A VLP Vaccine Induces Broad-Spectrum Cross-Protective Antibody Immunity against H5N1 and H1N1 Subtypes of Influenza A Virus

The recent threats of influenza epidemics and pandemics have prioritized the development of a universal vaccine that offers protection against a wider variety of influenza infections. Here, we demonstrate a genetically modified virus-like particle (VLP) vaccine, referred to as H5M2eN1-VLP, that increased the antigenic content of NA and induced rapid recall of antibody against HA(2) after viral infection. As a result, H5M2eN1-VLP vaccination elicited a broad humoral immune response against multiple viral proteins and caused significant protection against homologous RG-14 (H5N1) and heterologous A/California/07/2009 H1N1 (CA/07) and A/PR/8/34 H1N1 (PR8) viral lethal challenges. Moreover, the N1-VLP (lacking HA) induced production of a strong NA antibody that also conferred significant cross protection against H5N1 and heterologous CA/07 but not PR8, suggesting the protection against N1-serotyped viruses can be extended from avian-origin to CA/07 strain isolated in humans, but not to evolutionally distant strains of human-derived. By comparative vaccine study of an HA-based VLP (H5N1-VLP) and NA-based VLPs, we found that H5N1-VLP vaccination induced specific and strong protective antibodies against the HA(1) subunit of H5, thus restricting the breadth of cross-protection. In summary, we present a feasible example of direction of VLP vaccine immunity toward NA and HA(2), which resulted in cross protection against both seasonal and pandemic influenza strains, that could form the basis for future design of a better universal vaccine.     

Structural characterization and immuno-modulating activities of a polysaccharide from Ganoderma sinense

A protein-bound polysaccharide (GSP-4) with a molecular weight of 8.3×10(5)Da, was isolated from the water extract of the fruiting bodies of Ganoderma sinense. Chemical study revealed that this fraction was composed of mannose, glucose and galactose in the molar ratio of 4.7:27.1:1.0, with the sugar residues of t-, 1,3-, 1,4-, 1,6-, 1,3,4- and 1,3,6-linked Glcp, t-linked Galp, and 1,6-linked Manp. The immnomodulatory effects of GSP-4 were assessed using human peripheral blood mononuclear cells (PBMCs) and murine monocyte/macrophage cell line RAW 264.7. We found that GSP-4 could significantly stimulate the production of the immunomodulatory markers tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-12, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in PBMCs. This observation was further substantiated in RAW 264.7 cells, as indicated by the increase of nitric oxide (NO), TNF-α and IL-6 production. GSP-4 also enhanced the expression of inducible NO synthase mRNA in dose-dependent manner. Our current finding gives the first piece of evidence to support that GSP-4 possesses some promising immunomodulating effects and it could be a potential candidate to be further used in related cancer immunotherapy.     

Akt1 deficiency modulates reward learning and reward prediction error in mice

In contemporary reinforcement learning models, reward prediction error (RPE), the difference between the expected and actual reward, is thought to guide action value learning through the firing activity of dopaminergic neurons. Given the importance of dopamine in reward learning and the involvement of Akt1 in dopamine-dependent behaviors, the aim of this study was to investigate whether Akt1 deficiency modulates reward learning and the magnitude of RPE using Akt1 mutant mice as a model. In comparison to wild-type littermate controls, the expression of Akt1 proteins in mouse brains occurred in a gene-dosage-dependent manner and Akt1 heterozygous (HET) mice exhibited impaired striatal Akt1 activity under methamphetamine challenge. No genotypic difference was found in the basal levels of dopamine and its metabolites. In a series of reward-related learning tasks, HET mice displayed a relatively efficient method of updating reward information from the environment during the acquisition phase of the two natural reward tasks and in the reverse section of the dynamic foraging T-maze but not in methamphetamine-induced or aversive-related reward learning. The implementation of a standard reinforcement learning model and the Bayesian hierarchical parameter estimation show that HET mice have higher RPE magnitudes and that their action values are updated more rapidly among all three test sections in T-maze. These results indicate that Akt1 deficiency modulates natural reward learning and RPE. This study showed a promising avenue for investigating RPE in mutant mice and provided evidence for the potential link from genetic deficiency, to neurobiological abnormalities, to impairment in higher-order cognitive functioning.     

Lipase catalyzed acetylation of 3,5,4′-trihydroxystilbene: optimization and kinetics study

The use of immobilized lipase from Candida antarctica (Novozym(?) 435) to catalyze acetylation of trans-3,5,4'-trihydroxystilbene was investigated in this study. Response surface methodology and 5-level-4-factor central composite rotatable design were adopted to evaluate the effects of synthesis variables, including reaction time (24-72 h), temperature (25-65 °C), substrate molar ratio (1:15-1:75), and enzyme amount (600-3,000 PLU) on the percentage molar conversion of trans-4'-O-acetyl-3,5-dihydroxystilbene. The results showed that reaction temperature and enzyme amount were the most important parameters on percentage molar conversion. Based on ridge max analysis, the optimum conditions for synthesis were: reaction time 60 h, reaction temperature 64 °C, substrate molar ratio 1:56 and enzyme amount 2,293 PLU. The molar conversion of actual experimental values was 95% under optimal conditions. The synthesis product was analyzed using HPLC, mass and NMR. The results revealed that the major product was trans-4'-O-acetyl-3,5-dihydroxystilbene. The reaction kinetics was found to follow the Ping-Pong mechanism; substrate inhibition was not found at high vinyl acetate concentration.     

New Bapx1(Cre-EGFP) mouse lines for lineage tracing and conditional knockout studies

To gain insight into the roles of various genes in development and to circumvent embryonic lethality that hinders genetic studies, lineage tracing and conditional knockout techniques have been widely performed on mice using the increasing numbers of gene-targeted Cre mouse lines. Employing the internal ribosome entry site (IRES) and the 2A peptide multicistronic expression strategies, we report two new Bapx1 mouse lines with functional Bapx1 whereby Cre and enhanced green fluorescence protein (EGFP) are expressed discretely under the control of the Bapx1 promoter. These mouse lines, when mated with the Rosa26R-lacZ reporter line, can be used to trace the lineage of Bapx1-expressing cells whereas stage-specific, spatial expression of Bapx1 can be visualized by the EGFP fluorescence. In addition, both of our Bapx1(Cre-EGFP) mouse lines can be used to enrich for Bapx1-specific cells and also serve as effective conditional knockout tools to investigate gene functions in the skeleton and/or visceral organs.     

Ion channels/transporters as epigenetic regulators? —a microRNA perspective

MicroRNA (miRNA) alterations in response to changes in an extracellular microenvironment have been observed and considered as one of the major mechanisms for epigenetic modifications of the cell. While enormous efforts have been made in the understanding of the role of miRNAs in regulating cellular responses to the microenvironment, the mechanistic insight into how extracellular signals can be transduced into miRNA alterations in cells is still lacking. Interestingly, recent studies have shown that ion channels/transporters, which are known to conduct or transport ions across the cell membrane, also exhibit changes in levels of expression and activities in response to changes of extracellular microenvironment. More importantly, alterations in expression and function of ion channels/transporters have been shown to result in changes in miRNAs that are known to change in response to alteration of the microenvironment. In this review, we aim to summarize the recent data demonstrating the ability of ion channels/transporters to transduce extracellular signals into miRNA changes and propose a potential link between cells and their microenvironment through ion channels/transporters. At the same time, we hope to provide new insights into epigenetic regulatory mechanisms underlying a number of physiological and pathological processes, including embryo development and cancer metastasis.     

Pycnidione, a fungus-derived agent, induces cell cycle arrest and apoptosis in A549 human lung cancer cells

Pycnidione, a small tropolone first isolated from the fermented broth of Theissenia rogersii 92031201, exhibits antitumor activities through an undefined mechanism. The present study evaluated the effects and mechanisms of pycnidione on the growth and death of A549 human lung cancer cells. Pycnidione significantly inhibited the proliferation of A549 cells in a concentration-dependent manner, with a 50% growth inhibition (GI(50)) value of approximately 9.3nM at 48h. Pycnidione significantly decreased the expression of cyclins D1 and E and induced G(1)-phase cell cycle arrest and a subsequent increase in the sub-G(1) phase population. Pycnidione also markedly reduced the expression of survivin and activated caspase-8 and -3, increased reactive oxygen species (ROS) generation, caused the collapse of the mitochondrial membrane potential (MMP), and enhanced PAI-1 production, thus triggering apoptosis in the A549 cells. Taken together, pycnidione exerts anti-proliferative effects on human lung cancer cells through the induction of cell cycle arrest and apoptosis. Therefore, testing of its effects in vivo is warranted to evaluate its potential as a therapeutic agent against lung cancer.     

384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through Z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types.