ObjectiveTo investigate the role of DEAD-box helicase 3 X-linked （DDX3X） in immune-mediated liver injury （ILI）， and to clarify its mechanism by regulating endoplasmic reticulum stress （ERS）-dependent apoptotic pathway and its association with the clinical progression of hepatitis B. MethodsMice were given injection of concanavalin A （ConA） via the caudal vein to establish a model of ILI， PBS （control group） and different concentrations of ConA were injected into the tail vein of hepatocyte-specific DDX3X-knockout mice （DDX3X^ΔHep and DDX3X-flox mice （DDX3X^fl/fl）， respectively.. The log-rank survival analysis， measurement of the serum levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT）， and HE staining of liver tissue were performed to assess liver injury， and qRT-PCR and Western Blot were used to measure the mRNA and protein expression levels of glucose-regulated protein 78 （GRP78）， CCAAT/enhancer-binding protein homologous protein （CHOP）， and DDX3X in liver tissue. Intraperitoneal injection of 4-phenylbutyric acid （4-PBA， 100 mg/kg） was performed to inhibit ERS. Serum samples （n=30） and liver tissue samples （n=6） were collected from healthy controls， chronic hepatitis B （CHB） patients， and hepatitis B virus-associated liver failure （HBV-LF） patients； ELISA was used to measure the serum level of DDX3X， and qRT-PCR/Western Blot was used to analyze the expression of targets in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the control group of mice， the expression of DDX3X in the liver of mice induced by ConA was significantly increased after liver injury （P<0.05）， and hepatocyte-specific DDX3X knockout increased the 72-hour survival rate of mice by 55% （compared with 20% in the DDX3X^fl/fl group）， with significant reductions in the serum levels of ALT and AST （P<0.000 1） and the expression levels of the ERS markers GRP78 and CHOP （P<0.05）. After ERS was inhibited by 4-PBA， there was alleviation of liver injury （with reductions in ALT and AST， P <0.001） and a reduction in DDX3X expression （P<0.01）. The analysis of clinical samples showed that the mRNA and protein expression levels of liver DDX3X in CHB patients and HBV-LF patients were significantly higher than those in healthy controls （all P<0.01）， and there was a significant increase in the serum level of DDX3X in HBV-LF patients （P<0.000 1）. ConclusionDDX3X exacerbates ILI by regulating the ERS-dependent apoptotic pathway （GRP78/CHOP）， and its expression is associated with the progression of hepatitis B. Therefore， it can be used as a potential therapeutic target.

Objective:To establish a rapid and accurate method for the detection of Klebsiella pneumoniae carbapenemase (KPC) carbapenemase gene based on recombinase aided amplification (RAA)-CRISPR-Cas13a (CRISPR-Cas13a) technology. Methods:Twenty-five clinical isolates of carbapenem-resistant Klebsiella pneumoniae (CRKP) and five carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains preserved in 2020-2021 in Beijing Chuiyangliu Hospital were randomly collected, and the total DNA samples of the strains was extracted. RAA primers specific for KPC DNA and CRISPR RNA (crRNA) were designed to establish a rapid and accurate method for the detection of KPC carbapenemase gene based on RAA-CRISPR-Cas13a technology. The method was evaluated by plasmids and clinical sample strains, and the detection was also performed by Quantitative real-time PCR (qPCR) method to compare the detection rate and consistency of the two methods. Results:The RAA-CRISPR-Cas13a method can detect KPC plasmids and samples with a sensitivity of 1 copy/μl, which is higher than that of qPCR (10 1 copies/μl). Among the 30 clinical strains (including 25 CRKP strains and 5 CSKP strains), 23 strains were detected to carry KPC gene by both RAA-CRISPR-Cas13a method and qPCR method, and 7 strains were not detected with KPC gene. The detection rate of KPC gene in the 25 CRKP strains was 92% (23/25). The positive coincidence rate of the two methods was 100% (23/23). Conclusions:This study combined RAA amplification technology with CRISPR-Cas13a technology to establish a rapid and accurate method for detecting KPC carbapenemase gene. The method is useful for accurate screening of KPC carbapenemase-producing strains. It has a wide application prospect in drug resistance monitoring and infection control.

Objective:To evaluate the effect of workshop-based incontinence associated dermatitis (IAD) training course for geriatric nurse specialists, and to provide references and evidence for the application of workshop teaching model in specialized nursing training.Methods:The convenience sampling method was used to select 40 participants in the 9th Chongqing Geriatric Nurse Specialist Training Program in May 2021 as the intervention group, and IAD training was conducted using the workshop method. The 42 participants in the 8th Chongqing Geriatric Nurse Specialist Training Program in May 2020 were selected as the control group, who received IAD training through traditional teaching methods. After the training, the achievement of teaching objectives and teaching satisfaction were compared between the two groups using questionnaire survey, and IAD nursing knowledge and attitudes were compared using scales. SPSS 27.0 was used for the t-test and chi-square test. Results:The questionnaire survey showed that the intervention group achieved higher teaching objectives in all dimensions compared to the control group ( P<0.05). Compared with the control group, the intervention group had higher scores in teaching satisfaction regarding teaching format, interest in learning, practical skills, theoretical understanding, integration of theory and practice, and problem-solving abilities ( P<0.05). Compared with the control group, the intervention group demonstrated significantly higher scores in IAD nursing knowledge [(11.32±1.56) vs. (9.02±2.26), P<0.05] and attitudes [(37.63±3.54) vs. (32.81±2.97), P<0.05]. Conclusions:The workshop training has been highly recognized by the trainees, contributing to improved implementation effectiveness of teaching, enhancing the scores of IAD nursing knowledge and attitudes. This approach offers a new perspective for training with a practical focus in the specialized nursing field.

Objective:To compare the antiplatelet effects of vicagrel and clopidogrel in patients with different cytochrome P450 (CYP) 2C19 genotypes.Methods:This is a post-hoc analysis of a phase Ⅱ clinical trial of vicagrel, which included patients with coronary heart disease who underwent percutaneous coronary intervention from August 2018 to June 2019 in 18 centers. Patients were categorized based on the presence of CYP 2C19 *2 or *3 loss-of-function (LOF) alleles into LOF carrier group ( n=111) and non-LOF carrier group ( n=90). Each group included patients received vicagrel 5 mg, 6 mg, 7.5 mg, or clopidogrel 75 mg for 28 days per study protocol. P2Y 12 reaction units (PRU) were measured using VerifyNow at baseline, 6 to 8 hours after loading dose, 7 to 10 days after randomization, and 28 days after randomization and the percentage inhibition of platelet aggregation (%IPA) was calculated. The primary endpoint was %IPA on day 28. Within the patients from the General Hospital of Northern Theater Command, 8 to 12 patients in each study arms were enrolled in a prespecified pharmacokinetic sub-study, measuring the time to reach maximum plasma concentration (T max), peak plasma concentration (C max), and area under the plasma concentration-time curve (AUC). Results:Among 201 patients, the age was (58.8±8.5) years, and 139 (69.2%) were male. In non-LOF carriers, there was no significant differences in PRU values and %IPA between the vicagrel 5 mg, 6 mg, 7 mg, and clopidogrel groups at all time points (all P>0.05). In LOF carriers, %IPA was significantly higher in the vicagrel-treated groups than in the clopidogrel group at 6-8 hours after loading dose (22.9 (14.2, 31.5)% vs. 19.8 (11.0, 28.6)% vs. 29.5 (20.9, 38.0)% vs. 12.9 (3.9, 21.9)%, P=0.038) and 7-10 days after randomization (22.4 (14.2, 30.5)% vs. 34.4 (26.1, 42.6)% vs. 39.8 (31.8, 47.9)% vs. 24.7 (16.3, 33.2)%, P=0.001), with a trend towards higher %IPA in the vicagrel-treated groups at day 28 (30.4 (21.3, 39.6)% vs. 36.5 (27.2, 45.7)% vs. 40.8 (31.8, 49.8)% vs. 30.7(21.2, 40.2)%, P=0.056). Pharmacokinetic results of 35 patients showed that the C max and AUC of the active metabolite M15-2 of vicagrel was similar to that of clopidogrel in non-LOF carriers, but AUC between vicagrel 5 mg, 6 mg, 7 mg and clopidogrel were significantly different in LOF carriers ((5.6±0.6) h·μg -1·L -1 vs. (6.8±2.7) h·μg -1·L -1 vs. (9.2±3.3) h·μg -1·L -1 vs. (4.2±1.9) h·μg -1·ml -1, P=0.020). Conclusion:Vicagrel and clopidogrel have similar antiplatelet effects in non-LOF carriers, but vicagrel exhibits superior antiplatelet effects in LOF carriers.

Microglia/physiology* ; Neurons/physiology* ; Cells, Cultured

Objective:To investigate the expression and role of asparte-specific cysteine protease (Caspase)-1, inflammasomes key molecule, in hepatitis B virus (HBV)-related diseases.Methods:HBV-related liver disease patients' serum (438 cases) and liver tissue (82 cases) samples were collected from Beijing You'an Hospital affiliated with Capital Medical University. The mRNA expression level of caspase-1 in liver tissue was detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein expression level of Caspase-1 in liver tissue was detected by the immunofluorescence method. The activity of Caspase-1 was detected using the Caspase-1 colorimetric assay kit. The level of Caspase-1 in the serum was detected by an ELISA kit.Results:The results of qRT-PCR showed that the mRNA level of Caspase-1 was downregulated in patients with chronic hepatitis B (CHB), cirrhosis (LC), and hepatocellular carcinoma (HCC), while up-regulated in patients with acute-on-chronic liver failure (ACLF) ( P＜0.01) compared with normal subjects. Immunofluorescence assays showed that Caspase-1 protein levels were elevated in ACLF patients, decreased in HCC and LC patients, and slightly elevated in CHB patients. The activity of Caspase-1 was slightly higher in liver tissue from CHB, LC, and HCC patients than in the normal control group, and there was no statistically significant difference between the groups. Additionally, compared with the control group, Caspase-1 activity was significantly reduced in the ACLF group ( P＜0.01). Serum Caspase-1 levels were significantly lower in patients with CHB, ACLF, LC, and HCC than in normal subjects, and serum Caspase-1 levels were lowest in patients with ACLF ( P＜0.001). Conclusion:Caspase-1, a key molecule of inflammasomes, plays an important role in HBV-related diseases and has significant differences, showing distinct features for ACLF than other HBV-related diseases.

ObjectiveTo establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). MethodsHBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups. ResultsThe HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×103, 1×102, and 1×101 copies/μl standard substances were 441%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ2=4.286, P=0038). ConclusionThe established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.

In view of given that the current development trend of nucleic acid molecular detection technology is gradually shifting from clinical laboratory to field detection, there is an urgent need to establish a new nucleic acid diagnostic tool with high sensitivity, high specificity, great efficiency and convenience portability to meet the needs of clinical point-of-care testing(POCT). The detection method based on clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein (Cas) is a promising new nucleic acid detection method. In this method, the Cas effector protein would recognize highly specific nucleic acid sequences, and it can be used for POCT in combination with a highly sensitive small biosensor. This method can successfully achieves the high-efficiency and convenient detection characteristics while satisfying high sensitivity and high specificity. This article reviews the developments progress in the field of miniaturized analytical sensors related to CRISPR/Cas technology in recent years, and discusses the research prospects and challenges of related POCT detection.

Objective To investigate the influences of culture conditions on the in vitro induction and maturation of dendritic cells by using different combinations of cytokines. -ethods Mouse bone mar-row cells were isolated and cultured in media containing varying combinations of cytokines, including granu-locyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and fms-like tyrosine kinase 3 ligand (Flt3L). After cultured at 37℃ for seven days, the attached bone marrow cells were collected and stained by fluorescence-labeled monoclonal antibodies (McAb) against CD11c, MHCⅡ and CD86 for flow cytometry analysis. In the parallel group, LPS was added on day 5 to a final concentration of 1μg/ml for DC maturation analysis by flow cytometry. Results In group Flt3L (20 ng/ml)/GM-CSF (20 ng/ml)/IL-4 (10 ng/ml), 90％ of bone marrow cells were CD11c-positive. Flt3L (100 ng/ml) could induce 88％ of bone marrow cells to express CD11c. Bone marrow cells positive for MHCⅡ accounted for 35. 4％ and 36. 1％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF, where both Flt3L and GM-CSF were used at a concentration of 20 ng/ml. After LPS stimulation, the positive rates of MHCⅡ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF were 58. 1％ and 59. 6％, which increased by 22. 7％ and 23. 5％, re-spectively. The percentages of CD86-positive bone marrow cells were 7. 1％ and 5. 5％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF. Bone marrow cells positive for CD86 grew by 7. 1％ and 6. 2％ in group Flt3L (20 ng/ml) and group GM-CSF/IL-4 after LPS stimulation. Conclusions Flt3L and GM-CSF probably dominated the differentiation and maturation of bone marrow-derived dendritic cells with a synergis-tic effect. Combined usage of Flt3L and GM-CSF at the concentration of 20 ng/ml would be an optimal proto-col for DC research.