Abstract: Objective　To investigate the influences of notoginsenoside R1 (NGR1) on cell injury and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway of alveolar epithelial cells infected by Klebsiella pneumoniae (Kp). Methods　A549 cells were grouped into five groups: control group (C group), infection group (Infect group), infection + low NGR1 group (Infect + L-NGR1 group), infection + high NGR1 group (Infect + H-NGR1 group), and infection+high NGR1+JAK2/STAT3 pathway inhibitor group (Infect+H-NGR1+SD-1029 group). Cell proliferation was measured using CCK8; ELISA kits were applied to detect the contents of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interferon γ (IFN-γ) in the culture medium; flow cytometry was applied to detect apoptosis; RT-qPCR was applied to detect the expressions of JAK2/STAT3; Western blot was applied to detect JAK2/STAT3 pathway, autophagy protein microtubule-associated protein 1 light chain 3 (LC3), autophagy-relatedgene5 (Atg5), autophagy-related gene (Atg) 6 (Beclin-1), apoptosis protein B-cell lymphoma 2 (Bcl-2), Bcl-2-accociated protein (Bax), cysteinyl aspartate specific proteinase (cleaved-caspase-3) proteins expression. Results　Compared with the C group, the 72 h cell viability, the protein levels of Bcl-2, LC3-II/I, Atg5, Beclin-1, the mRNA relative expressions and protein phosphorylation levels of JAK2, STAT3 in the Infect group were obviously decreased (P<0.05); the contents of IL-1β, TNF-α, IFN-γ, apoptosis rate, the protein levels of Bax and cleaved-caspase-3 were obviously increased (P<0.05). Compared with Infect group, the 72 h cell viability, the protein levels of Bcl-2, LC3-II/I, Atg5, Beclin-1, the mRNA relative expressions and protein phosphorylation levels of JAK2, STAT3 in the Infect+L-NGR1 group and Infect+H-NGR1 group were obviously increased (P<0.05); the contents of IL-1β, TNF-α, IFN-γ, apoptosis rate, the protein levels of Bax and cleaved-Caspase-3 were obviously decreased (P<0.05). Compared with Infect+H-NGR1 group, the 72 h cell viability, the protein levels of Bcl-2, LC3-II/I, Atg5, Beclin-1, the protein phosphorylation levels of JAK2, STAT3 in the Infect+H-NGR1+SD-1029 group were obviously decreased (P<0.05), and the contents of IL-1β, TNF-α, IFN-γ, apoptosis rate, the protein levels of Bax and cleaved-caspase-3 were obviously increased (P<0.05). Conclusions NGR1 can activate the JAK2/STAT3 signaling pathway, promote autophagy of alveolar epithelial cells, and inhibit Kp-induced inflammatory injury and apoptosis of alveolar epithelial cells.

ObjectiveToexplore the appearance ofmusclecrushinjuryatquantitative ultrasonographic elastography by supersonic shear imaging (SSI).MethodsThe animal experiment was done using a special balloon cuff device to create left hind leg crush injury with a force of 18.6 kPa.Twentythree New Zealand rabbits had crush injury of extremity and survived for 72 hours.SSI quantitative elastography was performed in crushed and no-crushed regions of each rabbit hind leg.Quantitative lesion elasticity was measured in terms of the Young modulus (in kilopascals) at 30 min,2 h,6 h,24 h and 72 h after the release of the crushing pressure.A receiver operating characteristic (ROC) curve analysis was used to assess diagnostic performance.ResultsThe area of crushed region in left hind leg accounted for 2.6％ -3.0％ of body surface area in 23 rabbits.The crushed regions exhibited maximum elasticity values of (19.51 ± 6.74)kPa,(21.47 ± 5.54) Pa,(11.36 ± 5.35)kPa,(15.09 ± 3.31)kPa and (13.72 ± 3.74) kPa,and mean elasticity values of (12.44 ± 3.77)kPa,(13.20 ± 3.60)kPa,(6.80±2.86)kPa,(10.04 ± 2.95)kPa and (6.94 ± 0.97)kPa at 30 min,2 h,6 h,24 h and 72 h after the release of the crushing pressure.Comparing with those of no-crushed regions,they were higher obviously (P＜0.001).ROC curves showed that extremity crush injury was diagnosed by using elasticity value,and the greater the elasticity value,the greater the diagnostic value.Conclusions SSI provides quantitative elasticity measurements,thus adding complementary information that potentially could help in crush injury characterization with conventional ultrasonography.

Aim To investigate the anticancer effect of a new xanthono-pyridine derivative N, N '-( 7-oxo-7H-chromeno[3,2-h] quinoline-5,9-diyl)-bis(2-( pyrroli-din-1-yl)acetamide) (XP-16) on human lung carcino-ma cell line A549 and the potential mechanism. Meth-ods Antiproliferative effect of XP-16 on A549 cells was evaluated by MTT assay, morphological examina-tion and colonial assay. Apoptosis detection was car-ried out using Hoechst 33258 and PI double-dyeing method. Intracellular Ca2+ concentration ( [ Ca2+] i ) and mitochondria membrane potential were detected by fluorospectrophotometer. A549 cells treated with XP-16 were collected for Bad and metallothionein 1 A ( MT-1 A ) transcript analysis by real-time reverse tran-scriptase-polymerase chain reaction ( qRT-PCR) . Re-sults XP-16 inhibited A549 cell proliferation in dose-and time-dependent manner. Typical apoptotic mor-
phology such as chromatin aggregation and nuclear fragmentation was observed in A549 cells treated with XP-16 for 24 h, and the apoptosis was showed in a dose-dependent manner. After treated with XP-16, [ Ca2+] i and mitochondria membrane potential of A549 cells were decreased, and relative mRNA level of Bad and MT-1A was up-regulated. Conclusions XP-16 has anticancer effect on A549 cells through apoptosis, which might be associated with decreasing intracellular Ca2+ concentration and mitochondria membrane poten-tial. Up-regulation of MT-1A expression might be the result of decreased [ Ca2+] i .

Objective To explore imaging feature of muscle crush injury of extremity on contrast enhanced ultrasound (CEUS) by an animal experiment.Methods Twenty-eight New Zealand rabbits were anesthetized by intramuscular pentobarbital sodium (30 mg/kg).A special balloon cuff device was used to create left hind leg crush injury with a force of (18.6 kPa).23 of the 28 rabbits had crush injury of extremity and survived for 72 hours.CEUS evaluated infusion model and contrast intensity in crush region of hind leg at the 0.5,2,6,24 and 72 hour after the release of the crushing pressure.Peak intensity (PI) was analyzed by comparing with serum creatinekinase (CK) and myoglobin (MYO).Results Twenty-three rabbits presented that the area of crushed region in left hind leg was 60 cm2,accounting for 2.6％-3.0％ of body surface area.In different time after decompression,the crushed regions showed rapid enhancement and the intensity was higher than that of no-crushed region.The time-intensity curves of crushed regions all appeared as fast wash-in and slow wash-out.PIs of rushed regions were higher obviously than those of nocrushed regions (P ＜0.001).ROC curves were employed in diagnosis value of PI for crushed region.And PIs showed positive correlation with CK and MYO and the correlation coefficient was 0.833-0.996.Conclusions CEUS presents that the micro-circulation perfusion of the crushed muscle increased obviously after the release of the crush pressure.PIs evaluated quantitatively the micro-circulation perfusion changes.It suggests a potential alternative for evaluating micro-circulation abnormality and ischemia reperfusion injury of the muscle crush injury to extremities.

Objective To explore contrast-enhanced ultrasonography (CEUS) in diagnosis renal injuries complicated with active bleeding of different velocity,and analysis the related factors of renal traumatic degree.Methods Thirty-four Ⅰ - Ⅴ grade lesions of renal injury were made in 4 dogs and 6 New Zealand rabbits.Two and three dimensional CEUS were used to observe traumatic extension,and traumatic position,involving in vascular as well.Then the injury condition was classified and assessed synthetically.Results The range of lesions observed by using 2D and 3D ultrasound had consistency with those of the pathologic sample (length-diameter:F =0.4724,P =0.6252; transverse diameter:F =1.6174,P =0.20490),3D-CEUS can display the vascular that involved by renal injury.In the same traumatic extension condition,the time of animal becoming shocked and injury severity was related to not only traumatic extension but also different velocity of active bleeding and involving in vascular.Conclusions Contrastenhanced ultrasound can objectively reflect renal injury severity,and more information can be provided to clinical for management.

Objective To quickly evaluate the traumatic degree of abdominal solid-organs using contrast-enhanced ultrasonography (CEUS) and analysis on related factors with clinical treatment.Methods 52 patients with abdominal traumatic were observed by CEUS,and the traumatic degree was judged according to American Association for the Surgery of Trauma (AAST).The change of peritoneal fluid was observed with ultrasonography,and active bleeding and involve adjacent vessels their branches were observed with CEUS.In this way,a method of quickly evaluate the traumatic degree was established,and the correlation between indifferent grade trauma and appropriate interventions that include surgical and conservative treatment was studied.Results 52 patients with 71 lesions,compound injuries accounted for 82.7％ (43/52).Among them,37 lesions were Ⅰ-Ⅲ grade trauma,34 lesions were severe trauma of Ⅳ-Ⅴ grade.The lesions complicated with active bleeding were 76.1％ (54/71).The amount of peritoneal fluid was increased significantly within 30 min (P ＜0.05) in traumatic lesions with rapid bleeding.Among of 50lesions associated with active bleeding,the surgical treatment was 24.0％ (12/50),the conservative treatment was 76.0％ (38/50).Among of trauma lesions involving the two following vessels,Ⅰ-Ⅲ grade was 97.3％ (36/37),Ⅳ-Ⅴ grade was 61.8％ (21/34).Trauma involvement above level 2 focal blood vessels,surgical treatment accounted for 23.1％ (12/52),conservative treatment accounted for 44.2％(23/52).Conclusions The severity of the trauma can not be a comprehensive response by AAST,becauce it is not only related to the scope of the traumatic lesions,vascular level,also involved with the trauma associated with active bleeding,bleeding speed and amount of peritoneal effusion and other factors.

OBJECTIVETo investigate the relationship between hepatitis B and gallstones using epidemiological methods.

METHODSThe analysed data from 510 patients with hepatitis B and 359 patients with other diseases treated in our hospital from the period January 1998 to June 2001 were retrospectively analysed. The patients with hepatitis B were classified into groups. The incidence of gallstones was determined in each group. Queue study method was used to calculate RR in each group and statistical analysis was conducted to determine difference among different groups.

RESULTSAs compared the patients gallstone with the patients with other diseases RR in those with hepatitis B (including those with chronic hepatitis or liver cirrhosis) was significantly higher (P<0.01). The value of RR was not markedly increased in patients with acute hepatitis or cholestatic hepatitis. After long-term administration of Chinese herbal medicine, the incidence of gallstones in patients with hepatitis B was decreased.

CONCLUSIONSChronic pathological changes in patients with hepatitis B may be one of the causes for gallstones. This may provide epidemiological basis for prevention and treatment of gallstones.

Adolescent ; Adult ; Aged ; Cholelithiasis ; epidemiology ; etiology ; Cohort Studies ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Hepatitis B ; complications ; drug therapy ; Humans ; Male ; Middle Aged ; Retrospective Studies

Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.
Codon ; Enzyme Precursors ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; Lipase ; biosynthesis ; genetics ; metabolism ; Pichia ; enzymology ; genetics ; Protein Engineering ; methods ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics ; Rhizopus ; enzymology ; genetics ; Substrate Specificity

Objective To explore the clinical application value and feasibility of contrast-enhanced ultrasonography (CEUS) in rapid diagnosis for bleeding of hepatocellular carcinoma. Methods From January 2009 to December 2012, 58 patients from the 302nd Hospital of the People′s Liberation Army underwent CEUS and conventional ultrasound, who were suspicioused primary bleeding of liver cancer or secondary bleeding of liver cancer after percutaneous radiofrequency ablation (RFA). The change of effusion volume in front of liver and in abdominal cavity, the perfusion time, range and characteristics of contrast agent, tumor vessel and the characteristic manifestation of active bleeding within the tumor and subcapsular were evaluated. Theχ2 test was used to compare the show rates of lesion boundary and the bleeding point by conventional ultrasound and CEUS. The t test was used to compare the change of effusion in front of liver and in abdominal cavity from 0 min to 30 min after bleeding were diagnosis by CEUS. Results For 58 cases of suspicious rupture and bleeding of liver cancer, 34 cases were conifrmed by clinical and imaging ifndings. The bleeding points were detected by conventional ultrasound in 5 cases. The bleeding points in 30 cases (including 4 cases of rapid bleeding, 7 cases of medium bleeding and 19 cases of slow bleeding) were detected by CEUS. The detection rate of lesion boundary, tumor vessel and bleeding point of CEUS were higher than those of conventional ultrasound, and the differences were statistically significant (χ2=10.350, P=0.001; χ2=4.300, P=0.034;χ2=36.790, P=0.0007). CEUS showed that contrast agent continuously concentrated at the bleeding point of lesions, and different degrees of bleeding were shown as′jet-like′,′linear′or′intermittent′spillover. The volume of effusion in front of the liver and in abdominal cavity at 30 min was more than those at 0 min after bleeding were diagnosis by CEUS, and the differences were statistically significant (the volume of effusion in abdominal cavity:t=-3.467, P=0.026;t=-12.895, P=0.000;t=-3.055, P=0.007;the volume of effusion in front of the liver:t=-8.110, P=0.001;t=-5.642, P=0.002;t=-5.981, P=0.000). Conclusions CEUS can show direct signs of rupture and bleeding of liver cancer, and the degree of bleeding can be evaluated according to the extravasation characteristics of contrast medium and the changes of lfuid volume. Therefore, CEUS can provid an objective basis for rapid diagnosis of ruptured hepatocellular carcinoma in the emergency scene and bedside.

In order to realize over-expression of Burkholderia cepacia (B. cepacia) lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene (T7 RNAP) from the BL21 (DE3) and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene (lipA) promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMdeltalipAlipB, pUCPCMdeltalipA, pUCPCMdeltalipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/mg, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMdeltalipB were enhanced to some extent. The specific activities of wild type B. cepacia and B. cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain.
Bacteriophage T7 ; genetics ; Burkholderia cepacia ; enzymology ; genetics ; Cloning, Molecular ; DNA-Directed RNA Polymerases ; genetics ; Escherichia coli ; enzymology ; genetics ; Lipase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transformation, Genetic ; Viral Proteins ; genetics