The auxin/indole-3-acetic acid (Aux/IAA) gene family is an important regulator for plant growth hormone signaling, involved in plant growth, development, as well as response to environmental stresses. In the present study, we identified SmIAA7 which is potentially associated with Salvia miltiorrhiza leaf development through comparatively analyzed the transcriptome data from different pinnate leaves. SmIAA7 was successfully isolated from S. miltiorrhiza using the specific primers. Then subsequent bioinformatic analysis, prokaryotic expression and purification, subcellular localization, and induction expression analysis under auxin and abiotic stress were performed. The full-length of SmIAA7 contained an ORF of 684 bp encoding a protein of 227 amino acid with a molecular weight of 25.3 kD. Conserved domain analysis showed that SmIAA7 contains the conserved Aux_IAA domain (pfam02309). Sequence analysis and phylogenetic tree analysis results indicated SmIAA7 was phylogenetically close to IAA7 and IAA14 from other plants, suggesting SmIAA7 involved in plant growth and development as well as response to environmental stresses. The prokaryotic expression vector pET28a-SmIAA7 was constructed and SmIAA7 recombinant protein was successfully expressed in E. coli Rosetta (DE3) strain. Subcellular localization experiment demonstrated that SmIAA7 localized in the nucleus of plant cells. Real-time fluorescence quantitative PCR results showed that the expression level of SmIAA7 was upregulated in response to auxin. Drought, low temperature, and salt stress significantly increased the transcript level of SmIAA7 gene. This study lays a foundation for further elucidating the role of SmIAA7 in leaf development, signal transduction, and stress defense in S. miltiorrhiza.

The correct pairing of disulfide bonds maintains the correct folding mode and high-level structure formation of peptides and protein drugs, which is crucial for the quality control of products. In order to ensure that the disulfide bonds are correctly paired, disulfide bond analysis is an essential part of peptides and protein drug characterization. Mass spectrometry can be used to analyze disulfide bonds. However, insulin and its analogues have two pairs of disulfide bonds without restriction enzyme cutting site. Conventional collision-induced dissociation (CID) and high-energy induced cleavage (HCD) cannot accurately locate the complex disulfide bond. In our study, three methods were used to localize the complex disulfide, including enzyme digestion combined with key peptide fragment in source decay (ISD) fragmentation method, enzyme digestion combined with partial reduction alkylation method, intact protein source ISD and electron transfer dissociation (ETD) cleavage method, The applicability of insulin aspart, insulin lispro and insulin glargine were also investigated. This study provides a new way for the quality control of disulfide bonding mode of insulin and its analogues, and also provides a reference for the disulfide bond localization of peptides or proteins containing this complex disulfide bond.

ObjectiveBody fluid stains left at crime scenes are frequently trace amounts, while the identification of body fluids through real time fluorogenic quantitative technique often necessitates the repeated detection within the limited sample, as multiple miRNA markers are the basis for the identification. Based on the goal of both the throughput and efficiency improvement of miRNA analysis in trace samples, a duplex real time fluorogenic quantitative PCR assay system was designed to accurately quantify two miRNAs simultaneously, and the system should be further verified by actual sample for the body fluid identification. MethodsThe duplex real time fluorogenic quantitative PCR system of miR-451a to miR-21-5p was established with specially designed primers and probes, and the concentrations of the primers and probes were both optimized. The specificity, sensitivity and reproducibility of the system were validated, while its capability for body fluid identification was assessed using the miR-451a to miR-21-5p ratio. ResultsThe optimized assay system exhibited excellent specificity and repeatability, with coefficients of variation consistently below 8% for both intra- and inter-batch variability. The amplification efficiency of miR-451a and miR-21-5p reached 71.77% and 74.81%, respectively, with high and relatively consistent results. By utilizing this duplex real time fluorogenic quantitative PCR assay system, a total of 58 body fluid samples were analyzed, exhibiting a discrimination rate of 100% between blood and non-blood samples, as well as between peripheral blood and menstrual blood samples. Moreover, the results, obtained from single real time fluorogenic quantitative PCR assay system and duplex real time fluorogenic quantitative PCR assay system, showed no statistically significant difference with randomly selected blood samples (n=20). Compared to previous single real time fluorogenic quantitative PCR assay system, the sensitivity of duplex real time fluorogenic quantitative PCR assay system exhibited remarkable improvement. A minimum input of only 0.1 ng total RNA was sufficient for accurate detection of peripheral blood and menstrual blood samples, while saliva, semen, and vaginal secretion required only 1 ng total RNA for precise identification purposes. Additionally, the duplex real time fluorogenic quantitative PCR assay system successfully differentiated between different types of body fluids in simulated samples under natural outdoor conditions. ConclusionThe duplex real time fluorogenic quantitative PCR assay system effectively reduced both the time and material costs by half compared to the single system, especially suitable for the examination of body fluid stains left at crime scenes, solving the contradiction between the trace amount and the multiple sample volumes demand of repeated real time fluorogenic quantitative PCR. The duplex real time fluorogenic quantitative PCR assay successfully distinguished blood and other body fluid, as well as peripheral blood and menstrual blood samples, which maintains an equivalent capability for body fluid identification with half sample, time and reagent consumption. This system provides an efficient tool for identifying suspicious body fluids, as well as a foundation for more multiplexed real time fluorogenic quantitative PCR assay system research.

Objective To evaluate the application value of artificial intelligence mixed reality(AI-MR)technology in the planning of ultrasound-guided percutaneous nephrolithotomy(PCNL)for special types of complex upper urinary stones.Methods The prospective single-center,single-arm clinical study involved 15 patients with complex upper urinary stones undergoing ultrasound-guided PCNL during Aug.2022 and May 2023,including 9 male and 6 female,3 cases of pelvic ectopic kidney stones,5 cases of horseshoe kidney stones,3 cases of renal stones combined with spinal deformity,and 4 cases of transplant kidney stones.Based on preoperative computed tomography urography(CTU)data,digital three-dimensional reconstruction was performed,and AI-MR was used to project surgery-related three-dimensional images in real space to obtain"perspective"information of the surgical area.This facilitated preoperative design and planning,including target calyx,number of channels,and auxiliary measures.The compliance of target calyx and number of channels,stone clearance rate,total operation time,time required to establish the percutaneous renal channel,decrease in hemoglobin level,surgical complications,and postoperative hospital stay were analyzed.Results All 15 patients underwent preoperative planning using AI-MR and successfully completed one-stage ultrasound-guided PCNL.Based on the preoperative planning,we utilized S-PCNL alone or combined with Needle-perc or antegrade/retrograde FURS/RIRS.Among all patients,4 underwent single-channel S-PCNL,3 multi-channel S-PCNL,and 8 S-PCNL combined with Needle-perc or FURS.The compliance of target calyx and number of channels was 86.7％,the one-stage stone clearance rate was 80.0％,the average time for establishing the channel was(2.3±0.3)minutes,the average total operation time was(61.5±12.2)minutes,the mean decrease in hemoglobin level was(9.6±1.2)g/L,and the average postoperative hospital stay was(4.6±0.5)days.There were no Clavien-Dindo grade ≥ Ⅱ complications,such as blood transfusion,organ injury,or urosepsis.Conclusion Before surgery,AI-MR can be used to quantitatively analyze imaging data for patients with special types of complex upper urinary stones,which can achieve three-dimensional fluoroscopy effects,formulate surgical plans,optimize puncture paths,effectively avoid the risk of damage to surrounding organs,reduce complications,shorten treatment cycle and improve the first-stage stone clearance rate.

Helicobacter pylori(Hp)is responsible for chronic gastritis,peptic ulcers,and even gastric cancers.Currently,there is no vaccine to prevent or treat Hp infections.Here,we described the chemical synthesis of α-1,6-glucans with different lengths(di-to hexasaccharide),which are present in the core oligosaccharide of Hp lipopolysaccharide(LPS).The 1,2-cis-glucosidic bonds were constructed successfully using a synergistic glycosylation strategy based on acyl remote participation and solvent effects.The results of glycan microarrays indicated that all synthesized α-1,6-glucan fragments possessed a strong binding to IgG antibodies in both rabbit serum immunized with Hp O1 LPS and patient serum infected with Hp.The α-1,6-linked trisaccharide exhibited strong binding affinity to anti-LPS rabbit IgG antibodies.The α-1,6-glucan trisaccharide and pentasaccharide elicited a strong response to IgG antibodies in sera of most Hp-infected patients.Some patients'sera exhibited strong binding activity with α-1,6-linked disaccharide.The results suggest that the α-1,6-glucan disaccharide,trisaccharide and pentasaccharide could be important carbohydrate antigen fragments in Hp lipopolysaccharide.

Objective:To report the results of national surveillance on the distribution and antimicrobial resistance profile of clinical Gram-negative bacteria isolates from bloodstream infections in China in 2022.Methods:The clinical isolates of Gram-negative bacteria from blood cultures in member hospitals of national bloodstream infection Bacterial Resistant Investigation Collaborative System（BRICS）were collected during January 2022 to December 2022. Antibiotic susceptibility tests were conducted by agar dilution or broth dilution methods recommended by Clinical and Laboratory Standards Institute（CLSI）. WHONET 5.6 and SPSS 25.0 software were used to analyze the data.Results:During the study period，9 035 strains of Gram-negative bacteria were collected from 51 hospitals，of which 7 895（87.4%）were Enterobacteriaceae and 1 140（12.6%）were non-fermenting bacteria. The top 5 bacterial species were Escherichia coli（ n=4 510，49.9%）， Klebsiella pneumoniae（ n=2 340，25.9%）， Pseudomonas aeruginosa（ n=534，5.9%）， Acinetobacter baumannii complex（ n=405，4.5%）and Enterobacter cloacae（ n=327，3.6%）. The ESBLs-producing rates in Escherichia coli， Klebsiella pneumoniae and Proteus spp. were 47.1%（2 095/4 452），21.0%（427/2 033）and 41.1%（58/141），respectively. The prevalence of carbapenem-resistant Escherichia coli（CREC）and carbapenem-resistant Klebsiella pneumoniae（CRKP）were 1.3%（58/4 510）and 13.1%（307/2 340）；62.1%（36/58）and 9.8%（30/307）of CREC and CRKP were resistant to ceftazidime/avibactam combination，respectively. The prevalence of carbapenem-resistant Acinetobacter baumannii（CRAB）complex was 59.5%（241/405），while less than 5% of Acinetobacter baumannii complex was resistant to tigecycline and polymyxin B. The prevalence of carbapenem-resistant Pseudomonas aeruginosa（CRPA）was 18.4%（98/534）. There were differences in the composition ratio of Gram-negative bacteria in bloodstream infections and the prevalence of main Gram-negative bacteria resistance among different regions，with statistically significant differences in the prevalence of CRKP and CRPA（ χ2=20.489 and 20.252， P<0.001）. The prevalence of CREC，CRKP，CRPA，CRAB，ESBLs-producing Escherichia coli and Klebsiella pneumoniae were higher in provinicial hospitals than those in municipal hospitals（ χ2=11.953，81.183，10.404，5.915，12.415 and 6.459， P<0.01 or <0.05），while the prevalence of CRPA was higher in economically developed regions（per capita GDP ≥ 92 059 Yuan）than that in economically less-developed regions（per capita GDP <92 059 Yuan）（ χ2=6.240， P=0.012）. Conclusions:The proportion of Gram-negative bacteria in bloodstream infections shows an increasing trend，and Escherichia coli is ranked in the top，while the trend of CRKP decreases continuously with time. Decreasing trends are noted in ESBLs-producing Escherichia coli and Klebsiella pneumoniae. Low prevalence of carbapenem resistance in Escherichia coli and high prevalence in CRAB complex have been observed. The composition ratio and antibacterial spectrum of bloodstream infections in different regions of China are slightly different，and the proportion of main drug resistant bacteria in provincial hospitals is higher than those in municipal hospitals.

AIM To establish an HPLC method for the simultaneous content determination of gallic acid,protocatechuic acid,morroniside,loganin,sweroside,paeoniflorin,hypericin,astragalin,salvianolic acid B,salvianolic acid A,epimedin C and icariin in Bushen Huoxue Sanjie Capsules.METHODS The analysis was performed on a 30℃thermostatic Agilent 5 TC-C18 column(250 mm×4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1%phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 240 nm.RESULTS Twelve constituents showed good linear relationships within their own ranges(r≥0.999 8),whose average recoveries were 97.11%-101.14%with the RSDs of 0.60%-2.65%.CONCLUSION This simple,accurate and reproducible method can be used for the quality control of Bushen Huoxue Sanjie Capsules.

Objective To clarify the high-touch surface in oral diagnosis and treatment procedures,provide basis and guidance for cleaning and disinfection.Methods The direct observation method was used to investigate the tou-ch time and frequency of environmental surfaces in 7 outpatient departments of a tertiary stomatology hospitals in Beijing.The average touch frequency,95％confidence interval and cumulative touch rate were calculated.Results In oral diagnosis and treatment procedures,the average touch frequency of the environmental surface was 26.75 times per procedure,with the highest in endodontics(46.25 times per procedure)and the lowest in the oral mucosal specialty(10.19 times per procedure).The high-touch surface consisted of the shadowless lamp handle,manipula-tion panel and handle on dental unit(doctor's side),computer keyboard and mouse,handle and line front end of three way syringe,as well as dental high speed handpiece and line front end,with average touch frequencies of 3.99,3.85,2.65,1.86,and 1.40 times per procedure.The high-touch surface in all stomatology specialties in-cluded the manipulation panel and handle on dental unit(doctor's side),75％of specialties included computer key-board and mouse,and the shadowless lamp handle has the highest touch frequency in 50％of specialties.The ave-rage touch frequency of the environmental surface was highest(113.50 times per procedure)during crown prepara-tion procedure,and the lowest(8.50 times per procedure)during the orthodontic consultations.Conclusion The high-touch surface of different dental specialties and different diagnosis and treatment procedures are different.Me-dical institutions should take corresponding cleaning,disinfection and management measures according to the actual situation of high-touch surface in stomatology departments,so as to effectively improve the quality of environmental cleaning and disinfection.

Objective:To observe the regulatory effect of microRNA-10b(miR-10b)on the immune effect of glioma cells and explore its mechanism.Methods:Human glioma cell U251 was cultured to obtain cells in logarithmic growth stage.The cell suspen-sion was prepared according to the concentration of 1.0×105 cells/ml,and the control group,overexpression group,low expression group and blank group were set up,with 6 wells in each group.The negative control,miR-10b mimics and miR-10b inhibitor were transfected by liposome transfection in control group,overexpression group and low expression group,respectively.The blank group was given the same amount of sterile normal saline.Natural killer(NK)cells from peripheral blood of a healthy volunteer was isolated and cultured.The killing activity of NK cells was detected by MTT method.The expression of NK cell activated receptor(NKG2D)on the surface of NK cells in each group were detected by flow cytometry,and the expression of major histocompatibility complex class Ⅰ chain-related gene A(MICA),UL16 binding protein 2(ULBP2)and UL16 binding protein 3(ULBP3)on the surface of U251 hu-man glioma cells in each group were detected.Results:The transfection efficiency of control group,overexpression group and low ex-pression group were(93.55±2.05)％,(95.67±3.14)％,(94.18±3.26)％,respectively.Compared with control group and blank group,the expression of miR-10b increased in overexpression group and decreased in low expression group,and the difference were statisti-cally significant(P<0.05).There was no significant difference in the expression of miR-10b between control group and blank group(P>0.05).Compared with control group and blank group,the killing activity of NK cells with different effect target ratios in overex-pression group decreased,the expression of NKG2D decreased,the killing activity of NK cells with different effect target ratios in low expression group increased,and the expression of NKG2D increased,and the difference were statistically significant(P<0.05).The killing activity of NK cells in each group increased with the increase of effect target ratio,and the difference were statistically signifi-cant(P<0.05),and there was no significant difference in NK cell killing activity and NKG2D expression between control group and blank group(P>0.05).Compared with control group and blank group,the expression of MICA,ULBP2 and ULBP3 on the surface of human glioma cell U251 in overexpression group decreased,and the expression of MICA,ULBP2 and ULBP3 on the surface of human glioma cell U251 in low expression group increased,the difference were statistically significant(P<0.05),and there was no signifi-cant difference in the expression of MICA,ULBP2 and ULBP3 on the surface of U251 glioma cells between control group and blank group(P>0.05).Conclusion:Inhibiting the expression of miR-10b can increase the expression of NKG2D on the surface of NK cells and MICA,ULBP2 and ULBP3 on the surface of human glioma cell U251,and enhance the killing activity of NK cells against human glioma cell U251.

Objective To study the pharmacokinetic characteristics of etoricoxib tablets in healthy Chinese subjects and to evaluate the bioequivalence and safety of the test and reference formulations.Methods In a randomised,single-dose,two-period,two-sequence crossover trial,28 healthy subjects were enrolled under the fasting and fed conditions,respectively,who received a single oral dose of 60 mg of etoricoxib tablets in the test or reference formulation.The concentration of etoricoxib in plasma was detected by LC-MS/MS,and the main pharmacokinetic parameters were calculated to evaluate bioequivalence and using WinNonlin 8.2 software.Results The main pharmacokinetic parameters of the test and reference preparations were as follows:The fasting condition Cmax of etoricoxib were(1 176.96±287.95)and(1 164.93±189.65)ng·mL-1;AUC0-t were(18 651.95±6 100.27)and(19 241.39±6 107.48)ng·h·mL-1;and AUC0-∞ were(19 939.15±7 553.27)and(20 536.31±7 223.40)ng·h·mL-1.The fed condition Cmax of etoricoxib were(913.50±184.72)and(878.59±164.35)ng·mL-1;and AUC0-t were(19 085.22±5 155.01)and(18 669.54±4 508.21)ng·h·mL-1;AUC0-∞ were(20 103.77±5 567.02)and(19 528.05±4 989.74)ng·h·mL-1.The 90％confidence intervals for the geometric mean ratios of the main pharmacokinetic parameters in the fasting and fed conditions fell between 80.00％and 125.00％.The incidence of adverse events in the fasting and fed conditions were 28.57％and 21.43％,respectively.Conclusion Two kinds of etoricoxib tablets are bioequivalent,and have similar safety in healthy Chinese subjects.