Objective To perform quantirative study of HEK293 cell genomic DNA by microfluidic chip-based digital PCR technology, and to provide new ideas for more accurate gene quantification and genomic DNA related detection.Methods Firstly, the HEK293 cell DNA was obtained by Spin Column, identified and analyzed for the purity by agarose gel electrophoresis. Then, the genomic DNA of HEK293 cells was quantified by traditional ultraviolet spectrophotometry, Qubit fluorescence quantification and microfluidic chip-based digital PCR technology and statistically analyzed.Results The concentration of genomic DNA in HEK293 cells was detected as 100. 08 ng/μL by ultraviolet spectrophotometry, 93. 98 ng/μL by Qubit fluorescence quantification and the copy number was determined as 29 722. 81 copies/μL by digital PCR. Roughly calculated based on human single-copy genome of about 3. 3 pg, the converted copy numbers of spectrophotometry and Qubit fluorescence quantification were about 30 327 and 28 479 copies/μL, respectively. The deviation of the digital PCR detection value from the spectrophotometry detection value was only 2%, and the deviation from the Qubit detection value was only4%.Conclusion In this study, HEK293 genomic DNA was detected and compared by digital PCR, spectrophotometry and Qubit fluorescence, which provides a new detection method and idea for DNA quantification, and also provides a certain reference for the quantification of other nucleic acids.

Objective To construct a plasmid reference for the detection of residual DNA from early region 1A(E1A) protein and simian virus 40 large T antigen(SV40LTA), and to explore a digital PCR(dPCR)-based analytical technique for copy number calibration of plasmid reference to quantify plasmid reference accurately, thereby providing a new idea for host cell DNA residual detection in biological products.Methods The plasmid pUC19-E1A-SV40LTA was constructed, and the whole gene was synthesized and then amplified to obtain sufficient reference plasmid. The digital PCR system was employed to determine the copy numbers using double fluorescence channels of E1A and SV40LTA primer-probe sets, respectively.The reference material calibrated by digital PCR was subsequently applied to qPCR to establish a method for detecting the specific E1A and SV40LTA sequences. The developed qPCR assay was systematically validated for plasmid reference linearity, accuracy, limit of quantification(LOQ), precision, specificity, applicability, and stability. Furthermore, the established qPCR system was utilized for the detection of recombinant adenovirus(rAdV) sample and recombinant adeno-associated virus(rAAV) sample.Results The copy numbers of E1A and SV40LTA targets in the plasmid reference were found to be essentially consistent through digital PCR using dual fluorescence channels. The detection values detected by E1A and SV40LTA primer-probe sets were 3. 55 × 109and 3. 48 × 109copies/μL, respectively. The coefficient of variation(CV) of these two values was 1. 42%, and the mean value of 3. 51 × 109copies/μL was taken as the calibration value of digital PCR.The qPCR system established by using digital PCR to calibrate copy number exhibited good linearity, accuracy, LOQ, precision, specificity, applicability and stability, and the recovery rates for rAdV and rAAV samples were between 70% and130%.Conclusion A reference plasmid for detecting E1A and SV40LTA residues in biological products was established,and a more sensitive and accurate digital PCR method was introduced for quantification, which can be used to detect the residual DNA of E1A and SV40LTA in gene therapy products produced with HEK293 and HEK293T as host cells.

Objective To explore the antioxidant capacity of the volatile oil components of the Mongolian medicine Zhenbaowan and the mechanism of action in the treatment of rheumatoid arthritis(RA)by using bioinformatics technology,GC-MS technology,and antioxidant activity experiment.Methods The volatile oil in Zhenbaowan was extracted using water vapor distillation and the volatile oil components were qualitatively analyzed by GC-MS.Based on the results of GC-MS analysis,bioinfor-matics analysis was used to investigate the main components,key targets,and pathways of the volatile oil of Zhenbaowan in pre-venting and treating RA.Meanwhile,the antioxidant activity of the volatile oil was determined and analyzed using the 2,2-Diphen-yl-1-picrylhydrazyl(DPPH)method.Results A total of 115 volatile oil components were identified in Zhenbaowan,of which the top 48 components accounted for 97.66%of the total volatile oil content.Bioinformatics analysis revealed that Zhenbaowan can regulate signal transduction,inflammation,and protein phosphorylation through 147 RA-related targets,and intervene in signaling pathways such as PI3K-Akt,MAPK,and RAS.Molecular docking results showed that the seven main components of Zhenbaowan volatile oil can spontaneously bind to the five core targets.Antioxidant activity tests have proved that the volatile oil of Zhenbaowan has a more significant antioxidant capacity.Conclusion Using GC-MS high-throughput analysis technology combined with bioinformatic analysis and antioxidant activity test,the pharmacodynamic substances and mechanism of action of the volatile oil components of Zhenbaowan,a Mongolian medicine,in the treatment of RA are hypothesized.It provides a theoretical basis for the further study of the volatile oil components of Zhenbaowan.

Objective:To evaluate the association between preoperative serum β 2-microglobulin (β 2MG) concentrations and postoperative delirium (POD) in elderly patients. Methods:The study selected patients who underwent knee or hip arthroplasty under spinal-epidural anesthesia on an elective basis at Qingdao Municipal Hospital from May 2021 to November 2022. The patients were divided into a POD group and a non-POD group based on the occurrence of POD. The study was conducted as part of the Perioperative Neurocognitive Impairment and Biomarkers Lifestyle Cohort, which was a nested case-control study. The study collected baseline data from two groups of patients and analyzed the differences between them. Logistic regression was used to identify the risk factors for POD. The stability of the regression model was tested using sensitivity analysis. The mediation model was used to examine whether cerebrospinal fluid (CSF) biomarkers mediated the relationship between β 2MG and POD. The receiver operating characteristic curve was drawn and the area under the curve was calculated to evaluate the accuracy of preoperative β 2MG concentrations and CSF biomarker concentration in predicting POD. Results:There were 57 cases in POD group and 449 cases in non-POD group. The results of logistic regression analysis showed that the increased β 2MG and CSF total tau protein (t-tau) concentrations were risk factors for POD, and the increased CSF β-amyloid 42 concentration was a protective factor for POD after adjustment for multiple confounders such as age, gender, education, Mini-Mental State Examination, history of hypertension and infusion volume ( P<0.05). The results of mediation analysis showed that the serum β 2MG′s effect on POD was partly mediated by t-tau (18.1%). The results of the receiver operating characteristic curve showed that the area under the curve of the β 2MG concentration combined with the CSF biomarker concentration was 0.742. Conclusions:Elevated preoperative serum β 2MG concentration is a risk factor for POD in elderly patients, and the relationship may be partly mediated by CSF t-tau.

Objective:To evaluate the relationship between preoperative serum bilirubin concentrations and postoperative delirium (POD) in the patients undergoing knee or hip replacement.Methods:Medical records from 413 patients undergoing knee or hip arthroplasty were selected from August 2020 to October 2023 at Qingdao Municipal Hospital using a nested case-control design based on the PNDABLE study cohort. The patients were divided into POD group ( n=77) and non-POD group ( n=336) according to whether POD occurred. Univariate analysis was used to analyze the influencing factors. Logistic regression was used to identify the risk factors for POD. The significance of mediation effect was tested. The receiver operating characteristic curve was drawn to evaluate the accuracy of risk factors in predicting POD. Results:There were significant differences in age, education time, ratio of diabetes history, Memorial Delirium Assessment Scale score, total bilirubin concentration, direct bilirubin concentration, indirect bilirubin concentration, Aβ 42 concentration, p-tau concentration, t-tau concentration, Aβ 42/p-tau ratio and Aβ 42/t-tau ratio between POD group and non-POD group ( P<0.05). The results of logistic regression analysis showed that preoperative serum total bilirubin, direct bilirubin and indirect bilirubin were risk factors for POD ( P<0.05). The results of mediation effects showed that the concentration of total tau protein in CSF partly mediated the relationship between high serum total bilirubin, direct bilirubin and indirect bilirubin concentrations and POD. The area under the receiver operating characteristic curve of total bilirubin, direct bilirubin and indirect bilirubin combined with CSF biomarker concentrations in predicting POD was 0.83 ( P<0.001). Conclusions:Preoperative elevated concentrations of total bilirubin, direct bilirubin and indirect bilirubin are risk factors for POD in the patients undergoing knee or hip replacement. CSF t-tau concentration has a partly mediating role in the association between serum total bilirubin, direct bilirubin and indirect bilirubin concentrations and the development of POD.

Objective To explore the effect of mixed antibiotics on the intestinal flora of mice to affect the immune regulation of the body,explore the role of intestinal flora in the development of obesity,and provide new ideas and ways for the prevention and treatment of obesity.Methods Seventy-two 10-week-old male C57BL/6 mice were randomly divided into blank control(Ctrl)group,high-fat diet(HF)group,antibiotic(ABX)group,and combined(COMB)group(n=18).At the first 2 weeks(lavage intervention weeks),Ctrl and HF group were given normal saline gavage;ABX and COMB group were given mixed antibiotics gavage,and the gavage volume was 0.2 mL/animal/day.For the following 8 weeks(feeding weeks),Ctrl and ABX group were fed with ordinary diet,HF and COMB group were fed with high-fat diet.Body weight was measured weekly,and fasting blood glucose was measured before and after gavage,and at the 4th and 8th week of feeding.Oral glucose tolerance test was performed at the end of the experiment.The organ coefficient was measured and the cell morphology of white and brown adipose tissue was observed.Serum was collected for the determination of free fatty acid,high-density lipoprotein,low-density lipoprotein,triglyceride,and total cholesterol.Serum TNF-α,IL-10,IL-4,IL-13,IL-33 and MCP-1 was detected by ELISA.The stool of mice was collected for second generation sequencing.Results High-fat diet increased body weight,serum total cholesterol,low-density lipoprotein,IL-13,IL-33,TNF-α,MCP-1 content,and decreased glucose tolerance and organ coefficient in mice(P＜0.05).From the first feeding week to the end of the experiment,body weight in COMB group was significantly lower than that in HF group(P＜0.05).The level of glucose tolerance,serum total cholesterol,low density lipoprotein,IL-13,IL-33,TNF-α and MCP-1 in COMB group was lower than those in HF group(P＜0.05).The α diversity of intestinal flora in ABX group was lower than that in Ctrl group(P＜0.05).Congestion and bleeding in WAT were obvious in HF group,but not in COMB group.The microbial community composition of ABX and HF group was similar to that of Ctrl and COMB group,respectively.Conclusion High-fat diet induces obesity,disorder of glucose and lipid metabolism and inflammation in mice.Short-term mixed antibiotic use can regulate the intestinal flora of mice,mediate increased expression of related anti-inflammatory factors,up-regulate host immunity,and improve glucose and lipid metabolism in mice.

Objective To analyze the incidence and disease burden of substance use disorder(SUD)in China from 1990 to 2019,to evaluate the impact of different ages,periods and birth cohorts on the disease burden of SUD,and to predict disease burden of SUD from 2020 to 2034,so as to provide strategies for the prevention of SUD.Methods Based on the Global Burden of Disease Study 2019(GBD 2019)database,the disease burden was described by incidence,years of life lost(YLLs),years lived with disability(YLDs)and disability-adjusted life years(DALYs).The Joinpoint regression model was used to analyze the trend of standardized incidence and standardized DALYs rate of SUD.Based on the age-period-cohort model,the age,period and cohort effects of SUD were discussed.The grey prediction model GM(1,1)was used to fit the trend of the incidence and standardized incidence of SUD and the trend of disease burden,and to predict the incidence and disease burden of SUD in 2020-2034.Results From 1990 to 2019,the standardized incidence of SUD of amphetamines[average annual percentage change(AAPC)=-0.9％]and cocaine(AAPC=-0.5％)in China showed a downward trend(P＜0.001),and the standardized incidence of SUD of cannabis(AAPC=0.9％)showed an increasing trend year by year(P＜0.001).The trend of standardized incidence of opioid abuse disorders was not obvious(P＞0.05).The DALYs rate caused by the 4 SUD showed a decreasing trend year by year(AAPCamphetamines=-2.2％,AAPCcocaine=-1.5％,AAPCcannabis=-1.0％,AAPCopioids=-1.0％,P＜0.001).The results of age-period-cohort effect showed that the peak incidence of amphetamine,cocaine,cannabis and opioid use disorders was in the 25-30 age group.The DALYs rate caused by cannabis SUD increased with age,while the DALYs rates of amphetamines,cocaine and opioids SUD reached the peak in the 25-29,30-34 and 35-39 age groups,respectively.The results of period effect showed that the risk of SUD in propylamines,cocaine and cannabis decreased first and then increased,while the risk of SUD in opioids increased and then decreased and increased again.The results of birth cohort effect showed that the risk of SUD of amphetamines,cocaine and opioids showed a decreasing trend as a whole except for a small fluctuation in individual birth cohorts.The risk of DALYs rate caused by SUD of amphetamines,cocaine and opioids showed a decreasing trend as a whole,while the risk of DALYs rate caused by SUD of cannabis showed an increasing trend year by year.The prediction results showed that the incidence of SUD of amphetamines,cocaine and opioids showed a downward trend from 2020 to 2034,and the incidence of SUD of cannabis showed a fluctuating upward trend.The DALYs attributed to SUD of amphetamines,cocaine,cannabis and opioids showed a decreasing trend year by year.Conclusion The disease burden of SUD in China is decreasing year by year in the future.The incidence and disease burden are affected by age effect,period effect and cohort effect to varying degrees.Early prevention and effective intervention are the key measures to control SUD.

Based on the genomic information of Emericella sp. 1454, in conjunction with literature analysis of its secondary metabolite emestrin, this study identified the biosynthetic precursors of emestrin and enhanced its production by supplementing the culture medium with these precursors. In this study, it was found for the first time that the addition of biosynthetic precursor, reduced glutathione, to the culture medium significantly increased emestrin yield. By incorporating 1.5 g of reduced glutathione into 50 g of rice culture medium and fermenting for 15 days, a yield of 30.82 mg of emestrin was obtained, which marked an 11.71-fold increase compared to the original fermentation approach. The method is both simple and cost-effective, establishing a solid foundation for the efficient synthesis of emestrin and similar compounds. Additionally, it serves as an important reference for enhancing the production of other epipolythiodioxopiperazine compounds.

Objective To study the effects of antibiotics and high-fat diet on energy metabolism and the browning of white adipose tissue (WAT) and brown adipose tissue (BAT) in mice, so as to provide new ideas for the possible mechanism of adipose tissue in the prevention and treatment of obesity. Methods A total of 80 10-week-old C57BL/6 male mice were fed with normal diet in the early stage, and the antibiotic gavage group (AG) and antibiotic high-fat group (AFG) were given mixed antibiotics by gavage. The blank group (BG) and the high-fat diet group (FG) were given normal saline intragastric solution for 2 weeks, and after the gavage operation, the FG group and the AFG group were given high-fat diet for obesity modeling, and the BG group and AG group continued to be fed with normal diet for 8 weeks (N=20). After the experiment, each group was injected with β3-adrenergic receptor agonists for 5 days, and the high-fat/ordinary diet remained unchanged. At the end of the experiment, basal metabolic rate (BMR), fasting blood glucose (FBG) and rectal temperature were measured, and feces, blood, subcutaneous white fat, epididymis and brown adipose tissue in the scapular area of mice were collected. The automatic biochemical analyzer was used to determine the blood biochemical indexes; reverse transcription polymerase chain reaction (RT-qPCR) was used to measure the expression of genes related to browning of WAT and BAT adipose tissue, respectively. Real-time quantitative polymerase chain reaction (qPCR) was used to determine the expression of WAT mitochondrial DNA (mt DNA). Results From the 4th week to the end of the experiment, the weight of the AFG group was significantly higher than that of the AG group and significantly lower than that of the FG group (P<0.05). The body weight, organ coefficient, serum TC level, rectal temperature and WAT cell diameter in the AFG group were significantly higher than those in the AG group. The serum levels of FBG, TC and LDL in the AFG group were significantly lower than those in the FG group (P<0.05). The overall BMR(mlO2/h) FG group was significantly higher than that of BG group, and the AFG group was significantly higher than that of AG. BMR per unit body weight (mlO2/h/g) AFG was significantly higher than that of FG group (P<0.05). The expressions of RIP140, PPAR-γ and UCP-1 in BAT in the AFG group were significantly higher than those in the FG group, and the mt DNA copy number of WAT in the AFG group was significantly higher than that in the FG group (P<0.05). Conclusion Antibiotic intervention can up-regulate the expression of brown fat-related genes in high-fat diet mice, increase brown fat activity, increase the relative mitochondrial number of white fat, increase the level of browning of white fat, promote thermogenesis, increase the BMR per unit body weight of adult obese mice, and then improve the overall energy metabolism of the body, and slow down the weight gain induced by high-fat diet to a certain extent.

Objective:To summarize the clinical effect of laparoscopic medial umbilical ligament dissection in two-stage Fowler-Stephens orchiopexy (FSO).Methods:A case-series study was conducted.Clinical data of children who underwent two-stage FSO from March 2020 to July 2022 in the Department of Urology, Children′s Hospital Affiliated to Zhengzhou University were retrospectively analyzed.Indexes such as operation time, amount of blood loss, degree of postoperative pain in the operative area, testicular position, size, texture and parental satisfaction at the operative side 1 month, 6 months and 1 year after surgery were collected.Measurement data of normal distribution were expressed as Mean± SD and compared using the independent sample and paired sample t-tests. Results:A total of 18 children with complete clinical data were included in this study.Their age ranged from 7 months to 10 years, with an average of 2 years.There were 10 cases of FSO on the left side and 8 cases on the right side.Twelve cases (group A) had the testis directly descending to the scrotum without amputation of the umbilical medial ligament during the operation, including 7 cases on the left side and 5 cases on the right side; 6 cases (group B) with testis not descending to the scrotum received amputation of the umbilical medial ligament, and the testicle was pulled into the scrotum without tension, including 3 cases on the left side and 3 cases on the right side.The age of patients in group A [(0.88±0.26) years] was lower than that in group B [(4.32±2.96) years] ( t=-4.11, P<0.05).The operation time of patients in group B was (47.50±1.87) min, slightly longer than that in group A [(46.17±1.47) min] ( t=-1.66, P>0.05).The intraoperative bleeding volume in group B was (5.18±0.42) mL, slightly higher than that in group A [(4.96±0.20) mL] ( t=-1.56, P>0.05).Patients in both groups had mild pain in the operative area, which could be tolerated by the children.There were no significant differences in testicular position, size, texture and satisfaction between the two groups 1 month after surgery.Reexamination at 6 months after operation showed that 1 case in group A had mild testicular retraction, located above the scrotum; no significant testicular retraction was observed in group B; the size and texture of testis in both groups had no significant change compared with those before operation, and the blood supply was normal according to the color ultrasound findings; parental satisfaction was high.Reexamination at 1 year after operation showed that 1 case of testis in group A was located above the scrotum, 2 cases had no obvious development, and the rest cases had increased testis size [(0.51±0.03) mL] compared with before operation [(0.31±0.02) mL] ( t=-22.97, P<0.05); all cases of testis in group B returned to normal position, 1 case had no obvious development, and the rest cases had increased testis size [(0.45±0.02) mL] compared with before operation [(0.24±0.01) mL] ( t=-32.87, P<0.05).There was no significant difference in testicular size increase between group A [(0.20±0.03) mL] and group B [(0.20±0.01) mL] ( t=-1.19, P>0.05). Conclusions:In the case that the medial umbilical ligament obviously affects the testicular descent, it can be dissected, which significantly shortens the distance of testis descending to the scrotum.The testicular position after surgery is normal, and the two-stage FSO does not affect the testicular development.