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Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widely distributed in air, water, and sediments; however, limited data are available regarding their potential adverse effects on the early life stages of fish. In this study, we evaluated the embryonic teratogenicity and developmental toxicity of BaP in Japanese medaka (Oryzias latipes) using a nanosecond pulsed electric field (nsPEF) technique and predicted their molecular mechanisms via transcriptome analysis. The gas chromatography/mass spectrometry analyses revealed that the BaP was efficiently incorporated into the embryos by nsPEF treatment. The embryos incorporating BaP presented typical teratogenic and developmental effects, such as cardiovascular abnormalities, developmental abnormalities, and curvature of backbone. DNA microarray analysis revealed several unique upregulated genes, such as those involved in cardiovascular diseases, various cellular processes, and neural development. Furthermore, the gene set enrichment and network analyses found several genes and hub proteins involved in the developmental effects of BaP on the embryos. These findings suggest a potential mechanism of teratogenicity and developmental toxicity caused by exposure to BaP. The nsPEF and transcriptome analyses in combination can be effective for evaluating the potential effects of chemical substances on medaka embryos.

Epidermal growth factor receptor gene (EGFR) mutations are associated with response to tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). We developed a novel, rapid EGFR mutation assay using a real-time droplet-polymerase chain reaction machine (EGFR d-PCR assay). The purpose of this study was to validate the performance of the EGFR d-PCR assay using fresh liquid cytology specimens. We analyzed three major EGFR mutations (L858R in exon 21, E746_A750del in exon 19 and T790M in exon 20) in 80 fresh liquid cytology specimens of adenocarcinoma (ADC) or NSCLC-not otherwise specified (NOS) via the EGFR d-PCR assay and conventional real-time PCR assay using the therascreen® EGFR RGQ PCR kit (Therascreen assay). In addition, we performed sensitivity assays using cell lines with EGFR mutations. The EGFR d-PCR assay detected 16 L858Rs, 8 E746_A750dels and 1 T790M mutation and the Therascreen assay detected 16 L858Rs, 11 deletions in exon 19 and 1 T790M mutation. The results were concordant between the two assays. The reaction time of the EGFR d-PCR assay was 8 min and 10 sec, but that of the Therascreen assay was 1 h and 45 min. Sensitivity, as assessed by the detection limit of the EGFR d-PCR assay was 0.5, 0.05 and 0.5% for L858R, E746_A750del and T790M, respectively. The EGFR d-PCR assay markedly reduced the detection time of major EGFR mutations with high sensitivity compared with the conventional Therascreen assay and is expected to expedite EGFR-TKI therapy for lung cancer patients, especially those in advanced stages.

Background Acute promyelocytic leukemia (APL) with the PML-RARA fusion gene can be effectively cured using molecular-targeted therapies, which require both detection and quantification of the PML-RARA fusion gene. Here, we developed a rapid assay for identifying and measuring the PML-RARA fusion gene in patients with APL using droplet-reverse transcription-polymerase chain reaction (droplet-RT-PCR) and instant quality-fluorescence in situ hybridization (IQ-FISH). Methods RNA for droplet-RT-PCR and fixed-cell suspensions for IQ-FISH were prepared from five patients with APL and three controls. We evaluated the amplification efficiency and reaction time with droplet-RT-PCR and signal clarity and hybridization time with IQ-FISH. Results The reaction using droplet-RT-PCR was completed in 26 min. The PML-RARA fusion gene was detected in all samples from the five patients. IQ-FISH yielded clear signals after 1 h of hybridization. There were no significant differences in signal clarity or positive signal ratios between IQ-FISH and conventional FISH. Conclusions Simultaneous droplet-RT-PCR and IQ-FISH, in addition to morphological examination of blood smears, can be used to diagnose patients as having APL within 4 h based on molecular/cytogenetic results. Rapid diagnosis can allow effective therapies to be started promptly.

Background Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. Methods SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). Results Droplet-AS-PCR could determine genotypes within 8 min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. Conclusion The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

Background Single nucleotide alterations such as single nucleotide polymorphisms (SNP) and single nucleotide mutations are associated with responses to drugs and predisposition to several diseases, and they contribute to the pathogenesis of malignancies. We developed a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) with our droplet-PCR machine (droplet-AS-PCR). Methods Using 8 SNP loci, we evaluated the specificity and sensitivity of droplet-AS-PCR. Buccal cells were pretreated with proteinase K and subjected directly to the droplet-AS-PCR without DNA extraction. The genotypes determined using the droplet-AS-PCR were then compared with those obtained by direct sequencing. Results Specific PCR amplifications for the 8 SNP loci were detected, and the detection limit of the droplet-AS-PCR was found to be 0.1–5.0% by dilution experiments. Droplet-AS-PCR provided specific amplification when using buccal cells, and all the genotypes determined within 9 min were consistent with those obtained by direct sequencing. Conclusions Our novel droplet-AS-PCR assay enabled high-speed amplification retaining specificity and sensitivity and provided ultra-rapid genotyping. Crude samples such as buccal cells were available for the droplet-AS-PCR assay, resulting in the reduction of the total analysis time. Droplet-AS-PCR may therefore be useful for genotyping or the detection of single nucleotide alterations.

Background The polymerase chain reaction (PCR) has been widely used for diagnosis of infection. Rapid detection of influenza virus is useful for therapeutic decisions. We attempted to develop a novel assay by real-time droplet-PCR machine for influenza virus. Methods RNA extracted from nasal swabs or primary swabs pretreated only were used for PCR analyses. We evaluated reaction time, amplification efficiency, sensitivity, and specificity of the novel droplet-PCR. Results The reaction time of the novel droplet-PCR was 28 min, whereas that of PCR using the conventional PCR machine was 80 min. The standard curve constructed from the amplification plots by the novel droplet-PCR was: y = − 3.6x + 42.9; that by PCR using the conventional PCR machine was: y = − 3.5x + 37.8. The sensitivity and specificity of the novel droplet-PCR were 86.7% and 91.7% for the influenza A and 100.0% and 100.0% for the influenza B, respectively. The novel droplet-PCR provided the specific amplification when using primary swabs without RNA extraction. Conclusions Our novel droplet-PCR markedly reduced the reaction time while showing same reactivity as that by PCR using the conventional PCR machine. Thus, the novel droplet-PCR assay can be used as a rapid assay for detection of influenza virus.

Cysteine proteases (CPs) with N-succinyl-Leu-Tyr-4-methylcoumaryl-7-amide (Suc-LY-MCA) cleavage activity were investigated in green and senescent leaves of spinach. The enzyme activity was separated into two major and several faint minor peaks by hydrophobic chromatography. These peaks were conventionally designated as CP1, CP2 and CP3, according to their order of elution. From the analyses of molecular mass, subunit structure, amino acid sequences and cDNA cloning, CP2 was a monomer complex (SoCP-CPI) (51 kDa) composed of a 41-kDa core protein, SoCP (Spinacia oleracea cysteine protease), and 14-kDa cystatin, a cysteine protease inhibitor (CPI), while CP3 was a trimer complex (SoCP-CPI)(3) (151 kDa) of the same subunits as SoCP-CPI and showed a wider range of specificity toward natural substrates than SoCP-CPI. Trimer (SoCP-CPI)(3) was irreversibly formed from monomers through association. The results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that mRNAs of CPI and SoCP are hardly expressed in green leaves, but they are coordinately expressed in senescent leaves, suggesting that these proteases involve in senescence. Purified recombinant CPI had strong inhibitory activity against trimer SoCP, (SoCP)(3) , which had a cystatin deleted with K(i) value of 1.33 × 10(-9) M. After treatment of the enzyme with a succinate buffer (pH 5) at the most active pH of the enzyme, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and activity analyses showed that cystatin was released from both monomer SoCP-CPI and trimer (SoCP-CPI)(3) complexes with a concomitant activation. Thus, the removal of a cystatin is necessary to activate the enzyme activity.

The effects of nanosecond pulsed electric fields (nsPEFs) on medaka eggs were examined. Although embryogenesis was not affected by nsPEF treatment alone, significant harmful effects were observed when the eggs were treated with nsPEFs in the presence of cycloheximide in the outer solution. Nanosecond PEF treatment affected the permeability of both the egg envelope (chorion) and the cell membrane, which resulted in intracellular incorporation of cycloheximide.

A sodium dodecyl sulfate (SDS)-dependent cysteine protease was investigated in green leaves of barley ( Hordeum vulgare L.) by measuring the release of 7-amino-4-methyl-coumarin (AMC) from a synthetic substrate, N -succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (Suc-LLVY-MCA). The enzyme named Hv CP3 ( Horedeum vulgare cysteine protease activated by 0.1%, w/v SDS) increased well with the development of leaves, but it decreased drastically during senescence. Hv CP3 was purified 146-fold with a yield of 7.74% from the crude extracts by four steps of chromatography. The enzyme showed a broad pH optimum at pH 7–8 and the enzyme activity was activated about 10-fold by 0.1% (w/v) SDS. The molecular weight of the native enzyme was estimated to be approximately 50 k. SDS–polyacrylamide gel electrophoresis of the protease suggested that the protein was a complex that consists of 33 k and 18 k subunits. The enzyme activity was specifically inhibited by cysteine protease inhibitors such as 10 μM trans -epoxysuccinyl- l -leucylamido-(4-guanidino) butane (E-64) and 100 μM leupeptin to 5% and 4%, respectively. A full-length cDNA of Hv CP3 was cloned and its sequence displayed high similarity to other plant cysteine proteases of the papain family (C1A). The functional role of this protease as a maintainer in cytosol is also discussed.

Background Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. Methods The K562 BCR–ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. Results One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR–ABL mRNA within 40 min, which was 103-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. Conclusions One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories.

Various studies have demonstrated the estrogenic effect of bisphenol A (BPA), a member of bisphenol analogs (BPs), in in vitro and in vivo assays. However, limited data are available on the estrogenic potentials and risks of other BPs in aquatic organisms. In addition, the estrogenic effect of chemicals is known to have species-specific responses in teleost fish. The objective of this study was to evaluate the potential estrogenic effects of BPs on the medaka (Oryzias latipes) and common carp (Cyprinus carpio) using in vivo and in silico assays. Our quantitative real-time PCR analyses revealed that the expression levels of several hepatic estrogen-responsive biomarker genes in male medaka responded to various types and concentrations of BPs in a dose–response manner. The order of in vivo estrogenic potencies of BPs was as follows: BPC≈BPAF>BPB>BPA⋙BPP. To further investigate the interaction potential of BPs with medaka estrogen receptor α (ERα) in silico, a three-dimensional model of the ERα ligand-binding domain (LBD) was built and docking simulations were performed. The docking simulation analysis revealed that BPC interaction potential for medaka ERα LBD was the most potent, followed by BPAF and BPA. Comparing this with carp ERα LBD revealed that the interaction potentials of these BPs to medaka ERα LBD were more stable than to carp ERα LBD. Furthermore, we identified key amino acid residues in medaka ERα LBD that interacted with BPC (Glu356, Arg397, and Cys533), BPAF (Thr350 and Glu356), and BPA (Glu356 and Met424), and found some differences in these key amino acid residues between medaka and carp ERα LBDs. These results of in vivo and in silico analyses showed potential estrogenic effects of BPs in teleost fish, and they also indicated that the differences in interaction potentials and key amino acid residues between medaka and carp ERα LBDs may be due to the differences between the species and estrogenic potencies of the selected BPs.

Background Acute promyelocytic leukemia (APL) is an aggressive disease requiring prompt diagnosis and treatment. Rapid detection of the PML – RARA fusion gene provides the molecular basis for a highly effective therapy with all- trans retinoic acid. We developed a rapid assay by novel droplet-reverse transcriptase-polymerase chain reaction (droplet-RT-PCR) for the detection of the PML – RARA fusion gene in APL patients. Methods RNA was extracted from 7 samples obtained from 5 APL patients with the PML – RARA fusion gene confirmed by nested RT-PCR and fluorescence in situ hybridization. Using these 7 samples, we evaluated the reaction time and amplification efficiency of the droplet-RT-PCR. Results Using the droplet-RT-PCR, we could detect the PML – RARA fusion gene in all 7 samples. The reaction time for 50 cycles of droplet-RT-PCR was 27 min. The amplification by the droplet-RT-PCR assay was considered positive for the PML – RARA fusion gene in less than 22 min, at the point when the fluorescence exceeded the threshold level. Conclusions Our novel droplet-RT-PCR assay is specific for the detection of the PML – RARA fusion gene and has a markedly reduced reaction time. Thus, the novel droplet-RT-PCR assay contributes to the rapid diagnosis of APL without lagging behind the morphological assessment.

A novel steroid compound, (17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), was found to be a partial agonist of the androgen receptor (AR) in an androgen responsive element (ARE)-luciferase reporter assay. YK11 accelerates nuclear translocation of AR. Furthermore, YK11 does not induce amino/carboxyl-terminal (N/C) interaction and prevents 5-α-dihydrotestosterone (DHT)-mediated N/C interaction. Thus, YK11 activates AR without causing N/C interaction, which may in turn be responsible for the partially agonistic nature of YK11 observed in the ARE-luciferase reporter system. YK11 acts as a gene-selective agonist of AR in MDA-MB 453 cells. The effect of YK11 on gene expression relative to that of androgen agonist varies depending on the gene context. YK11 activated the reporter gene by inducing the translocation of the AR into the nuclear compartment, where its amino-terminal domain (NTD) functions as a constitutive activator of AR target genes. Our results suggest that YK11 might act as selective androgen receptor modulator (SARM).

The effects of magnesium aluminum silicate (MAS) addition on the sorption of bromhexine HCl to polyethylene film in tablets were studied. The addition of MAS prevented the sorption of bromhexine HCl to polyethylene film. In order to investigate the mechanism, the interaction between bromhexine HCl and MAS was studied by the powder X-ray diffraction method. It was observed that bromhexine HCl was preferentially adsorbed to the surface of MAS rather than to polyethylene film. The adsorption was accelerated at high temperature and reduced pressure conditions. The sorption of bromhexine base and bromhexine HCl to packaging material were compared using tablet dosage forms. The sorption of bromhexine base to polyethylene film was greater than that of bromhexine HCl.

The interaction between polyethylene containers and bromhexine·HCl solid dosage forms prepared by different methods was studied. It was found that bromhexine·HCl tablets prepared by the wet method (kneading) had more drug remaining than those prepared by the dry method. The sorption of drug to polyethylene was influenced by the kneading solvents used, and the most effective solvent in preventing sorption was methanol, which has high solubility for bromhexine·HCl. A group mixture of bromhexine·HCl with crystalline cellulose was effective inhibiting the sorption of bromhexine to polyethylene. This was explained in terms of the monomolecular dispersion of bromhexine molecules within the network of cellulose molecules in the ground mixture.

Here we report on the in vivo estrogenic effects of two fluorotelomer alcohols, such as 1 H ,1 H ,2 H ,2 H -perfluorooctan-1-ol (6:2 FTOH) and 1 H ,1 H ,2 H ,2 H -perfluorodecan-1-ol (8:2 FTOH), in male medaka ( Oryzias latipes ). An in vitro yeast two-hybrid assay indicated a significant, dose-dependent interaction between medaka estrogen receptor α (ERα) and coactivator TIF2 upon treatment with 6:2 FTOH, 8:2 FTOH or 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluoro-1-decanol (NFDH). The relative ranks of tested chemicals on the estrogenic effects for medaka ERα descended in the order of estradiol-17β (100) ≫ 6:2 FTOH (0.16) > NFDH (0.016) > 8:2 FTOH (0.0044). In contrast, no interaction with the ERα was observed upon treatment with perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDA) or perfluoroundecanoic acid (PFUnDA). Expression analysis of hepatic vitellogenin (VTG) protein showed estrogenic potentials with, 6:2 FTOH and 8:2 FTOH, indicative of the induction of VTG synthesis in the livers of male medaka. We also investigated mRNA expression levels of two ER subtypes (ERα and β) and two VTGs (VTG I and VTG II) in the livers of male medaka following exposure to FTOHs. Quantitative real-time polymerase chain reaction analyses revealed that hepatic ERα, VTG I, and VTG II mRNA responded rapidly to FTOHs such as 6:2 FTOH and 8:2 FTOH after 8-h exposure, whereas no effects of these compounds on ERβ mRNA transcription were observed. These results from both in vitro and in vivo assays strongly suggest that certain FTOHs, such as 6:2 FTOH and 8:2 FTOH, induce hepatic VTG through activation of ERα in male medaka.

1. The action of thiamine and related compounds on the isolated heart of clam (Meretrix meretrix lusoria) was investigated.2. S-benzoylthiamine (SBT) and thiamine propyl disulfide (TPD) among these related compounds displayed a very remarkable positive inotropic action, whereas thiamine, acetylthiamine, thiamine diphosphate, thiamine triphosphate, benzoylthiamine monophosphate and ATP did not practically display any clear action.3. SBT and TPD showed a remarkable antagonistic action against the negative inotropic action of acetylcholine, but have no effect on the action of 5-hydroxytryptamine.4. The positive inotropic action of SBT and TPD was retained in K-free medium, but it completely disappeared in Na-free or Cafree medium. When more or less Na+ or Ca2+ was present in the medium, the action of SBT and TPD appeared as in the normal medium.5. The positive inotropic action of SBT and TPD, therefore, is believed to be closely related to the transport system of the cellular membrane of the clam heart muscle, in which Na+ and Ca2+ are involved.