One of the technical bottlenecks limiting the high yield of 1,4-butanediamine is the insufficient tolerance of strains to 1,4-butanediamine. Enhancing the tolerance of strains to 1,4-butanediamine is therefore a primary challenge that needs to be addressed for the construction of strains with high yields of 1,4-butanediamine. Staphylococcus pasteuri 326180 exhibits exceptional tolerance to high-concentration 1,4-butanediamine, serving as both an ideal model for studying the mechanism underlying the 1,4-butanediamine tolerance and a novel host for constructing strains capable of efficiently producing 1,4-butanediamine. However, for both the research on the tolerance mechanism and the modification of chassis strains, gene editing of S. pasteuri needs to be carried out at the molecular level. The research objective of this paper is to establish a genetic manipulation system for S. pasteuri, laying foundation for subsequent studies on tolerance mechanism and the modification of chassis strains. This study systematically optimized the electroporation conditions, including key parameters such as the growth phase of cells, electric field strength, electroporation buffer, and recovery medium, successfully establishing an electroporation method for S. pasteuri. Additionally, we constructed the gene editing plasmid pCpfOA by replacing the resistance expression cassette, optimized the selection markers for gene editing, and finally established a CRISPR/Cpf1-based gene editing technology for S. pasteuri, achieving an editing efficiency of 90%. The genetic manipulation system of S. pasteuri established in this study provides technical support for research into the tolerance mechanism of this bacterium and the genetic modification of chassis strains.
Staphylococcus/drug effects* ; Gene Editing/methods* ; Electroporation/methods* ; Plasmids/genetics* ; CRISPR-Cas Systems ; Genetic Engineering/methods*

Objective:To evaluate the short-term efficacy of parallel overlapping anastomosis in 3D laparoscopic radical resection for transverse colon cancer.Methods:A retrospective analysis of clinical data was conducted on 73 patients who underwent 3D laparoscopic radical resection and parallel overlapping anastomosis for reconstruction of digestive tract transverse colon cancer at He'nan Provincial People's Clinical Medical School of Zhengzhou University between Apr 2019 and Jul 2023.Results:The average operation time was (165.3±28.9) min, the parallel overlapping anastomosis time was (15.6±2.6) min, the intraoperative blood loss was (47.7±19.4) ml, the first postoperative exhaust time was(2.2±0.9) d, the first time getting out of bed was (1.1±0.4) d, the number of lymph node dissection was (18.2±5.1), the first postoperative oral fluid diet time was (4.4±0.8) d, the postoperative hospital stay was (6.4±1.8) d. Anastomotic leakage occurred in one patient , who was discharged after enterostomy; Two patients with poor wound healing were cured after wound debridement and dressing change; Pulmonary infection in one patient was healed by anti-infection treatment. The postoperative complication rate was 6%, and no complications such as anastomotic bleeding, anastomotic stenosis, intestinal obstruction, abdominal infection occurred.Conclusion:Parallel overlapping anastomosis is a safe and reliable anastomotic procedure for the reconstruction of digestive tract in patients undergoing 3D laparoscopic radical resection of transverse colon cancer.

AIM:To investigate the protective effect and mechanism of zinc ions on lung ischemia/reperfusion injury(LIRI)in rats.METHODS:SPF SD rats aged 6～8 weeks were divided randomly into 4 groups:control(control)group,ischemia/reperfusion(I/R)group,zinc chloride(ZnCl2)+I/R group,and PI3K inhibitor(LY294002)+ZnCl2+I/R group.Inductively coupled plasma mass spectrometry(ICP-MS)was used to measure the concentration of zinc ions in lung tissues,while the degree of lung tissue injury was assessed by HE staining,the alveolar damage index,and the lung wet/dry weight ratio.qPCR was used to detect the mRNA expression of solute carrier family 39 member 8(SLC39A8/ZIP8),with the TUNEL assay used to determine the level of apoptosis in lung tissue.The phosphorylation levels of caspase3,PI3K,AKT,GSK-3β,ZIP8,and solute carrier family 30 member 9(SLC30A9/ZNT9)were detected by Western blot,while the mitochondrial membrane potential was measured by the mitochondrial extraction kit and JC-1 mitochondrial mem-brane potential detection kit.RESULTS:Compared with the I/R group,the zinc ion level in the ZnCl2+I/R group in-creased(P<0.01),with the qPCR results showing that the expression level of ZIP8 also increased(P<0.01).The West-ern blot results demonstrated that the phosphorylation levels of PI3K/AKT/GSK-3β and cleaved caspase-3/pro were both in-creased(P<0.01 or P<0.05).In addition,the level of caspase-3 was decreased(P<0.01),the ZIP8 level was increased(P<0.05),whereas the level of ZNT9 was not significantly different(P>0.05).The mitochondrial membrane potential was increased(P<0.01)and the level of apoptosis was decreased(P<0.01).The results of HE staining,total lung water(TLW),lung index of quantitative assessment of histology(IQA),and lung tissue wet/dry weight ratio showed that the de-gree of injury was reduced significantly(P<0.05 or P<0.01).Compared with the ZnCl2+I/R group,the LY294002+Zn-Cl2+I/R group had a significant reduction in zinc ion levels(P<0.05),while qPCR showed a significant reduction in ZIP8 expression(P<0.01).Western blot showed that the phosphorylation level of PI3K/AKT/GSK-3β was decreased(P<0.01),the level of caspase-3/pro-caspase-3 was increased(P<0.01)the level of ZIP8 was decreased(P<0.05),al-though there was no significant difference in ZNT9(P>0.05).Measurements of the mitochondrial membrane potential demonstrated a significant decrease(P<0.01),while the TUNEL results showed that the level of apoptosis had increased(P<0.05).HE staining,TLW,IQA and lung tissue wet/dry weight ratio indicated that the degree of injury was aggravated significantly(P<0.05 or P<0.01).CONCLUSION:Administration of zinc chloride in rats has a protective role in lung ischemia/reperfusion injury by activating the PI3K/AKT pathway,leading to inactivation of GSK-3β,stabilization of the mitochondrial membrane potential level,and inhibition of cell apoptosis.

AIM:To investigate the protective effect and mechanism of zinc ions on lung ischemia/reperfusion injury(LIRI)in rats.METHODS:SPF SD rats aged 6～8 weeks were divided randomly into 4 groups:control(control)group,ischemia/reperfusion(I/R)group,zinc chloride(ZnCl2)+I/R group,and PI3K inhibitor(LY294002)+ZnCl2+I/R group.Inductively coupled plasma mass spectrometry(ICP-MS)was used to measure the concentration of zinc ions in lung tissues,while the degree of lung tissue injury was assessed by HE staining,the alveolar damage index,and the lung wet/dry weight ratio.qPCR was used to detect the mRNA expression of solute carrier family 39 member 8(SLC39A8/ZIP8),with the TUNEL assay used to determine the level of apoptosis in lung tissue.The phosphorylation levels of caspase3,PI3K,AKT,GSK-3β,ZIP8,and solute carrier family 30 member 9(SLC30A9/ZNT9)were detected by Western blot,while the mitochondrial membrane potential was measured by the mitochondrial extraction kit and JC-1 mitochondrial mem-brane potential detection kit.RESULTS:Compared with the I/R group,the zinc ion level in the ZnCl2+I/R group in-creased(P<0.01),with the qPCR results showing that the expression level of ZIP8 also increased(P<0.01).The West-ern blot results demonstrated that the phosphorylation levels of PI3K/AKT/GSK-3β and cleaved caspase-3/pro were both in-creased(P<0.01 or P<0.05).In addition,the level of caspase-3 was decreased(P<0.01),the ZIP8 level was increased(P<0.05),whereas the level of ZNT9 was not significantly different(P>0.05).The mitochondrial membrane potential was increased(P<0.01)and the level of apoptosis was decreased(P<0.01).The results of HE staining,total lung water(TLW),lung index of quantitative assessment of histology(IQA),and lung tissue wet/dry weight ratio showed that the de-gree of injury was reduced significantly(P<0.05 or P<0.01).Compared with the ZnCl2+I/R group,the LY294002+Zn-Cl2+I/R group had a significant reduction in zinc ion levels(P<0.05),while qPCR showed a significant reduction in ZIP8 expression(P<0.01).Western blot showed that the phosphorylation level of PI3K/AKT/GSK-3β was decreased(P<0.01),the level of caspase-3/pro-caspase-3 was increased(P<0.01)the level of ZIP8 was decreased(P<0.05),al-though there was no significant difference in ZNT9(P>0.05).Measurements of the mitochondrial membrane potential demonstrated a significant decrease(P<0.01),while the TUNEL results showed that the level of apoptosis had increased(P<0.05).HE staining,TLW,IQA and lung tissue wet/dry weight ratio indicated that the degree of injury was aggravated significantly(P<0.05 or P<0.01).CONCLUSION:Administration of zinc chloride in rats has a protective role in lung ischemia/reperfusion injury by activating the PI3K/AKT pathway,leading to inactivation of GSK-3β,stabilization of the mitochondrial membrane potential level,and inhibition of cell apoptosis.

Objective:To evaluate the short-term efficacy of parallel overlapping anastomosis in 3D laparoscopic radical resection for transverse colon cancer.Methods:A retrospective analysis of clinical data was conducted on 73 patients who underwent 3D laparoscopic radical resection and parallel overlapping anastomosis for reconstruction of digestive tract transverse colon cancer at He'nan Provincial People's Clinical Medical School of Zhengzhou University between Apr 2019 and Jul 2023.Results:The average operation time was (165.3±28.9) min, the parallel overlapping anastomosis time was (15.6±2.6) min, the intraoperative blood loss was (47.7±19.4) ml, the first postoperative exhaust time was(2.2±0.9) d, the first time getting out of bed was (1.1±0.4) d, the number of lymph node dissection was (18.2±5.1), the first postoperative oral fluid diet time was (4.4±0.8) d, the postoperative hospital stay was (6.4±1.8) d. Anastomotic leakage occurred in one patient , who was discharged after enterostomy; Two patients with poor wound healing were cured after wound debridement and dressing change; Pulmonary infection in one patient was healed by anti-infection treatment. The postoperative complication rate was 6%, and no complications such as anastomotic bleeding, anastomotic stenosis, intestinal obstruction, abdominal infection occurred.Conclusion:Parallel overlapping anastomosis is a safe and reliable anastomotic procedure for the reconstruction of digestive tract in patients undergoing 3D laparoscopic radical resection of transverse colon cancer.

Fibroblast growth factor 21 (FGF21) has been only reported to prevent type 1 diabetic nephropathy (DN) in the streptozotocin-induced type 1 diabetes mellitus (T1DM) mouse model. However, the FVB (Cg)-Tg (Cryaa-Tag, Ins2-CALM1) 26OVE/PneJ (OVE26) transgenic mouse is a widely recommended mouse model to recapture the most important features of T1DM nephropathy that often occurs in diabetic patients. In addition, most previous studies focused on exploring the preventive effect of FGF21 on the development of DN. However, in clinic, development of therapeutic strategy has much more realistic value compared with preventive strategy since the onset time of DN is difficult to be accurately predicted. Therefore, in the present study OVE26 mice were used to investigate the potential therapeutic effects of FGF21 on DN.

Objective: To understand the needs of primary and secondary school students, parents and teachers for comprehensive sexuality education content, and to explore the applicability of comprehensive sexuality education content in China. Methods: By using the method of stratified cluster sampling, 5 571 primary and middle school students and 4 835 parents from Beijing, Chongqing, Heilongjiang (Harbin and Jiamusi) and Hubei (Wuhan and Xiaogan) were selected for questionnaire survey. Depending on the physical health monitoring points of various provinces and cities, 176 sexuality education related teachers in nine regions were surveyed. The development of the questionnaire was based on the content framework of comprehensive sexuality education proposed in the International Technical Guidelines for Sexuality Education. Results: Students had a higher demand for knowledge on the concepts of "relationship" (53.4% for primary school students, 57.6% for junior high school students) and "health and well-being skills" (71.1% for primary school students, 55.9% for junior high school students). Parents had higher demand for most comprehensive sexuality education content with the exception of lower need for "sex and sexual behavior (74.7% of primary school parents, 80.8% of junior high school parents)" and "sexual and reproductive health (primary parents 74.3%, junior high school parents 75.8%)". Teachers had a high demand for most comprehensive sexuality education content, but primary school teachers had a lower need for "values, rights, culture and sex(65.9%)", "sex and sexuality(60.1%)" and "sexual and reproductive health(66.8%)". The results of correspondence analysis showed that junior middle school teachers had the highest demand for comprehensive education content, followed by primary school parents, junior middle school parents, primary school teachers, junior middle school students and primary school students. Conclusion Teachers and parents have a higher demand for most comprehensive sexuality education content, while lower demand for some relatively sensitive content. Students have a relatively low demand for comprehensive sexuality education content. It is recommended to further improve the content of school sexuality education based on the actual needs of China.

Objective: To understand the awareness of sexual knowledge among primary and secondary school students, as well as their attitudes towards school sex education and to provide a reference for school-based sex education. Methods: A total of 5 531 primary and secondary school students were selected from Beijing, Chongqing, Heilongjiang (Harbin and Jiamusi) and Hubei (Wuhan and Xiaogan) by stratified cluster sampling and were investigated with self-reported questionnaire. Results: Sex-related knowledge scored 62.33±19.35, with a pass rate of 61.9%, and the excellent rate 17.5%. Sexual knowledge of junior high school students scored 64.30±19.07, with the pass rate of 64.7%, and the excellent rate 22.0%. A large proportion of primary students reported unaware of pubertal growth and HIV/AIDS-related knowledge, and a large proportion of junior high students unaware of reproductive physiology. Multivariate Logistic regression analysis showed region, grade, gender, and suburban areas were related to the level of sexual knowledge(P<0.05). More than 76.0% primary students and 85.9% middle school students agreed on the importance of school sexuality education. 59.7% of primary school students and 73.3% of junior high school students hoped to include school sexuality education in compulsory education courses; 59.8% of primary school students and 68.3% of junior high school students felt that school sexuality education should be equipped with specialized teachers. Conclusion Chinese primary and secondary school students are lack of sexual knowledge. As students have strong wills to get more information on sexual knowledge, schools should provide them with more comprehensive and effective sex education through appropriate courses and activities.

Objective To summarize the experience of emergency treatment of primary liver cancer rupture bleeding and improve the level of diagnosis and treatment of the disease.Methods From October 2008 to December 2012.The clinical data of 11 patients with spontaneous rupture of primary liver cancer were analyzed retrospectively.Results Three cases were given conservative treatment.Among them,1 case died,2 bleeding cases were hemostasis,8 cases were operated.Surgical operation is the main method of emergency treatment.The effect after operation was satisfactory.1 case with liver tumor resection died in the first days after the operation,1 case was transferred to higher level hospitals for further treatment because massive ascities in third days after operation,6 cases were successfully discharged,survival rate was over 3 months.Conclusions In the process of emergency treatment we should pay attention to reduce the rate of misdiagnosis and missed diagnosis.When the diagnosis was confirmed,we must treat the shock and plan an active operation scheme.Operation mode can be selected of tumor resection or simple hemostatic gauze tamponade suture hemostasis,saving lives of patients in the shortest time is the goal of emergency treatment.