Over-expression of uPA increases risk of liver injury in pAAV-HBV transfected mice

AIM:To investigate the relationship between overexpression of urokinase plasminogen activator(uPA) and hepatitis B virus(HBV) related liver diseases in a transgenic mouse model.METHODS:Albumin-tetracycline reverse transcriptional activator and tetO-uPA transgenic mice were generated respectively through pronuclear injection and crossed to produce the double transgenic in-alb-uPA mice,for which doxycycline(Dox)-inducible and liver-specific over-expression of uPA can be achieved.Hydrodynamic transfection of plasmid adeno-associated virus(AAV)1.3HBV was performed through the tail veins of the Dox-induced in-alb-uPA mice.Expression of uPA and HBV antigens were analyzed through double-staining immunohistochemical assay.Cytokine production was detected by enzyme linked immunosorbent assay and α-fetoprotein(AFP) mRNA level was evaluated through real-time quantitative polymerase chain reaction.RESULTS:Plasmid AAV-1.3HBV hydrodynamic transfection in Dox-induced transgenic mice not only resulted in severe liver injury with hepatocarcinoma-like histological changes and hepatic AFP production,but also showed an increased serum level of HBV antigens and cytokines like interleukin-6 and tumor necrosis factor-α,compared with the control group.CONCLUSION:Over-expression of uPA plays a synergistic role in the development of liver injury,inflammation and regeneration during acute HBV infection.

Detection of Antibody to Hepatitis Delta Virus in Human Serum by Double Antigen Sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein’s purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.

The pseudospectrum and spectrum(in)stability of quantum corrected Schwarzschild black hole

In this study,we investigate the pseudospectrum and spectrum(in)stability of quantum corrected Schwarzschild black hole.Methodologically,we use the hyperboloidal framework to cast the quasinormal mode(QNM)problem into an eigenvalue problem associated with a non-selfadjoint operator,and then the spectrum and pseudospectrum are depicted.Besides,the invariant subspace method is exploited to improve the computational efficiency for pseudospectrum.The investigation into the spectrum(in)stability entails two main aspects.On the one hand,we calculate the spectra of the quantum corrected black hole,then by the means of the migration ratio,the impact of the quantum correction effect on the Schwarzschild black hole has been studied.The results indicate that the so-called“migration ratio instability”will occur for small black holes with small angular momentum number l.In the eikonal limit,the migration ratios remain the same for each overtone.On the other hand,we study the spectrum(in)stability of the quantum corrected black hole by directly adding some particular perturbations into the effective potential,where perturbations are located at the event horizon and null infinity,respectively.There are two interesting observations under the same perturbation energy norm.First,perturbations at infinity are more capable of generating spectrum instability than those at the event horizon.Second,we find that the peak distribution can lead to the instability of QNM spectrum more efficiently than the average distribution.

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonu-clease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, cir-cularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.