Akt finds its new path to regulate cell cycle through modulating Skp2 activity and its destruction by APC/Cdh1

Skp2 over-expression has been observed in many human cancers. However, the mechanisms underlying elevated Skp2 expression have remained elusive. We recently reported that Akt1, but not Akt2, directly controls Skp2 stability by interfering with its association with APC/Cdh1. As a result, Skp2 degradation is protected in cancer cells with elevated Akt activity. This finding expands our knowledge of how specific kinase cascades influence proteolysis governed by APC/Cdh1 complexes. However, it awaits further investigation to elucidate whether the PI3K/Akt circuit affects other APC/Cdh1 substrates. Our results further strengthen the argument that different Akt isoforms might have distinct, even opposing functions in the regulation of cell growth or migration. In addition, we noticed that Ser72 is localized in a putative Nuclear Localization Sequence (NLS), and that phosphorylation of Ser72 disrupts the NLS and thus promotes Skp2 cytoplasmic translocation. This finding links elevated Akt activity with the observed cytoplasmic Skp2 staining in aggressive breast and prostate cancer patients. Furthermore, it provides the rationale for the development of specific Akt1 inhibitors as efficient anti-cancer therapeutic agents

Simple and robust downstream purification process for cell-derived influenza vaccines

New emerging influenza viruses with pandemic potentials were occurred in recent years, e.g. H5N1 in 1997, H1N1 in 2009, and H7N9 in 2013. The demand of producing pandemic influenza vaccines for human use with quick supply is high. For the cell-based pandemic influenza vaccines, we proposed a flow-through chromatography purification process. This process has only involved few purification steps and is easy to operate. Vero- and MDCK- cell derived avian influenza viruses including H5N1 and H7N9 were purified efficiently by the process proposed. The presented purification process consisted of clarification, inactivation, concentration, anion exchange chromatography (Capto Q), size exclusion and adsorption chromatography (Capto Core 700), diafiltration and sterile filtration. In the chromatography steps, cell DNA and protein were removed remarkably, and the virus were flowed through these columns. The flow rate was set as fast as 250 cm/min. The loading volume of virus solution was up to 50 times of column volume (CV).The DNA was removed over 90% after using Capto Q column, and was further removed by Capto Core 700 column. The overall removal rate of cellular DNA was more than 99%. The HA recovery rates of H5N1 and H7N9 influenza virus from Vero and MDCK cells were 20 to 40%. The DNA concentration of all purified bulks met the regulatory requirement of 10ng per dose. The developed purification process is simple and efficient, and it is suitable for purification of various influenza virus strains and can be used for the pandemic influenza vaccine production

Macrophage Polarization Status Impacts Nanoceria Cellular Distribution but Not Its Biotransformation or Ferritin Effects

The innate immune system is the first line of defense against external threats through the initiation and regulation of inflammation. Macrophage differentiation into functional pheno- types influences the fate of nanomaterials taken up by these immune cells. High-resolution electron microscopy was used to investigate the uptake, distribution, and biotransformation of nanoceria in human and murine M1 and M2 macrophages in unprecedented detail. We found that M1 and M2 macrophages internalize nanoceria differently. M1-type macrophages predominantly sequester nanoceria near the plasma membrane, whereas nanoceria are more uniformly distributed throughout M2 macrophage cytoplasm. In contrast, both macrophage phenotypes show identical nanoceria biotransformation to cerium phosphate nanoneedles and simultaneous nanoceria with ferritin co- precipitation within the cells. Ferritin biomineralization is a direct response to nanoparticle uptake inside both macrophage phenotypes. We also found that the same ferritin biomineralization mecha- nism occurs after the uptake of Ce-ions into polarized macrophages and into unpolarized human monocytes and murine RAW 264.7 cells. These findings emphasize the need for evaluating ferritin biomineralization in studies that involve the internalization of nano objects, ranging from particles to viruses to biomolecules, to gain greater mechanistic insights into the overall immune responses to nano object

Analytical High-Resolution Electron Microscopy Reveals Organ-Specific Nanoceria Bioprocessing

This is the first utilization of advanced analytical electron microscopy methods, including high-resolution transmission electron microscopy, high-angle annular dark field scanning transmission electron microscopy, electron energy loss spectroscopy, and energy-dispersive X-ray spectroscopy mapping to characterize the organ-specific bioprocessing of a relatively inert nanomaterial (nanoceria). Liver and spleen samples from rats given a single intravenous infusion of nanoceria were obtained after prolonged (90 days) in vivo exposure. These advanced analytical electron microscopy methods were applied to elucidate the organ-specific cellular and subcellular fate of nanoceria after its uptake. Nanoceria is bioprocessed differently in the spleen than in the liver

Architecting the Communication and Navigation Networks for NASA's Space Exploration Systems

NASA is planning a series of short and long duration human and robotic missions to explore the Moon and then Mars. A key objective of the missions is to grow, through a series of launches, a system of systems communication, navigation, and timing infrastructure at minimum cost while providing a network-centric infrastructure that maximizes the exploration capabilities and science return. There is a strong need to use architecting processes in the mission pre-formulation stage to describe the systems, interfaces, and interoperability needed to implement multiple space communication systems that are deployed over time, yet support interoperability with each deployment phase and with 20 years of legacy systems. In this paper we present a process for defining the architecture of the communications, navigation, and networks needed to support future space explorers with the best adaptable and evolable network-centric space exploration infrastructure. The process steps presented are: 1) Architecture decomposition, 2) Defining mission systems and their interfaces, 3) Developing the communication, navigation, networking architecture, and 4) Integrating systems, operational and technical views and viewpoints. We demonstrate the process through the architecture development of the communication network for upcoming NASA space exploration missions

From Dose to Response: In Vivo Nanoparticle Processing and Potential Toxicity

Adverse human health impacts due to occupational and environmental exposures to manufactured nanoparticles are of concern and pose a potential threat to the continued industrial use and integration of nanomaterials into commercial products. This chapter addresses the inter-relationship between dose and response and will elucidate on how the dynamic chemical and physical transformation and breakdown of the nanoparticles at the cellular and subcellular levels can lead to the in vivo formation of new reaction products. The dose-response relationship is complicated by the continuous physicochemical transformations in the nanoparticles induced by the dynamics of the biological system, where dose, bio-processing, and response are related in a non-linear manner. Nanoscale alterations are monitored using high-resolution imaging combined with in situ elemental analysis and emphasis is placed on the importance of the precision of characterization. The result is an in-depth understanding of the starting particles, the particle transformation in a biological environment, and the physiological response

Nanoceria Distribution and Effects Are Mouse-Strain Dependent

Prior studies showed nanoparticle clearance was different in C57BL/6 versus BALB/c mice, strains prone to Th1 and Th2 immune responses, respectively. Objective: Assess nanoceria (cerium oxide, CeO2 nanoparticle) uptake time course and organ distribution, cellular and oxidative stress, and bioprocessing as a function of mouse strain. Methods: C57BL/6 and BALB/c female mice were i.p. injected with 10 mg/kg nanoceria or vehicle and terminated 0.5 to 24 h later. Organs were collected for cerium analysis; light and electron microscopy with elemental mapping; and protein carbonyl, IL-1β, and caspase-1 determination. Results: Peripheral organ cerium significantly increased, generally more in C57BL/6 mice. Caspase-1 was significantly elevated in the liver at 6 h, to a greater extent in BALB/c mice, suggesting inflammasome pathway activation. Light microscopy revealed greater liver vacuolation in C57BL/6 mice and a nanoceria-induced decrease in BALB/c but not C57BL/6 mice vacuolation. Nanoceria increased spleen lymphoid white pulp cell density in BALB/c but not C57BL/6 mice. Electron microscopy showed intracellular nanoceria particles bioprocessed to form crystalline cerium phosphate nanoneedles. Ferritin accumulation was greatly increased proximal to the nanoceria, forming core-shell-like structures in C57BL/6 but even distribution in BALB/c mice. Conclusions: BALB/c mice were more responsive to nanoceria-induced effects, e.g. liver caspase-1 activation, reduced liver vacuolation, and increased spleen cell density. Nanoceria uptake, initiation of bioprocessing, and crystalline cerium phosphate nanoneedle formation were rapid. Ferritin greatly increased with a macrophage phenotype-dependent distribution. Further study will be needed to understand the mechanisms underlying the observed differences

Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain

Phagocyte recognition and clearance of bacteria play essential roles in the host response to infection. In an on-going forward genetic screen, we identify the Drosophila melanogaster scavenger receptor Croquemort as a receptor for Staphylococcus aureus, implicating for the first time the CD36 family as phagocytic receptors for bacteria. In transfection assays, the mammalian Croquemort paralogue CD36 confers binding and internalization of Gram-positive and, to a lesser extent, Gram-negative bacteria. By mutational analysis, we show that internalization of S. aureus and its component lipoteichoic acid requires the COOH-terminal cytoplasmic portion of CD36, specifically Y463 and C464, which activates Toll-like receptor (TLR) 2/6 signaling. Macrophages lacking CD36 demonstrate reduced internalization of S. aureus and its component lipoteichoic acid, accompanied by a marked defect in tumor necrosis factor-α and IL-12 production. As a result, Cd36−/− mice fail to efficiently clear S. aureus in vivo resulting in profound bacteraemia. Thus, response to S. aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain, which initiates TLR2/6 signaling

In Vivo Processing of Ceria Nanoparticles inside Liver: Impact on Free-Radical Scavenging Activity and Oxidative Stress

The cytotoxicity of ceria ultimately lies in its electronic structure, which is defined by the crystal structure, composition, and size. Despite previous studies focused on ceria uptake, distribution, biopersistance, and cellular effects, little is known about its chemical and structural stability and solubility once sequestered inside the liver. Mechanisms will be presented that elucidate the in vivo transformation in the liver. In vivo processed ceria reveals a particle-size effect towards the formation of ultrafines, which represent a second generation of ceria. A measurable change in the valence reduction of the second-generation ceria can be linked to an increased free-radical scavenging potential. The in vivo processing of the ceria nanoparticles in the liver occurs in temporal relation to the brain cellular and protein clearance responses that stem from the ceria uptake. This information is critical to establish a possible link between cellular processes and the observed in vivo transformation of ceria. The temporal linkage between the reversal of the pro-oxidant effect (brain) and ceria transformation (liver) suggests a cause-effect relationship