Preparation of Metal-Immobilized Methacrylate-Based Monolithic Columns for Flow-Through Cross-Coupling Reactions.

With the aim of developing efficient flow-through microreactors for high-throughput organic synthesis, in this work, microreactors were fabricated by chemically immobilizing palladium-, nickel-, iron-, and copper-based catalysts onto ligand-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) [poly(GMA-co-EDMA)] monoliths, which were prepared inside a silicosteel tubing (10 cm long with an inner diameter of 1.0 mm) and modified with several ligands including 5-amino-1,10-phenanthroline (APHEN), iminodiacetic acid (IDA), and iminodimethyl phosphonic acid (IDP). The performance of the resulting microreactors in Suzuki-Miyaura cross-coupling reactions was evaluated, finding that the poly(GMA-co-EDMA) monolith chemically modified with 5-amino-1,10-phenanthroline as a binding site for the palladium catalyst provided an excellent flow-through performance, enabling highly efficient and rapid reactions with high product yields. Moreover, this monolithic microreactor maintained its good activity and efficiency during prolonged use.

Identification of genes for sulfolipid synthesis in primitive red alga Cyanidioschyzon merolae.

Sulfoquinovosyl diacylglycerol is one of the lipids that construct thylakoid membranes, and is distributed from cyanobacteria to plastids in plants including a red lineage. One of the most primitive red algae, Cyanidioschyzon melorae, similar to cyanobacteria and green plants, possesses homologs of the SQD1 and SQD2 genes that code for UDP-sulfoquinovose and sulfoquinovosyl diacylglycerol synthases, respectively, for the synthesis of sulfoquinovosyl diacylglycerol. We here revealed the structural properties of SQD1 and SQD2 homologs in C. melorae intrinsic to those of the authentic proteins, and verified their enzymatic functions through heterologous expression in cyanobacterial disruptants as to the corresponding genes. The results demonstrated that the system of sulfoquinovosyl diacylglycerol synthesis could have been conserved through evolution of cyanobacteria to plastids in a red lineage, which is compatible with the monophyletic origin of plastids.

Hydrophilic interaction chromatography-type sorbent prepared by the modification of methacrylate-base resin with polyethyleneimine for solid-phase extraction of polar compounds.

Hydrophilic interaction chromatography (HILIC)-type sorbents were newly developed for the solid-phase extraction (SPE) of polar compounds. Two methacrylate-base resins with different cross-linking monomers and pore properties were synthesized, and three polyethyleneimines (PEIs) with different molecular weights were modified onto each base resin. In both cases, PEIs with a molecular weight of 10,000 (PEI-10,000) exhibited the highest adsorption properties for polar compounds (uracil, uridine, adenosine, cytidine, and guanosine). To control the water-enriched layer at the surface of the PEI-10,000-modified sorbents, the additive amount of PEI-10,000 in the modified reaction was also optimized. When 10 times the amount of PEI-10,000 to each base resin was added, an improvement in adsorption property was observed. Moreover, the use of a nonaqueous sample solution (100% acetonitrile) during the sample loading process drastically improved adsorption, especially for uracil (about 80%) and adenosine (100%). These results indicate that the formation of a strong water-enriched layer at the surface of sorbents with an effective expression of hydrophilic interaction was an important factor in the adsorption properties of polar compounds in HILIC mode-SPE.

Novel chemically cross-linked self-molding particulate sorbents as solid-phase extraction media.

Here, we describe novel, chemically cross-linked, self-molding particulate polymer sorbents that are utilized as a molding-type solid-phase extraction medium (M-SPEM), which exhibits high permeability and rigidness. To fabricate such M-SPEM, first, polyethyleneimine (PEI)-modified reversed-phase (RP)-type particulate sorbents were synthesized, thereafter, they were chemically cross-linked by a polymer having many epoxy groups together with additional PEI. By optimizing the binding conditions of the particulate sorbents, the resultant M-SPEM has almost the same adsorption properties as the corresponding unmolded particulate sorbent for some polar (e.g., uracil and adenine) compounds. The binding technique proposed here is expected to facilitate the fabrication of molding-type sorbents and improve the performance of the SPE procedure.

Atomic mineral characteristics of Indonesian osteoporosis by high-resolution inductively coupled plasma mass spectrometry.

Clinical research indicates that negative calcium balance is associated with low bone mass, rapid bone loss, and high fracture rates. However, some studies revealed that not only calcium is involved in bone strengthening as risk factor of fracture osteoporosis. Thus, in this report, the difference of metallic and nonmetallic elements in osteoporosis and normal bones was studied by high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). The influence of these elements on bone metabolic processes is also discussed. Inclusion criteria of bone samples consist of postmenopausal woman, trabecular bone fracture, normal and osteoporosis BMD value, and no history of previous disease. The results showed that the concentration of B, Al, S, V, Co, Mo, Te, Ba, La, Ni, As, and Ca/P ratio is higher in osteoporosis than normal. These atomic minerals have negative role to imbalance between bone resorption and bone formation activity. Conversely, concentrations of Na, Mg, P, K, Ca, Cr, Pd, Ag, Mn, Fe, Cu, Zn, Rb, Sr, Pb, and Se are lower in osteoporosis than in normal bones. Among these atoms, known to have important roles in bone structure, we found involvement of atomic mineral and calcium which are considerable to contribute to osteoporotic phenomena.

Preparation and evaluation of molding-type solid-phase extraction media binding with commercially available adhesives.

A fabrication method of molding-type solid-phase extraction media (M-SPEM) bound with commercially available adhesive is presented. Six pieces of M-SPEM were prepared by heating each kneaded product of a particulate sorbent and an adhesive inserted into a six-hole cylindrical mold for hardening under an open system and normal pressure. The particulate sorbent contained in M-SPEM was divinylbenzene-based reversed-phase mode solid-phase extractants that we have reported. An examination of several adhesives showed that the moldability of M-SPEM depended on the composition and properties of the adhesive. The optimized procedure can be used to prepare an M-SPEM containing an 85 wt% particulate sorbent (particulate sorbent/adhesive, 100 mg/17 mg; particle diameter, 90-150 µm), and the M-SPEM has a specific surface area of about 500 m2/g. The established procedure in this study can bind particulate sorbents together, which showed almost no reductions in the adsorption property and liquid permeability compared with those of the particulate sorbent.

Development of a surface plasmon resonance sensor using an optical fiber prepared by electroless displacement gold plating and its application to immunoassay.

A simple and low-cost method of fabricating an optical fiber for a surface plasmon resonance (SPR) sensor was proposed. The method is based on the electroless nickel plating and subsequent displacement gold plating of the core of the optical fiber. The thickness of the nickel and gold thin films deposited on the core of the optical fiber could be controlled by measuring the reflected light intensity from the tip of the optical fiber during the plating processes. The sensitivity and resolution of the SPR sensor with the fabricated optical fiber in the refractive index range from 1.333 to 1.348 were 1324.3 nm/RIU and 7.6 × 10-4 RIU, respectively. The developed SPR sensor was successfully used in the determination of immunoglobulin A (IgA) in human saliva. The IgA quantification results obtained by the SPR sensor were in excellent agreement with those obtained by conventional enzyme-linked immunosorbent assay using a 96-well microtiter plate.

Development of a fluorescence microplate reader using an organic photodiode array with a large light receiving area.

We developed a small fluorescence microplate reader with an organic photodiode (OPD) array. The OPD array has nine OPDs that have a large light receiving area (9.62 mm2 per one OPD). Since the OPD array is fabricated on a flat glass plate, it can be placed just below microwells and can detect fluorescence emitted through the entire surface of the microwell bottom. The analytical performance of the developed plate reader was evaluated by measuring an aqueous solution of resorufin. The limit of detection (LOD) for resorufin (0.01-0.05 µM) was lower than that obtained with a plate reader equipped with nine inorganic photodiodes developed in a previous study (0.30 µM) and a commercially available microplate reader (0.16 µM). These results indicate that the large light receiving area improves the detection performance of the system. In addition, the developed reader was successfully used to quantify immunoglobulin A (IgA) in human saliva. The LOD for IgA was estimated to be 1.2 ng/mL, which is low enough to objectively evaluate human stress.

Estimation of the distribution of intravenously injected adipose tissue-derived stem cells labeled with quantum dots in mice organs through the determination of their metallic components by ICPMS.

Adipose tissue-derived stem cells (ASCs) have shown promise in cell therapy because of their ability to self-renew damaged or diseased organs and easy harvest. To ensure the distribution and quantification of the ASCs injected from tail vein, several whole-body imaging techniques including fluorescence optical imaging with quantum dots (QDs) have been employed, but they may suffer from insufficient sensitivity and accuracy. Here, we report quantitative distribution of ASCs in various organs (heart, lung, liver, spleen, and kidney) of mice, which were intravenously injected with QDs-labeled ASCs (QDs-ASCs), through the detection of QDs-derived metallic components by inductively coupled plasma mass spectrometry (ICPMS). For accurate and precise determination, each organ was harvested and completely digested with a mixture of HNO(3) and H(2)O(2) in a microwave oven prior to ICPMS measurement, which was equipped with a microflow injection system and a laboratory-made capillary-attached micronebulizer. After optimization, 16 elements including major components (Cd, Se, and Te) of QDs and essential elements (Na, K, Mg, Ca, P, S, Mn, Fe, Co, Cu, Zn, Se, Sr, and Mo) were successfully determined in the organs. As compared to untreated mice, QDs-ASCs-treated mice showed significantly higher levels of Cd and Te in all organs, and as expected, the molar ratio of Cd to Te in each organ was in good agreement with the molar composition ratio in the QDs. This result indicates that the increment of Cd (or Te) can be used as a tracer for calculating the distribution of ASCs in mice organs. As a result of the calculation, 36.8%, 19.1%, 0.59%, 0.49%, and 0.25% of the total ASCs injected were estimated to be distributed in the liver, lung, heart, spleen, and kidney, respectively.

The ER cholesterol sensor SCAP promotes CARTS biogenesis at ER-Golgi membrane contact sites.

In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here, we show that in cholesterol-fed cells, ER-localized SCAP interacts through Sac1 phosphatidylinositol 4-phosphate (PI4P) phosphatase with a VAP-OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER-Golgi membrane contact sites (MCSs). SCAP knockdown inhibited the turnover of PI4P, perhaps due to a cholesterol transport defect, and altered the subcellular distribution of the VAP-OSBP complex. As in the case of perturbation of lipid transfer complexes at ER-Golgi MCSs, SCAP knockdown inhibited the biogenesis of the trans-Golgi network-derived transport carriers CARTS, which was reversed by expression of wild-type SCAP or a Golgi transport-defective mutant, but not of cholesterol sensing-defective mutants. Altogether, our findings reveal a new role for SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis via ER-Golgi MCSs, depending on the ER cholesterol.

HPLC with electrochemical detection for determining homogentisic acid and its application to urine from rats fed tyrosine-enriched food.

A highly sensitive method for determining urine homogentisic acid (HGA) is required to provide adequate diagnosis and therapy for alkaptonuria in early stages. In this study, we developed a highly sensitive high-performance liquid chromatography with electrochemical detection (HPLC-ECD) for determining HGA in urine. In order to obtain a chromatogram of HGA by HPLC-ECD, an oxidation current was monitored at +0.5 V vs. Ag/AgCl. The peak heights of HGA showed linearity (r = 0.999) ranging from 4.2 ng/mL to 168 ng/mL, and the detection limit was 1.2 ng/mL (signal-to-noise ratio, S/N = 3). In recovery tests using human control urine spiked with an HGA standard, the recoveries of HGA were more than 93.2 %, and the relative standard deviations (n = 6) were less than 1.9 %. As an in vivo application using male Wistar rats, the level of urine HGA, which was metabolized from tyrosine in tyrosine-enriched food, was determined by this HPLC-ECD method. The determination of HGA in urine by this HPLC-ECD method requires only 0.1 mL of a rat urine specimen and simple sample preparation consisting of dilution and filtration.

Molding-type Solid-phase Extraction Media Glued with Commercially Available Adhesives.

The handling of a particulate sorbent for solid-phase extraction is often troublesome because it causes static clinging and scattering. To overcome this problem, a production method for a simple molding-type solid-phase extraction medium (M-SPEM) was developed in this study by using commercially available adhesives. The content of a particulate sorbent can increase to as much as 85 wt% in the M-SPEM. Because of the high content, the proposed M-SPEMs have a higher specific surface area than previous monolithic media.

Development and Evaluation of HILIC-type Sorbents Modified with Hydrophilic Copolymers for Solid-phase Extraction.

Hydrophilic interaction chromatography (HILIC) has attractive attention for the separation of water-soluble compounds via HPLC. There are, however, few studies on the pretreatment of the HILIC-type solid-phase extraction (SPE) due to the difficulty of obtaining the HILIC-type sorbent. Therefore, the development of HILIC-type sorbents for SPE is essential. In this study, four different hydrophilic copolymers, namely diallylamine-maleic acid copolymer (DAM), diallylamine-acrylamide copolymer (DAA), allylamine-maleic acid copolymer (MAM), and partly methylcarbonylated allylamine acetate copolymer (MAC), were immobilized on glycidyl methacrylate (GMA)-base resin, and their adsorptive properties were evaluated. The results of the physical and adsorptive properties indicated that a balance between the water content of the water-enriched layer on sorbent and the amount of hydrophilic copolymer immobilized on the GMA-base resin was vital for the adsorption in HILIC-type sorbent for SPE.

Evaluation of the adsorption properties of nucleobase-modified sorbents for a solid-phase extraction of water-soluble compounds.

We developed hydrophilic interaction chromatography (HILIC)-type sorbents modified with nucleobases for solid phase extraction (SPE). The synthesized hydrophilic base resins were modified by each nucleobase (adenine, guanine, and cytosine). The measurement of the amount of water content indicated that each nucleobase-modified sorbent had a water layer. To evaluate the adsorption properties in the HILIC mode, we chose two nucleobases (uracil and adenine) and four nucleosides (uridine, adenosine, cytidine, guanosine) as water-soluble analytes, which were loaded into an SPE cartridge packed with the nucleobase-modified sorbent. Firstly, 95% acetonitrile (ACN) solutions were used in the process of conditioning and sample loading of the above polar analytes. High recoveries of the analytes were observed in each nucleobase-modified sorbent, and the Diol-type sorbent (no modification with any of the nucleobases) did not adsorb each water-soluble analyte. On the basis of this result, a 98% ACN solution was used during the process of conditioning and sample loading to decrease the concentration of water in the sample, which potentially inhibited the formation of hydrogen bonding between each analyte and the modified nucleobase. Considerable improvements of recoveries were observed in Adenine- and Cytosine-modified sorbents. These results were possibly attributed to the effective expression of hydrogen bonding by decreasing water concentration in the sample solution. Although a non-aqueous (100% ACN) sample solution can be expected to obtain higher recoveries compared with the 98% ACN solution, a decrease in recoveries was observed in Adenine-modified sorbent. From these results, the highest adsorption property was observed in Adenine-modified sorbent using 98% ACN as a sample condition, and the combination of this sample condition and sorbent is effective for high adsorption under HILIC condition. Moreover, we also revealed that a balance between the thickness of water layer and the modification amount of nucleobase is important for retention in the HILIC-type sorbent.

Multiple-channel Concentric Grid Nebulizer for Online Standard Addition in Inductively Coupled Plasma Optical Emission Spectrometry.

A multiple channel-type concentric grid nebulizer (m-CGrid) was developed for realizing efficient online standard addition in inductively coupled plasma optical emission spectrometry (ICP-OES) without premixing of liquids before nebulization. The m-CGrid can nebulize two independent liquids without premixing due to a unique structure, having two independent liquid-flow capillaries inside a single nozzle and a grid screen (over 350 mesh per inch) placed on the hole of the nozzle. The grid acts as both a flow damper and sieve; two liquids are well-mixed with a gas flow in a small space just before the grid screen, and the mixture breaks up into small droplets by passing through the grid. The m-CGrid nebulizer provides almost the same or better spray performance compared with a conventional nebulizer, such as Meinhard nebulizer; the primary aerosols were much finer (D50: 2.9 and 3.1 µm for two channels) than those generated with Meinhard nebulizer type C (D50: 19.5 µm). The signal intensities in ICP-OES obtained with two liquid channels were almost the same and were 2- to 3-fold higher than that obtained with the Meinhard nebulizer for 23 elements. The performance of m-CGrid in an online standard addition was demonstrated in the analysis of NIST SRM1577b bovine liver and NIES No. 3 Chrorella. The analytical results were in good agreement with their certified values.

Quantification of trace elements in natural samples by electrospray ionization mass spectrometry with a size-exclusion column based on the formation of metal-aminopolycarboxylate complexes.

Metal ions were determined by ESI-MS in the negative ion mode as monovalent negative ions of their aminopolycarboxylic acid (APC) complexes, e.g., [Al(cydta)](-), [Pb(Hcydta)](-), where excess amounts of the APC agents were added to sample solutions. Among several APCs studied, we chose trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA) as the best chelating agent because of higher stabilities and higher sensitivities of the complexes. The ionization efficiency of these metal complexes was strongly affected by the presence of matrix salts, e.g., NaCl, KNO(3), and etc. Thus, a size exclusion column (Sephadex G-10) was used for the online separation of the metal-APC complexes from other matrix salts. This method was successfully applied to the quantitative analyses for total amounts of Al, Ni, Cu, Zn, and Pb in the biological certified reference materials, olive leaves (BCR-062) and plankton (BCR-414). The detection limits of the present methods for these elements were several to several ten nanomolar levels. Moreover, this approach was extended to determine ultratraces of fluoride based on the formation of the ternary complex of aluminum, fluoride and nitrilotriacetic acid (NTA), i.e., [AlF(nta)](-). Its detection limit was 10 nM and was 2 orders of magnitude better than that of a fluoride ion selective electrode method. This method was applied to determine fluoride in tap water, river water, and green tea samples.

Short-lived long noncoding RNAs as surrogate indicators for chemical stress in HepG2 cells and their degradation by nuclear RNases.

Long noncoding RNAs (lncRNAs) are non-protein-coding transcripts >200 nucleotides in length that have been shown to play important roles in various biological processes. The mechanisms underlying the induction of lncRNA expression by chemical exposure remain to be determined. We identified a novel class of short-lived lncRNAs with half-lives (t1/2) ≤4 hours in human HeLa Tet-off cells, which have been suggested to express many lncRNAs with regulatory functions. As they may affect various human biological processes, short-lived lncRNAs may be useful indicators of the degree of stress on chemical exposure. In the present study, we identified four short-lived lncRNAs, designated as OIP5-AS1, FLJ46906, LINC01137, and GABPB1-AS1, which showed significantly upregulated expression following exposure to hydrogen peroxide (oxidative stress), mercury II chloride (heavy metal stress), and etoposide (DNA damage stress) in human HepG2 cells. These lncRNAs may be useful indicators of chemical stress responses. The levels of these lncRNAs in the cells were increased because of chemical stress-induced prolongation of their decay. These lncRNAs were degraded by nuclear RNases, which are components of the exosome and XRN2, and chemical exposure inhibited the RNase activities within the cells.

Investigation of adverse effect of coexisting aminopolycarboxylates on the determination of rare earth elements by ICP-MS after solid phase extraction using an iminodiacetate-based chelating-resin.

When iminodiacetic acid chelating-resin solid phase extraction (SPE) was used for the preconcentration of rare earth elements (REEs) in river water samples around sewage treatment plant (STP), low recovery values for heavy REEs were observed. In order to find out the reason for the low recovery, in the present paper, organic ligands in the STP effluent, which may compete with iminodiacetic acids, were analyzed by GC-NPD. It was found that EDTA was contained in the STP effluent at several-100 nM level and interfered with the adsorption of REEs, especially heavy REEs (present at pM level) on the chelating-resin due to the formation of stable complexes. Therefore, acid treatment was applied to decompose EDTA molecules. As a result of acid treatment with HNO3 and H2O2 at 170 °C for 4 h, all REEs were almost quantitatively recovered from the STP effluent with chelating-resin SPE with good reproducibility. After the acid treatment and subsequent 40-times preconcentration with SPE, all REEs in river water samples were precisely determined by ICP-MS at several-10 to sub pg mL-1 levels.

Identification of aluminum species in an aluminum-accumulating plant, hydrangea (Hydrangea macrophylla), by electrospray ionization mass spectrometry.

The use of electrospray ionization mass spectrometry (ESI-MS) in negative ion mode was investigated as a direct probe for identifying Al species in Al-accumulating hydrangea (Hydrangea macrophylla) samples. Cell sap solutions of hydrangea leaves were purified using Sephadex G-10 liquid chromatography and each fraction was analyzed using ESI-MS and ESI-MS/MS to identify Al species. In hydrangea leaves, a 1:1 Al-citrate complex was found as [AlH(-1)cit](-) (m/z 215), where H(3)cit denotes citric acid. This result is consistent with that of Ma et al. who used (27)Al-NMR.

Electrochemically active bacteria sense electrode potentials for regulating catabolic pathways.

Electrochemically active bacteria (EAB) receive considerable attention for their utility in bioelectrochemical processes. Although electrode potentials are known to affect the metabolic activity of EAB, it is unclear whether EAB are able to sense and respond to electrode potentials. Here, we show that, in the presence of a high-potential electrode, a model EAB Shewanella oneidensis MR-1 can utilize NADH-dependent catabolic pathways and a background formate-dependent pathway to achieve high growth yield. We also show that an Arc regulatory system is involved in sensing electrode potentials and regulating the expression of catabolic genes, including those for NADH dehydrogenase. We suggest that these findings may facilitate the use of EAB in biotechnological processes and offer the molecular bases for their ecological strategies in natural habitats.