OBJECTIVE To identify the chemical constituents of Rhamni Songoricae Fructus and to establish their fingerprints and the method for simultaneous determination of four constituents to comprehensively evaluate the quality of Rhamni Songoricae Fructus. METHODS The chemical constituents in Rhamni Songoricae Fructus were qualitatively analyzed by ultra-performance liquid chromatography quadrupole-time-of-flight tandem mass spectrometry. The fingerprints of 15 batches of Rhamni Songoricae Fructus were established by HPLC and chemometric analysis was performed by using SPSS 26.0 and SIMCA 14.1 software； the contents of quercetin， kaempferol， kaempferide and emodin were determined by the same method. RESULTS A total of 35 constituents were identified， including 28 kinds of flavonoids， 5 kinds of anthraquinones and 2 kinds of organic acids. A total of 19 common peaks were identified in the HPLC fingerprints， recognizing quercetin， kaempferol， kaempferide and emodin. The similarities between HPLC fingerprints of 15 batches of samples and control chromatograms were greater than 0.9. The results of cluster analysis showed that 15 batches of samples were divided into 2 classes， of which S1-S5， S7 and S9 were one class and the rest were one class， similar to the results of principal component analysis. The results of the orthogonal partial least squares- discriminant analysis showed that the variable importance projections of peaks No. 2， 6， 1， 11 （quercetin）， 3， 14， 8， 10， 19 （emodin）， 5 were all greater than 1. The contents of quercetin， kaempferol， kaempferide and emodin ranged from 0.710 to 10.478 mg/g， 0.236 to 0.660 mg/g， 0.334 to 3.039 mg/g， and 0.261 to 0.504 mg/g. CONCLUSIONS The constructed chemical constituent identification， fingerprint and content determination methods are simple， feasible and reproducible， which combined with chemometric analysis can be used for comprehensive evaluation of the quality of Rhamni Songoricae Fructus.

Objective This study aimed to identify PAX9 variants in non-syndromic tooth agenesis families of Chi-na,as well as to analyze the genotype-phenotype of non-syndromic tooth agenesis caused by PAX9 variants,which can provide a basis for the genetic diagnosis of tooth agenesis.Methods We collected the data of 44 patients with non-syn-dromic oligodontia who underwent treatment at Stomatological Hospital of Hebei Medical University between 2018 and 2023.Whole-exome sequencing was performed on the peripheral blood of the proband and its core family members,and the variants were verified by Sanger sequencing.Pathogenicity analysis and function prediction of the variants were per-formed using bioinformatics tools.The correlation between the genotype of PAX9 variant and its corresponding pheno-type was examined by reviewing 55 publications retrieved from PubMed.The studies involved 232 tooth agenesis pa-tients with PAX9 variants.Results A novel PAX9 c.447delG(p.Pro150Argfs*62)and a reported PAX9 c.406C>T(p.Gln136*)were identified in two Chinese families.Through bioinformatics analysis and three-dimensional structural mod-eling,we postulated that the frameshift variant was pathogenic.The outcome was the premature cessation of PAX9 pro-tein,which caused severe structural and functional deficiencies.Summarizing the PAX9 genotype-phenotype relationship revealed that patients carrying the PAX9 variant commonly led to loss of the second molars.Conclusion We identified the novel PAX9 c.447delG(p.Pro150Argfs*62)in a Chinese family of non-syndromic oligodontia,expanding the known variant spectrum of PAX9.The most susceptible tooth position for PAX9 variants of tooth agenesis was the second mo-lars and the deciduous molars during the deciduous dentition.

Background::One of the significant challenges for cell therapies, such as chimeric antigen receptor (CAR)-T cell therapy, is the poor infiltration of immune cells into tumor tissues. CAR-monocytes/macrophages (CAR-M) are promising therapies because of their enrichment in the tumor microenvironment. Thus, we constructed a novel CAR-M to facilitate the infiltration of T cells and other immune cells.Methods::The suicide gene inducible caspase-9 ( iCasp9) and anti-erb-b2 receptor tyrosine kinase 2 (HER2) CAR elements were transfected into THP1 (an immortalized human monocyte cell line) by lentivirus. The suicide efficiency and specific anti-tumor efficacy were assessed using flow cytometry, inCucyte, and tumor-bearing BALB/c-nude mouse models. The activation of related signaling pathways in CAR-THP1 activation was explored by transcriptome sequencing. Finally, the synergistic therapeutic efficacy of CAR-THP1 combined with RAK cell treatment was demonstrated in tumor-bearing NOD.CB17-Prkdc scid Il2rg tm1/Bcgen mouse models. Results::We developed a novel CAR-THP1, which incorporated iCasp9, CD3ζ, and CD147 intracellular segments, based on the first-generation HER2-CAR backbone. By constructing and comparing a series of CARs with different permutations, CAR-CD3ζ-CD147-iCasp9-THP1 was selected as the optimal combination. CAR-CD3ζ-CD147-iCasp9-THP1 initiated suicide quickly and efficiently under the control of iCasp9 gene, which enabled us to achieve controlled proliferation of CAR-THP1. CAR-THP1 also exhibited robust specific anti-tumor efficacy independently of T cells in vitro and in vivo. Through transcriptional sequencing, we found that CAR-THP1 tended to differentiate into the M1 phenotype and bridged innate and adaptive immunity. A combination of CAR-THP1 and Retronectin actived killer cells (RAKs) showed better therapeutic efficiency, as the metalloproteinases (MMPs) secreted by CAR-THP1 facilitated the degradation of the dense tumor matrix. This further assisted intratumoral infiltration of T cells and augmented the anti-tumor immune response. Conclusion::CAR-THP1 might be effective against HER2-positive tumor cells and has great potential for combination therapy with other immune cells.

Objective:To discuss the inhibitory effect of diosmetin(DIO)on the ferroptosis induced by the glutathione peroxidase(GSH-Px)inhibitor(1S,3R)-RSL3(RSL3)in spermatocytes GC-2 of the mice,and to clarify the mechanism.Methods:The GC-2 cells were divided into control group,RSL3 group,RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+ferroptosis inhibitor Ferrostain-1(Fer-1)group(200 nmol·L-1 Fer-1).The cells were treated with 0,1,5,10,50,100,500,and 1 000 nmol·L-1 RSL3 solutions,and 0,0.5,0.1,1.0,5.0,10.0,and 50.0 μmol·L-1 DIO solutions,respectively.Additionally,the GC-2 cells were divided into blank group,model group,and treatment group.The GC-2 cells in treatment group were further divided into 0.8,4.0,and 20.0 nmol·L-1 DIO groups,as well as RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,and RSL3+20.0 nmol·L-1 DIO group.MTT method was used to detect the survival rates of the GC-2 cells in various groups.The GC-2 cells were treated with 100 nmol·L-1 RSL3 for 0,6,12,24,36,and 48 h;Western blotting method was used to detect the expression levels of ferroptosis-related proteins in the GC-2 cells in various groups;kits were used to detect the activities of superoxide dismutase(SOD),levels of malondialdehyde(MDA),and ratios of glutathione(GSH)to glutathione disulfide(GSSG)in the GC-2 cells in various groups;immunofluorescence method was used to detect the fluorescence intensities of acyl-CoA synthetase long-chain family member 4(ACSL4)protein in the GC-2 cells in various groups.Results:The MTT method results showed that compared with 0 nmol·L-1 RSL3 group,the survival rates of the GC-2 cells in 50,100,500,and 1 000 nmol·L-1 RSL3 groups were significantly decreased(P<0.01);compared with 0 μmol·L-1 DIO group,the survival rates of the GC-2 cells in 0.5,1.0,5.0,10.0,and 50.0 μmol·L-1 DIO groups were significantly decreased(P<0.01),and 100 nmol·L-1 RSL3 with DIO concentration<0.1 μmol·L-1 were selected for the subsequent experiments.Compared with blank group,the survival rates of the GC-2 cells in model group was significantly decreased(P<0.01);compared with model group,the survival rates of the GC-2 cells in RSL3+20.0 nmol·L-1 DIO group was significantly increased(P<0.01).The Western blotting results showed that compared with 0 h,the expression level of GPX4 protein in the GC-2 cells was significantly decreased after treated with RSL3 for 6 h(P<0.01),and the expression level of HO-1 protein was significantly increased after treated with RSL3 for 12 h(P<0.05);after treated with RSL3 for 12 h,the expression levels of GPX4 and FTH1 proteins were significantly decreased(P<0.05 or P<0.01);after treated with RSL3 for 24 h,the expression levels of GPX4 and HO-1 proteins were significantly decreased(P<0.05 or P<0.01);after treated with RSL3 for 36 and 48 h,the expression levels of HO-1 protein were significantly decreased(P<0.01).Therefore,100 nmol·L-1 RSL3 and for 12 h were selected as the experimental condition for the subsequent experiments.Compared with control group,the MDA level in the GC-2 cells in RSL3 group was significantly increased(P<0.01),and the SOD activity and GSH/GSSG ratio were significantly decreased(P<0.05).Compared with RSL3 group,the SOD activities in the cells in RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+Fer-1 group were significantly increased(P<0.05 or P<0.01).The MDA levels in the cells in RSL3+20.0 nmol·L-1 DIO group and RSL3+Fer-1 group were significantly decreased(P<0.05 or P<0.01),and the GSH/GSSG ratio in the cells in RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+Fer-1 group were significantly increased(P<0.05 or P<0.01).The immunofluorescence observation results showed that compared with control group,the fluorescence intensity of ACSL4 protein in the GC-2 cells in RSL3 group was significantly increased;compared with RSL3 group,the fluorescence intensities of ACSL4 protein in the cells in RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+Fer-1 group were significantly decreased.The Western blotting results showed that compared with control group,the expression level of HO-1 protein in the cells in RSL3 group was increased(P<0.05),and the expression levels of GPX4 and FTH1 proteins were significantly decreased(P<0.05 or P<0.01);compared with RSL3 group,the expression levels of HO-1 protein in the cells in RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+Fer-1 group were significantly decreased(P<0.05 or P<0.01),and the expression levels of GPX4 and FTH1 proteins were significantly increased(P<0.05 or P<0.01).Conclusion:DIO can alleviate the RSL3-induced ferroptosis in the GC-2 spermatocytes of the mice,and its mechanism may be related to the inhibition of HO-1 protein expression and the upregulation of expressions of GPX4 and FTH1 proteins.

Objective To use color Doppler ultrasound to measure the hemodynamic indexes,and to es-tablish the diagnostic prediction model of inflammatory fetal distress.Methods A total of 213 pregnant women admitted to the obstetrics department of the First Affiliated Hospital of Air Force Military Medical U-niversity were collected as the research subjects and divided into the control group and case group according to whether or not fetal distress occurred,including 93 cases in the control group and 120 cases in the case group.The predictive value of PI,RI,S/D values of middle cerebral artery,umbilical artery and uterine artery for pre-dicting fetal distress was analyzed The diagnostic model was constructed by logistic regression analysis.The receiver operating characteristic(ROC)curve,calibration curve and clinical decision curve were adopted to an-alyze and evaluate the diagnostic efficiency of the model for adverse perinatal outcome and the clinical benefit of the patients.Results The univariate analysis results showed that MCA-PI,MCA-RI,MCA,S/D and CPR in the case group were lower than those in the control group,while UA-RI,UA,S/D and UtA-RI were higher than those in the control group.The multivariate regression analysis further showed that MCA-PI,MCA-RI and CPR were the independent protective factors for predicting fetal distress,while UA-R1 and UA-S/D served as the independent risk factors affecting the fetal outcome.Based on five independent influencing fac-tors,the risk prediction model was constructed,and the area under the receiver operating characteristic curve was 0.880(95％CI:0.834-0.925).The sensitivity,specificity and accuracy were 0.93,0.70 and 0.83 respec-tively,and the goodness of fit was good.Conclusion The hemodynamic indexes measured by color Doppler ul-trasound have good predictive value for the diagnosis of fetal distress.The risk prediction model established by the combined indexes has a certain reference value for the intervention in advance of pregnant women with fe-tal distress occurence.

Objective To understand the changing distribution and antimicrobial resistance profiles of Proteus,Morganella and Providencia in hospitals across China from January 1,2015 to December 31,2021 in the CHINET Antimicrobial Resistance Surveillance Program.Methods Antimicrobial susceptibility testing was carried out following the unified CHINET protocol.The results were interpreted in accordance with the breakpoints in the 2021 Clinical & Laboratory Standards Institute(CLSI)M100(31 st Edition).Results A total of 32 433 Enterobacterales strains were isolated during the 7-year period,including 24 160 strains of Proteus,6 704 strains of Morganella,and 1 569 strains of Providencia.The overall number of these Enterobacterales isolates increased significantly over the 7-year period.The top 3 specimen source of these strains were urine,lower respiratory tract specimens,and wound secretions.Proteus,Morganella,and Providencia isolates showed lower resistance rates to amikacin,meropenem,cefoxitin,cefepime,cefoperazone-sulbactam,and piperacillin-tazobactam.For most of the antibiotics tested,less than 10％of the Proteus and Morganella strains were resistant,while less than 20％of the Providencia strains were resistant.The prevalence of carbapenem-resistant Enterobacterales(CRE)was 1.4％in Proteus isolates,1.9％in Morganella isolates,and 15.6％in Providencia isolates.Conclusions The overall number of clinical isolates of Proteus,Morganella and Providencia increased significantly in the 7-year period from 2015 to 2021.The prevalence of CRE strains also increased.More attention should be paid to antimicrobial resistance surveillance and rational antibiotic use so as to prevent the emergence and increase of antimicrobial resistance.

Objective:To assess the capability of seven reference medical laboratories to detect BCR::ABL1 p210 transcription levels and to compare the results among those laboratories.Methods:The interlaboratory comparison was carried out in two stages. The samples were prepared by the reference laboratory. The quantitative values of BCR::ABL1 p210 of the comparison samples covered 0.001%-0.01%, 0.01%-0.1%, 0.1%-1%, 1%-10% and>10% in each stage. Real-time quantitative PCR (RT-PCR) and dPCR (digital PCR) were used to examine the samples. The conversion factor (CF) was calculated and validated for each laboratory.Results:In the RT-PCR comparison, one laboratory was failed to detect BCR::ABL1 p210 in fourteen samples at the first stage. The results of the other six laboratories were qualified with the bias <±1.2 folds (-0.133-0.338) and 95% limits of agreement within ±5 folds (upper limit 0.147-0.785, lower limit -0.770--0.109), and the corresponding CF values were calculated and validated. In the dPCR comparison, one laboratory did not report results at the second stage. The results of the other six laboratories were qualified with the bias <±1.2 folds (-0.026-0.267) and 95% limits of agreement within±5 folds (upper limit 0.084-0.991, lower limit -0.669--0.135), and the corresponding CF values were calculated and validated. The samples with BCR::ABL1 p210 quantitative values of 0.01%-0.1%, 0.1%-1%, 1%-10% and >10% could be detected by both RT-PCR and qPCR. When the quantitative value of BCR::ABL1 p210 was 0.001%-0.01%, the detection rate of dPCR was higher than that of RT-PCR (85.56% vs. 68.00%).Conclusions:A good consistency is present among various laboratories. The quantitative value of BCR::ABL1 p210 is comparable among laboratories as shown by the CF value conversion. For quantitative detection of BCR::ABL1 p210 deep molecular reaction, dPCR has a higher positive detection rate and more advantages than RT-PCR. To ensure the accuracy and reproducibility of the BCR::ABL1 p210 test, it is imperative for every laboratory to enhance their daily quality control practices.

Objective:To search, evaluate and integrate the best evidence of the best evidence for emergency service surge capacity.Methods:According to the "6S" model of evidence resources, the related evidence on emergency service surge capacity in Guidelines International Network, National Guideline Clearinghouse, Canadian Medical Association CPG Infobase, Scottish Intercollegiate Guidelines Network, European Society for Emergency Medicine, the American College of Emergency Physicians, Emergency Nurses Association, Cochrane Library, PubMed, Embase, CINAHL, Web of Science, BMJ Best Practice, UpToDate,CKNI, Wanfang, and VIP database were searched by computer. The retrieval time limit was from the establishment of the database to Dec 31, 2023. Literature quality assessment and data extraction were performed by 2 researchers.Results:A total of 11 articles were included in this study, including 1 guideline, 7 expert consensuses and 3 systematic reviews, which summarized 43 pieces of evidence involving 7 categories, namely core elements, organizational management, space management, personnel allocation, material allocation, education and training, and support services.Conclusions:The best evidence summarized in this study can provide a reference for emergency service to improve surge capacity. In clinical application, emergency departments should focus on organizational, space, personnel and materials management, combined with the type of emergency events, to maximize their routine, emergency and crisis response capabilities, so as to respond to medical surges effectively.

Objective:To investigate the expression and prognostic value of ANO1 in oral squamous cell carcinoma(OSCC)tissues.Methods:Immunohistochemistry(IHC,n=163)and Quantitative real-time PCR(qRT-PCR,n=42)were employed to detect the expression level of ANO1 protein and mRNA in OSCC tissues and paracancerous normal tissues.The relationship between ANO1 ex-pression and clinicopathological features(n=163)and prognosis(n=93)of the patients were analyzed,and the results were compared with those in TCGA database.Results:IHC and qRT-PCR confirmed that ANO1 was highly expressed in OSCC(P＜0.05),which was consistent with the results of the TCGA database.Cox regression analysis showed that ANO1 expression was significantly correlated with T stage,lymph node metastasis and TNM stage and poor prognosis(P＜0.05).By Cox regression analysis,ANO1 overexpression(P=0.002)and differentiation degree(P=0.034)were independent prognostic factors.Conclusion:ANO1 is highly expressed in OSCC and is correlated with poor prognosis,which can be used as a novel biomarker for poor prognosis of OSCC patients.

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.
Animals ; Swine ; Circovirus/genetics* ; Vaccines, DNA/genetics* ; Replicon/genetics* ; Genetic Vectors/genetics* ; Plasmids/genetics*