miR­139­5p enhances cisplatin sensitivity in non­small cell lung cancer cells by inhibiting cell proliferation and promoting apoptosis via the targeting of Homeobox protein Hox­B2.

The development of chemotherapeutic dug resistance hinders the clinical treatment of cancer. MicroRNAs (miRNAs/miRs) have been revealed to serve essential roles in the drug resistance of numerous types of cancer. miR­139­5p was previously reported to be associated with cisplatin (DDP) sensitivity in human nasopharyngeal carcinoma cells and colorectal cancer cells. However, the effect and underlying mechanism of miR­139­5p in DDP sensitivity in non­small cell lung cancer (NSCLC) cells has not yet been fully elucidated. In the present study, the expression of miR­139­5p and Homeobox protein Hox­B2 (HOXB2) in NSCLC tissues was examined by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting. Subsequently, the effect of miR­139­5p on the DDP sensitivity of NSCLC cells in vitro was investigated. Cell proliferation was examined using a Cell Counting Kit­8 assay. Western blotting was used to evaluate the protein expression of HOXB2, phosphorylated (p)­PI3K, p­AKT, caspase­3 and cleaved­caspase­3, and RT­qPCR was used to evaluate the expression of miR­139­5p, and the mRNA expression levels of HOXB2, PI3K, AKT and caspase­3. The apoptotic rate of the cells was detected using flow cytometry. miR­139­5p expression in NSCLC tissues was shown to be significantly lower compared with that in adjacent tissues. Additionally, miR­139­5p increased cell apoptosis and inhibited NSCLC cell proliferation induced by DDP in vitro via modulating the PI3K/AKT/caspase­3 signaling pathway. Furthermore, HOXB2 was identified to be a target of miR­139­5p, and miR­139­5p was revealed to sensitize NSCLC cells to DDP via the targeting of HOXB2. Taken together, the results of the present study demonstrated that regulating the expression of miR­139­5p could provide a novel approach to reverse DDP resistance and increase chemosensitivity in the treatment of NSCLC.

Synergistic effect of halofuginone and dexamethasone on LPS­induced acute lung injury in type II alveolar epithelial cells and a rat model.

Acute lung injury (ALI) is characterized by neutrophilic infiltration, uncontrolled oxidative stress and inflammatory processes. Despite various therapeutic regimes having been performed, there remains no effective pharmacotherapy available to treat ALI. Halofuginone (HF), a ketone isolated from Dichroa febrifuga, exhibits significant anti­inflammatory and antifibrotic effects. Dexamethasone (DEX), a synthetic glucocorticoid, has been routinely used as an adjuvant therapy in treating inflammatory diseases, including ALI. The present study aimed to investigate the effects of the combination of HF and DEX in the treatment of ALI. The present results suggested that the simultaneous administration of HF and DEX markedly decreased the level of pro­inflammatory cytokines and increased the level of anti­inflammatory cytokines, as assessed by western blot analysis. In addition, HF and DEX effectively decreased nuclear factor­κB activity via suppressing the phosphorylation of P65 in lipopolysaccharide (LPS)­induced human pulmonary alveolar epithelial cells (HPAEpiC) and lung tissues extracted from ALI rats, as determined by immunofluorescence. Furthermore, in vivo experiments demonstrated that the combination of HF and DEX in LPS­induced ALI rats defended against lung fibrosis, perivascular inflammation, congestion and edema of pulmonary alveoli, as assessed by histopathological analysis, TUNEL staining and immunohistochemistry assay. Taken together, the present study indicated the synergistic effect of HF and DEX on LPS­induced ALI in HPAEpiC cells and a rat model. These results offer a novel therapeutic approach for the treatment of ALI.

Development and evaluation of rapid point-of-care tests for detection of Enterovirus 71 and Coxsackievirus A16 specific immunoglublin M antibodies.

Two colloidal gold immunochromatographic assays (CGIAs) for detection of EV71- and CA16- immunoglobulin M (IgM) were developed and evaluated. A total of 1465 sera collected from children with hand, foot, and mouth disease (HFMD), non-HFMD patients and healthy children. The sensitivity of IgM CGIA tests for EV71 and CA16 were 97.6% (330/338) and 91.6% (296/323) respectively, compared to those who were viral RNA positive by PCR. Their performances were comparable to those of commercial ELISA kits, with agreement of 98.1% for EV71-IgM and 97.3% for CA16-IgM. In addition, for EV71- and CA16-IgM CGIAs, the results of whole blood samples were 99.6% (248/249) and 100% (191/191) concordant to those with serum samples, respectively. As rapid point-of-care (POC) tests, the two CGIAs were suitable to be used in community clinic units, especially in resource-poor areas and will facilitate the control of HFMD.

Development of an enzyme-linked immunosorbent spot assay to measure serum-neutralizing antibodies against coxsackievirus B3.

Coxsackievirus B3 (CVB3) is the most common pathogen that induces acute and chronic viral myocarditis in children. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) and the plaque reduction neutralization test (PRNT) are the most common methods for measuring neutralizing antibody titers against CVB3 in blood serum samples. However, these two methods are inefficient for CVB3 vaccine clinical trials, which require the testing of a large number of serum specimens. In this study, we developed an efficient neutralization test based on the enzyme-linked immunospot (Nt-ELISPOT) assay for measuring CVB3-neutralizing antibodies. This modified ELISPOT assay was based on the use of a monoclonal antibody against the viral capsid protein VP1 to detect the cells that are infected with CVB3, which, after immunoperoxidase staining, are counted as spots using an automated ELISPOT analyzer. Using the modified ELISPOT assay, we characterized the infection kinetics of CVB3 and divided the infection process of CVB3 on a cluster of cells into four phases. The stability of the Nt-ELISPOT was then evaluated. We found that over a wide range of infectious doses (10(2) to 10(6.5)× 50% tissue culture infectious dose [TCID(50)] per well), the neutralizing titers of the sera were steady as long as they were tested during the log phase or the first half of the stationary phase of growth of the spots. We successfully shortened the testing period from 7 days to approximately 20 h. We also found that there was a good correlation (R(2) = 0.9462) between the Nt-ELISPOT and the Nt-CPE assays. Overall, the Nt-ELISPOT assay is a reliable and efficient method for measuring neutralizing antibodies in serum.

Immunogenicity and safety of hepatitis E vaccine in healthy hepatitis B surface antigen positive adults.

A recombinant hepatitis E vaccine, Hecolin, has been proven safe and effective in healthy adults. As hepatitis B surface antigen (HBsAg) positive individuals have a higher risk of poor prognosis after super-infection with hepatitis E virus (HEV), the safety and immunogenicity of Hecolin in this population should be assessed. The present study is an extending analysis of data from a large randomized controlled clinical trial of Hecolin. Healthy participants (n = 14,065) without current or previous evidence of chronic liver disease were randomized to receive Hecolin or placebo (hepatitis B vaccine) and donated their blood samples before vaccination and subsequently over 31 mo. Most of the adverse events were mild and comparable between participants with and without baseline hepatitis B surface antigen (HBsAg). No vaccine-related serious adverse events were reported. Rates of serious adverse events in HBsAg (+) or HBsAg (-) participants were also comparable between both groups. Almost all participants in the Hecolin group seroconverted to anti-HEV one month after full vaccination. The antibody response rates and levels were similar in HBsAg (+) and HBsAg (-) participants (98.38%, 19.32 Wu/mL vs. 98.69%, 19.00 Wu/mL). The two-year antibody dynamics of HBsAg (+) participants overlapped perfectly with those of HBsAg (-) participants. In conclusion, the safety and immunogenicity of Hecolin for HBsAg (+) adults is very similar to that for the general population.

Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot (ELISPOT) assay.

The baculovirus expression vector system (BEVS) is one of the most powerful methods for production of recombinant proteins for research or commercial purposes. Titration of viable virus in insect cell culture is often required when BEVS is used for basic research or bioprocessing. An enzyme-linked immunosorbent spot (ELISPOT) assay using monoclonal antibodies against the major capsid protein VP39 of both Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) was developed for baculovirus quantitation at 48h post-infection. The titer was determined by visualizing infected insect cells as blue spots and automated spot counting was achieved with ELISPOT hardware and software. Log-scale comparison of the results between the ELISPOT assay and a conventional end point dilution assay using a fluorescent marker showed a good correlation for both AcMNPV (R(2)=0.9980, p<0.05) and BmNPV (R(2)=0.9834, p<0.05). In conclusion, a novel, rapid and semi-automated procedure for titrating baculovirus was developed based on the specific immunostaining of infected cells followed by automated spot counting.

Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts.

The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein-protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368-606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH4)2SO4], sodium sulfate (Na2SO4), sodium chloride (NaCl), and ammonium chloride (NH4Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH4)2SO4 was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28-aa region (aa368-395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³7², Leu³75, and Leu³95 of p239 was found to be critical for particle assembly, which was revealed by site-directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions.

Homology model and potential virus-capsid binding site of a putative HEV receptor Grp78.

P239, a truncated construct of the hepatitis E virus (HEV) ORF2 protein, has been proven able to bind with a chaperone, Grp78, in both an in vitro co-immune precipitation test and an in vivo cell model. We previously solved the crystal structure of E2s--the C-terminal domain of p239 involved in host interactions. In the present study, we built a 3D structure of Grp78 using homology modeling methods, and docked this molecule with E2s using the Zdockpro module of the InsightII software package. The modeled Grp78 structure was deemed feasible by profile 3D evaluation and molecular dynamic simulations. The docking result consists of six clusters of distinct complexes and C035 was selected as the most reasonable. The interacting interface of the predicted complex is comprised of the Grp78 linker region and nucleotide binding domain along with the E2s groove region and surrounding loops. Using energy, hydrogen bond and solvent accessible surface analyses, we identified a series of key residues that may be involved in the Grp78:E2s interaction. By comparing with the known structure of the Hsp70:J complex, we further concluded that the interaction of Grp78 and E2s could interrupt binding of Grp78 with the J domain, and in turn diminish or even eliminate the binding ability of the Grp78 substrate binding domain. The predicted series of key residues also provides clues for further research that should improve our understanding of the fundamental molecular mechanisms of HEV infection.

Induction of cross-reactive antibodies against mimotopes of H5N1 hemagglutinin.

A sub-library based on peptide mimic 125 was designed and constructed, and 18 phagotopes specifically binding 8H5mAb were isolated. Antisera against three phagotopes, containing peptide 12MH-1, 12MH-5 and 12MH-8 reacted with 3 different H5N1 virus strains, but not with 2 H1N1 and 2 H3N2 viruses by Dot blots. The affinity of 12MH-8 was approximately eight times more than 12MH-1 or 12MH-5 or parent peptide 125. Furthermore, synthesized 12MH-1 and 12MH-8 could block the 8H5mAb binding with 4 H5N1 virus strains via hemagglutinin inhibition. These results suggest that these 3 mimotopes closely mimics the native 8H5 epitopes.

[Production of human papillomavirus type 16 virus-like particles and its immunogenicity].

HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.

Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus-host interaction.

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV-host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV.

Broad cross-protection against H5N1 avian influenza virus infection by means of monoclonal antibodies that map to conserved viral epitopes.

BACKGROUND: Passive immunization with human H5 antisera or H5-specific monoclonal antibodies (MAbs) has potential as an effective treatment for acute H5N1 influenza virus infection, but its efficacy against antigenically diverse H5N1 viruses is unconfirmed. METHODS: Cross-protection against antigenically diverse H5N1 strains with H5-specific MAbs, generated by successive immunization of antigenically distinct strains, was evaluated in mice. RESULTS: A panel of 52 broadly cross-reactive H5 specific MAbs were generated and characterized. One of these MAbs, 13D4, has been demonstrated to protect mice against lethal challenge by 4 H5N1 strains representing the current major genetic populations, clades 1, 2.1, 2.2, and 2.3, even at a stage of infection when H5N1 virus has disseminated beyond the pulmonary system. Complete neutralization of virus in lung tissue of infected animals was observed 24 h after treatment with 13D4. Mapping of this MAb with escape mutants showed it to bind to 2 conserved, possibly critical, sites of H5N1 hemagglutinin, 152 and 182. CONCLUSION: Generation of broadly cross-protective MAbs against H5N1 influenza virus may be optimized by selecting MAbs that target conserved sites in hemagglutinin. H5 MAbs such as 13D4 may prove to have therapeutic value in controlling infection due to current and future H5N1 variants.

[The preliminary analysis of the recognition epitopes of anti-HEV monoclonal antibodies on HEV ORF2].

Western blot, capture-PCR, blocking ELISA and synthetic polypeptides were used to systematically study the recognition epitopes on HEV ORF2 of 23 anti-HEV monoclonal antibodies(McAbs) which were previously generated in our laboratory directed against HEV ORF2. Results showed that seven McAbs recognized linear epitopes that located at aa408-458 of HEV ORF2 and 16 conformation-dependent McAbs, most of which recognized the surface epitopes of native HEV, located at aa459-606 of HEV ORF2. The systematical study of the recognition epitopes of anti-HEV McAbs on HEV ORF2 provides important information for the investigation of virus receptor and HEV infection mechanism, as well as its vaccine and diagnostics development.

[Improving baculovirus transduction of mammalian cells by spinoculation].

Recently many reports have described that recombinant baculovirus could serve as a new gene transfer vehicle for mammalian cells with many unique advantages. In this study, the constructed recombinant baculovirus BacV-CMV-EGFPA containing the enhanced green fluorescent protein (eGFP) gene driven by CMV promoter was used to explore the feasibility of improving the efficiencies of transduction experiment in CV-1 cells by centrifugal method. Refer to the centrifugal transduction protocol of recombinant lentivirus, CV-1 cells were incubated with the culture supematant of Sf-21 cells infected by BacV-CMV-ECFPA (moi = 30) and then centrifuged at 600g for 1 h at RT, reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM) 48h post transduction. Results showed that centrifugal method can achieve higher gene delivery and expression efficiencies than transduction by simple virus-cell mixing for 4h at 27 t with least impairment to cell viability. The centrifugal transduction protocol was further optimized by testing different centrifugal times, post-centrifugation incubation times and surrounding solutions. We found that centrifugation at 600g for 1 h at RT is sufficient to achieve the highest transduction efficiencies in target cells and PBS is more suitable than other surrounding solutions. Compared with previous protocol in which tranduction occurs for 4 - 8h at 27 degrees C, centrifugal method developed in this study could achieve more higher transduction efficiencies in more shorter time. Nine different mammalian cell lines (CV-1, 293FT, HepG2, 293T, CHO, C127, MT4, H9, Molt-4) were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the BacV-CMV-EGFPA supernatant (moi = 30) at 600g for 1h in PBS surrounding solution at RT. Results showed that most mammalian cell lines used in this study could be effectively transduced with recombinant baculoviruses by centrifugal method, and more higher and satisfactory transduction efficiencies could be achieved in primate adherent culture cells than in suspended culture cells. These results show that the baculovirus centrifugal transduction protocol have notable advantages: more rapid, efficient and nontoxic, and could be easily used in daily common experiments.

Abundant presence of the gamma-like Proteobacterial pufM gene in oxic seawater.

Known anoxygenic photosynthetic bacteria (APB) affiliated to Gammaproteobacteria usually use anaerobic metabolism and are restricted to oxygen-free habitats. Here, we report abundant (average of 34.5%) presence of diverse APB related to gamma-like Proteobacteria in oxic oceanic surface water as indicated by the pufM gene, that encodes the M subunit of the light reaction centre complex. Thus, our sequences were most likely derived from aerobic anoxygenic phototrophs (AAnP). Two genetically distinct genotypes were revealed: one was from the oligotrophic North Pacific Ocean Gyre and the other, was from the trophic East China Sea and Bering Sea. The discovery of abundant presence of novel gamma-like Proteobacterial pufM gene in the oxic seawater extends the functional ecotypes of AAnP.

Diversity and distribution of pigmented heterotrophic bacteria in marine environments.

A systematic investigation of marine pigmented heterotrophic bacteria (PHB) based on the cultivation method and sequencing analysis of 16S rRNA genes was conducted in Chinese coastal and shelf waters and the Pacific Ocean. Both the abundance of PHB and the ratio of PHB to CFU decreased along trophic gradients from coastal to oceanic waters, with the highest values of 9.9 x 10(3) cell mL(-1) and 39.6%, respectively, in the Yangtze River Estuary. In contrast to the total heterotrophic bacteria (TB) and CFU, which were present in the whole water column, PHB were primarily confined to the euphotic zone, with the highest abundance of PHB and ratio of PHB to CFU occurring in surface water. In total, 247 pigmented isolates were obtained during this study, and the phylogenetic analysis showed a wide genetic diversity covering 25 genera of six phylogenetic classes: Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacilli, Flavobacteria and Sphingobacteria. PHB belonging to Alphaproteobacteria, Flavobacteria and Sphingobacteria were obtained mainly from the South China Sea and East China Sea; PHB from the Pacific Ocean water were predominantly affiliated with Gammaproteobacteria, and most isolates from the Yangtze River Estuary fell into the classes Actinobacteria and Bacilli. The isolates exhibited various colours (e.g. golden, yellow, red, pink and orange), with genus or species specificity. Furthermore, the pigment of PHB cells absorbed light mainly in the wavelength range between 450 and 550 nm. In conclusion, our work has revealed that PHB with broad genetic diversity are widely distributed in the marine environment, and may account for up to 39.6% of culturable bacteria, equivalent to 1.4% of the total microbial community. This value might even be underestimated because it is probable that not all pigmented bacteria were isolated. Their abundance and genetic distribution are heavily influenced by environmental properties, such as light and nutrition, suggesting that they have important roles in the marine ecosystem, especially in the absorption of visible light.