Objective To explore the effect of fluid shear stress on the expression of glucose regulatory protein 78(GRP78)and C/EBP homologous protein(CHOP)in human umbilical vein endothelial cells(HUVECs).Methods HUVECs were used as experimental cells,and a fluid dynamics simulation experimental system was designed and constructed.Different fluid shear stresses were applied to the experimental cells by controlling the flow rate of the perfusion fluid in the experimental system.According to the fluid shear stress experienced by the experimental cells in the experimental system,they were divided into low shear stress group(group A;0.4Pa),medium shear stress group(group B;0.8 Pa)and high shear stress group(group C;1.2 Pa).Each group of HUVECs consisted of 3 cell slides,and each slide was repeatedly circulated through the perfusion solution of the experimental system for 12 h.Western blotting was used to detect the levels of GRP78 and CHOP proteins,and real-time quantitative reverse transcription polymerase chain reaction was used to determine the levels of GRP78 and CHOP and their messenger RNA(mRNA)in each group.GraphPad Prism 8.0 software was used to statistically analyze the data.Results(1)The relative expression levels of GRP78 protein in groups A,B and C were 1.33±0.46,0.93±0.34,0.64±0.30,respectively,the difference among groups was statistically significant(F=36.17,P＜0.05).The relative expression level of GRP78 protein in group A was higher than that in group B and group C(both P＜0.01),and the relative expression level of GRP78 protein in group B was higher than that in group C(P=0.0013).The relative expression levels of CHOP protein in the three groups were 1.29±0.38 in group A,0.90±0.34 in group B,and 0.59±0.29 in group C,the difference among groups was statistically significant(F=41.27,P＜0.05).The relative expression level of CHOP protein in group A was higher than that in group B and group C(both P＜0.01),and the relative expression level of CHOP protein in group B was higher than that in group C(P=0.0 004).(2)The relative expression levels of GRP78 mRNA in groups A,B,and C were 18.3±3.4,11.3±1.8,5.4±2.2,respectively,the difference among groups was statistically significant(F=189.20,P＜0.05).The relative expression level of GRP78 mRNA in group A was higher than that in group B and group C(both P＜0.01),and the relative expression level of GRP78 mRNA in group B was higher than that in group C(P＜0.01).The relative expression levels of CHOP mRNA in the three groups were 20.4±3.8 in group A,14.2±2.1 in group B,and 7.8±1.3 in group C,the difference among groups was statistically significant(F=171.80,P＜0.05).The relative expression level of CHOP mRNA in group A was higher than that in group B and group C(both P＜0.01),and the relative expression level of CHOP mRNA in group B was higher than that in group C(P＜0.01).Conclusion Low fluid shear stress may increase the protein and mRNA expression levels of GRP78 and CHOP in HUVECs.

Objective To investigate the antimicrobial resistant mechanisms of high level carbapenem resistant Klebsiella pneumoniae infection of burn patients .Methods A retrospective study was conducted on totally 18 non-repetitive high level CR-KP which were isolated from burn patients hospitalized between July 2014 and June 2015.MIC of antibiotics were determined by using the GN 13 cards and agar dilution method.The specific PCR and DNA sequence analysis were performed to confirm the β-lactamase type.Plasmid conjugation transfer experiments and southem hybridization were applied to study the mode of carbapenem resistance transmission .Outer membrane proteins ( Omps) were isolated and examined by PCR and ( sodium dodecyl sulfate polyacrylamide gel electrophores ) SDS-PAGE.Pulsed-field gel electrophoresis( PFGE ) and Multilocus sequence typing ( MLST ) was used to determine the genotypes . Results Susceptibility of antimicrobial agents indicated that all these strains with multiple drug resistance . The resistance rate to piperacillin/tazobactam, ceftriaxone, levofloxacin, ciprofloxacin, gentamicin, tobramycin, cefotetan, ceftazidime, cefepime, aztreonam, imipenem, and meropenem was 100％ (18/18).Moreover, the resistance rate of CR-KP isolates to amikacin was 72.2％ ( 13/18 ) , compound sulfamethoxazole was 61.1％(11/18), tigecycline was 0％(0/18).Conjugation study with Escherictda coli J53 resulted in the transfer of significant reduced carbapenem susceptibility from donors (MICs increased at least 8-fold).By PCR, eighteen strains of Klebsiella pneumoniae carried NDM-1 gene, 5 strains carried KPC-2 gene.The blaNDM-1 was transferable by plasmids.Southern blot hybridization indicated that the blaNDM-1 gene was located on plasmid in size of 46 kb.The plasmid belonged to incompatibility group IncX 3.Seven types of CR-KP were detected by PFGE.In addition, MLST assigned them to sequence type ( ST)11, ST395, ST17, ST37, ST263, ST14 and ST76 types.SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that there was absence of outer membrane proteins OmpK 36 in ST11, ST395, ST37 strains.However, the other STs strains expressed lower quantities of OmpK 36.Conclusions High level carbapenem resistance in K.pneumoniae causing infection in burn patients is attributable to production of plasmid-mediated metallo· β-laetamase NDM-1 combined with porin OmpK36 deficiency or low expression .The K.pneumoniae with NDM-1 and KPC-2 carbapenemase were detected .

Objective: The consistency of 24-hour oropharyngeal Dx-pH monitoring and proton pump inhibitor(PPI) test in the diagnosis of laryngopharyngeal reflux disease (LPRD) was investigated. Methods: Sixty patients with laryngopharyngeal reflux (LPR) related symptoms who had never received PPI treatment were assessed by reflux symptom index (RSI) and reflux finding score (RFS) between October 2017 and October 2018, including 28 males and 38 females, aged from 16 to 72 years, with a medium age of 38 years. Prior to treatment, all patients were evaluated with 24 hours oropharyngeal Dx-pH monitoring(Restech). After empiric therapy with PPI twice-daily for 8 weeks, the efficacy was evaluated according to posttreatment RSI score.The data was analysed with Kruskal-Wallis test, Student Newman Keuls test and consistency check. Results: (1)Among all 60 patients,13 patients (21.7%) had pathologic Ryan score and all resulted responsive to PPI;27 patients (45.0%) with a negative Ryan score were unresponsive to PPI; 20 patients (33.3%) despite a negative Ryan score resulted responsive to PPI therapy. Considering responsiveness to PPI therapy as the gold standard for the diagnosis of LPRD, the sensitivity, specificity, positive predictive value and negative predictive value of Ryan score were 39.4%, 100%, 100% and 57.4% respectively. The Kappa value was 0.369 (P<0.01). (2)Among 34 patients (56.7%) with positive Dx-pH results (24-hour oropharyngeal acid reflux events≥ 3 times), 29 patients were positive and 5 patients were negative in PPI test. Among 26 patients with negative Dx-pH results (24-hour oropharyngeal acid reflux events<3 times), 4 patients were positive and 22 patients were negative in PPI test. Considering responsiveness to PPI therapy as the gold standard for the diagnosis of LPRD, the sensitivity, specificity, positive predictive value and negative predictive value of 24-hour oropharyngeal acid reflux events were 87.9%, 81.5%, 85.3% and 84.6% respectively. The Kappa value was 0.696(P<0.01). Conclusions There is a positive correlation between 24-hour oropharyngeal Dx-pH monitoring positive results (24-hour oropharyngeal acid reflux events≥3 times) and PPI test in the diagnosis of LPRD. The 24-hour oropharyngeal Dx-pH monitoring can be a promising tool for the diagnosis of suspected LPRD patients, and more sensitive and accurate Dx-pH diagnostic index will be required in the clinic.

Objective:To investigate the clinicopathological features, immunophenotypic and molecular genetic characteristics and differential diagnosis of fibrous hamartoma of infancy (FHI).Methods:Thirty-three cases of surgically removed FHI were collected from the Department of Pathology, Henan Provincial People′s Hospital from October 2011 to December 2020, the clinical and pathologic data with follow-up were collected and analyzed. Next-generation sequencing (NGS) and quantitative real time polymerase chain reaction (q-PCR) were used to study the molecular genetics.Results:The FHI cases occurred in 21 males and 12 females (mean age 16.7 months, range 6 months to 6 years). The sites included trunk ( n=21), limb ( n=11), and neck (n=1). All patients had painless solitary superficial soft tissue masses, the size was 1.5-9.0 cm (mean 3.8 cm). Microscopically, they were composed of mature adipose tissue, fibroblast/myofibroblast bundle and primitive mesenchymal cells in different proportions; giant cell fibroblastoma-like areas were seen in 14 cases. Immunohistochemistry showed variable expression of EGFR in the spindle cells and primitive mesenchymal components. In most cases, the spindle cells were positive for CD34 and SMA; giant cell fibroblastoma-like areas were strongly positive for CD34; and S-100 protein was expressed by adipocytes in all cases. Ki-67 labeling index ranged 1%-5%. There were recurrent somatic EGFR exon 20 insertion/duplication mutations in six cases tested by NGS, and there were three different mutation types: p.Asn771_His773dupAsnProHis, p.Pro772_His773insProProHis, and p.His773_Val774insThrHis. All the above 6 and another 15 tested cases showed EGFR exon 20 insertion/duplication mutations by q-PCR. Conclusions:FHI is a rare benign fibroblast/myofibroblast tumor. The characteristic histologic feature is organoid triphasic morphology, and the molecular feature is somatic mutation of EGFR exon 20 (insertion/duplication).