Quantification of Intracellular Thiols by HPLC-Fluorescence Detection.

Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and N-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R2 > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5-153 pmol/1 × 106 cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.

Fast analysis using pillar array columns: Quantification of branched-chain α-keto acids in human plasma samples.

Branched-chain α-keto acids (BCKAs, namely, α-ketoisovaleric acid (KIV), α-ketoisocaproic acid (KIC), and α-keto-ß-methylvaleric acid (KMV)) are related to many diseases such as myeloid leukemia, liver cancer, and diabetes mellitus. A rapid quantitative analytical method for BCKAs using pillar array columns was developed. α-Keto acids were labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB), followed by their separation on octadecylsilane-treated pillar array columns with MeOH/H2O as the mobile phase. Five DMB-labelled α-keto acids including the internal standard were separated in 160 s. The lower limits of quantification for DMB-α-keto acids were 2-5 µM. The intra- and interday precisions were 2.9-6.6 % and 5.2-10.7 %, respectively. The developed method was applied to BCKA quantification in human plasma samples; KIV, KIC, and KMV concentrations were determined to be 13.8, 24.2, and 15.2 µM, respectively. The method realized rapid, sensitive, and precise analysis of BCKAs and can be applied for clinical diagnosis.

Analysis of intracellular α-keto acids by HPLC with fluorescence detection.

Branched-chain keto acids and branched-chain amino acids are metabolites of branched-chain amino acid aminotransferases (BCATs), which catalyzes reversible transamination between them. We found that BCAT1 plays an important role in the progression of myeloid leukaemia, and a method for the analysis of intracellular α-keto acids including branched-chain keto acids was necessary to further investigate their role. In this study, we developed a method to analyze six α-keto acids (α-ketoglutaric acid (KG), pyruvic acid, α-ketobutyric acid, α-ketoisovaleric acid, α-ketoisocaproic acid, and α-keto-ß-methylvaleric acid) in K562 cells by HPLC with fluorescence detection, using 1,2-diamino-4,5-methylenedioxybenzene (DMB) as a derivatization reagent. Because split peaks of DMB-KG were observed when injection samples were too acidic, the derivatization solution was diluted with NaOH solution to obtain a single peak. Limits of detection and limits of quantification were 1.3-5.4 nM and 4.2-18 nM, respectively. Intracellular concentrations of α-keto acids were 1.55-316 pmol/1 × 106 K562 cells. The developed method realized reproducible and sensitive analysis of intracellular α-keto acids. Thus, the method could be used to elucidate the role of BCAT in myeloid leukaemia.

Photo-reactive oligodeoxynucleotide-embedded nanovesicles (PROsomes) with switchable stability for efficient cellular uptake and gene knockdown.

A photo-responsive nanovesicle is fabricated by polyion complex (PIC) formation between poly(ethylene glycol) (PEG)-block-polypeptides and photo-reactive oligodeoxynucleotides (PROs)/anti-sense oligonucleotides (ASOs). The ultraviolet (UV) light triggers reversible crosslinking between PROs and ASOs in the vesicular membrane, providing the nanovesicle with switchable stability under physiological conditions. The resulting nanovesicle allows efficient cellular internalization, leading to significant UV-triggered gene knockdown in cultured cells.

Extraction of urinary cell-free DNA by using triamine-modified silica particles for liquid biopsy.

The presence of approximately 200-bp cell-free DNA (cfDNA) in the urine has attracted attention as a biomarker for liquid biopsy. However, it is currently useful only for diagnoses of cancers in which a large amount of cfDNA is excreted in the urine. Therefore, the development of an efficient method for extracting cfDNA existing in small amounts in the urine is essential for diagnosing many other diseases. We examined the effect of particle size, small pore size (surface area), and surface modification of porous silica particles on the efficiency of DNA extraction. Our observations suggested that cfDNA could be captured by tertiary amine-modified particles and then removed from the particles by repeatedly washing with sodium bicarbonate (pH 11). Using this method with 30 mg of triamine-modified particles, we succeeded in extracting a few hundred nanograms of cfDNA from 15 mL urine. Furthermore, we could detect ~ 67 fg/mL caries DNA (211 bp) in 15 mL urine sample, suggesting that this method may be suitable for the extraction of genetic biomarkers for cfDNA-based liquid biopsy.

Optimization of tris(2-carboxyethyl) phosphine reduction conditions for fast analysis of total biothiols in mouse serum samples.

In this study, we investigated suitable conditions for the reduction of disulfides in mouse serum samples by tris(2-carboxyethyl) phosphine (TCEP) for fast analysis of total biothiols. Disulfides were reduced with TCEP, and then, thiols were derivatized with the fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Interference peaks on chromatograms of mouse serum samples disappeared when the TCEP reaction was conducted on ice instead of at room temperature, which is used classically. Low-molecular-weight disulfides, such as cystine and glutathione disulfide, were nearly completely reduced by TCEP on ice. Six SBD-biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, γ-glutamylcysteine, and N-acetylcysteine) were separated within 7.5 min on a sulfoalkylbetain-type column (ZIC-HILIC: 150 × 2.1 mm i.d., 3.5 µm), without interference peaks. The developed method showed good linearity and reproducibility, with inter- and intra-day precisions of less than 3%.

Extraction of cell-free DNA from urine, using polylysine-coated silica particles.

DNA analysis is used for a variety of purposes, including disease diagnosis and DNA profiling; this involves extracting DNA from living organisms. In this study, we prepared polycationic silica particles to extract DNA that has the negatively charged phosphate backbone from solution. The coated particles were prepared by mixing conventional silica gel particles and poly-Lys; these particles could efficiently extract 1.3 µg of cell-free DNA from 50 mL of (male) urine. It is expected that these easily prepared particles (just a mixture of two commercially available chemicals) can be used as a noninvasive diagnostic tool for genetic disorders such as cancer, diabetes, and hypertension. Graphical abstract Effective extraction method of cfDNA from urine was developed that used commercially available silica gel particles and poly-Lys.

ASK1 signalling regulates brown and beige adipocyte function.

Recent studies suggest that adult humans have active brown or beige adipocytes, the activation of which might be a therapeutic strategy for the treatment of diverse metabolic diseases. Here we show that the protein kinase ASK1 regulates brown and beige adipocytes function. In brown or white adipocytes, the PKA-ASK1-p38 axis is activated in response to cAMP signalling and contributes to the cell-autonomous induction of genes, including Ucp1. Global and fat-specific ASK1 deficiency leads to impaired metabolic responses, including thermogenesis and oxygen consumption, at the cell and whole-body levels, respectively. Our data thus indicate that the ASK1 signalling axis is a regulator of brown and beige adipocyte gene expression and function.

Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform.

Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel ß-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes.

Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry.

Biothiols such as homocysteine, cysteine, and glutathione play many biologically important roles, especially in reduction-oxidation homeostasis and resistance to oxidative stress, and the measurement of their concentrations in model animal fluids is important in clarifying the pathology of thiol-related diseases. In this study, an analytical method for total biothiols in mouse serum using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection was developed. Mouse serum samples were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), after reduction by tris(2-carboxyethyl)phosphine. Five biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine) in the mouse sera were separated within 16 min on an amide-type HILIC column. The method possessed good linearity, good reproducibility with an intra-day variance of less than 3%, and low detection limits of 0.2-4 nM. Concentrations of homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine in the mouse serum samples were calculated as 6.7 ± 0.3, 227.7 ± 16.9, 1.2 ± 0.4, 77.5 ± 29.2, and 8.2 ± 0.9 µM, respectively (mean ± S.D., n = 4). Furthermore, HILIC-negative electrospray ionization-mass spectrometry (MS) analysis using a high-resolution mass spectrometer was conducted to determine the exact masses of two unknown peaks, which were found in the mouse serum samples with high signal intensity and were not detected in human plasma samples. The exact masses of the unknown compounds were determined as 1184.519 and 800.281 (as SBD-derivatized negative ions), which possessed a product ion common to SBD-thiols (m/z 230.954, as [SBD-SH](-)) upon tandem MS spectrometric analysis.

Nascent SecM chain outside the ribosome reinforces translation arrest.

SecM, a bacterial secretion monitor protein, contains a specific amino acid sequence at its C-terminus, called arrest sequence, which interacts with the ribosomal tunnel and arrests its own translation. The arrest sequence is sufficient and necessary for stable translation arrest. However, some previous studies have suggested that the nascent chain outside the ribosome affects the stability of translation arrest. To clarify this issue, we performed in vitro translation assays with HaloTag proteins fused to the C-terminal fragment of E. coli SecM containing the arrest sequence or the full-length SecM. We showed that the translation of HaloTag proteins, which are fused to the fragment, is not effectively arrested, whereas the translation of HaloTag protein fused to full-length SecM is arrested efficiently. In addition, we observed that the nascent SecM chain outside the ribosome markedly stabilizes the translation arrest. These results indicate that changes in the nascent polypeptide chain outside the ribosome can affect the stability of translation arrest; the nascent SecM chain outside the ribosome stabilizes the translation arrest.

Label-free quantification of microRNAs using ligase-assisted sandwich hybridization on a DNA microarray.

MicroRNAs (miRNAs) can be used as biomarkers for cancer and other human diseases; therefore, high-throughput and reliable miRNA-quantification methods are required to exploit these markers for diagnostic testing. In this report, we describe the construction of a platform for miRNA-quantification using ligase-assisted sandwich hybridization (LASH) without miRNA-labeling. T4 DNA ligase was used to compensate for the low affinity between miRNAs and two short complementary DNA probes, and it improved the hybridization yield ∼50,000 times. The LASH assay enabled synthesized miR-143 to be quantified at concentrations ranging from 30 fM to 30 pM. The LASH assay could also quantify endogenous miR-143 released from cultured cells as well as some miRNAs in total RNAs derived from blood. Furthermore, multi-color detection enabled us to distinguish between the highly homologous miR-141 and miR-200a. This simple label-free quantification technique is an easy-to-use approach that can be applied to disease diagnosis.

Analytical methods involving separation techniques for determination of low-molecular-weight biothiols in human plasma and blood.

Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5-15, 200-300, and 1-5 µM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry.

Fast and simultaneous analysis of biothiols by high-performance liquid chromatography with fluorescence detection under hydrophilic interaction chromatography conditions.

A method for analyzing biothiols based on high-performance liquid chromatography (HPLC)-fluorescence detection under hydrophilic interaction chromatography (HILIC) conditions has been developed. Thiols were derivatized with nonfluorescent ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), which selectively reacts with the thiol groups to furnish the corresponding fluorescent SBD-thiols. Among the six different kinds of HILIC columns examined, the ZIC-HILIC column with sulfobetaine groups in the stationary phase proved to be the best for the separation of SBD-thiols. Eight thiols-N-acetylcysteine, cysteamine, homocysteine, cysteine, cysteinylglycine, glutathione, γ-glutamylcysteine, and internal standard N-(2-mercaptopropionyl)glycine-were baseline-separated within 10 min. The detection sensitivity was improved partly due to the increase in the SBD-thiol fluorescence owing to the acetonitrile-rich mobile phase used. The detection limits at a signal-to-noise ratio of 3 were 0.02-3.4 nmol l(-1). The method could successfully quantify six thiols in a human plasma sample, while cysteamine could not be detected. Both the intra- and interday precisions were below 4% for homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine except for N-acetylcysteine. This method should be a useful tool for investigating the relationship between sulfur metabolism and related diseases, since a multicomponent system consisting of different thiol compounds could be analyzed simultaneously with high sensitivity within a short time.

Single-molecule observation of protein folding in symmetric GroEL-(GroES)2 complexes.

The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding.

Real-time single-molecule observation of green fluorescent protein synthesis by immobilized ribosomes.

The dynamics of full protein synthesis and the co-translational folding processes are not fully understood. We have developed a novel method, using a combination of ribosome display and single-molecule techniques, for monitoring the synthesis, co-translational folding, and maturation of a complete polypeptide chain at the single-molecule level. This method enabled us to observe the appearance of green fluorescent protein fluorescence after de novo synthesis of the complete protein. Here, we provide the information necessary to reproduce this method, which will be valuable in revealing the dynamics of the co-translational folding and maturation of nascent polypeptides.

Biocompatible, surface functionalized mesoporous titania nanoparticles for intracellular imaging and anticancer drug delivery.

Mesoporous titania nanoparticles (MTNs) with excellent biocompatibility (LC(50)≈ 400 µg mL(-1)) and a large surface area (ca. 237.3 m(2) g(-1)) were synthesized and further functionalized with a phosphate-containing fluorescent molecule (i.e. flavin mononucleotide; FMN) and loaded with an anticancer drug (i.e. Doxorubicin) for successful intracellular bioimaging and drug delivery, respectively, in human breast cancer cells BT-20.

Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2'-O-methyl RNA probes in living cells.

Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

Denatured proteins facilitate the formation of the football-shaped GroEL-(GroES)2 complex.

Controversy exists over whether the chaperonin GroEL forms a GroEL-(GroES)2 complex (football-shaped complex) during its reaction cycle. We have revealed previously the existence of the football-shaped complex in the chaperonin reaction cycle using a FRET (fluorescence resonance energy transfer) assay [Sameshima, Ueno, Iizuka, Ishii, Terada, Okabe and Funatsu (2008) J. Biol. Chem. 283, 23765-23773]. Although denatured proteins alter the ATPase activity of GroEL and the dynamics of the GroEL-GroES interaction, the effect of denatured proteins on the formation of the football-shaped complex has not been characterized. In the present study, a FRET assay was used to demonstrate that denatured proteins facilitate the formation of the football-shaped complex. The presence of denatured proteins was also found to increase the rate of association of GroES to the trans-ring of GroEL. Furthermore, denatured proteins decrease the inhibitory influence of ADP on ATP-induced association of GroES to the trans-ring of GroEL. From these findings we conclude that denatured proteins facilitate the dissociation of ADP from the trans-ring of GroEL and the concomitant association of ATP and the second GroES.