ObjectiveThe U6 promoter is an essential element for driving sgRNA expression in the clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9（CRISPR/Cas9）gene editing system in dicotyledonous plants. Endogenous U6 promoters typically exhibit higher transcriptional activity， which can significantly improve gene editing efficiency. This study aims to identify endogenous U6 promoters in Artemisia annua to optimize its CRISPR/Cas9 gene editing system， which holds significant importance for its molecular breeding. MethodsOn the basis of the highly conserved U6 snRNA sequences in Arabidopsis thaliana， endogenous U6 promoters were screened in the A. annua genome. Expression vectors were constructed with candidate AaU6 promoter driving the firefly luciferase （LUC） reporter gene， and then transiently transformed into Nicotiana benthamiana. Transcriptional activities of the promoters were measured and compared by in vivo imaging and the Dual Luciferase Reporter assay. ResultsEight endogenous U6 promoters were successfully cloned from A. annua. Sequences alignment revealed that all these promoters contained the two conserved cis-acting elements， upstream sequence element （USE） and TATA-box， which affected their transcriptional activity. Dual-luciferase activity assays indicated that the transcriptional activities of AaU6-3， AaU6-1， and AaU6-5 were significantly higher than that of the Arabidopsis AtU6-26 promoter， with AaU6-3 exhibiting the highest activity. ConclusionThis study identified three endogenous AaU6 promoters with high transcriptional activity in A. annua， providing key functional elements for establishing an efficient gene editing system in A. annua. These findings will contribute to advancing precision molecular breeding and high-quality germplasm innovation in A. annua.

OBJECTIVE To explore the specific application and evaluation effect of objective structured clinical examination(OSCE)-guided scenario-based practical teaching mode in training clinical pharmacists. METHODS Fifty-six trainees who participated in the clinical pharmacist training program in the Second Xiangya Hospital of Central South University from October 2020 to September 2022 were selected as the research objects. OSCE-guided teaching was conducted, and the application effect of OSCE-guided teaching mode in clinical pharmacist training was explored and analyzed by using theoretical examination results and OSCE assessment results as evaluation indicators. RESULTS Through comparative analysis, it was found that the OSCE-guided teaching mode not only enabled students to better grasp the theoretical knowledge points required by the training outline, but also improved their clinical thinking ability, problem-solving ability, and communication and coordination skills to varying degrees. CONCLUSION For clinical pharmacist trainees, the OSCE teaching mode is conducive to the comprehensive improvement of clinical pharmacist skills and is suitable for cultivating clinical pharmacists who are capable of independently carrying out clinical pharmacy services in the new situation.

This study constructed a LHCGR-CRE-luc-HEK293 transgenic cell line according to the activation of the cAMP signaling pathway after recombinant human chorionic gonadotropin binding to the receptor. The biological activity of recombinant human chorionic gonadotropin was assayed using a luciferase assay system. The relative potency of the samples was calculated using four-parameter model. And the method conditions were optimized to validate the specificity, relative accuracy, precision and linearity of the method. The results showed that there was a quantitative potency relationship of human chorinonic gonadotropin (hCG) in the method and it was in accordance with the four-parameter curve. After optimization, the conditions were determined as hCG dilution concentration of 2.5 μg·mL^-1, dilution ratio of 1∶4, cell number of 10 000-15 000 cells/well, and induction time of 6 h. The method had good specificity, relative accuracy with relative bias ranging from -8.9% to 3.4%, linear regression equation correlation coefficient of 0.996, intermediate precision geometric coefficient of variation ranging from 3.3% to 15.0%, and linearity range of 50% to 200%. This study successfully established and validated a reporter gene method to detect hCG biological activity, which can be used for hCG biological activity assay and quality control.

Objective To summarize the epidemiological and clinical features of infectious diseases of the central nervous system(CNS)by a single-center analysis.Methods A retrospective analysis was conducted on the data of 1247 cases of CNS infectious diseases diagnosed and treated in the First Medical Center of PLA General Hospital from 2001 to 2020.Results The data for this group of CNS infectious diseases by disease type in descending order of number of cases were viruses 743(59.6％),Mycobacterium tuberculosis 249(20.0％),other bacteria 150(12.0％),fungi 68(5.5％),parasites 18(1.4％),Treponema pallidum 18(1.4％)and rickettsia 1(0.1％).The number of cases increased by 177 cases(33.1％)in the latter 10 years compared to the previous 10 years(P<0.05).No significant difference in seasonal distribution pattern of data between disease types(P>0.05).Male to female ratio is 1.87︰1,mostly under 60 years of age.Viruses are more likely to infect students,most often at university/college level and above,farmers are overrepresented among bacteria and Mycobacterium tuberculosis,and more infections of Treponema pallidum in workers.CNS infectious diseases are characterized by fever,headache and signs of meningeal irritation,with the adductor nerve being the more commonly involved cranial nerve.Matagenomic next-generation sequencing improves clinical diagnostic capabilities.The median hospital days for CNS infectious diseases are 18.00(11.00,27.00)and median hospital costs are ￥29,500(￥16,000,￥59,200).The mortality rate from CNS infectious diseases is 1.6％.Conclusions The incidence of CNS infectious diseases is increasing last ten years,with complex clinical presentation,severe symptoms and poor prognosis.Early and accurate diagnosis and standardized clinical treatment can significantly reduce the morbidity and mortality rate and ease the burden of disease.

Objective To investigate the predictive value of new simplified insulin resistance(IR)assessment indexes in identifying subclinical left ventricular systolic function impairment in patients with type 2 diabetes mellitus(T2DM).Methods A total of 150 T2DM patients with preserved left ventricular ejection fraction(LVEF≥50%)who were admitted to Department of Endocrinology of the First Affiliated Hospital of Air Force Medical University from June 2021 to December 2021 were retrospectively analyzed.All patients underwent two-dimensional speckle tracking echocardiography to measure left ventricular global longitudinal strain(GLS).According to GLS value,the subjects were divided into the normal group(GLS≥18%group,n=80)and the impaired group(GLS<18%group,n=70).Some new simplified IR assessment indicators were calculated and compared between the two groups,including body mass index(BMI),TG/HDL-C ratio,triglyceride-glucose(TyG)index,TyG-BMI index,TyG-WHR and metabolic score for IR(METS-IR).Correlation between the GLS and the new simplified IR assessment indexes was analyzed.The receiver operating characteristic(ROC)curve was used to analyze the diagnostic efficacy of different simplified IR assessment indexes,with the area under the curve(AUC)calculated.Furthermore,according to whether the subjects were complicated with hypertension,binary logistics regression analysis was performed to explore the independent correlation between the simplified IR assessment index and GLS<18%.Results Total 150 were included with aged(54.5±13.7)years with 96(64.0%)men and 54(36.0%)women.Compared with the GLS≥18%group,the TG/HDL-C ratio,TyG index,TyG-BMI,and METS-IR of subjects in the GLS<18%group were significantly increased(P<0.05).Pearson correlation analysis showed that TG/HDL-C ratio,TyG index,TyG-BMI,TyG-WHR,and METS-IR were negatively correlated with GLS(P<0.05).ROC analysis showed that TyG index had a certain predictive value for the evaluation of GLS<18%(AUC=0.678,95%CI 0.591-0.765,P<0.001).Stratification based on hypertension and further adjusting for confounding factors,TyG index remains significantly associated with GLS<18%(OR=3.249,95%CI 1.045-10.103,P=0.042).Conclusions The novel simplified insulin resistance evaluation indexes are closely associated with left ventricular subclinical systolic dysfunction in T2DM patients with preserved ejection fraction.TyG index is an effective index to identify left ventricular subclinical dysfunction in these populations.

Objective:To compare the surgical outcomes between scarless endoscopic thyroidectomy-lateral neck dissection (SET-LND) and open LND for papillary thyroid cancer (PTC) with lymph node metastasis and to share the experience in the prevention and treatment of lymphatic leakage after LND.Methods:Totally 90 PTC patients (25 males) who underwent LND in the Second Affiliated Hospital of Zhejiang University School of Medicine from Jan. 2021 to Aug. 2022 were retrospectively enrolled, with an average age of (28.06±4.89) years. They were divided into the SET-LND group ( n=50) and the open LND group ( n=40). The intraoperative blood loss, operation time, drainage volume on the first and the second days, postoperative lymphatic leakage, tumor (T), and node (N) were observed. The categorical variable data were compared between groups by χ 2 test, and the continuous variable data confirming normal distribution were compared between groups by independent sample t-test. Results:The operation time of the SET-LND group was (284.00±74.65) min, significantly longer than that of the open LND group (145.38±43.26) min ( t=-10.42, P<0.001). The intraoperative blood loss ( t=-1.309, P=0.194), postoperative hospital stay ( t=-0.136, P=0.892), drainage volume on the first day ( t=-1.074, P=0.286), and drainage volume on the second day ( t=-1.595, P=0.114), postoperative lymphatic leakage ( χ2=0.001, P=0.989), T ( t=0.367, P=0.714), N ( t=-1.614, P=0.110) were not significantly different between the two groups. Conclusion:Two surgical methods of LND have similar therapeutic effects and no significant difference was observed in the incidence of postoperative lymphatic leakage between the two groups.

Objective:This study evaluates the performance of chemiluminescence assay, which is designed to detect Hepatitis D Virus (HDV) Immunoglobulin G (IgG) antibodies.Methods:A comparative analysis was conducted among chemiluminescence anti-HDV IgG reagent, the magnetic particle-based domestic reagent A and domestic reagent B, and the Robo Gene HDV RNA kit, using 1909 HBsAg-positive plasma samples. This comparison aimed to delineate clinical specificity and detection accuracy. The anti-HDV IgG reagent precision was assessed at three different concentration levels following the Clinical Laboratory Standards Institute EP5-A2 guidelines. The specificity of the assay was validated using 200 HAV IgM positive, 545 HBsAg-positive but anti-HDV IgG-negative, 350 anti HCV positive plasma samples and 200 healthy human blood samples. Additionally, a concordance study was conducted with 545 HBsAg-positive and 37 anti-HDV IgG-positive plasma samples, comparing the anti-HDV IgG reagent against reagent A.Results:1 909 HBsAg-positive plasma samples were tested using 3 anti HDV IgG reagent and 1 HDV RNA reagent, 19 samples were identified as anti-HDV IgG-positive. The anti-HDV IgG demonstrated superior accuracy and specificity. The assay exhibited excellent precision, with intra-assay coefficient of variation (CV) values ranging from 1.57% to 4.30%, and inter-assay CV values between 1.71% and 4.67% for detecting samples at high, medium, and low concentration levels. Concordance with Reagent A showed consistent results in both positive and negative detections.Conclusion:In this study, the anti-HDV IgG reagent (chemiluminescence method) displayed outstanding specificity in detecting clinical samples and exhibited a high conformity rate with commercialized reagents, making it potentially suitable for screening anti-HDV IgG in HBsAg-positive samples.

Objective:This study aims to evaluate the quality and explore the preliminary clinical applications of a domestically developed hepatitis D virus nucleic acid quantification reagent (abbreviated as"domestic HDV RNA reagent").Methods:The sensitivity and accuracy of the reagent were evaluated in accordance with the WHO HDV RNA international standard, employing the Bio-Rad CFX Opus 96 real-time fluorescence quantitative PCR analysis system. Serial dilutions of pseudo-viruses or cell culture-derived virus were used to determine the linear range of the domestic HDV RNA reagent. Specificity was assessed using positive samples of HAV, HBV, HCV infection, and HEV national reference materials. Precision was evaluated with samples at both high and low concentrations. In a comparative analysis, 30 HDV IgG positive samples were tested using both the domestic HDV RNA reagent and the RoboGene HDV RNA kit based on the ABI 7500 FAST DX system. The Pearson correlation coefficient (r) was used to examine the correlation between the two reagents.Results:The domestic HDV RNA reagent demonstrated a high sensitivity of up to 6 IU/ml, consistent with that of the comparator reagent. The calibration curve for WHO HDV RNA standards had a slope of -3.286, with an amplification efficiency of 101.6%. The linear detection range spanned from 10 to 10 8 IU/ml for eight HDV genotypes. The domestic HDV RNA reagent exhibited exceptional specificity, without cross-reactivity observed with HAV, HBV, HCV, or HEV. Accuracy assessments at five concentration levels met the required standards, with intra-assay precision coefficient of variation ( CV) ranging from 1.20% to 4.20%, and inter-assay precision CV from 1.20% to 7.90%. The detection results for HDV IgG positive samples were highly correlated with the comparator reagent ( r=0.984, P<0.001), achieving a diagnostic accuracy of 100% compared to sequencing results. Conclusion:In this study, the domestic HDV RNA reagent possesses excellent specificity, accuracy, precision, and a broad linear range, attaining a sensitivity level on par with international reagents of the same type.

Abnormal origin of coronary artery is a rare clinical manifestation in cardiovascular disease,among which the abnormal origin of left circumflex artery accounts for 0.022％-0.024％of all coronary angiography patients.Cases of abnormal origin of the left circumflex artery combined with acute occlusion are even rarer.Timely and effective strategies and methods for finding abnormal openings during surgery are crucial for completing interventional treatment.This case reports a rare left circumflex artery anomaly originating from the absence of coronary sinus and acute occlusion of the left circumflex artery leading to myocardial infarction during coronary intervention treatment.The patient experienced sudden chest pain and was admitted to the hospital.The electrocardiogram showed elevated ST segments in the lower and posterior walls.The acute coronary angiography showed only the left main trunk,left anterior descending branch,and right coronary artery(thrombolysis in myocardial infarction blood flow grade Ⅲ),but no left circumflex branch No left circumflex branch was found in the left and right coronary sinuses.Non selective angiography was performed at the root of the ascending aorta,and an abnormal opening of the left circumflex branch was finally discovered in the non coronal sinus.This case discusses common sites of abnormal origin of the left circumflex branch,strategies,methods,and interventional treatments for finding abnormal openings.

Objective To observe the influence of different energy windows of the medium-energy general-purpose(MEGP)collimator on image quality,so as to optimize the energy window of yttrium-90(90Y)bremsstrahlung SPECT imaging.Methods 90Y bremsstrahlung spectrum was acquired,and the sensitivity,percentage of the source counts in useful field of view(S/FOV%)and signal-to-background ratio(S/B)of 90Y bremsstrahlung SPECT imaging at MEGP under different energy windows were compared.Results The energy spectrum of 90Y bremsstrahlung was a continuous curve,with the peak of 76.2 keV with MEGP collimator.The images obtained with MEGP collimator were clear,and no significant differences of S/FOV%nor S/B was found between 10%and 20%window width groups(both P＞0.05),but the sensitivities of the latter was higher than the former(P＜0.05).The sensitivity of 70-90 keV images was relatively high,while the S/FOV%and S/B had decreased.The S/FOV%and S/B of images ranging from 40-60 keV were high,but the sensitivity was low.Images acquired with 100 keV±20%showed fairly high sensitivity,S/FOV%and S/B,which was 69.73%,0.62 and 1.64,respectively.Conclusion When performing 90Y bremsstrahlung SPECT with MEGP collimator,the image quality at 20%window width was better than at 10%window width,and 100 keV±20%showed fairly high sensitivity and not significantly decreased S/FOV%and S/B.