Quantitative assessment of nano-plastic aerosol particles emitted during machining of carbon fiber reinforced plastic.

Focusing on the relatively unexplored presence of micro- and nano-plastic aerosol particles, this study quantitatively assessed the emission of nano-plastic particles during the machining of carbon fiber reinforced plastic (CFRP) in the working environment. Measurements of aerosol particles smaller than 1 µm in size were performed by aerosol mass spectrometry. The findings revealed that concentrations of carbonous aerosol particles (organic aerosol and refractory black carbon (rBC)) were higher during working hours than during non-working hours. Positive matrix factorization identified CFRP particles as a significant source, contributing an average of approximately 30% of concentration of carbonous aerosol particles during working hours. This source apportionment was corroborated by the presence of bisphenol A and F fragments, principal components of the epoxy resins used in CFRP, and was corroborated by similarities to the carbon cluster ion distribution observed in rBC during CFRP pipe-cutting operations. Further, the particle size distribution suggested the existence of plastic aerosol particles smaller than 100 nm. This study established the method to quantitatively distinguish nano-plastic aerosol particles from other aerosol particles in high temporal resolution and these techniques are useful for accurately assessing exposure to nano-plastic aerosol particles in working environments.

Factory site analysis of respirable fibers generated during the process of cutting and grinding of carbon fibers-reinforced plastics.

OBJECTIVES: Carbon fibers are used in a variety of industrial applications, based on their lightweight and high stiffness properties. There is little information on the characteristics and exposure levels of debris generated during the factory processing of carbon fibers or their composites. This study revisits the general assumption that carbon fibers or their debris released during composite processing are considered safe for human health. METHODS: The present interventional study was conducted at a factory located in Japan, and involved on-site collection of debris generated during the industrial processing of polyacrylonitrile (PAN)-based carbon-fiber-reinforced plastic (CFRP). The debris were collected before being exhausted locally from around different factory machines and examined morphologically and quantitatively by scanning electron microscopy. The levels of exposure to respirable carbon fibers at different areas of the factory were also quantified. RESULTS: The collected debris mainly contained the original carbon fibers broken transversely at the fiber's major axis. However, carbon fiber fragments morphologically compatible with the WHO definition of respirable fibers (length: > 5 µm, width: < 3 µm, length/width ratio: > 3:1) were also found. The concentrations of respirable fibers at the six examined factory areas under standard working conditions in the same factory were below the standard limit of 10 fibers/L, specified for asbestos dust-generating facilities under the Air Pollution Control Law in Japan. CONCLUSIONS: Our study identified potentially dangerous respirable fibers with high aspect ratio, which was generated during the processing of PAN-based CFRP. Regular risk assessment of carbon fiber debris is necessary to ensure work environment safety.

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

An IC-MS/MS Method for the Determination of 1-Hydroxyethylidene-1,1-diphosphonic Acid on Uncooked Foods Treated with Peracetic Acid-Based Sanitizers.

A rapid, sensitive, and specific analytical method for the determination of 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) on uncooked foods after treatment with a peracetic acid-based sanitizer (PAS) was developed. The method involves simple sample preparation steps and analysis using ion chromatography (IC) coupled with tandem mass spectrometry (MS/MS). The quantification limits of HEDP on uncooked foods are 0.007 mg/kg for vegetables and fruits and 0.2 mg/kg for meats. The recovery and relative standard deviation (RSD) of HEDP analyses of uncooked foods ranged from 73.9 to 103.8% and 1.9 to 12.6%, respectively. The method's accuracy and precision were evaluated by inter-day recovery tests. The recovery for all samples ranged from 93.6 to 101.2%, and the within-laboratory repeatability and reproducibility were evaluated based on RSD values, which were less than 6.9 and 11.5%, respectively. Analyses of PAS-treated fruits and vegetables using the developed method indicated levels of HEDP ranging from 0.008 to 0.351 mg/kg. Therefore, the results of the present study suggest that the proposed method is an accurate, precise, and reliable way to determine residual HEDP levels on PAS-treated uncooked foods.

Ginger hexane extract suppresses RANKL-induced osteoclast differentiation.

Osteoporosis is a debilitating disease caused by decreased bone density. Compounds with anti-osteoclastic activity, such as bisphosphonates, may help in the prevention and treatment of osteoporosis. Herein, we determined the inhibitory effects of ginger hexane extract (GHE) on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. The results showed that GHE (1) suppressed osteoclast differentiation and the formation of actin rings; (2) inhibited the expression of Nfatc1, a master transcriptional factor for osteoclast differentiation, in a dose-dependent manner (10-20 µg/mL); and (3) inhibited other osteoclastogenesis-related genes, such as Oscar, Dc-stamp, Trap, and Mmp9. These findings suggest that GHE may be used to prevent and treat osteoporosis by inhibiting osteoclast differentiation.

Identification of target antigens of naturally occurring autoantibodies in cerebrospinal fluid.

Naturally occurring autoantibodies have natural physiologic functions related to normal cell processes. However, the repertoire of naturally occurring autoantibodies against neuronal antigens in CSF is unclear. The purpose of this study was to identify naturally occurring autoantibodies against neuronal antigens in CSF from patients with various neurologic diseases by proteomics-based analysis. The CSF samples were collected from 77 patients with various neurologic disorders. The antigen source for 2-dimensional immunoblotting was the SH-SY5Y human neuroblastoma cell line. There were 8 spots recognized in CSF from more than one-fourth of the 77 patients including all patient groups and these spots were recognized in intravenous immunoglobulin preparations. These antigen spots were identified as heat shock 105-kDa/110-kDa protein 1, isoform CRA_b, 78-kDa glucose-regulated protein, heat shock cognate 71-kDa protein, tubulin beta chain, vimentin (2 spots), and 60-kDa heat shock protein, mitochondrial; we could not identify the protein name corresponding to 1 of the 8 spots. In summary, there were 6 proteins identified that were main target antigens that reacted with naturally occurring autoantibodies in CSF from patients with varied neurologic disorders; the functions of autoantibodies against the identified antigens are unknown and may be clarified with further studies. BIOLOGICAL SIGNIFICANCE: Naturally occurring autoantibodies may have important functions in tissue homeostasis. In this study, we identified 6 common target antigens that reacted with autoantibodies in cerebrospinal fluid (CSF) from patients, independent of disease type. These findings may clarify the importance of naturally occurring autoantibodies in CSF and the use of these antibodies potentially may be a novel therapy for various neurologic disorders.

Specific detection by the polymerase chain reaction of potentially allergenic salmonid fish residues in processed foods.

Salmonid fish is one of the allergenic items that are recommended to be labeled in the Japanese allergen-labeling system. This study develops a salmonid-specific polymerase chain reaction (PCR) method. A new primer pair, SKE-F/SKE-R, was designed to specifically detect the salmonid fish gene encoding mitochondrial DNA cytochrome b. Genomic DNAs extracted from 58 kinds of seafood and 11 kinds of processed food were individually subjected to PCR by using the primer pair, and a salmonid-specific fragment of 212 bp was only amplified in the salmonid samples and salmonid-containing processed foods. The detection limit of the PCR method was as low as 0.02 fg/µL of salmonid fish DNA (corresponding to 10 copies). There is no ELISA method for salmonid fish, making our PCR method the only reliable measure for detecting salmonid fish in processed foods.

Quantification of pork, chicken and beef by using a novel reference molecule.

A standard plasmid was constructed as a novel reference molecule for use in real-time quantitative PCR assays to verify the identity of beef, pork, chicken, mutton, and horseflesh. The plasmid contained a target domain of the cytochrome b (cyt b) gene and an artificial DNA sequence. Primers CO-F and CO-R, and probe CO-P were specifically designed to detect the artificial sequence. The calculated R² values of the standard curves (10³-107 copies per reaction) for the five species ranged between 0.998 and 0.999 in the quantification analysis. The constructed plasmid provides a universal method for measuring the copy number of cyt b DNA in minced meat. This method would be a useful procedure for verifying food labels.

Specific detection of banana residue in processed foods using polymerase chain reaction.

Specific polymerase chain reaction (PCR) methods were developed for the detection of banana residue in processed foods. For high banana specificity, the primer set BAN-F/BAN-R was designed on the basis of the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) genes of chloroplasts and used to obtain amplified products specific to banana by both conventional and real-time PCR. To confirm the specificity of these methods, genomic DNA samples from 31 other species were examined; no amplification products were detected. Subsequently, eight kinds of processed foods containing banana were investigated using these methods to confirm the presence of banana DNA. Conventional PCR had a detection limit of 1 ppm (w/w) banana DNA spiked in 50 ng of salmon testis DNA, whereas SYBR Green I real-time semiquantitative PCR had a detection limit as low as 10 ppm banana DNA. Thus, both methods show high sensitivity and may be applicable as specific tools for the detection of trace amounts of banana in commercial food products.

A real-time quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods.

A rapid real-time quantitative PCR method to detect trace amounts of pork, chicken, beef, mutton, and horseflesh in foods was developed. The primers and TaqMan MGB (minor groove binder) probes were designed on the gene encoding cytochrome b for the specific detection of each species. The limit of quantification of this method was found to be 100 fg/microl of each mitochondrial DNA in 10 ng/microl of the wheat mitochondrial DNA matrix. The calculated R(2) values of the standard curves for the five species ranged between 0.994 and 0.999. This method would be particularly useful in the detection of hidden meat mince in processed foods, which would verify food labeling and gain consumers' trust.

Detection of walnut residues in processed foods by polymerase chain reaction.

A sensitive qualitative detection method for walnut (Juglans regia) using polymerase chain reaction (PCR) was developed. For detection of walnuts with high specificity, the primer pair WAL-F/WAL-R was designed based on walnut matK genes. Trace amounts of walnuts in commercial food products can be qualitatively detected using this method.

Increased levels of tissue endostatin in human malignant gliomas.

PURPOSE: Malignant gliomas are typically angiogenic and express greater amounts of angiogenic factors. We examined glioma tissues for their expression of an endogenous inhibitor of angiogenesis, endostatin, a COOH-terminal fragment of collagen XVIII. EXPERIMENTAL DESIGN: We examined frozen tissues from 51 patients with astrocytic tumors (grade 2, 13; grade 3, 9; and grade 4, 29). Frozen tissues were subjected to immunoblot analysis and immunohistochemistry for endostatin. Tumor vascular density was determined by calculating the percentage of tumor capillary vessel areas/tissue section area. Tissue concentrations of vascular endothelial growth factor and basic fibroblast growth factor were examined by enzyme immunoassay. RESULTS: The levels of endostatin protein estimated by immunoblotting were significantly higher in grade 4 than lower-grade glioma tissues. The immunoreactive bands for endostatin were identified as the fragment derived from noncollagenous domain 1 of collagen XVIII, a peptide 15 residues longer than endostatin toward the NH(2)-terminal end, by NH(2)-terminal amino acid sequencing. In addition to an intense immunoreactivity for endostatin in tumor blood vessels, sections from malignant gliomas showed widely distributed immunoreactivity around tumor cells near the hyperplastic microvessels. The tumor vascular density and the levels of vascular endothelial growth factor in grade 4 glioma tissues were significantly higher than grade 2 and grade 3 gliomas, whereas the levels of basic fibroblast growth factor were the same. CONCLUSIONS: The results indicate a positive correlation between the levels of tissue endostatin and malignancy grades in gliomas. The endostatin may be released near the tumor blood vessels with hyperplasia to counteract angiogenic stimuli in malignant gliomas.

Rho-dependent agonist-induced spatio-temporal change in myosin phosphorylation in smooth muscle cells.

Agonist-induced translocation of RhoA and the spatio-temporal change in myosin regulatory light chain (MLC20) phosphorylation in smooth muscle was clarified at the single cell level. We expressed green fluorescent protein-tagged RhoA in the differentiated tracheal smooth muscle cells and visualized the translocation of RhoA in a living cell with three-dimensional digital imaging analysis. The stimulation of the cells by carbachol initiated the translocation of green fluorescent protein-tagged wild type RhoA to the plasma membrane within a minute. The change in MLC20 phosphorylation level after carbachol stimulation was monitored by using phospho-Ser-19-specific antibody recognizing the phosphorylated MLC20 in single cells. Cells expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC20 phosphorylation during the prolonged phase (>300 s), whereas the maximum phosphorylation level (reached at 10 s after stimulation) of these cells was not significantly different from the control cells. The kinetics of RhoA translocation was consistent with that of sustained myosin phosphorylation, suggesting the involvement of a RhoA pathway. Carbachol stimulation increased myosin phosphorylation within a minute both at the cortical and the central region. On the other hand, during prolonged phase, myosin phosphorylation was sustained at the cortical region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at the central region of the cells after the stimulation but not at the cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but not the central region. The results clearly show that the myosin light chain kinase pathway and the Rho pathway distinctly change myosin phosphorylation in smooth muscle cells in both a temporal and spatial manner.