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This study demonstrates bioluminescent indicators for determining stress hormones in mammalian cells. A genetically encoded bioluminescent probe for stress sensing was first synthesized with a LXXLL, motif-linked ligand binding domain of the glucocorticoid receptor (GR LBD), which was then sandwiched between the fragments of Gaussia luciferase (GLuc). This prototype of the bioluminescent indicators was carefully modified with a circular permutation (CP) and/or a corepressor motif. The first notable appearance by cofusion of a corepressor motif to the probe was the biphasic dose-response curves of the indicator to cortisol. A CP largely improved the detection limit of the indicator to cortisol up to 100 times. Fabrication of both CP and the attachment of a corepressor motif in the indicator synergistically contributes to (i) the lower detection limit and wider dynamic range and (ii) the enhanced absolute luminescence and ligand selectivity. This study is the first example that contribution of core-pressor motifs to single-chain probes was investigated. This study also provides new insight into improving the sensorial properties of single-chain probes with CP.

The goal of the present study is to develop a small luminescence template, in which any protein may be incorporated, for illuminating the dynamics of protein signaling. We first determined optimal fragmentation sites of Gaussia princeps-derived luciferase (GLuc). The utility of the template was demonstrated with calmodulin (CAM) and a peptide-linked CAM, which were sandwiched between the fragments of split GLuc dissected at Q105. Living mammalian cells with the probe quickly emitted luminescence in response to endo- and exogenous [Ca.sup.2+] levels. The applicability of the template was expanded to the visualization of the phosphorylation of the estrogen receptor and interaction of the androgen receptor with a peptide via an intramolecular complementation between GLuc fragments. It also provides the smallest bioluminescence template ever synthesized. Considering that conformational changes of proteins generally occur for signal transductions, the present luminescence template will contribute to the exploration of intracellular molecular events with signaling proteins.

A new method for phylogenetic classification of bacterial swains using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is proposed. This method was developed using a bioinformatics-based approach to the rapid identification of bacteria as previously proposed by Demirev and co-workers, which uses ribosomal proteins composed of ~50 subunit proteins as biomarkers. Although the amino acid sequences of ribosomal proteins are highly conserved, slight sequence variations can occur at the strain level. Since ribosomal subunit proteins are a complex of housekeeping proteins that have different phylogenetic evolution rates, sequence variation detected as mass differences by MALDI-MS may be useful for the phylogenetic classification of bacteria at strain level. In our proposed method, the first step is the selection of reliable biomarkers through characterization of the expressed ribosomal subunit proteins of a reference strain (usually a genome-sequenced strain) by MAIDI-MS. The observed masses in the MALDI mass spectra of cell lysates of sample strains are then compared with the biomarker masses of the reference swain. The biomarkers for each sample strain were designated as present or absent at the reference masses, indicated by 1 or 0, respectively, which were summarized in a table. This table is processed by cluster analysis, generating a phylogenetic tree. In this study, the success of this approach was confirmed by classification of Pseudomonas putida strains because its classification is much more complicated than that of other bacterial strains. Forty-three reliable biomarkers were selected from ribosomal subunit proteins of a genome-sequenced strain, P. putida KT2440. The numbers and kinds of biomarkers observed for 16 strains of P. putida, including different biovars, were markedly different, reflecting the variety of the strains. The classification results by the proposed method were highly comparable to those based on the DNA gyrase subunit B gene (gyrB) sequence analysis, suggesting our proposed method would be a useful high-throughput method for phylogenetic classification of newly isolated bacteria.

A novel ionization platform for matrix-free laser desorption/ionization-mass spectrometry (LDI-MS) was developed using self-assembled germanium nanodots (GeNDs) of uniform size (~150-200-nm width and ~50-nm height) grown on a silicon wafer produced by molecular beam epitaxy. The performance of LDI-MS using GeNDs (GeND-MS) was investigated through measurements of a broad range of analytes, including peptides, proteins, synthetic oligomers, and polymer additives. Mass spectra of tryptic digests were clearly observed even for the mass range lower than m/z 800 without obstructive peaks. A detection limit of subfemtomole level was achieved for angiotensin-I. The upper limit of detectable mass range was ~17 kDa (myoglobin). GeND-MS also has potential for application to the characterization of industrial compounds. Almost accurate molecular weight distribution was obtained for a nonionic surfactant (Triton X-100) and for poly(ethylene glycol) oligomer. Furthermore, a brominated flame retardant, tetrabromobisphenol-A bis(2,3-dibromopropyl ether), was successfully ionized with less fragmentation, a result not obtainable by matrix-assisted laser desorption/ionization-mass spectrometry or desorption/ionization on porous silicon-mass spectrometry.

Click beetle luciferase (CBLuc) is insensitive to pH, temperature, and heavy metals, and emits a stable, highly tissue-transparent red light with luciferin in physiological circumstances. Thus, the luminescence signal is optimal for a bioanalytical index reporting the magnitude of a signal transduction of interest. Here, we validated a single-molecule-format complementation system of split CBLuc to study signal-controlled protein--protein (peptide) interactions. First, we generated 10 pairs of N- and C-terminal fragments of CBLuc to examine respectively whether a significant recovery of the activity occurs through the intramolecular complementation. The ligand binding domain of androgen receptor (AR LBD) was connected to a functional peptide sequence through a flexible linker. The fusion protein was then sandwiched between the dissected N- and C-terminal fragments of CBLuc. Androgen induces the association between AR LBD and a functional peptide and the subsequent complementation of N- and C-terminal fragments of split CBLuc inside the single-molecule-format probe, which restores the activities of CBLuc. The examination about the dissection sites of CBLuc revealed that the dissection positions next to the amino acids D412 and I439 admit a stable recovery of CBLuc activity through an intramolecular complementation. The ligand sensitivity and kinetics of the single molecular probe with split CBLuc were discussed in various cell lines and in different proteinpeptide binding models. The probe is applicable to developing biotherapeutic agents on the AR signaling and for screening adverse chemicals that possibly influence the signal transduction of proteins in living cells or animals.

A novel postcolumn photooxidation reactor with remarkably high efficiency was developed. It was installed into a system of liquid chromatography (LC) combined with inductively coupled plasma mass spectrometry (ICPMS) using a hydride generation (HG) technique to enable sensitive speciation of organic arsenicals. An LC eluent was passed into a reaction tube that runs through a low-pressure mercury lamp of a high-efficiency photooxidation (HEPO) reactor. It provided efficient irradiation with vacuum ultraviolet light at 185 nm, which induced oxidative decomposition of persistent organic arsenicals, for example, arsenobetaine, to the final product, arsenate, for an extremely short time, 3.5 s, without using any oxidizing agents. That arsenate was subsequently detectable by HG-ICPMS. In contrast, conventional photooxidation reactors require more than 100 s for completion, even when using a peroxodisulfate solution as an oxidizing agent. Because of the absence of broadening and dilution of the LC-separated peaks caused by mixing with the oxidant solution, the detection limits for 10 arsenic species including the persistent organic arsenicals by the proposed LC-HEPO-HG-ICPMS ranged from 2.3 to 18 ng of As/L, which were about one-tenth as low as those by LC-ICPMS. The proposed method revealed for the first time the occurrence of 20 arsenic species, including the unknown trace species, which were undetectable by LC-ICPMS and LC-HG-ICPMS, in human urine.

Inorganic arsenic (InAs) is a ubiquitous metalloid that has been shown to exert multiple adverse health outcomes. Urinary InAs and its metabolite concentration has been used as a biomarker of arsenic (As) exposure in some epidemiological studies, however, quantitative relationship between daily InAs exposure and urinary InAs metabolites concentration has not been well characterized. We collected a set of 24-h duplicated diet and spot urine sample of the next morning of diet sampling from 20 male and 19 female subjects in Japan from August 2011 to October 2012. Concentrations of As species in duplicated diet and urine samples were determined by using liquid chromatography-ICP mass spectrometry with a hydride generation system. Sum of the concentrations of urinary InAs and methylarsonic acid (MMA) was used as a measure of InAs exposure. Daily dietary InAs exposure was estimated to be 0.087 µg kg−1day−1(Geometric mean, GM), and GM of urinary InAs+MMA concentrations was 3.5 ng mL−1. Analysis of covariance did not find gender-difference in regression coefficients as significant (P> 0.05). Regression equation Log10[urinary InAs+MMA concentration] = 0.570× Log10[dietary InAs exposure level per body weight] + 1.15 was obtained for whole data set. This equation would be valuable in converting urinary InAs concentration to daily InAs exposure, which will be important information in risk assessment. [ABSTRACT FROM AUTHOR]

As a result of the growing potential industrial and medical applications of multi-walled carbon nanotubes (MWCNTs), people working in or residing near facilities that manufacture them may be exposed to airborne MWCNTs in the future. Because of concerns regarding their toxicity, quantitative data on the long-term clearance of pristine MWCNTs from the lungs are required. We administered pristine MWCNTs well dispersed in 0.5 mg ml−1 Triton-X solution to rats at doses of 0.20 or 0.55 mg via intratracheal instillation and investigated clearance over a 12-month observation period. The pristine MWCNTs pulmonary burden was determined 1, 3, 7, 28, 91, 175 and 364 days after instillation using a method involving combustive oxidation and infrared analysis, combined with acid digestion and heat pretreatment. As 0.15- and 0.38-mg MWCNTs were detected 1 day after administration of 0.20 and 0.55 mg MWCNTs, respectively, approximately 30% of administrated MWCNTs may have been cleared by bronchial ciliary motion within 24 h of administration. After that, the pulmonary MWCNT burden did not decrease significantly over time for up to 364 days after instillation, suggesting that MWCNTs were not readily cleared from the lung. Transmission electron microscopy (TEM) showed that alveolar macrophages internalized the MWCNTs and retained in the lung for at least 364 days after instillation. MWCNTs were not detected in the liver or brain within the 364-day study period (<0.04 mg per liver, < 0.006 mg per brain). Copyright © 2015 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]