Self-Amplifying RNA Vaccines for Venezuelan Equine Encephalitis Virus Induce Robust Protective Immunogenicity in Mice.

Venezuelan equine encephalitis virus (VEEV) is a known biological defense threat. A live-attenuated investigational vaccine, TC-83, is available, but it has a high non-response rate and can also cause severe reactogenicity. We generated two novel VEE vaccine candidates using self-amplifying mRNA (SAM). LAV-CNE is a live-attenuated VEE SAM vaccine formulated with synthetic cationic nanoemulsion (CNE) and carrying the RNA genome of TC-83. IAV-CNE is an irreversibly-attenuated VEE SAM vaccine formulated with CNE, delivering a TC-83 genome lacking the capsid gene. LAV-CNE launches a TC-83 infection cycle in vaccinated subjects but eliminates the need for live-attenuated vaccine production and potentially reduces manufacturing time and complexity. IAV-CNE produces a single cycle of RNA amplification and antigen expression without generating infectious viruses in subjects, thereby creating a potentially safer alternative to live-attenuated vaccine. Here, we demonstrated that mice vaccinated with LAV-CNE elicited immune responses similar to those of TC-83, providing 100% protection against aerosol VEEV challenge. IAV-CNE was also immunogenic, resulting in significant protection against VEEV challenge. These studies demonstrate the proof of concept for using the SAM platform to streamline the development of effective attenuated vaccines against VEEV and closely related alphavirus pathogens such as western and eastern equine encephalitis and Chikungunya viruses.

S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products.

BACKGROUND: Airway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated. METHODS: Rat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay. RESULTS: Results showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose-dependent manner with the maximal effect at 1 µg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE. CONCLUSIONS: Our study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products

BACKGROUND: Airway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated. METHODS: Rat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay. RESULTS: Results showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose- dependent manner with the maximal effect at 1 µg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE. CONCLUSIONS: Our study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

Effect of nitrogen limitation on cell growth, lipid accumulation and gene expression in Chlorella sorokiniana

The influence of nitrogen (N) limitation on the cell growth, chlorophyll content, intracellular lipid production and expression levels of two pathway genes in Chlorella sorokiniana was investigated in mixotrophic culture in this study. The maximum biomass concentration of C. sorokiniana cultured in modified BG11 medium containing 3, 6, 9, 12 and 15 mM N were 1.60, 2.21, 2.74, 3.18 and 3.21 g/L of dry cell weight, respectively with maximum specific growth rate of 0.180, 0.198, 0.201, 0.203, 0.206 day−1 during culture with an initial N feed of 3, 6, 9, 12 and 15 mM in the first eight days, respectively. The maximum lipid content was 51 % with 3 mM N. However, the maximum lipid productivity of 0.00884 g L−1 day−1 was achieved with 6 mM N. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit) and accI (homomeric acetyl-CoA carboxylase) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflected the increased lipid content in stationary phase. In contrast, expression of the acc1 gene always remained low, showing that the gene might not be critical for lipid accumulation.

Long circulating micelles of an amphiphilic random copolymer bearing cell outer membrane phosphorylcholine zwitterions.

Polymeric micelles with cell outer membrane mimetic structure were prepared in water from amphiphilic random copolymers bearing both the hydrophilic phosphorylcholine zwitterions and hydrophobic octadecyl side chains of cell outer membrane. The polymeric micelles showed sizes ranging from 80 nm to 120 nm in hydrodynamic diameter and zeta-potentials from -6.4 mV to -2.4 mV by dynamic light scattering measurements. The micelles loaded with 6-coumarin as a fluorescence probe were stable to investigate their blood circulation and biodistribution. The in vitro phagocytosis results using murine peritoneal macrophages showed 10-fold reduction compared with a reference micelle. The in vivo blood circulation half-life of the polymeric micelles following intravenous administration in New Zealand Rabbits was increased from 0.55 h to 90.5h. More interestingly, tissue distribution results showed that the concentration of the micelles in the kidney is 4-fold higher than that in the liver and other organs 48 h after administration. The results of this work show great promise for designing more effective stealth drug carriers that can minimize reticuloendothelial system clearance and circulate for long time to reach target by using simple cell membrane mimetic random copolymer micelles.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells.

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Natural and semisynthetic mammea-type isoprenylated dihydroxycoumarins uncouple cellular respiration.

In an effort to identify natural product-based molecular-targeted antitumor agents, mammea-type coumarins from the tropical/subtropical plant Mammea americana were found to inhibit the activation of HIF-1 (hypoxia-inducible factor-1) in human breast and prostate tumor cells. In addition to the recently reported mammea E/BB (15), bioassay-guided fractionation of the active extract yielded 14 mammea-type coumarins including three new compounds, mammea F/BB (1), mammea F/BA (2), and mammea C/AA (3). The absolute configuration of C-1' in 1 was determined by the modified Mosher's method on a methylated derivative. These coumarins were evaluated for their effects on mitochondrial respiration, HIF-1 signaling, and tumor cell proliferation/viability. Acetylation of 1 afforded a triacetoxylated product (A-2) that inhibited HIF-1 activation with increased potency in both T47D (IC(50) 0.83 µM for hypoxia-induced) and PC-3 cells (IC(50) 0.94 µM for hypoxia-induced). Coumarins possessing a 6-prenyl-8-(3-methyloxobutyl) substituent pattern exhibited enhanced HIF-1 inhibitory effects. The O-methylated derivatives were less active at inhibiting HIF-1 and suppressing cell proliferation/viability. Mechanistic studies indicate that these compounds act as anionic protonophores that potently uncouple mitochondrial electron transport and disrupt hypoxic signaling.

Mammea E/BB, an isoprenylated dihydroxycoumarin protonophore that potently uncouples mitochondrial electron transport, disrupts hypoxic signaling in tumor cells.

The mammea-type coumarin mammea E/BB (1) was found to inhibit both hypoxia-induced and iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in human breast tumor T47D cells with IC(50) values of 0.96 and 0.89 µM, respectively. Compound 1 suppressed the hypoxic induction of secreted VEGF protein (T47D cells) and inhibited cell viability/proliferation in four human tumor cell lines. Compound 1 (at 5 and 20 µM) inhibited human breast tumor MDA-MB-231 cell migration. While the mechanisms that underlie their biological activities have remained unknown, prenylated mammea coumarins have been shown to be cytotoxic to human tumor cells, suppress tumor growth in animal models, and display a wide variety of antimicrobial effects. Mechanistic studies revealed that 1 appears to exert an assemblage of cellular effects by functioning as an anionic protonophore that potently uncouples mitochondrial electron transport and disrupts mitochondrial signaling in human tumor cell lines.