTGS2, TNF, MAPK14 and PPARG targets, thereby affecting the regulation of lipopolysaccharide-mediated signaling pathway, positive regulation of nitric oxide biosynthetic process, caveola, and enzyme binding, and improving cardiac hypertrophy.

AIM: To investigate the effect of oxypeucedanin (OPD) on doxorubicin resistance in human breast cancer MCF-7/DOX cells and its possible mechanism. METHODS: MCF-7/DOX cells were cultured in vitro, MTT assay was used to detect the effect of OPD on the survival of MCF-7/DOX cells, and the effect of OPD combined with different concentrations of doxorubicin on the proliferation of MCF-7/DOX cells were investigated. The effect of OPD combined with doxorubicin on the expression of genes including MDR1, MRP1, AGPAT2, CHKA, CEPT1, DGKA, PCYT1A, PLA2G15 in MCF-7/DOX cells was measured by qRT-PCR. The effect of OPD combined with doxorubicin on the protein expression of MDR1, MRP1, CHKA and CCTα in MCF-7/DOX cells was determined by Western blot. RESULTS: The IC