Acerola exosome-like nanovesicles to systemically deliver nucleic acid medicine via oral administration.

Extracellular vesicles derived from mammalian cells could be useful carriers for drug delivery systems (DDSs); however, with regard to clinical application, there are several issues to be overcome. Acerola (Malpighia emarginata DC.) is a popular health food. In this study, the feasibility of orally administered nucleic acid drug delivery by acerola exosome-like nanoparticles (AELNs) was examined. AELNs were recovered from acerola juice using an affinity column instead of ultracentrifugation. MicroRNA (miRNA) was sufficiently encapsulated in AELNs by 30-min incubation on ice and was protected against RNase, strong acid, and base treatments. The administration of an AELN/miRNA mixture in cells achieved downregulation of the miRNA's target gene, and this mixture showed cytoplasmic localization. AELNs orally delivered small RNA to the digestive system in vivo. The target gene-suppressing effect in the small intestine and liver peaked 1 day after administration, indicating potential for use as an oral DDS for nucleic acid in the digestive system.

Theoretical Justification of Single-Ended Dislocation-Source-Controlled Deformation of Micropillar fcc Crystals.

It was established at the beginning of the 21st century that the critical resolved shear stress of small-sized (diameter from 50 nm to 10 µm) metallic crystals fabricated from bulk crystals increases drastically with decreasing specimen diameter. Dou and Derby [Scr. Mater. 61, 524 (2009)SCMAF71359-646210.1016/j.scriptamat.2009.05.012] showed that, the critical shear stresses of small-sized single crystals of various fcc metals obeyed a universal power law of specimen size with an exponent of -0.66. In this study, we succeeded in reproducing almost perfectly the above universal relation without any adjustable parameters, based on a deformation process controlled by the operation of single-ended dislocation sources.

Low-intensity pulsed ultrasound promotes the expression of immediate-early genes in mouse ST2 bone marrow stromal cells.

PURPOSE: The effects of low-intensity pulsed ultrasound (LIPUS) on the expression of immediate-early genes (IEGs) in bone marrow stromal cells (BMSCs) were evaluated to elucidate the early cellular response to LIPUS. METHODS: Mouse ST2 BMSCs were treated with LIPUS (ISATA, 12-34 mW/cm2 for 20 min), then cultured at 37 °C. The expression levels of four IEGs (Fos, Egr1, Jun, and Ptgs2) and ERK1/2, a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), were assessed using real-time quantitative PCR and Western blot analyses, respectively. RESULTS: A single exposure of LIPUS at an intensity of 25 mW/cm2 significantly and transiently increased the expression levels of all four IEGs, and the peak expression was detected at 30-60 min after LIPUS stimulation. LIPUS exposure also significantly increased the phosphorylation level of ERK1/2. U0126, an inhibitor of MAPK/ERK, significantly prevented LIPUS-induced expression of Fos and Egr1, but not that of Jun and Ptgs2. On the other hand, treatment of the cells with LIPUS did not affect cell growth or alkaline phosphatase activity, a marker of osteoblast differentiation. CONCLUSION: These results suggest that LIPUS exposure significantly induces expression of IEGs such as Fos and Egr1 via the MAPK/ERK pathway in ST2 BMSCs.

TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase.

TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs.

SIPA1 promotes invasion and migration in human oral squamous cell carcinoma by ITGB1 and MMP7.

Signal-induced proliferation-associated protein 1 (SIPA1) is known to be a GTPase activating protein. Overexpressed SIPA1 is related to metastatic progression in breast and prostate cancers; however, the relevance of SIPA1 in oral squamous cell carcinoma (OSCC) is still unknown. The aim of this study was to examine SIPA1 expression and its functional mechanisms in OSCC. SIPA1 mRNA and protein expressions were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. The expressions of SIPA1 were up-regulated significantly in vitro and in vivo. Moreover, SIPA1 expression was correlated with regional lymph node metastasis. We next assessed the cellular functions associated with tumoral metastasis using SIPA1 knockdown (shSIPA1) cells and analyzed the downstream molecules of SIPA1, i.e., bromodomain containing protein 4(BRD4), integrin beta1 (ITGB1), and matrix metalloproteinase 7 (MMP7). The shSIPA1 cells showed decreased invasiveness and migratory activities, however cellular adhesion ability was maintained at a high level. In addition, ITGB1 expression was greater in shSIPA1 cells, whereas MMP7 expression was lower than in control cells. This research is the first to establish that SIPA1 promotes cancer metastasis by regulating the ITGB1 and MMP7. Therefore, SIPA1 might be a novel therapeutic target for patients with lymph node metastasis of OSCC.

Electroreductive intermolecular coupling of 3-methoxycarbonylindoles with ketones.

The electroreductive coupling of 1-alkoxycarbonyl-3-methoxycarbonylindoles with aromatic ketones in the presence of chlorotrimethylsilane gave cis-adducts stereoselectively. The cis-adducts were readily transformed to trans-adducts by treatment with catalyst DBU. On the other hand, the electroreductive coupling of 1-methyl-3-methoxycarbonylindole with aliphatic ketones in isopropanol afforded trans-adducts exclusively. The adducts are the precursors for the synthesis of 2-substituted 3-methoxycarbonylindoles and indolines.

PAHs concentration and toxicity in organic solvent extracts of atmospheric particulate matter and sea sediments.

The concentration of polycyclic aromatic hydrocarbons (PAHs) and the toxicity to marine bacteria (Vibrio fischeri) were measured for the organic solvent extracts of sea sediments collected from an urban watershed area (Hiroshima Bay) of Japan and compared with the concentrations and toxicity of atmospheric particulate matter (PM). In atmospheric PM, the PAHs concentration was highest in fine particulate matter (FPM) collected during cold seasons. The concentrations of sea sediments were 0.01-0.001 times those of atmospheric PM. 1/EC50 was 1-10 L g(-1) PM for atmospheric PM and 0.1-1 L g(-1) dry solids for sea sediments. These results imply that toxic substances from atmospheric PM are diluted several tens or hundreds of times in sea sediments. The ratio of the 1/EC50 to PAHs concentration ((1/EC50)/16PAHs) was stable for all sea sediments (0.1-1 L µg(-1) 16PAHs) and was the same order of magnitude as that of FPM and coarse particulate matter (CPM). The ratio of sediments collected from the west was more similar to that of CPM while that from the east was more similar to FPM, possibly because of hydraulic differences among water bodies. The PAHs concentration pattern analyses (principal component analysis and isomer ratio analysis) were conducted and the results showed that the PAHs pattern in sea sediments was quite different to that of FPM and CPM. Comparison with previously conducted PAHs analyses suggested that biomass burning residues comprised a major portion of these other sources.

Transmission electron microscopy study on the effects of the ultrasound contrast agent Levovist on hepatic cells.

PURPOSE: The Ultrasound Equipment and Safety Committee of The Japan Society of Ultrasonics in Medicine performed experiments to confirm whether contrast-enhanced ultrasonography damages liver cells. METHODS: Rats were injected with 0.1 ml of 300 mg/ml ultrasound contrast agent (UCA). Diagnostic ultrasound pulses with a center frequency of 6 MHz and a mechanical index of 1.9 were applied to rat livers with a water bag as a coupler to maintain a distance of 2-6 cm between the ultrasound probe surface and the liver. Contrast-enhanced ultrasonography was carried out for 10 s to visualize the entire liver. Then, specimens of liver tissue were fixed using two types of fixation: immersion and perfusion fixation. RESULTS: Although some variations were found in electron micrographs of liver tissue fixed using immersion fixation, none of three blinded readers found any significant differences between micrographs of liver tissue from rats receiving UCA with sonication and those from sham-treated control rats. Changes observed were not thought to be group-specific but instead due to differences between individual rats. When the livers were fixed using perfusion fixation and the hepatic vein was cut after injection of physiological saline for perfusion, a large number of vacuoles ≥2 µm in diameter were observed. This finding suggested that hepatic cell damage observed in this study was caused by high perfusion pressure during the liver fixation process rather than by sonication with UCA. CONCLUSION: Blinded readings of electron micrographs showed no clear evidence that the use of Levovist in ADI mode ultrasonography causes significant damage to liver tissue.

Structural insights into the hot spot amino acid residues of mushroom tyrosinase for the bindings of thujaplicins.

Tyrosinase inhibitors are important agents for cosmetic products. We examined here the inhibitory effects of three isomers of thujaplicins (α, ß and γ) on mushroom tyrosinase and analyzed their binding modes using a homology model from the crystal structure of Streptomyces castaneoglobisporus tyrosinase (PDB ID: 1wx2). All the thujaplicins were found to be competitive inhibitors and γ-thujaplicin has the most potent inhibitory activity (IC(50)=0.07µM). It is noted that there are good correlations between their observed IC(50) values and their binding free energies calculated by MM-GB/SA. The binding modes of thujaplicins were predicted to be similar to that of Tyr98 of caddie protein (ORF378), which was co-crystallized with S. castaneoglobisporus tyrosinase. Furthermore, free energy decomposition analysis indicated that the potent inhibitory activity of γ-thujaplicin is due to the interactions with His242, Val243 and Pro257 (hot spot amino acid residues) at the active site of tyrosinase. These results provide a novel structural insight into the hot spot of mushroom tyrosinase for the specific binding of γ-thujaplicin.