Objective To analyze the prognostic significance and biological effects of cytochrome P450 family 27 subfamily A member 1(CYP27A1)in hepatocellular carcinoma(HCC),and to preliminarily explore its molecular mechanism of regulating the malignant growth of HCC.Methods The Cance Genome Atlas(TCGA)database was used to analyze the expression level of CYP27A1 and its prognostic effect on HCC patients.The samples were divided into CYP27A1 high-expression group(n=170)and low-expression group(n=170)based on the median expression of CYP27A1 in HCC,gene set enrichment analysis(GSEA)was performed to investigate gene sets associated with CYP27A1 expression.The subcellular localization of CYP27A1 was detected by immunofluorescence staining and search database.The over-expression plasmid of CYP27A1 was constructed and then transfected into the HCC cells MHCC-97H and HCCLM3 cell lines,including two groups,namely control group(transfecting empty vector)and CYP27A1 over-expression group(transfecting CYP27A1 over-expressed vector).CCK-8,flow cytometer,and reactive oxygen species(ROS)fluorescence probe were applied to detect the effects of CYP27A1 over-expression on cell viability,apoptosis and ROS levels in HCC cells.Combining bioinformatics to analyze the correlation between CYP27A1 and the expression of ROS generation-related genes and HCC proliferation-related genes.Results Compared with the normal liver tissue,the expression level of CYP27A1 mRNA in HCC tissue was significantly reduced(P<0.01).The expression of CYP27A1 was significantly correlated with sex,T stage,tumor grade and tumor stage of HCC patients(P<0.05).Compared to the CYP27A1 high-expression group,patients in CYP27A1 low-expression group had lower survival rate(P<0.01).GSEA enrichment analysis revealed that the levels of HCC stem cell-related gene clusters and HCC proliferation gene clusters were remarkably increased in CYP27A1 low-expression group.The immunofluorescence showed that CYP27A1 was mainly located in nucleus in MHCC-97H and HCCLM3,whereas CYP27A1 was mainly located in mitochondria in HepG2.CYP27A1 over-expression attenuated cell viability(P<0.01),and reduced the ROS levels(P<0.05),whereas it had no effects on the apoptosis in HCC cells(P>0.05).The expression of CYP27A1 and the expression of inhibiting ROS generation-related genes were positively correlated(P<0.05),while the expression of inhibiting ROS generation-related genes and the expression of HCC proliferation-related genes were negatively correlated(P<0.05).Conclusions The expression of CYP27A1 was decreased in HCC,and down-regulated CYP27A1 promoted cell growth by enhancing ROS generation,although the precise mechanism requires future educidation.

Objective To evaluate the bioequivalence and safety of ezetimibe tablets in healthy Chinese subjects.Methods The study was designed as a single-center,randomized,open-label,two-period,two-way crossover,single-dose trail.Subjects who met the enrollment criteria were randomized into fasting administration group and postprandial administration group and received a single oral dose of 10 mg of the subject presparation of ezetimibe tablets or the reference presparation per cycle.The blood concentrations of ezetimibe and ezetimibe-glucuronide conjugate were measured by high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS),and the bioequivalence of the 2 preparations was evaluated using the WinNonlin 7.0 software.Pharmacokinetic parameters were calculated to evaluate the bioequivalence of the 2 preparations.The occurrence of all adverse events was also recorded to evaluate the safety.Results The main pharmacokinetic parameters of total ezetimibe in the plasma of the test and the reference after a single fasted administration:Cmax were(118.79±35.30)and(180.79±51.78)nmol·mL-1;tmax were 1.40 and 1.04 h;t1/2 were(15.33±5.57)and(17.38±7.24)h;AUC0-t were(1 523.90±371.21)and(1 690.99±553.40)nmol·mL-1·h;AUC0-∞ were(1 608.70±441.28),(1 807.15±630.00)nmol·mL-1·h.The main pharmacokinetic parameters of total ezetimibe in plasma of test and reference after a single meal:Cmax were(269.18±82.94)and(273.93±87.78)nmol·mL-1;Tmax were 1.15 and 1.08 h;t1/2 were(22.53±16.33)and(16.02±5.84)h;AUC0_twere(1 463.37±366.03),(1 263.96±271.01)nmol·mL-1·h;AUC0-∞ were(1 639.01±466.53),(1 349.97±281.39)nmol·mL-1·h.The main pharmacokinetic parameters Cmax,AUC0-tand AUC0-∞ of the two preparations were analyzed by variance analysis after logarithmic transformation.In the fasting administration group,the 90％CI of the log-transformed geometric mean ratios were within the bioequivalent range for the remaining parameters in the fasting dosing group,except for the Cmax of ezetimibe and total ezetimibe,which were below the lower bioequivalent range.The Cmax of ezetimibe,ezetimibe-glucuronide,and total ezetimibe in the postprandial dosing group was within the equivalence range,and the 90％CI of the remaining parameters were not within the equivalence range for bioequivalence.Conclusion This test can not determine whether the test preparation and the reference preparation of ezetimibe tablets have bioequivalence,and further clinical trials are needed to verify it.

Objective To investigate the value of the human chorionic gonadotropin(hCG)stimulation test in the diagnosis of disorder of sexual development(DSD)in children.Methods A retrospective analysis was conducted on 132 children with DSD.According to the karyotype,they were divided into three groups:46,XX group(n=10),46,XY group(n=87),and sex chromosome abnormality group(n=35).The above groups were compared in terms of sex hormone levels before and after hCG stimulation test,and the morphological manifestation of the impact of testicular tissue on the results of the hCG stimulation test was analyzed.Results There was no significant difference in the multiple increase of testosterone after stimulation among the three groups(P>0.05).In the 46,XY group,the children with 5α-reductase type 2 deficiency had a testosterone-to-dihydrotestosterone ratio higher than that of the 46,XY DSD children with other causes.Morphological analysis showed that DSD children with testicular tissue demonstrated a significantly higher multiple increase in testosterone after stimulation compared to children without testicular tissue(P<0.05).Conclusions The hCG stimulation test has an important value in assessing the presence and function of testicular interstitial cells in children with different types of DSD,and it is recommended to perform the hCG stimulation test for DSD children with unclear gonadal type.[Chinese Journal of Contemporary Pediatrics,2024,26(2):158-163]

Objective To evaluate the in vitro effect of combinations of 5 β-lactam antibiotics with different β-lac-tamase inhibitors on the activity of multidrug-resistant Mycobacterium tuberculosis(MDR-TB),and identify the most effective combination of β-lactam antibiotics and β-lactamase inhibitors against MDR-TB.Methods MDR-TB strains collected in Henan Province Antimicrobial Resistance Surveillance Project in 2021 were selected.The mini-mum inhibitory concentrations(MIC)of 5 β-lactam antibiotics or combinations with different β-lactamase inhibitors on clinically isolated MDR-TB strains were measured by MIC detection method,and the blaC mutation of the strains was analyzed by polymerase chain reaction(PCR)and DNA sequencing.Results A total of 105 strains of MDR-TB were included in the analysis.MIC detection results showed that doripenem had the highest antibacterial activity against MDR-TB,with a MIC50 of 16 μg/mL.MIC values of most β-lactam antibiotics decreased significantly after combined with β-lactamase inhibitors.A total of 13.33％(n=14)strains had mutations in blaC gene,mainly 3 nu-cleotide substitution mutations,namely AGT333AGG,AAC638ACC and ATC786ATT.BlaC proteins Ser111 Arg and Asn213Thr enhanced the synergistic effect of clavulanic acid/sulbactam and meropenem on MDR-TB compared with synonymous single-nucleotide mutation.Conclusion The combination of doripenem and sulbactam has the strongest antibacterial activity against MDR-TB.Substitution mutations of BlaC protein Ser111 Arg and Asn213Thr enhances the sensitivity of MDR-TB to meropenem through the synergy with clavulanic acid/sulbactam.

Objective:To investigate the expression of kinase-like gene CT10 regulatory fac-tor(CRKL)in intrahepatic cholangiocarcinoma(ICC)and its effect on the proliferation and invasion of intrahepatic cholangiocarcinoma cells.Methods:qRT-PCR detected CRKL transcription in ICC pa-tient tissues and adjacent tissues.Immunohistochemistry assessed CRKL expression in ICC tissues and adjacent tissues,and analyzed its relationship with clinicopathological features.For the inhibition of CRKL expression in QBC939 cells by siRNA technology,the effect of CRKL silencing on AKT and ERK signaling pathway was measured by Western blot.CRKL's influence on QBC939 cell prolifera-tion and invasion was analyzed by MTT,clonogenesis,and Transwell assays.Results:The expres-sion of CRKL in ICC tissues was up-regulated,and there were statistically significant differences in CRKL expression in ICC with different clinicopathological features such as tumor size,lymph node metastasis and TNM stage.Cellular experiments showed a significant decrease in the phosphoryla-tion of AKT and ERK upon inhibition of CRKL,and the proliferation and invasion ability of ICC cells was significantly diminished(P＜0.05).Conclusion:CRKL promotes ICC cell proliferation and invasion through AKT and ERK pathways,offering new molecular targets and directions for targeted therapy.

AIM To investigate the effects of total Astragalus saponins on the improvement of sarcopenia in a rat model of type 2 diabetes mellitus(T2DM).METHODS The rats were divided into the normal group for a normal feeding and the model group for the feeding of high-sugar and high-fat diet combined with intraperitoneal injection of STZ to establish a T2DM model.The successful model rats were randomly divided into the model group,the metformin group(0.2 g/kg)and the total Astragalus saponins group(80 mg/kg),and given corresponding doses of drugs by gavage.After 12 weeks administration,the rats had their FBG,postprandial blood glucose(PG2h)and wet weight of skeletal muscle measured;their serum levels of INS,C-peptide(C-P),IGF-1,TNF-α and IL-1β detected by ELISA;their morphological changes of skeletal muscle observed by HE staining;their protein expressions of PI3K,p-Akt,mTOR,S6K1,FoxO1 and Murf1 in skeletal muscle detected by Western blot;and their mRNA expressions of Pi3k,Akt and mtor in skeletal muscle detected by RT-qPCR method.RESULTS Compared with the model group,the total Astragalus saponins group displayed decreased levels of FBG,PG2h,OGTT-AUC,HOMA-IR,TNF-α and IL-1β(P＜0.01);increased levels of INS,C-P,IGF-1 and wet weight of skeletal muscle(P＜0.05,P＜0.01);improved skeletal muscle atrophy and increased protein expressions of PI3K,p-Akt,mTOR and S6K1 in skeletal muscle(P＜0.05,P＜0.01);decreased protein expressions of FoxO1 and Murf1(P＜0.05,P＜0.01);and increased mRNA expressions of Pi3k,Akt and mtor(P＜0.01).CONCLUSION The improvement effects of total Astragalus saponins on sarcopenia in T2DM rats may be associated with the regulation of PI3K/Akt/mTOR and PI3K/Akt/FoxO1 pathways.

1-Deoxy-D-xylulose-5-phosphate synthase (DXS), the first key enzyme in 2-methyl-D-erythritol-4-phosphate (MEP) pathway, catalyzes the condensation of glyceraldehyde-3-phosphate with pyruvate to 1-deoxy-xylose-5-phosphate (DXP). In this study, PgDXS1, PgDXS2, and PgDXS3 genes were cloned from the root of Platycodon grandiflorum (P. grandiflorum). The open reading frame (ORF) of PgDXS1, PgDXS2, and PgDXS3 were 2 160, 2 208, and 2 151 bp in full length, encoding 719, 735, and 716 amino acids, respectively. Homologous alignment results showed a high identity of PgDXSs with DXS in Hevea brasiliensis, Datura stramonium and Stevia rebaudiana. The recombinant expression plasmids of pET-28a-PgDXSs were constructed and transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and the induced proteins were successfully expressed. Subcellular localization results showed that PgDXS1 and PgDXS2 were mainly located in chloroplasts, and PgDXS3 was located in chloroplasts, nucleus and cytoplasm. The expression of three DXS genes in different tissues of two producing areas of P. grandiflorum were assayed via real-time fluorescence quantitative PCR, and the results showed that all of them were highly expressed in leaves of P. grandiflorum from Taihe. Under methyl jasmonate (MeJA) treatment, the expression levels of three PgDXS genes showed a trend of first decreasing and then increasing at different time points (3 - 48 h), and the activity of DXS showed a trend of first increasing and then decreasing in three tissues of P. grandiflorum. This study provides a reference for further elucidating the biological function of PgDXS in terpenoid synthesis pathway in P. grandiflorum.

Objective: To observe the association between clinical phenotypes of hypertrophic cardiomyopathy (HCM) patients and a rare calcium channel and regulatory gene variation (Ca²⁺ gene variation) and to compare clinical phenotypes of HCM patients with Ca²⁺ gene variation, a single sarcomere gene variation and without gene variation and to explore the influence of rare Ca²⁺ gene variation on the clinical phenotypes of HCM. Methods: Eight hundred forty-two non-related adult HCM patients diagnosed for the first time in Xijing Hospital from 2013 to 2019 were enrolled in this study. All patients underwent exon analyses of 96 hereditary cardiac disease-related genes. Patients with diabetes mellitus, coronary artery disease, post alcohol septal ablation or septal myectomy, and patients who carried sarcomere gene variation of uncertain significance or carried>1 sarcomere gene variation or carried>1 Ca²⁺ gene variation, with HCM pseudophenotype or carrier of ion channel gene variations other than Ca²⁺ based on the genetic test results were excluded. Patients were divided into gene negative group (no sarcomere or Ca²⁺ gene variants), sarcomere gene variation group (only 1 sarcomere gene variant) and Ca²⁺ gene variant group (only 1 Ca²⁺ gene variant). Baseline data, echocardiography and electrocardiogram data were collected for analysis. Results: A total of 346 patients were enrolled, including 170 patients without gene variation (gene negative group), 154 patients with a single sarcomere gene variation (sarcomere gene variation group) and 22 patients with a single rare Ca²⁺ gene variation (Ca²⁺ gene variation group). Compared with gene negative group, patients in Ca²⁺ gene variation group had higher blood pressure and higher percentage of family history of HCM and sudden cardiac death (P<0.05); echocardiographic results showed that patients in Ca²⁺ gene variation group had thicker ventricular septum ((23.5±5.8) mm vs. (22.3±5.7) mm, P<0.05); electrocardiographic results showed that patients in Ca²⁺ gene variation group had prolonged QT interval ((416.6±23.1) ms vs. (400.6±47.2) ms, P<0.05) and higher RV5+SV1 ((4.51±2.26) mv vs. (3.50±1.65) mv, P<0.05). Compared with sarcomere gene variation group, patients in Ca²⁺ gene variation group had later onset age and higher blood pressure (P<0.05); echocardiographic results showed that there was no significant difference in ventricular septal thickness between two groups; patients in Ca²⁺ gene variation group had lower percentage of left ventricular outflow tract pressure gradient>30 mmHg (1 mmHg=0.133 kPa, 22.8% vs. 48.1%, P<0.05) and the lower early diastolic peak velocity of the mitral valve inflow/early diastolic peak velocity of the mitral valve annulus (E/e') ratio ((13.0±2.5) vs. (15.9±4.2), P<0.05); patients in Ca²⁺ gene variation group had prolonged QT interval ((416.6±23.1) ms vs. (399.0±43.0) ms, P<0.05) and lower percentage of ST segment depression (9.1% vs. 40.3%, P<0.05). Conclusion: Compared with gene negative group, the clinical phenotype of HCM is more severe in patients with rare Ca²⁺ gene variation; compared with patients with sarcomere gene variation, the clinical phenotype of HCM is milder in patients with rare Ca²⁺ gene variation.
Humans ; Cardiac Surgical Procedures/methods* ; Cardiomyopathy, Hypertrophic/genetics* ; Echocardiography ; Electrocardiography ; Phenotype ; Sarcomeres/genetics* ; Adult

Objective: To determine the feasibility of using temporary permanent pacemaker (TPPM) in patients with high-degree atrioventricular block (AVB) after transcatheter aortic valve replacement (TAVR) as bridging strategy to reduce avoidable permanent pacemaker implantation. Methods: This is a prospective observational study. Consecutive patients undergoing TAVR at Beijing Anzhen Hospital and the First Affiliated Hospital of Zhengzhou University from August 2021 to February 2022 were screened. Patients with high-degree AVB and TPPM were included. Patients were followed up for 4 weeks with pacemaker interrogation at every week. The endpoint was the success rate of TPPM removal and free from permanent pacemaker at 1 month after TPPM. The criteria of removing TPPM was no indication of permanent pacing and no pacing signal in 12 lead electrocardiogram (EGG) and 24 hours dynamic EGG, meanwhile the last pacemaker interrogation indicated that ventricular pacing rate was 0. Routinely follow-up ECG was extended to 6 months after removal of TPPM. Results: Ten patients met the inclusion criteria for TPPM, aged (77.0±11.1) years, wirh 7 females. There were 7 patients with third-degree AVB, 1 patient with second-degree AVB, 2 patients with first degree AVB with PR interval>240 ms and LBBB with QRS duration>150 ms. TPPM were applied on the 10 patients for (35±7) days. Among 8 patients with high-degree AVB, 3 recovered to sinus rhythm, and 3 recovered to sinus rhythm with bundle branch block. The other 2 patients with persistent third-degree AVB received permanent pacemaker implantation. For the 2 patients with first-degree AVB and LBBB, PR interval shortened to within 200 ms. TPPM was successfully removed in 8 patients (8/10) at 1 month without permanent pacemaker implantation, of which 2 patients recovered within 24 hours after TAVR and 6 patients recovered 24 hours later after TAVR. No aggravation of conduction block or permanent pacemaker indication were observed in 8 patients during follow-up at 6 months. No procedure-related adverse events occurred in all patients. Conclusion: TPPM is reliable and safe to provide certain buffer time to distinguish whether a permanent pacemaker is necessary in patients with high-degree conduction block after TAVR.
Female ; Humans ; Atrioventricular Block/therapy* ; Feasibility Studies ; Transcatheter Aortic Valve Replacement ; Pacemaker, Artificial ; Bundle-Branch Block

Objective: To study the diagnostic value of different detection markers in histological categories of endocervical adenocarcinoma (ECA), and their assessment of patient prognosis. Methods: A retrospective study of 54 patients with ECA in the Cancer Hospital, Chinese Academy of Medical Sciences from 2005-2010 were performed. The cases of ECA were classified into two categories, namely human papillomavirus-associated adenocarcinoma (HPVA) and non-human papillomavirus-associated adenocarcinoma (NHPVA), based on the 2018 international endocervical adenocarcinoma criteria and classification (IECC). To detect HR-HPV DNA and HR-HPV E6/E7 mRNA in all patients, we used whole tissue section PCR (WTS-PCR) and HPV E6/E7 mRNA in situ hybridization (ISH) techniques, respectively. Additionally, we performed Laser microdissection PCR (LCM-PCR) on 15 randomly selected HR-HPV DNA-positive cases to confirm the accuracy of the above two assays in identifying ECA lesions. Receiver operating characteristic (ROC) curves were used to analyze the efficacy of markers to identify HPVA and NHPVA. Univariate and multifactorial Cox proportional risk model regression analyses were performed for factors influencing ECA patients' prognoses. Results: Of the 54 patients with ECA, 30 were HPVA and 24 were NHPVA. A total of 96.7% (29/30) of HPVA patients were positive for HR-HPV DNA and 63.3% (19/30) for HR-HPV E6/E7 mRNA, and 33.3% (8/24) of NHPVA patients were positive for HR-HPV DNA and HR-HPV E6/E7 mRNA was not detected (0/24), and the differences were statistically significant (P<0.001). LCM-PCR showed that five patients were positive for HR-HPV DNA in the area of glandular epithelial lesions and others were negative, which was in good agreement with the E6/E7 mRNA ISH assay (Kappa=0.842, P=0.001). Analysis of the ROC results showed that the AUC of HR-HPV DNA, HR-HPV E6/E7 mRNA, and p16 to identify HPVA and NHPVA were 0.817, 0.817, and 0.692, respectively, with sensitivities of 96.7%, 63.3%, and 80.0% and specificities of 66.7%, 100.0%, and 58.3%, respectively. HR-HPV DNA identified HPVA and NHPVA with higher AUC than p16 (P=0.044). The difference in survival rates between HR-HPV DNA (WTS-PCR assay) positive and negative patients was not statistically significant (P=0.156), while the difference in survival rates between HR-HPV E6/E7 mRNA positive and negative patients, and p16 positive and negative patients were statistically significant (both P<0.05). Multifactorial Cox regression analysis showed that International Federation of Obstetrics and Gynecology (FIGO) staging (HR=19.875, 95% CI: 1.526-258.833) and parametrial involvement (HR=14.032, 95% CI: 1.281-153.761) were independent factors influencing the prognosis of patients with ECA. Conclusions: HR-HPV E6/E7 mRNA is more reflective of HPV infection in ECA tissue. The efficacy of HR-HPV E6/E7 mRNA and HR-HPV DNA (WTS-PCR assay) in identifying HPVA and NHPVA is similar, with higher sensitivity of HR-HPV DNA and higher specificity of HR-HPV E6/E7 mRNA. HR-HPV DNA is more effective than p16 in identifying HPVA and NHPVA. HPV E6/E7 mRNA and p16 positive ECA patients have better survival rates than negative.
Female ; Humans ; Papillomavirus Infections/diagnosis* ; Retrospective Studies ; Uterine Cervical Neoplasms/pathology* ; Prognosis ; Oncogene Proteins, Viral/genetics* ; Human Papillomavirus Viruses ; Adenocarcinoma/pathology* ; RNA, Messenger/genetics* ; Papillomaviridae/genetics* ; RNA, Viral/genetics*