Objective To investigate the role of protein kinase C (PKC) in mechanical ventilation-induced lung injury in rats.Methods Thirty healthy male Wistar rats,weighing 250-300 g,were randomly divided into 5 groups (n =6 each):control group (group C),small tidal volume group (group S),small tidal volume and PKC inhibitor group (group S + P),large tidal volume group (group L),and large tidal volume and PKC inhibitor group (group L + P).VT =42 ml/kg,RR =40 bpm,I∶E =1∶ 2,PEEP =0,FiO2 =21％ in groups L and L + P,while VT=7 ml/kg,RR=40 bpm,I∶E=1∶2,PEEP=0,FiO2 =21％ in groups S and S+P.The rats were only tracheostonized in group C,while the rats were mechanically ventilated for 4 h in the other four groups.PKC inhibitor bisindolylmaleinide Ⅰ 0.12 mg/kg was injected intramuscularly 1 h before anesthesia in groups S + P and L + P.The animals were sacrificed immediacy after tracheotomy in group C,and at 4 h of ventilation in the other four groups and lungs were removed for calculation of wet/dry lung weight ratio (W/D ratio) and for microscopic examination.The expression of occludin was determined in the lung tissues by Western blot.Results Compared with group C,W/D ratio was significantly increased and the expression of occludin was down-regulated in the other four groups (P ＜ 0.05).Compared with group S,W/D ratio was significantly increased and the expression of occludin was down-regulated in group L,and W/D ratio was decreased and the expression of occludin was up-regulated in group S + P (P ＜ 0.01).W/D ratio was significantly lower and the expression of occludin was higher in group L + P than in group L (P ＜ 0.01).The pathological changes were attenuated in groups S + P and L + P as compared with groups S and L.Conclusion PKC is involved in mechanical ventilation-induced lung injury in rats.

ObjectiveTo understand the current status of Cronobacter sakazakii (Cronobacter spp.) infection and its molecular epidemiological characteristics among patients with diarrhea, so as to provide evidence for the prevention and control of diarrhea disease caused by infection with Cronobacter spp. in Changping District, Beijing. Methods760 stool samples were collected from the diarrhea patients in a sentinel hospital in 2019, for the detection of Cronobacter spp., Salmonella, diarrheogenic Escherichia coli (DEC), and Vibrio Parahaemolyticus. Meanwhile, drug sensitivity experiment and pulsed-field gel electrophoresis (PFGE) typing analysis were conducted on the Cronobacter spp. strains isolated. ResultsA total of 20 Cronobacter spp. strains (2.63%) were isolated, with a lower detection rate than that of Salmonella and Vibrio Parahaemolyticus (χ²=9.052, P=0.011). However, there were no statistically significant differences between the detection rates in Cronobacter spp. and DEC (χ²=1.076, P=0.300). Seasonal characterization analysis showed that Cronobacter spp. could be detected in spring (1.00%), summer (4.17%), autumn (3.00%) and winter (1.67%), and the differences were statistically significant (χ²=662.700, P<0.001). The PFGE analysis showed that 20 PFGE banding patterns were found in 20 Cronobacter spp. strains, with a similarity coefficient ranging from 56.30% to 90.09% and a diverse PFGE banding pattern. The drug sensitivity experiment results showed that 18 (90.00%) strains were resistant to cefazolin, and2 (10.00%) strains were intermediate. While, as for cefoxitin, 2 (10.00%) strains were resistant to it, and 5 (25.00%) strains were intermediate. All the 20 strains were 100.00% sensitive to the other 11 antibiotics. ConclusionIn the study, Cronobacter spp. is detected in all seasons through the year, with a high resistance rate to cefazolin, no multi-drug resistant bacteria appeared, and diverse PFGE banding patterns.

Objective To investigate the impact of oral contrast agent for assisting in outlining the small bowel on pelvic volumetric modulated arc therapy (VMAT) dose in patients with cervical cancer.Methods Nine cervical cancer patients for postoperative radiotherapy underwent CT scans,and the target volumes and organs at risk including the small bowel were contoured.The VMAT plan was designed for each case.Then another plan was generated by re-calculating the radiation dose after changing the electron density of the small bowel.The first plan (plan A) was the conventional VMAT plan,and the second one (plan B) specified the electron density of the small bowel.Paired t-test was used to compare the dose distribution between the two plans.Results The Dg8,D5o,conformity index,and homogeneity index of plans A and B were 4 989.1 vs.5 000.1 cGy (P =0.026),5 208.6 vs.5 191.6 cGy (P =0.005),0.766 vs.0.765 (P =0.920),and 0.081 vs.0.074(P =0.055),respectively.The volumes of the small bowel receiving at least 30 Gy for plans A and B were 309.3 vs.314.3 cm3(P =0.207),while bladder V45 of the two plans was 52.4％ vs.51.1％ (P =0.168).To achieve the same prescribed dose,plan A and plan B needed 893.3 MU and 865.8 MU (P =0.093).Conclusions The contrast agent filling the small bowel does not lead to a significant increase in the pelvic VMAT dose in patients with cervical cancer after surgery.

Objective To evaluate the role of Src tyrosine kinase in damage to alveolar epithelial cells caused by mechanical stretch.Methods MLE-12 cells cultured in vitro were randomly divided into 3 groups using a random number table:mechanical stretch group (group S),dimethyl sulfoxide control group (group D),and Src tyrosine kinase inhibitor PP2 group (group P).In D and P groups,dimethyl sulfoxide 30 μl/ml and PP2 100 μmol/L were added to the culture medium,respectively,and the cells were then cultured for 30 min.The cells underwent mechanical stretch for 8 h with frequency of0.5 Hz and amplitude of 20％ in the three groups.At 0,2,4 and 8 h of mechanical stretch,MLE-12 cells in 3 wells of each group were collected for determination of cell apoptosis with flow cytometry and expression of occludin using Western blot.The apoptosis rate was calculated.Results Compared with S group,no significant changes were found in the apoptosis rate and expression of occludin at each time point in group D,and the apoptosis rate was significantly decreased,and the expression of occludin was up-regulated at 2,4 and 8 h of mechanical stretch in group P.Conclusion The activation of Src tyrosine kinase is involved in damage to alveolar epithelial cells caused by mechanical stretch.

Objective To evaluate the role of pl20-catenin protein (p120) in mechanical stretchinduced transferring of E-cadherin to cytoplasm in mouse alveolar epithelial cells.Methods Experiment Ⅰ Mouse alveolar epithelial cells (MLE-12 cells) were seeded in 6-well cell stretch plates at a density of (1.0-1.5) ×106 cells/well and divided into 3 groups (n=12 each) using a random number table:control group (group C),cyclic stretch for 2 h group (group CS2) and cyclic stretch for 4 h group (group CS4).The cells underwent 20％ cyclic stretch at 0.5 Hz (stretch:intermittence =1 ∶ 1) for 2 and 4 h in CS2 and CS4 groups,respectively.The cells underwent no cyclic stretch in group C.The expression of p120,E-cadherin and phosphorylated Src kinase (p-Src) and expression of E-cadherin in cytomembrane and cytoplasma were detected by Western blot.Experiment Ⅱ MLE-12 cells were seeded in 6-well cell stretch plates at a density of (1.0-1.5)× 106 cells/well and divided into 4 groups (n =6 each) using a random number table:control group (group C),cyclic stretch group (group CS),p120 small interfering RNA (siRNA) transfection group (group p120 siRNA),and p120 siRNA transfection plus cyclic stretch group (group p120 siRNA+CS).The cells were transfected with scramble siRNA in C and CS groups,and 24 h later the cells underwent 20％ cyclic stretch for 2 h at 0.5 Hz (stretch:intermittence =1 ∶ 1) in group CS.The cells were transfected with p120 siRNA in p120 siRNA and p120 siRNA+CS groups,and 24 h later the cells underwent 20％ cyclic stretch for 2 h at 0.5 Hz (stretch ∶ intermittence =1 ∶ 1) in group p120 siRNA+CS.The expression of E-cadherin in cytomembrane and cytoplasm was detected by Western blot after the end of treatment in each group.Results Experiment Ⅰ Compared with group C,the expression of p120 and E-cadherin was significantly down-regulated,the expression of p-Src was up-regulated,the expression of E-cadherin in cytomembrane was down-regulated,and the expression of E-cadherin in cytoplasm was up-regulated in CS2 and CS4 groups (P ＜ 0.05).Compared with group CS2,the expression of p120 and E-cadherin was significantly down-regulated,the expression of p-Src was up-regulated,the expression of E-cadherin in cytomembrane was down-regulated,and the expression of E-cadherin in cytoplasm was upregulated in group CS4 (P ＜ O.05).Experiment Ⅱ Compared with group C,the expression of E-cadherin in cytomembrane was significantly down-regulated,and the expression of E-cadherin in cytoplasm was up-regulated in CS,p120 siRNA and p120 siRNA+CS groups (P＜ 0.05).Compared with group CS or group p120 siRNA,the expression of E-cadherin in cytomembrane was significantly down-regulated,and the expression of E-cadherin in cytoplasm was up-regulated in group p120 siRNA+CS (P＜0.05).Conclusion The degradation of p120 can promote mechanical stretch-induced transferring of E-cadherin to cytoplasm in mouse alveolar epithelial cells.

Objective To evaluate the effect of recombinant human annexin A5 on the expression of phosphorylated protein kinase C alpha (p-PKCα) and p120-catenin during endotoxin-induced damage to cardiomyocytes.Methods H9c2 cells cultured in vitro were randomly divided into 3 groups (n=18 each) using a random number table:control group (group C),endotoxin group (group L),and recombinant human annexin A5 group (group A).Recombinant human annexin A5 (final concentration 5 ng/ml) was added,and the cells were incubated for 2 h in group A,and then lipopolysaccharide (final concentration 1 μg/ml) was added,and the cells were incubated for 4 h in L and A groups.At 4 h of incubation,cell apoptosis was detected using the cell apoptosis detection kit,the intercellular space was measured using the confocal microscopy,and the expression of p-PKCα and p120-catenin was determined by Western blot.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,the intercellular space was significantly widened,the expression of p120-catenin was significantly down-regulated,and the expression of p-PKCα was significantly up-regulated in group L (P＜0.05).Compared with group L,the apoptosis rate and intercellular space were significantly decreased,the expression of p120-catenin was significantly up-regulated,and the expression of p-PKCα was significantly down-regulated in group A (P＜0.05).Conclusion Recombinant human annexin A5 can inhibit phosphorylation of PKCα and up-regulate the expression of p120-catenin,thus attenuating endotoxin-induced damage to cardiomyocytes.

Objective To evaluate the relationship between the expression of Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) in nerve tissues and mitochondrial function during mechanical stretch-induced damage to mouse alveolar epithelial cells.Methods Alveolar epithelial type Ⅱ cell line MLE-12 cells were divided into 3 groups (n=13 each) using a random number table:control group (group C),cyclic stretch group (group CS) and cyclic stretch plus NLRP3 inhibitor TAK-242 group (group CS+T).MLE-12 cells underwent 20％ cyclic stretch at a frequency of 0.5 Hz (stretch ∶ intermittence =1 ∶ 1) for 4 h in group CS.In group CS+T,after being incubated for 1 h with 1 μ mol/L TAK-242,MLE-12 cells underwent 20％ cyclic stretch for 4 h,and the parameters were similar to those previously described in group CS.The reactive oxygen species (ROS) content and mitochondrial membrane potential (△ΨM) were measured using chemiluminescence assay.Enzyme-linked immunosorbent assay was used to determine the concentration of interleukin-1 beta (IL-1β) in the supernatant.The expression of NLRP3 in MLE-12 cells was detected using Western blot.Results Compared with group C,the △ΨM of cells was significantly decreased,the ROS content and IL-1β concentration were increased,and the expression of NLRP3 was up-regulated in group CS,and the △ΨM of cells was significantly decreased,the ROS content was increased,and the expression of NLRP3 was up-regulated in group CS+T (P＜0.05).Compared with group CS,the △ΨM of cells was significantly increased,the ROS content and IL-1β concentration were decreased,and the expression of NLRP3 was down-regulated in group CS+T (P＜ 0.05).Conclusion Mechanical stretch induces damage to mitochondria through up-regulating the expression of NLRP3,thus leading to damage to mouse alveolar epithelial cells.

OBJECTIVEDuring 2003-2005, an outbreak of meningitis due to Neisseria meningitidis serogroup C occurred in China. With the aim to find strain clues result in the final epidemics, the ancestral strain 053442, a clinical isolate, and a carrier strain 053426 with different gene type were analyzed.

METHODSClinical strain 053442 and carrier strain 053426 were cultured on GC agar plates under the same condition. Two-dimensional electrophoresis was performed using the pH 3-10 nonlinear IPG strips of 24 cm length, and all the protein spots were identified by matrix-assisted laser desorption/ionization time of flight spectrometry.

RESULTS502 and 380 protein spots were identified in 053426 and 053442 respectively, relating to 266 and 202 different genes covering a wide range of cellular functions. The express volume and number of proteins involved in energy metabolism, protein synthesis and amino acid biosynthesis in 053426 were higher than in 053442. Virulence factor Opa, Opc and a series of proteins involved in pilus assembly and retraction were identified in 053442, which appear to be of primary importance in colonization and invasion of human cells. Compared to 053442, virulence protein species were less in 053426, with lower express volumes too. No Opa and Opc were detected in 053426.

CONCLUSIONSThe different protein expression profiles of the clinical strain 053442 and carrier strain 053426 in the present study provide some clues of the different pathogenicity of the two strains, which may account for result in the final epidemics.

Bacterial Proteins ; analysis ; Bacterial Typing Techniques ; China ; epidemiology ; Disease Outbreaks ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Meningitis, Meningococcal ; cerebrospinal fluid ; epidemiology ; microbiology ; Neisseria meningitidis, Serogroup C ; classification ; isolation & purification ; Proteome ; analysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

BACKGROUND: Low-density computed tomography (LDCT) improved early lung cancer diagnosis but introduces an excess of false-positive pulmonary nodules data. Hence, accurate diagnosis of early-stage lung cancer remains challenging. The purpose of the study was to assess the feasibility of using circulating tumour cells (CTCs) to differentiate malignant from benign pulmonary nodules. METHODS: 122 patients with suspected malignant pulmonary nodules detected on chest CT in preparation for surgery were prospectively recruited. Peripheral blood samples were collected before surgery, and CTCs were identified upon isolation by size of epithelial tumour cells and morphological analysis. Laser capture microdissection, MALBAC amplification, and whole-exome sequencing were performed on 8 samples. The diagnostic efficacy of CTCs counting, and the genomic variation profile of benign and malignant CTCs samples were analysed. RESULTS: Using 2.5 cells/5 mL as the cut-off value, the area under the receiver operating characteristic curve was of 0.651 (95% confidence interval: 0.538-0.764), with a sensitivity and specificity of 0.526 and 0.800, respectively, and positive and negative predictive values of 91.1% and 30.3%, respectively. Distinct sequence variations differences in DNA damage repair-related and driver genes were observed in benign and malignant samples. TP53 mutations were identified in CTCs of four malignant cases; in particular, g.7578115T>C, g.7578645C>T, and g.7579472G>C were exclusively detected in all four malignant samples. CONCLUSIONS CTCs play an ancillary role in the diagnosis of pulmonary nodules. TP53 mutations in CTCs might be used to identify benign and malignant pulmonary nodules.
Humans ; Lung Neoplasms ; Exome Sequencing ; Multiple Pulmonary Nodules ; Carcinoma ; DNA Repair