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.

Deep venipuncture catheterization is a routine and basic operation in the treatment of critically ill patients, and it is the most effective way to quickly correct the shock. Clinical B-ultrasound guided deep vein catheters can improve the success rate of puncture, but in the process of operation, the short axis needs to be replaced by the long axis. In the replacement process, the stability of the novice is insufficient, the positioning is difficult, and the operation time is too long. If only short axis puncture is used, it is impossible to know whether the current position of the puncture needle, and the puncture may be too deep and stray into the artery. The accuracy of the 45 degree angle of the injection point requires a very experienced operator. In view of the above shortcomings, doctors in the department of critical care medicine of Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine designed a B-ultrasound puncture equipment, which has obtained the National Invention Patent of China (ZL 2016 1 0571557.X). The device is composed of B-ultrasound probe fixing frame, sliding scale plate, simulation slide rule, puncture needle, sliding device. By sliding device the angle of the pinhole channel, it is conducive to the accurate positioning of the puncture target, optimizing the operation procedure, improving the puncture speed and accuracy, effectively reducing the occurrence of puncture complications, ensuring patient safety, reducing unnecessary waste of human and material resources. It can reduce the workload of medical staff and is worthy of clinical practice.
Humans ; Catheterization, Central Venous/methods* ; Ultrasonography, Interventional/methods* ; Ultrasonography ; Punctures/methods* ; Needles

Objective:To explore the expectations and values of patients with malocclusion on participation in shared decision-making of orthognathic surgical protocols, and to provide references for further development of clinical shared decision-making models.Methods:Based on the expected value theory and descriptive qualitative research methods, using purposive sampling, 13 patients with malocclusion in the Ninth People's Hospital of Shanghai Jiao tong University School of Medicine from May to August in 2021 were selected for semi-structured interviews. The interview data were sorted, classified and refined by traditional content analysis.Results:Two themes were extracted: patients' ability beliefs about their ability to participate in shared decision making for orthognathic surgery(decision support ability, psychological coping ability and environmental adaptability), and task values for shared decision making for orthognathic surgery(interest value, acquisition value).Conclusions:Low level of patients′ ability beliefs in shared decision-making, active physician guidance facilitates patient participation, but the depth of patient participation is influenced by factors such as information support, cultural climate, and physical space for shared decision making. It is suggested that the magnetic role of physicians should be actively played, the shared decision-making team should be strengthened, at the same time, hospital manager should enhance shared decision making propaganda to increase the acceptance and participation of patients in shared decision making so as to improve the quality of shared decision-making.

Objective:To explore the application value of apparent diffusion coefficient (ADC) of magnetic resonance diffusion weighted imaging (DWI) parameters in glioma classification and glioma microstructure evaluation.Methods:From June 2017 to November 2019, 38 patients with glioma confirmed by surgery and pathology in Haikou Hospital Affiliated to Xiangya Medical College of Central South University were retrospectively analyzed. According to the pathological results, they were divided into low-grade (WHO Ⅰ-Ⅱ, 15 cases) glioma group and high-grade (WHO Ⅲ-Ⅳ, 23 cases) glioma group. They received magnetic resonance imaging (MRI) plain scan and DWI scan respectively, and the ADC value and microstructure of different grades of glioma were compared. The correlation between ADC value of glioma and the percentage of vascular endothelial growth factor (VEGF)-positive cells, cell density and integrated optical density (IOD) value of aquaporin 1 (AQP1) expression was analyzed.Results:(1) MRI examination showed that the signals of low-grade glioma were more uniform, with no or slight peritumoral edema and space occupying effect, and the enhancement was more non enhanced or slightly enhanced. The signals of high-grade glioma were more heterogeneous due to necrosis and bleeding, and the peritumoral edema and space occupying effect were more obvious, showing uneven obvious enhancement or irregular ring enhancement; (2) The percentage of VEGF positive cells, cell density and the IOD value of AQP1 expression in high-grade glioma were significantly higher than that in low-grade glioma, and the ADC value was lower than that in low-grade glioma (all P<0.05); (3) The ADC value of glioma patients was negatively correlated with the percentage of VEGF-positive cells, cell density, and the IOD value of AQP1 expression ( r=-0.55, -0.65, -0.63, all P<0.05). Conclusions:The ADC value of glioma can indirectly reflect the expression of VEGF, cell density and AQP1 positive expression level, which is helpful for preoperative glioma classification and evaluation of glioma microstructure and biological characteristics.

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.

Objective:To retrieve, evaluate and summarize the evidence for the prevention of pressure injury in adult patients with acute respiratory distress syndrome (ARDS) and mechanical ventilation in prone position, to provide a basis for clinical nurses in the management of pressure injury in patients with mechanical ventilation in the prone position of ARDS.Method:UpToDate, BMJ Best Practice, Guidelines International Network, National Guideline Clearinghouse, National Institute for Health and Clinical Excellence, Joanna Briggs Institute (JBI) Evidence-based Health Care Centre, Cochrane Library, Medlive, EMBASE, PubMed, BMJ, CNKI, Wanfang Database and SinoMed were systematically retrieved to search evidence on the prevention of pressure injury in adults with ARDS and mechanical ventilation in prone position, including clinical decision-making, guidelines, evidence summary, systematic evaluation, Meta-analysis, RCT and quasi-experimental studies. The search time limit was from December 1, 2010 to December 1, 2020. The literature quality was evaluated by 4 personnel, and the included literature was extracted and summarized by 2 personnel.Result:Finally, 7 articles were included, including 1 clinical decision, 3 guidelines, 2 RCTs and 1 quasi-experimental study. A total of 12 pieces of evidence were extracted from 5 aspects, such as evaluation, intervention measures, supervision, professional knowledge training of nursing staff and principles of prevention and compliance.Conclusions:The best evidence for the prevention of pressure injury in adult ARDS patients with prone mechanical ventilation summarized in this study is convenient for clinical nurses to conduct more targeted and scientific evaluation and care of pressure injury for patients.

Objective:To investigate the mechanism of action of peroxisome proliferator-activated receptor α (PPARα)-mediated CCAAT/enhancer binding protein homologous protein (CHOP) signaling molecule with response to inflammation in mice with acute liver failure.Methods:C57BL/6 mice were used as the research subjects, and D-galactose (D-GalN) combined with lipopolysaccharide (LPS) was injected intraperitoneally to establish a mouse model of acute liver failure. PPARα was activated by Wy-14643. CHOP expression was promoted by plasmids. Liver pathological changes and serum transaminases (ALT and AST) were detected in mice to evaluate liver function. The mRNA expression level of inflammatory factors in liver tissue was detected by real-time fluorescence quantitative PCR. LPS-stimulated macrophage was used to establish an inflammation model. PPARα and CHOP expression was inhibited by siRNA. The mRNA expression level of inflammatory factors in the cells was detected by real-time fluorescence quantitative PCR.Results:Promoted PPARα activation had inhibited liver hemorrhage and inflammation in mice with acute liver failure induced by D-GalN/LPS. In addition, the serum level of transaminases and genetic level of inflammatory factors in liver tissues were reduced ( P < 0.01). CHOP accelerated expression had reversed the hepatoprotective effect of PPARα activation, aggravated liver injury, and increased inflammatory factors expression ( P < 0.01). At the cellular level, the inhibition of PPARα activation had accelerated the increase of inflammatory factors ( P < 0.01), while the inhibition of CHOP activation had all over again decreased the inflammatory factors ( P < 0.01). Conclusion:PPARα and CHOP are important signaling molecules to regulate the inflammatory response in acute liver failure and liver injury. PPARα acceleration can down-regulate CHOP to inhibit inflammatory factors, which might play a protective role in mice with acute liver failure.

Objective: To investigate the endoplasmic reticulum stress (ERS) role in the course of liver failure induced by severe hepatitis B virus (HBV) infection and its related mechanism. Methods: Liver tissue samples and clinical data [chronic hepatitis B patients (12 cases, chronic hepatitis B group), hepatic failure induced by severe hepatitis B virus (12 cases, severe hepatitis B virus liver failure group), and normal subjects (8 cases, control group)] were collected from the Beijing You'an Hospital affiliated to Capital Medical University between 2009 to 2011. Statistical analysis was performed on the clinical indicators of each group. The structure of endoplasmic reticulum in liver tissue was observed by transmission electron microscopy. Western blot and qRT-PCR were used to detect the expression of endoplasmic reticulum stress and apoptosis-related factors, including glucose-regulated protein (Grp), and C/EBP homologous protein (CHOP). Frozen sections of liver tissues were prepared for immunofluorescence test. All data were expressed as mean ± standard deviation. LSD-t test was used to compare the results between groups. A p value < 0.05 was considered as statistically significant. Results: Transmission electron microscopy showed that the morphological structure of the endoplasmic reticulum was damaged in both groups (chronic hepatitis B and liver failure induced by severe hepatitis B virus), and liver failure induced by severe hepatitis B virus group was more critical. Western blot and qRT-PCR showed that Grp78, Grp94 and Caspase-4 were highly expressed in normal group and chronic hepatitis B group, and the relative protein expressions were 1.20 ± 0.13 and 0.78 ± 0.11, 0.90 ± 0.06 and 0.11 ± 0.01, 0.15 ± 0.02 and 0.22 ± 0.04, respectively. The expression of protein was weakened in liver failure induced by severe hepatitis B virus group (relative protein expression was 0.01 ± 0, 0.01 ± 0, and 0.11 ± 0.02, respectively).There was a statistically significant difference between the two groups (P < 0.05). The expression of CHOP was consistent with the results of immunofluorescence, and increased with the stressing of injury. Conclusion During the course of severe hepatitis B infection, dysregulated endoplasmic reticulum stress activated mild stress in chronic hepatitis B group, while severe stress in hepatic failure induced by severe hepatitis B virus group. Therefore, endoplasmic reticulum stress plays an important and complex role in the pathogenesis of hepatic failure induced by severe hepatitis B virus.

Objective: To evaluate the clinical value of high-risk factors in combination with stratification method in predicting the clinical prognosis of patients diagnosed with N_2-3M₀ nasopharyngeal carcinoma (NPC). Methods: A total of 226 N_2-3M₀ NPC patients who underwent IMRT from November 2013 to May 2015 were enrolled in this study. The relationship between tumor volume, cervical metastatic lymph node characteristics (necrosis and fusion) and T and N staging was analyzed. The high-risk factors that affected the survival were identified. The value of high-risk factors combined with stratification method in predicting the clinical prognosis was assessed. Results: N₃ staging, V_n≥47.15cm³ and lymph node fusion (LNF) were the high-risk factors for distant metastasis in patients with stage N_2-3M₀ NPC. All patients were classified into the low-risk, medium-risk, high-risk and extremely high-risk groups according to high-risk factors. For patients in the low-risk, medium-risk, high-risk and extremely high-risk groups, the 3-year overall survival rates were 84.2%, 76.7%, 58.7% and 36.4%(all P<0.001), 87.3%, 85.2%, 54.5% and 12.1% for the distant metastasis-free survival (DMFS) rates (all P<0.001), 76.8%, 74.3%, 49.2% and 12.1%for the progression-free survival (PFS) rates (all P<0.001), and 89.2%, 88.5%, 91.5% and 88.3% for the loco-regional recurrence-free survival (LRRFS) rates (P=0.914), respectively. The risk stratification method showed the best curve separation for DMFS compared to the V_n, N staging and LNF classification groups (all P<0.05). Conclusion High-risk factors in combination with stratification method has the highest clinical value in predicting the clinical prognosis of N_2-3M₀ NPC patients.