Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis

Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing "nanotoxicity" are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surface-coating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of "nanotoxicity".     

Evolution of a new nonclassical MHC class I locus in two Old World primate species

HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecule that is expressed only in the human placenta, suggesting that it plays an important role at the fetal-maternal interface. In rhesus monkeys, which have similar placentation to humans, the HLA-G orthologue is a pseudogene. However, rhesus monkeys express a novel placental MHC class I molecule, Mamu-AG, which has HLA-G-like characteristics. Phylogenetic analysis of AG alleles in two Old World primate species, the baboon and the rhesus macaque, revealed limited diversity characteristic of a nonclassical MHC class I locus. Gene trees constructed using classical and nonclassical primate MHC class I alleles demonstrated that the AG locus was most closely related to the classical A locus. Interestingly, gene tree analyses suggested that the AG alleles were most closely related to a subset of A alleles which are the products of an ancestral interlocus recombination event between the A and B loci. Calculation of the rates of synonymous and nonsynonymous substitution at the AG locus revealed that positive selection was not acting on the codons encoding the peptide binding region. In exon 4, however, the rate of nonsynonymous substitution was significantly lower than the rate of synonymous substitution, suggesting that negative selection was acting on these codons.     

A three-dimensional,clothed human thermal model

We have developed a three-dimensional clothed human thermal model for predicting physiological responses to temperature and humidity within clothing by considering the thermal properties of fabrics, clothing ensembles, and their designs. The model is a coupled simulation system of a human thermal model and a clothing thermal model. The model performance was investigated by comparing its predictions with experimental data. The following results were obtained. (1) The model predictions of skin temperature distributions and the micro-climate within clothing basically agreed with experimental data. (2) The skin temperatures of clothed body parts were higher than the corresponding body parts in the nude condition, and the skin temperatures of the hand and foot were higher than those in the nude condition, both in the model predictions and in the experimental data. (3) The model prediction of total moisture transfer was almost the same as obtained from the experimental data. 1998 Scripta Technica, Heat Trans Jpn Res, 26(8): 554–566, 1997     

Overview of Ferroptosis and Synthetic Lethality Strategies

Ferroptosis, a term first proposed in 2012, is iron-dependent, non-apoptotic regulatory cell death induced by erastin. Ferroptosis was originally discovered during synthetic lethal screening for drugs sensitive to RAS mutant cells, and is closely related to synthetic lethality. Ferroptosis sensitizes cancer stem cells and tumors that undergo epithelial−mesenchymal transition and are resistant to anticancer drugs or targeted therapy. Therefore, ferroptosis-inducing molecules are attractive new research targets. In contrast, synthetic lethal strategies approach mechanisms and genetic abnormalities that cannot be directly targeted by conventional therapeutic strategies, such as RAS mutations, hypoxia, and abnormalities in the metabolic environment. They also target the environment and conditions specific to malignant cells, have a low toxicity to normal cells, and can be used in combination with known drugs to produce new ones. However, the concept of synthetic lethality has not been widely adopted with ferroptosis. In this review, we surveyed the literature on ferroptosis-related factors and synthetic lethality to examine the potential therapeutic targets in ferroptosis-related molecules, focusing on factors related to synthetic lethality, discovery methods, clinical application stages, and issues in drug discovery.     

No. 3Vol. 42, No. 2, pp. 94–101, 2008Effect of Birch (Betula platyphylla Sukatchev var. japonica Hara) sap on cultured human epidermal keratinocyte differentiation

The beauty of ideal skin texture is closely associated with dermal moisture factors. The key factors of skin moisture are NMF (natural moisturizing factor) and skin normal barrier function. The former keeps dermal surface moisture, and the later protects from excess water loss. So we have searched for the ingredient that improves these factors. Birch sap has been widely used as an effective drink for anti-fatigue and anti-stress. However, the effect of birch sap on skin as a cosmetic agent has not been known entirely. In this study, we investigated the effects of birch ( Betula platyphylla Sukatchev var. japonica Hara) sap on human skin. Birch sap induced epidermal keratinocyte differentiation properties in vitro . We assessed two epidermal differentiation agents. Filaggrin is a precursor protein of NMF, and involucrin is one of the precursor proteins of the cornified cell envelope (CE), which is related to normal barrier function. We have evaluated the production of these proteins where birch sap was applied to human normal keratinocytes. Birch sap not only increased mRNA expression of filaggrin and involucrin, but also accelerated these proteins production. Otherwise, birch sap did not have any influence for IL-6 production, which is related to inflammatory and aberrant keratinocyte proliferation. The results of induced differentiation properties on birch sap-treated keratinocytes are very similar to the differentiation induced by calcium in vitro . This similarity suggested that birch sap has a differentiation inducible property on in vitro cultured keratinocytes. Our study suggested that birch sap is able to control both moisturizing- and barrier-related factor production. From these effects, birch sap provides appropriate epidermal functions and skin homeostasis, and revealed itself as a very useful ingredient in the cosmetic field.     

New method for testing phototoxicity of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs), widespread environmental pollutants, were recently reported to show photomutagenesis. As contaminants in the environment are usually exposed to sunlight, a way to evaluate the phototoxic characteristics of pollutants is required. We have previously found that phosphorylation of histone H2AX (gamma-H2AX), which accompanied the induction of DNA double strand breaks (DSBs), was significantly induced by low concentrations of benzo[a]pyrene (10(-9)-10(-7) M) and UVA (0.6 J/cm2) in CHO-K1 cells. Higher concentrations have been required for the detection of DSBs. The aim of the present study is to investigate the applicability of gamma-H2AX in a new phototoxicity assay of PAHs. The human keratinocytes, HaCaT, were treated with four model PAHs (naphthalene, phenanthrene, pyrene, and benzo[a]pyrene, 10(-11)-10(-7) M) and/or UVA (5 J/cm2), and the induction of gamma-H2AX was assessed. Furthermore, DSBs were directly detected using a biased sinusoidal field gel electrophoresis, and the cell viability was examined as a general assay of phototoxicity. The induction of gamma-H2AX was detected in the presence of all the PAHs except naphthalene at concentrations of 10(-9)-10(-7) M, whereas neither DSBs nor cell death could be detected at those concentrations, and higher concentrations were required for the detection. Naphthalene showed no phototoxicity in any of the three different assays. These findings suggest that histone H2AX is a potential moleculartargetfor detecting the phototoxicity of PAHs more sensitively than the detection of cell viability and DSBs.     

Sensitive analyses of neutral N-glycans using anion-doped liquid matrix G3CA by negative-ion matrix-assisted laser desorption/ionization mass spectrometry

Negative-ion fragmentation of N-glycans has been proven to be more informative than that of positive-ion. In particular, it defines structural features such as the specific composition of the two antennae and the location of fucose. However, negative-ion formation of neutral N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) remains a challenging task, and the detection limit of N-glycans in negative-ion mode is merely at the subpicomole level. Thus, practical applications are limited. In this study, combinations of five liquid matrices and nine anions were used to ionize N-glycans as anionic adducts, and their performances for sensitive analyses were evaluated. The best results were obtained with anion-doped liquid matrix G(3)CA, which consists of p-coumaric acid and 1,1,3,3-tetramethylguanidine; the detection limits of anion adducted N-glycans were 1 fmol/well for NO(3)(-), and 100 amol/well for BF(4)(-). Negative-ion MS(2) spectra of 1 fmol N-glycans were successfully acquired with a sufficient signal-to-noise ratio and were quite useful for MS-based structural determination. The anion-doped G(3)CA matrix opens the way for sensitive and rapid analysis of neutral N-glycans in negative-ion MALDI at a low femtomole level.     

Detection of pathogenic bacteria by using zinc finger protein fused with firefly luciferase

We constructed a novel bacterial genome detection system using zinc finger protein (ZF) fused with firefly luciferase (ZF-luciferase). Taking advantage of the direct recognition of double-stranded DNA (dsDNA) by ZF, we previously constructed bacterial genome detection systems that did not require dehybridization processes. To detect polymerase chain reaction (PCR) products rapidly and with a high sensitivity, we constructed two kinds of ZF-luciferase, Sp1-fused luciferase (Sp1-luciferase), and Zif268-fused luciferase (Zif268-luciferase). ZF-luciferase not only maintains luciferase activity but also shows dsDNA-binding ability and specificity. Furthermore, we succeeded in the detection of 10 copies of the genome of Legionella pneumophila and Escherichia coli O157. ZF-luciferase would be a useful tool for highly sensitive detection of pathogenic bacterial genome.