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.

To investigate the effects of ORF9 gene of porcine circovirus type 2(PCV2)on PK-15,eu-karyotic expression plasmid was constructed and transfected into PK-15 cells,and the effects of overexpression of ORF9 on proliferation,apoptosis and immunization of PK-15 cells were exam-ined by flow cytometry and qRT-PCR.The results showed that ORF9 gene overexpression signifi-cantly up-regulated the expression levels of the ER stress marker gene GRP78,increased the num-ber of S phase cells,accelerated cell cycle progression,increased the apoptosis rate of PK-15 cells,up-regulated the expression levels of apoptosis-related genes caspase-3,caspase-8,caspase-9,p53 and Bax(P＜0.01),down-regulated the expression levels of apoptosis-related genes Bcl-2,up-reg-ulated the expression levels of immune-related genes 1L-8,IL-10,NF-κB and TNF-α(P＜0.01),and down-regulated the expression levels of immune-related genes IL-2,IFN-β and IL-12(P＜0.01).The above results indicate that ORF9 gene may promote the proliferation and apoptosis of PK-15 cells and play a role in the escape process of PK-15 cells.

In order to understand the genomic characteristics and genetic variation and strain type of infectious bursal disease virus(IBDV)isolate GZGY2022,which caused the death of chickens in Guizhou farm,primers were designed to amplify the whole genome of the isolate,and genetic evo-lution and strain type analysis were performed after cloning and sequencing.The results showed that the A and B segments of IBDV genome were 3 260,2 827 bp,respectively,encoding VP2-VP5 and VP1 genes.The nucleotide sequence homology between the A and B segments of this strain and the VvIBDV were 96.2％-98.7％and 87.7％-98.9％,respectively,which is the highest with NN1172 strain,83.1％-94.7％and 90.1％-91.0％with other strains.The results of genetic evolution and strain type study showed that IBDV strains can be divided into 6 branches according to antigen and virulence,and the A and B segments of the strain were clustered in the evolutionary branch of VvIBDV,and the strain was A3B3 genotype according to the new genotype classification method.The results of amino acid sequence analysis showed that there were 3 and 7 unique amino acid site variations in the A and B segments of the strain,respectively,and 13 unique characteristic amino acid sites in the coding region of the full-length genome were consistent with VvIBDV.The VP2 sequence of segment A has 19 characteristic amino acid identical with VvIBDV,among which hyper variable regions 222A,242I,253Q,256I,279D,284A,294I and 299S were characteristic ami-no acid sites of the VvIBDV,and the heptapeptide region sequence SWSASGS was consistent with the virulent strain.The VP1 sequence of segment B has 10 characteristic amino acid identical with VvIBDV,among which 61I,145T and 287A were the characteristic amino acid sites of the VvIB-DV.In addition,the nucleotide sequence GGTGCC of 777-782 did not form the restriction endo-nuclease site of Kpn Ⅰ,and combined with the triplet site 145/146/147(TEG),the segment B was consistent with the NN1172 strain,showed that its virulence was slightly weaker than that of the B2 strain of VvIBDV.The results of recombination analysis showed that there were no breaks and recombination sites in the sequence of the strain,and no recombination event occurred.In summa-ry,this study found that GZGY2022 strain belonged to the A3B3 genotype non-recombinant VvIB-DV strain,and its special amino acid sites were consistent with the molecular characteristics of VvIBDV.This study lays the foundation for further exploring the genomic characteristics and path-ogenicity of VvIBDV.

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 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 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 explore the clinical value of intracranial translucency (HT) in open spina bifida at 11-13 +6 weeks of gestation.Methods Abdominal ultrasound was performed in 200 cases of normal fetus and 6 cases of confirmed open spina bifida at 11-13 +6 weeks of gestation to compare the morphology of IT,diencephalon and midbrain.Results Fetal IT was readily recognized in all 200 normal cases,with diencephalons and midbrain showing number "8" shape.In 6 cases of open spina bifida,fetal IT cannot be identified,and the expected " 8" shape of diencephalon and midbrain was distorted.During 11-13 +6 weeks of pregnancy,the fetal brain is caused by intracranial negative pressure,resulting in morphological changes in the intracranial hyaline,diencephalon and mesencephalon.Conclusions Fetal brain characteristics including intracranial translucency and the shape of diencephalon and midbrain in 11-13 +6 weeks gestation are valuable ultrasound screening indicators for opens pina bifida.

It is complicated to decide the treatment plan of hopeless anterior teeth in esthetic zone due to severe periodontitis, periodontal-endodontic combined lesion or teeth trauma.The optional treatment plan for this kind of teeth includes retention after periodontal treatment, extraction and implant treatment, extraction and prosthodontic treatment and so on.To make an appropriate treatment plan, patients'' periodontal conditions, periodontal biotype, local anatomy, esthetic demand, economic condition and social psychological status should be comprehensively considered.A combine of periodontal, endodontic and orthodontic therapy may achieve a good treatment effect in hopeless anterior teeth with severe periodontal destruction, tooth extrusion and occlusal trauma.In this case, a 20-year-old female who presented with symptoms of bleeding on brushing and upper incisors loosening for 1 month came to the Department of Periodontology, Peking University School and Hospital of Stomatology.The clinical examinations revealed that the patient''s right upper incisor had signs of mobility (Ⅲ°), intrusion of 1-2 mm, and probing depth (PD) of 9-10 mm.The periapical radiograph showed that the alveolar bone of right upper incisor absorbed horizontally to the apex.And the patients showed Angle Ⅱ° malocclusion with Ⅱ° overbite in anterior teeth and maxillary protrusion.A diagnosis of aggressive periodontitis and Angle Ⅱ° malocclusion was made.The treatment of this patient lasts for 5 years which include periodontal initial therapy, orthodontic therapy, guided tissue regeneration (GTR) of right upper incisor and supportive periodontal therapy and the clinical result is fine.A hopeless upper incisor was successfully retained and the longtime clinical condition was stable.The strategy of retention of hopeless upper anterior teeth, the relationship of periodontal treatment and orthodontic treatment, and the indications of periodontal and orthodontic combined therapy were also discussed on the basis of this case.Generally, the positive factors in retention of hopeless teeth includes young age, absence of systemic conditions, strong motivation for maintaining the tooth, single root anatomy, integrated dentition, good response to cause-related therapy, intrabony alveolar bone defect, thick periodontal biotype, and regular supportive periodontal therapy.And in the progress of orthodontic therapy, regular supportive periodontal therapy and good plaque control is extremely important.