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Objective The peptide drug T20 (enfuvirtide), derived from the C-terminal heptad repeat region of HIV-1 gp41, is the only membrane fusion inhibitor available for treatment of viral infection; however, its mechanism of action remains elusive and its structural basis is lacking. Design We focused on determining the crystal structure of T20 in complex with N39, a target mimic peptide derived from the N-terminal heptad repeat region of gp41. On the basis of the structural information, the mechanisms of action of T20 and its resistance were further characterized. Methods A panel of peptides was synthesized. The T20/N39 complex was assembled for crystallization studies. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), native polyacrylamide gel electrophoresis (N-PAGE), and mutational analysis were applied to analyze the structural and functional properties. Results A crystal structure of six-helical bundle (6-HB) structure formed by T20 and N39 was determined with a resolution limit of 2.3 A, which revealed the critical intrahelical and interhelical interactions underlying the mechanism of action of T20 and its resistance mutations. Although the structural properties in the C-terminal tryptophan-rich motif (TRM) of T20 and the fusion peptide proximal region (FPPR) of N39 could not be finely defined by the structure, the data from biophysical and mutational analyses verified the essential roles of the TRM and FPPR motifs for the binding and inhibitory activities of T20. Conclusion For the first time, our studies provide a structural basis of T20, which help our understanding on the mechanisms of HIV-1 fusion and its inhibition.

Extracellular plaque deposits of β-amyloid peptide (Aβ) are one of the main pathological features of Alzheimer's disease (AD). The aggregation of Aβ

Enfuvirtide (T20) is the only viral fusion inhibitor approved for clinical use, but it has relatively weak anti-HIV activity and easily induces drug resistance. In succession to T20, T1249 has been designed as a 39-mer peptide composed of amino acid sequences derived from HIV-1, HIV-2, and simian immunodeficiency virus (SIV); however, its development has been suspended due to formulation difficulties. We recently developed a T20-based lipopeptide (LP-40) showing greatly improved pharmaceutical properties. Here, we generated a T1249-based lipopeptide, termed LP-46, by replacing its C-terminal tryptophan-rich sequence with fatty acid. As compared with T20, T1249, and LP-40, the truncated LP-46 (31-mer) had dramatically increased activities in inhibiting a large panel of HIV-1 subtypes, with IC(50) values approaching low picomolar concentrations. Also, LP-46 was an exceptionally potent inhibitor against HIV-2, SIV, and T20-resistant variants, and it displayed obvious synergistic effects with LP-40. Furthermore, we showed that LP-46 had increased helical stability and binding affinity with the target site. The crystal structure of LP-46 in complex with a target surrogate revealed its critical binding motifs underlying the mechanism of action. Interestingly, it was found that the introduced pocket-binding domain in LP-46 did not interact with the gp41 pocket as expected; instead, it adopted a mode similar to that of LP-40. Therefore, our studies have provided an exceptionally potent and broad fusion inhibitor for developing new anti-HIV drugs, which can also serve as a tool to exploit the mechanisms of viral fusion and inhibition.

S100A4, a member of the S100 calcium-binding protein family, has been identified in a subpopulation of liver macrophages and promotes liver fibrosis via hepatic stellate cell activation. However, the specific role of S100A4 in alcoholic liver disease (ALD) has not been well investigated. Here, S100A4 knockout (S100A4-/-) mice were used in a chronic-binge ethanol model for studying the role of S100A4 and its related molecular mechanism in ALD. S100A4 expression was increased in ethanol-induced liver tissues of wild-type (WT) mice. Macrophage-derived S100A4 promoted liver inflammation but suppressed lipid accumulation under the ethanol feeding condition. S100A4 deficiency promoted ethanol-induced liver injury and hepatic fat accumulation. Further mechanistic studies found that S100A4 inhibited liver fat accumulation mainly by activating the STAT3 pathway and downregulating lipogenic gene expression, especially that of SREBP-1c. In AML-12 cells, a STAT3 inhibitor abolished STAT3 levels and decreased the expression of SREBP1c. Furthermore, the administration of a neutralizing S100A4 antibody to WT mice significantly promoted ethanol-induced liver injury and fatty accumulation. Thus, S100A4 may represent a potential candidate target for the prevention and treatment of ethanol-induced fatty liver. In this study, we discovered the special role of S100A4 in alcoholic liver disease. S100A4 deficiency attenuated ethanol-induced hepatitis and promoted hepatic fat accumulation in ethanol-induced liver tissues. Further mechanistic studies have found that S100A4 promotes early alcoholic hepatitis mainly by activating the STAT3 pathway and its downstream proinflammatory gene expression. Interestingly, activation of the STAT3 pathway downregulates lipogenic gene expression, especially SREBP-1c. KEY MESSAGES: In this study, we discovered the special role of S100A4 in alcoholic liver disease. S100A4 deficiency attenuated ethanol-induced hepatitis and promoted hepatic fat accumulation in ethanol-induced liver tissues. Further mechanistic studies have found that S100A4 promotes early alcoholic hepatitis mainly by activating the STAT3 pathway and its downstream proinflammatory gene expression. Interestingly, activation of the STAT3 pathway downregulates lipogenic gene expression, especially SREBP-1c.

To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3).We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy.We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3.This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.

Fluorescence-based methods are promising for measuring amyloid beta (Aβ) oligomers, given their capacity to analyse a sample at the single-molecule level. As the attachment of fluorescent labels may influence the biochemical properties of the Aβ oligomers, the effects of fluorescent labels on Aβ oligomers must be evaluated. In this paper, we compared the impacts of five different fluorescent dyes on the aggregation of Aβ

The deep hydrophobic pocket of HIV-1 gp41 has been considered a drug target, but short-peptides targeting this site usually lack potent antiviral activity. By applying the M-T hook structure, we previously generated highly potent short-peptide fusion inhibitors that specifically targeted the pocket site, such as MT-SC22EK, HP23L, and LP-11. Here, the crystal structures of HP23L and LP-11 bound to the target mimic peptide N36 demonstrated the critical intrahelical and interhelical interactions, especially verifying that the hook-like conformation was finely adopted while the methionine residue was replaced by the oxidation-less prone residue leucine, and that addition of an extra glutamic acid significantly enhanced the binding and inhibitory activities. The structure of HP23L bound to N36 with two mutations (E49K and L57R) revealed the critical residues and motifs mediating drug resistance and provided new insights into the mechanism of action of inhibitors. Therefore, the present data help our understanding for the structure-activity relationship (SAR) of HIV-1 fusion inhibitors and facilitate the development of novel antiviral drugs.

Human respiratory syncytial virus (RSV) is the most significant cause of acute lower respiratory infection in children. However, there is no licensed vaccine available. Here, we investigated the effect of five or 20 copies of C-Class of CpG ODN (CpG-C) motif incorporated into a plasmid DNA vaccine encoding RSV fusion (F) glycoprotein on the vaccine-induced immune response. The addition of CpG-C motif enhanced serum binding and virus-neutralizing antibody responses in BALB/c mice immunized with the DNA vaccines. Moreover, mice vaccinated with CpG-modified vaccines, especially with the higher 20 copies, resulted in an enhanced shift toward a Th1-biased antibody and T-cell response, a decrease in pulmonary pathology and virus replication, and a decrease in weight loss after RSV challenge. This study suggests that CpG-C motif, cloned into the backbone of DNA vaccine encoding RSV F glycoprotein, functions as a built-in adjuvant capable of improving the efficacy of DNA vaccine against RSV infection.

The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition. IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.

Human respiratory syncytial virus (RSV) is an important pediatric pathogen causing acute viral respiratory disease in infants and young children. However, no licensed vaccines are currently available. Virus-like particles (VLPs) may bring new hope to producing RSV VLP vaccine with high immunogenicity and safety. Here, we constructed the recombinants of matrix protein (M) and fusion glycoprotein (F) of RSV, respectively into a replication-deficient first-generation adenoviral vector (FGAd), which were used to co-infect Vero cells to assemble RSV VLPs successfully. The resulting VLPs showed similar immunoreactivity and function to RSV virion in vitro. Moreover, Th1 polarized response, and effective mucosal virus-neutralizing antibody and CD8+ T-cell responses were induced by a single intranasal (i.n.) administration of RSV VLPs rather than intramuscular (i.m.) inoculation, although the comparable RSV F-specific serum IgG and long-lasting RSV-specific neutralizing antibody were detected in the mice immunized by both routes. Upon RSV challenge, VLP-immunized mice showed increased viral clearance but decreased signs of enhanced lung pathology and fewer eosinophils compared to mice immunized with formalin-inactivated RSV (FI-RSV). In addition, a single i.n. RSV VLP vaccine has the capability to induce RSV-specific long-lasting neutralizing antibody responses observable up to 15 months. Our results demonstrate that the long-term and memory immune responses in mice against RSV were induced by a single i.n. administration of RSV VLP vaccine, suggesting a successful approach of RSV VLPs as an effective and safe mucosal vaccine against RSV infection, and an applicable and qualified platform of FGAd-infected Vero cells for VLP production.

Human respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory tract infection in infants, the elderly, and the immunocompromised population. There is no licensed vaccine against RSV until now. It has been reported that targeting antigen to DEC205, a phagocytosis receptor on dendritic cells (DCs), could induce enhanced CD4+ and CD8+ T cell responses in mice. To develop RSV DNA vaccine and target the encoded antigen protein to DCs, the ectodomain of fusion glycoprotein (sF, amino acids: 23-524) of RSV was fused with anti-DEC205 single-chain Fv fragment (scDEC) and designated scDECF. Following successful expression from the recombinant plasmid of pVAX1/scDECF, the recombinant protein of scDECF was found capable of specifically binding to DEC205 receptor on CHOmDEC205 cells, and facilitating uptake of RSV F by DC2.4 cells in vitro. Furthermore, the higher levels of RSV-specific IgG antibody responses and neutralization antibody titers, as well as RSV F-specific CD8+ T cell responses were induced in mice immunized intramuscularly by pVAX1/scDECF than by the control plasmid of pVAX1/scISOF encoding sF protein fused with isotype matched control single-chain Fv fragment (scISO). Compared with pVAX1/scISOF, both the ratio of IgG2a/IgG1, >1, and the enhanced IFN-γ cytokine were induced in mice following pVAX1/scDECF immunization, which exhibited a Th1 dominant response in pVAX1/scDECF vaccinated mice. Notably, the elevated efficiency of RSV F protein bound by DCs in vivo could also be observed in mice inoculated by pVAX1/scDECF. Collectively, these results demonstrate the enhanced IgG and CD8+ T cell immune responses have been induced successfully by DNA vaccine against RSV by targeting F antigen to DCs via the DEC205 receptor, and this DC-targeting vaccine strategy merits further investigation.

The effects of the properties of casting solution on the surface structure of poly(methyl methacrylate) (PMMA) films were systematically investigated. It was observed that the hydrophobicity of PMMA films increased with increasing viscosity of the corresponding polymer solution regardless of the film-formation techniques that were utilized. The ratio of the C-H symmetric stretching vibrations of methylene groups (hydrophobic components, with a peak at 2910 cm(-1)) to those of the ester methyl groups (relative hydrophilic components, with a peak at 2955 cm(-1)) from sum frequency generation (SFG) vibrational spectra, A2910/A2955, was used as a parameter to evaluate the structure on the film surface, which was related to the surface wettability of the films. The results showed that A2910/A2955 of cast PMMA films increased linearly with ηsp(0.3) (ηsp, the specific viscosity of the casting solution), whereas that of the corresponding spin-coated films showed a linear relationship defined as ηsp(0.3)E(0.26), where E is the average number of entanglement points per molecule (E = Mw/Me). These results indicate that a relative equilibrium conformation on the PMMA film surface, adopted from the perspective of thermodynamics, was easily achieved during film formation, when the conformation of the polymer chains in the corresponding casting solution was close to that in the bulk. For the spin-coated films, the chain entanglement structure in the casting solution was a more important factor for the resulting film to reach a relative equilibrium state, since this structure was in favor of maintaining the pristine conformation in casting solution under centrifugal force during spin-coating. This work may help to enhance the fundamental understanding of the formation of the film surface structure from polymer solution to the resulting solid film, which will affect not only the corresponding surface properties, but also the dynamics of the resulting thin films.

Information technology brought organizations revolution of business process reengineering. ASCM is a core strategy of enterprises. How to apply IOT into ASCM is a hot problem in management. This paper constructed a general model of ASCM based on IOT, and put forward the evaluation method through entropy-DEA. To demonstrate the theoretical model and it’s evaluation method, we took a case as empirical analysis, and gave some advices from the conclusions.

In order to investigate antibody responses by musosal DNA vaccines encoding the codon-optimized F protein of human respiratory syncytial virus (RSV) delivered with attenuated Salmonella typhimurium aroA strain SL7207 (SL7207) via intranasal or intragastric routes.After the codon-optimized F gene was synthesized, we constructed eukaryotic expression plasmid pcDNA3.1/Fsyn and transformed it into SL7207 to get recombinant SL7207 of SL7207/pcDNA3.1/Fsyn. Then, SL7207/pcDNA3.1/Fsyn was exploited to evaluate its immunogenicity in BALB/c mice administered intranasally or intragastrically. The resultant serum and mucosal antibody responses were analyzed by indirect ELISA.Compared with intragastric immunization group, intranasal immunization group achieved higher level of RSV specific serum IgG and secretion IgA (SIgA) (P0.05). Moreover, F protein expressed by the codon-optimized F gene of Fsyn was of elevated immunogenicity, compared with wild type F (Fwt) (P0.05).Intranasal immunization and codon optimization approaches can improve antibody responses of RSV DNA vaccine delivered by attenuated Salmonella typhimurium aroA strain SL7207.

Knowledge innovation was one of targets and cores of knowledge management, organization knowledge innovation was an open complicated system work, which was affected by several internal and external factors. First, elements of knowledge innovation system were analyzed; Second, factors of organization knowledge innovation system were discussed; Third, key organizational factors and model of organization knowledge innovation were concluded through empirical research; Last, some conclusions and advices were proposed.

Previous studies reported that astrocyte response to amyloid-β (Aβ) before obvious neuronal damage could be detected in Alzheimer's disease (AD). It is suggested that astrocytes play a key role in AD pathologies. In this study, we investigated the effects of Aβ(42) oligomer on the proliferation and activation of astrocytes by in vitro experiments. The results showed that Aβ(42) oligomers could convert astrocytes to responsive astrocytes. It was revealed by MTT and ELISA assays that the viability of astrocytes gradually decreased, and the release of brain-derived neurotrophic factor increased with elevated Aβ(42) concentration and prolonged duration. Reverse-transcription polymerase chain reaction (RT-PCR) assay indicated that Aβ(42) oligomers increased the expression of glial fibers acid protein and interleukin-1β in a dose-dependent but not time-dependent manner. It was showed that A8, a mouse monoclonal antibody, was able to protect the cultured astrocytes against the toxicity of Aβ(42) oligomers. The result demonstrated that A8 could inhibit Aβ(42) oligomers toxic effects on astrocytes and that, alone, A8 could promote the proliferation of astrocytes in certain time. The present study laid a theoretical foundation for further understanding the effects of Aβ(42) on astrocytes and, hence, is conducive to the theoretical understanding and clinical therapies of AD progression.

To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector (HDAd) in vitro, we constructed a HDAd encoding enhanced green fluorescent protein (EGFP), denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP (FGAd/EGFP) in vitro. Therefore, we infected A549 cells with 2000 virus particles (vp) per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in early infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.

Based on knowledge fermenting theory, this paper analyzed the current situation and obstacles of organizational learning (OL) in its implementation process. Firstly, it analyzed the external driving force and the internal driving force of OL from the angle of knowledge enzyme. Secondly, it analyzed the knowledge source of OL from the concept of knowledge Sourdough and knowledge matrix. Thirdly, the impact of various elements in the fermentation bar on OL is analyzed. Finally, it explored the contents and the technologies of OL. The correct assessment of the actuality make for bringing forward correct strategy for OL, therefore this paper would provide the useful reference for the smooth implementation of OL.

To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.

The reverse genetics technology is an important way to develop genetically engineered attenuated living human respiratory syncytial virus (RSV) vaccine candidates. As the pilot experiment,it is necessary to prepare RSV minireplicon and investigate its biological activity.After the gene start and gene end (GSGE) fragment containing the sequence of leader (Le), gene start (GS), multiple cloning sites (MCS), gene end (GE) and trailer (Tr) was synthesized and positioned under the control of T7 RNA promoter, we cloned this fragment further into px8deltaT vector. Then, the enhanced green fluorescent protein (EGFP) was cloned into the above recombinant px8deltaT vector, and RSV minireplicon plasmids px8deltaT/GSGE1/EGFP and px8deltaT/GSGE2/EGFP were finally obtained. Meanwhile, two ORFs of nucleocapsid proteins of large protein (L) and transcription elongation/antitermination factor (M2-1) were cloned to construct pcDNA3.1/L and pcDNA3.1/M2-1. At the end, we co-transfected a RSV minireplicon plasmid and four nucleocapsid protein plasmids into BSR T7/5 cell lines expressing T7 RNA polymerase by lipofectamine 2000, and analyed the expression of EGFP by inverted fluorescent microscopy and fluorescence activated cell sortor (FACS), respectively.The px8deltaT/GSGE1/EGFP, px8deltaT/GSGE2/EGFP, pcDNA3.1/L and pcDNA3.1/M2-1 were successfully constructed. After co-transfecting, EGFP can be observed in the transfected BSR T7/5 cells under fluorescent microscopy and by FACS.The constructed RSV minireplicon is able to replicate and transcript, which provides a solid foundation for further RSV vaccine development and drug selection by RSV reverse genetics.

Helper-dependent adenoviral vector (HDAd) lacking all viral coding sequences with the advantages of minimal immunogenicity, negligible chronic-toxicity, and durable transgene expression over first-generation adenoviral vector (FGAd). HDAd vehicles have demonstrated tremendous potential for gene therapy in animal models for inherited diseases, neurodegenerative diseases and cancer etc. Additionally, the large cloning capacity of HdAd, up to 37 kb, permits the delivery of whole genemic loci, multiple transgenes. In this review we characterize the basic features of HdAd and summarize some of their experimental and potential clinical applications both at present and in future.

Quality improvement and quality innovation are the important approach to enhance competitive force for enterprises. Quality improvement is a process of knowledge innovation in nature which must be actualized by organizational learning. This article starts from the relations among quality improvement, quality innovation and the ability of organizational learning, analyzes the knowledge moving rule in the interior process of quality improvement and innovation and expatiates on the creation, development and diffusion mechanisms of quality knowledge in the quality flow based on the knowledge fermenting theory of organizational learning. Furthermore, taking the quality control (QC) group as an example, this article analyzes the behaviors and functions of nuclear factors such as quality knowledge sourdough, quality knowledge matrix and quality knowledge enzyme in the process of quality knowledge fermenting, and these nuclear factors function mutually in the quality knowledge fermenting bar. This article also puts forward five types of quality knowledge fermentation and analyzes their characters respectively, points out the implementation of knowledge fermentation possesses meanings to enhance the level of quality improvement and quality innovation for enterprises.

With the globalization of the world economy, especially after China entering the WTO, it is an unavoidable trend for China!? small & medium-sized enterprises to join the international competition.According to the analysis on the advantages and disadvantages of China!?s small & medium-sized enterprises, industrial cluster should be an efficient way for the small medium-sized enterprises to reach the target of internationalization. By establishing and improving specialized industry clusters with regional uniqueness, China!?s small & medium-sized enterprises could have formed their core competing powe in global competition and carry out their strategy of internationalization.

Background By using the hepatitis C virus (HCV) genotype 2a JFH-1 or its chimeric strains, a HCV infection system has been previously developed through several methods– such as in vitro-transcribed JFH1-RNA transfection or stable transfection of the JFH1 cDNA into human hepatoma Huh-7 cell line or its derivatives. However, other reliable methods for delivery of the HCV genome into cells are still worth trying. The helper-dependent adenovirus (HDAd) is devoid of all viral coding sequences and has a package capacity of 37 kb, which is suitably large for the delivery of the HCV genome. Here we report a new method for delivery of the HCV genome into Huh-7 and HepG2 cells by using the HDAd vector. Results Our results demonstrated that the infection of Huh-7 cells with the HDAdJFH1 virus led to efficient HCV replication and virion production. We found that the HCV viral RNA levels could reach 107 copies per milliliter (ml) in the culture medium. HDAdJFH1-infected Huh-7 cells could be cultured for 8 passages with the culture medium remaining infectious for naïve Huh-7 cells throughout this period. This infection system proved effective for evaluating the anti-HCV effects of IFN-α in Huh-7 cells. Co-infection of HepG2 cells with the HDAdJFH1 and HDAdmiR-122 virus also resulted in HCV expression and replication. Conclusion This is the first report of an HDAd-based strategy for HCV replication and production in vitro.

Liquid chromatography-mass spectrometry (LC-MS) combining with both highly efficient separation and detection is widely applied to chemistry, medicine, pharmacy biochemistry etc. Particularly, the application of LC-MS to clinic chemistry opened a new chapter for diagnosis technologies. Alzheimer' s disease (AD), also called as senile dementia, is the fourth leading cause of death in modern society. With the rapid aging of population, the prevention and treatment of AD need to be strengthened. In this review, we briefly introduce the new LC-MS developments and discuss the applications of LC-MS techniques in AD research in the last three years.