Mitochondria, the power house of cells, are important organelles in eukaryotic cells. Having their own unique and complete DNA (mtDNA) and genetic system, mitochondria play an essential role in cellular energy metabolism, intracel?lular signaling and apoptotic pathways, as well as many other biological functions, which are closely related with cellular met?abolic network. A disruption of mitochondrial genes can therefore result in mitochondrial dysfunction and human diseases, thus they have been widely used in molecular biology, development biology, genetics, forensic identification and clinical diag?nosis. Consequently, sequencing mitochondrial genome has shown great significance in mitochondrial structure and function research. In this review, research progress in mitochondrial genome sequencing method is summarized, mainly focusing on Sanger sequencing, long-PCR and next-generation sequencing. Also rolling circle amplification and indirect sequencing of mtDNA are reviewed. The ambiguities caused by numts in indirect sequencing are mentioned and resolved.

Objective To establish a hepatocyte line from human for bioartificial liver support system and hepatocyte transplantation. Methods Hepatocytes isolated from donor liver of a 25 year old brain death male were transfected with pcDNA3.1(-) recombined vector containing the genes encoding Simian Virus 40 large T antigen. Characteristics of the immortalized hepatocyte line were evaluated by morphologic and functional methods. Results One of the hepatocyte clones displayed highly differentiated liver function and immortalized characteristics has been screened after 42 day's selection of 700～300 ?g/ml G418. The immortalized hepatocyte appeared epithelial and displayed morphologic characteristics of liver parenchymal cells. ALB, AST, ALT and LDH secreted from hepatocyte line were detected. Transmission electron microscope examination revealed that immortalized cells contained a round nucleus with nucleoli, moderate numbers of mitochondria, numerous rough endoplasmic reticulums, and some vacuoles. Adjacent cells were joined with junctional complexes. RT PCR and Western blot detection revealed that immortalized hepatocyte could express ALB mRNA and showed immunoreactive of cytochrome P450 2E1. Conclusions The new established hepatocyte line from human retained some specificbiologic functions and morphological cha racteristics of primary cultured hepatocytes.

Objective To investigate the efficacy and safety of adefovir dipivoxil (ADV) in treatment of chronic hepatitis B (CHB) patients with lamivudine (LAM) resistance. Methods There were treatment group (32 CHB patients with LAM resistance) and historical control group (24 CHB patients with LAM resistance) in this study. The treatment group received ADV 10 mg/d and LAM 100 mg/d for 48 weeks; the historical control group continued to use LAM monotherapy. During the treatment causes, serum HBV DNA levels, liver function and HBV serology were monitored regularly, and safety assessments were also conducted. Results In treatment group, mean HBV DNA levels decreased by 2.56 log10 eopies/ml and 2.93 log10 copies/ml, virus response rates were 50. 0% and 75.0%, ALT normalization rates were 53.1% and 68.8% after 24 and 48 weeks of treatment, respectively. The histological improvement rate was 65.6% after 48 weeks. Comparing with those in control group, the differences were statistically significant ( P <0. 05), while there was no significant statistical differences in HBeAg loss rate and HBeAg seroconversion rate between two groups. There was no severe adverse event during the treatment. Conclusion ADV is effective and safe in treatment of lamivudine-resistant CHB.

Objective To investigate the expression profile of mRNAs in brain samples collected from pronuclear transfer (PNT) mice. Methods Female CD-1 mice were superovulated, and zygotes were collected after mating with adult male mice. Zygotes with two pronuclei were selected for pronuclear transfer manipulation, and then the reconstructed zygotes were transferred into the oviduct of pseudopregnant female mice. The infant mice obtained from pronuclear transfer were called PNT group, while the embryoes that were not performed pronuclear transfer was regarded as control group. Total RNA were extracted from brain samples of both PNT and control mice, and cDNA were labeled with fluorescent dye. Genes that were differentially expressed were identified using the Agilent mouse mRNA array. Gene ontology analysis and pathway analysis were also completed. Results Compared with control group, 392 mRNAs were expressed differentially, which showed more than 2.0 times variation and statistical significance, accounting for 1.7% of all mRNAs. Among those 366 mRNAs were up-regulated and 26 mRNAs were down-regulated. Eleven mRNAs came to 4.0 times variation in total. Gene ontology analysis indicated that differentially expressed genes were significantly enriched in alternative mRNA splicing, small GTPase mediated signal transduction, regulation of insulin receptor signaling pathway, hydrolase activity, transmembrane transporter activity and pyrophosphatase activity. Significant enriched pathway terms contained ion channel transport, fatty acid metabolism, butanoate metabolism, triacylglycerol and ketone body metabolism. Conclusion Pronuclear transfer might influence some key metabolism process in mouse brain.

Objective To establish normally immortalized porcine hepatocyte lines by ectopic expression of simian virus 40 large T (SV40LT) antigen and the human telomerase reverse transcriptase(hTERT). Methods Primary porcine Hepatoeyte cells were transfeeted with recombinant retrovirus containing SV40LT or hTERT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Immortalized porcine hepatocyte was confirmed by examination. Results The morphological phenotype of the transfected cells was similar to the primary porcine hepatocyte. One clone, HepLP, has been maintained in cultue for half year, and expanded by more than 60 passages. SV40 LT and hTERT could be detected in transfected porcine hepatocyte. Pig albumin mRNA was also detected by RT-PCR. No tumor formation occurred when HepLP cells were injected into Balb/c nude mice. Conclusions The immortalized, nontumorigenic, porcine hepatoeytes maintained the properties of porcine primary hepatocytes such as the albumin secretion. This generation of immortalized porcine hepatocyte may be helpful for bioartifical liver support system, hepatocytes transplantation, drug/toxicological studies, and liver biologic studies.

Objective To establish immortalized human hepatocyte lines for studies of bioartificial liver,hepatocyte transplantation,and drug metabolism in vitro.Methods Primary human hepatocytes were isolated by 4-step perfusion technique with collagenase and transfected with recombinant retrovirus containing Simian virus 40 large T antigen(SV40 LT).Subsequently,immortalized human hepatocytes were evaluated by analysis of gene expression and functional characteristics in vitro.Results Two immortalized human hepatocyte lines,HepLi2 and HepLi3,were obtained after primary human hepatocytes being infected by SV40 LT containing recombinant retrovirus for 3-4 weeks.The immortalized human hepatocytes showed classical appearance of hepatocyte observed by phase contrast microscope.The protein expression of SV40 LT in HepLi2 and HepLi3 cells were detected by Western blotting.The mRNA expressions of albumin(Alb),glutathione S-transferase(GST-p),human blood coagulation factor X(HBCF-X)and β-actin in HepLi2 and HepLi3 cells were detected by reverse transcription polymerase chain reaction(RT-PCR),and the mRNA expressions of cytochrome(CY)450 subtypes(CYP3A5,CYP2E1,CYP2C8-19 and CYP3A4)in HepLi2 and HepLi3 cells were also observed by RT-PCR.Levels of alanine transaminase (ALT),lactate dehydrogenase(LDH)and Alb were detected in the supernatant of immortalized human hepatoeyte culture.Conclusions The immortalized human hepatocyte lines have the biological characteristics of primary human hepatocytes and have the CYP450 functions of hepatocytes,which may be heIDful for the studies of bioartificial liver,heoatocvte transplantation and drug metabolism in vitro.

Objective:To compare the diagnostic efficacies of Sonazoid contrast-enhanced ultrasound (CEUS) and contrast-enhanced magnetic resonance imaging (CE-MRI) in the diagnosis of focal liver lesions (FLLs), and to evaluate the clinical value of Sonazoid.Methods:A total of 58 FLLs in 50 patients who underwent Sonazoid-CEUS and CE-MRI examinations from July 2019 to January 2021 in the First Affiliated Hospital of Zhejiang University School of Medicine were enrolled in this study according to the inclusion criteria. The final diagnostic reference standard was decided by surgical pathology or ultrasound-guided biopsy pathology. Sonazoid-CEUS and CE-MRI features of benign and malignant FLLs were analyzed, and the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic coincidence rate of the two tests were calculated respectively.Results:There was a statistically significant difference between benign and malignant FLLs in the imaging pattern of homogeneous or heterogeneous intratumoral enhancement in the artery-dominant phase and washout images in the late phase( P<0.001).9.8%(4/41) of the malignant lesions did not decrease until the late phase but decreased in the post-vascular phase. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic coincidence rate of the two tests were 97.6%, 52.9%, 83.3%, 90.0%, 84.5%(Sonazoid-CEUS) and 85.4%, 64.7%, 85.4%, 64.7%, 79.3%(CE-MRI), the differences of sensitivity and specificity were not statistically significant ( P=0.125, P=0.687). Conclusions:The vascular phase in Sonazoid-CEUS is still an important diagnostic sign of FLLs, and the unique Kupffer phase can provide additional information for the diagnosis. Sonazoid-CEUS has the same important value as CE-MRI in the diagnosis of FLLs.

Objective:To investigate the prognostic value of serum free triiodothyronine (FT3) in patients with hepatitis E-related acute liver failure (HEV-ALF).Methods:Clinical data of 88 patients with HEV-ALF and 86 patients with acute hepatitis E (AHE) were collected from the member hospitals of Chinese Consortium for the Study of Hepatitis E between January 2016 and December 2021; the data of 100 health subjects who underwent health check-up in Suzhou Municipal Hospital were also collected as healthy control (HC) group. Serum FT3 levels were analyzed in all subjects. HEV-ALF patients were divided into survival group ( n=73) and death group ( n=15) according to their 30 day survival. Correlation between serum FT3 level and prognosis of HEV-ALF patients were analyzed by Cox regression and orthogonal partial least squares discriminant analysis (OPLS-DA). The receiver operating characteristic (ROC) curve and decision curve analysis (DCA) were used to assess the predictive value of serum FT3 levels for predicting the prognosis of patients, and its prediction efficacy was compared with conventional Model for End-Stage Liver Disease (MELD), King’s College Hospital criteria (KCH) and Child-Pugh models. Results:The levels of serum FT3 in HEV-ALF patients were significantly lower than those in AHE patients and HC group ( P=0.006 or <0.001). Cox regression analysis showed that international standardized ratio ( HR=17.984, 95% CI 2.804-115.362), hepatic encephalopathy ( HR=12.895, 95% CI 2.386-69.695) and total cholesterol ( HR=2.448, 95% CI 1.108-5.409) were independent risk factors for death in HEV-ALF patients, and serum FT3 level ( HR=0.323, 95% CI 0.119-0.876) was a protective factor. OPLS-DA results showed serum FT3 levels had high predictive value. ROC curve analysis results showed that the area under the curve was 0.828 (95% CI 0.733-0.900, P<0.001), the sensitivity was 80.00%, and the specificity was 78.08%. DCA showed that FT3 has good prediction ability and decision-making level serum FT3 levels in patients with improvement and fluctuation were significantly higher than those in the patients with deterioration ( P<0.05 or <0.01). Conclusion:Serum FT3 levels are closely related to the prognosis of HEV-ALF patients and it may be used as a biomarker for the prognosis of patients with HEV-ALF.

Objective:To investigate the serotype distribution, drug resistance and the virulence gene profiles of clinical isolates of Salmonella in Zhejiang Province. Methods:A retrospective study of 463 clinical isolates of Salmonella in nine regions of Zhejiang Province from 2010 to 2017 was conducted. Their serotypes were detected using the Kauffmann-White scheme based on serological detection. Antibiotics susceptibility tests were carried out using K-B disk diffusion method. Eleven virulence genes were detected by polymerase chain reaction. Results:A total of 35 serotypes were identified among the 463 strains of Salmonella. The dominant serotypes were Salmonella typhimurium (41.90%(194/463)) and Salmonella enteritis (22.25%(103/463)). The resistance rate of Salmonella to ampicillin was the highest (66.52%(308/463)), followed by ampicillin/sulbactam (46.87%(217/463)), while low resistance rate to piperacillin/tazobactam was observed (3.24%(15/463)). All strains were sensitive to imipenem and meropenem. Finally, 188 strains (40.60%) of multi-drug resistance were found. The virulence genes hilA, ssrB, marT, siiD, sopB and pagN were found in all Salmonella strains. The virulence gene vexA was found only in Salmonella typhi and Salmonella Dublin; virulence gene icmF was mainly found in Salmonella enteritis. The virulence plasmid gene spvB and pefA were mainly found in Salmonella enteritidis and Salmonella typhimurium and invariably appeared in pairs. The virulence gene cdtB was mainly found in Salmonella typhi and Salmonella paratyphi A. Conclusions:Salmonella typhimurium and Salmonella enteritis are the main clinically isolated Salmonella strains in Zhejiang Province. The situation of multi-drug resistance is severe and a variety of virulence genes are highly detected.

‍Hepatitis E is a type of viral hepatitis caused by hepatitis E virus （HEV） infection. There are four main genotypes of HEV， namely genotypes 1‍ — ‍4. Compared with hepatitis B and hepatitis C， previous research on hepatitis E has made slow progress； however， in recent years， hepatitis E has gradually attracted more attention in China， achieving the transition from behind the scenes to the front stage. First， China has a large population base， a large number of people infected with hepatitis E， and a heavy disease burden， and thus it is necessary to attach great importance to the prevention， diagnosis， and treatment of hepatitis E. Second， the World Health Organization （WHO） has put forward the ambitious goal of eliminating viral hepatitis by 2030， and China has made remarkable achievements in the prevention and treatment of hepatitis B and hepatitis C. At present， the incidence and mortality rates of hepatitis E in China have exceeded those of hepatitis A， and the prevention and treatment of hepatitis E in China is of great significance to actively respond to the call of the WHO. Finally， Chinese researchers in the field of hepatitis E have formed Chinese Consortium for the Study of Hepatitis E， formulated related expert consensus， and established international cooperation platforms， which further promotes the research on hepatitis E in China.