First Measurements on Triple Oxygen Isotopes of Dissolved Inorganic Phosphate in the Hydrosphere.

Although dissolved inorganic phosphate (DIP) is an important nutrient in the hydrosphere, it is difficult to quantitatively clarify the dynamics of DIP in the hydrosphere using the δ18O value of DIP as a tracer. In this study, we quantified the triple oxygen isotopic compositions (Δ'17O) of DIP relative to VSMOW with the reference line with a slope of 0.528 as an additional tracer to clarify the sources and dynamics of DIP in the hydrosphere. We found significant variation in the Δ'17O values of riverine DIP in urban areas, ranging from -107 × 10-6 to +3 × 10-6, while those of DIP in the effluents from wastewater treatment plants (WWTP) and DIP extracted from the chemical fertilizers showed -56 ± 5 × 10-6 (1SD) and -98 ± 5 × 10-6, respectively. We conclude that both the DIP supplied directly from the artificial loads (the WWTP effluent and chemical fertilizers) showing 17O-depleted Δ'17O values and the DIP turned over via the aquatic biosphere showing 17O-enriched Δ'17O values similar to ambient H2O were the major sources of riverine DIP. High-precision determination of the Δ'17O value of DIP can contribute to quantitative clarification of the dynamics of DIP in the hydrosphere.

In Situ Control of Crystallinity of 3D Colloidal Crystals by Tuning the Growth Kinetics of Nanoparticle Building Blocks.

Colloidal crystals (CCs) constructed from inorganic nanoparticle (NP) building blocks exhibit properties that cannot be realized from isolated NPs or corresponding bulk counterparts. Because the arrangement of NPs in CCs is crucial in the CC's collective properties, development of a procedure to modulate the assembly of NP constituents is important. We demonstrate rapid formation of nickel (phosphide) CCs with tunable crystallinity through van der Waals force-driven spontaneous self-assembly of NPs in a facile one-pot colloidal synthesis. The quantity of size-regulating reagent (tri-n-octylphosphine) modulates the assembly of NPs from ordered close-packed to a disordered configuration in CCs. Synchrotron-based in situ small-angle X-ray scattering revealed that the size uniformity of the NPs determines the crystallinity of CCs, indicating the importance of regulating the growth kinetics of NPs during the synthesis. Our work will be useful for universal scalable preparation of CCs from a variety of materials and structures, with tunable concerted properties.

Determination of the triple oxygen isotopic composition of tropospheric ozone in terminal positions using a multistep nitrite-coated filter-pack system.

RATIONALE: The triple oxygen isotopic composition (Δ17 O) of tropospheric ozone (O3 ) is a useful tracer for identifying the source and is essential for clarifying the atmospheric chemistry of oxidants. However, the single nitrite-coated filter method is inaccurate owing to the nitrate blank produced through the reaction of nitrite and oxygen compounds other than O3 . METHODS: A multistep nitrite-coated filter-pack system is newly adopted to transfer the O-atoms in terminal positions of O3 to nitrite on each filter to determine the Δ17 O of O3 in terminal positions (denoted as Δ17 O(O3 )term ). The NO3 - produced by this reaction is chemically converted into N2 O, and continuous-flow isotope ratio mass spectrometry (CF-IRMS) is used to determine the oxygen isotopic compositions. RESULTS: The reciprocal of the NO3 - quantities on the nitrite-coated filters in each sample showed a strong linear relationship with Δ17 O of NO3 - . Using the linear relation, we corrected the changes in Δ17 O of NO3 - on the filters. We verified the accuracy of the new method through the measurement of artificial O3 with known Δ17 O(O3 )term value that had been determined from the changes in Δ17 O of O2 . The Δ17 O(O3 )term of tropospheric O3 was in agreement with previous studies. CONCLUSIONS: We accurately determined the δ18 O and Δ17 O values of tropospheric O3 by blank correction using our new method. Measurements of Δ17 O(O3 )term of the ambient troposphere showed 1.1 ± 0.7‰ diurnal variations between daytime (higher) and nighttime (lower) due likely to the formation of the temperature inversion layer at night.

Water-Soluble closo-Docecaborate-Containing Pteroyl Derivatives Targeting Folate Receptor-Positive Tumors for Boron Neutron Capture Therapy.

Water-soluble pteroyl-closo-dodecaborate conjugates (PBCs 1-4), were developed as folate receptor (FRα) targeting boron carriers for boron neutron capture therapy (BNCT). PBCs 1-4 had adequately low cytotoxicity with IC50 values in the range of 1~3 mM toward selected human cancer cells, low enough to use as BNCT boron agents. PBCs 1-3 showed significant cell uptake by FRα positive cells, especially U87MG glioblastoma cells, although the accumulation of PBC 4 was low compared with PBCs 1-3 and L-4-boronophenylalanine (L-BPA). The cellular uptake of PBC 1 and PBC 3 by HeLa cells was arrested by increasing the concentration of folate in the medium, indicating that the major uptake mechanisms of PBC 1-3 are primarily through FRα receptor-mediated endocytosis.

Folate receptor-targeted novel boron compound for boron neutron capture therapy on F98 glioma-bearing rats.

Folic acid (FA) has high affinity for the folate receptor (FR), which is limited expressed in normal human tissues, but over-expressed in several tumor cells, including glioblastoma cells. In the present work, a novel pteroyl-closo-dodecaborate conjugate (PBC) was developed, in which the pteroyl group interacts with FR, and the efficacy of boron neutron capture therapy (BNCT) using PBC was investigated. Thus, in vitro and in vivo studies were performed using F98 rat glioma cells and F98 glioma-bearing rats. For the in vivo study, boronophenylalanine (BPA) was intravenously administered, while PBC was administered by convection-enhanced delivery (CED)-a method for direct local drug infusion into the brain of rats. Furthermore, a combination of PBC administered by CED and BPA administered by intravenous (i.v.) injection was also investigated. In the biodistribution experiment, PBC administration at 6 h after CED termination showed the highest cellular boron concentrations (64.6 ± 29.6 µg B/g). Median survival time (MST) of untreated controls was 23.0 days (range 21-24 days). MST of rats administered PBC (CED) followed by neutron irradiation was 31 days (range 26-36 days), which was similar to that of rats administered i.v. BPA (30 days; range 25-37 days). Moreover, the combination group [PBC (CED) and i.v. BPA] showed the longest MST (38 days; range 28-40 days). It is concluded that a significant MST increase was noted in the survival time of the combination group of PBC (CED) and i.v. BPA compared to that in the single-boron agent groups. These findings suggest that the combination use of PBC (CED) has additional effects.

Improvement of human cell line activation test (h-CLAT) using short-time exposure methods for prevention of false-negative results.

Recently, animal testing has been affected by increasing ethical, social, and political concerns regarding animal welfare. Several in vitro safety tests for evaluating skin sensitization, such as the human cell line activation test (h-CLAT), have been proposed. However, similar to other tests, the h-CLAT has produced false-negative results, including in tests for acid anhydride and water-insoluble chemicals. In a previous study, we demonstrated that the cause of false-negative results from phthalic anhydride was hydrolysis by an aqueous vehicle, with IL-8 release from THP-1 cells, and that short-time exposure to liquid paraffin (LP) dispersion medium could reduce false-negative results from acid anhydrides. In the present study, we modified the h-CLAT by applying this exposure method. We found that the modified h-CLAT is a promising method for reducing false-negative results obtained from acid anhydrides and chemicals with octanol-water partition coefficients (LogKow) greater than 3.5. Based on the outcomes from the present study, a combination of the original and the modified h-CLAT is suggested for reducing false-negative results. Notably, the combination method provided a sensitivity of 95% (overall chemicals) or 93% (chemicals with LogKow > 2.0), and an accuracy of 88% (overall chemicals) or 81% (chemicals with LogKow > 2.0). We found that the combined method is a promising evaluation scheme for reducing false-negative results seen in existing in vitro skin-sensitization tests. In the future, we expect a combination of original and modified h-CLAT to be applied in a newly developed in vitro test for evaluating skin sensitization.

Disturbance of deep-sea environments induced by the M9.0 Tohoku Earthquake.

The impacts of the M9.0 Tohoku Earthquake on deep-sea environment were investigated 36 and 98 days after the event. The light transmission anomaly in the deep-sea water after 36 days became atypically greater (∼35%) and more extensive (thickness ∼1500 m) near the trench axis owing to the turbulent diffusion of fresh seafloor sediment, coordinated with potential seafloor displacement. In addition to the chemical influx associated with sediment diffusion, an influx of (13)C-enriched methane from the deep sub-seafloor reservoirs was estimated. This isotopically unusual methane influx was possibly triggered by the earthquake and its aftershocks that subsequently induced changes in the sub-seafloor hydrogeologic structures. The whole prokaryotic biomass and the development of specific phylotypes in the deep-sea microbial communities could rise and fall at 36 and 98 days, respectively, after the event. We may capture the snap shots of post-earthquake disturbance in deep-sea chemistry and microbial community responses.

Stable hydrogen isotopic analysis of nanomolar molecular hydrogen by automatic multi-step gas chromatographic separation.

We have developed a new automated analytical system that employs a continuous flow isotope ratio mass spectrometer to determine the stable hydrogen isotopic composition (δD) of nanomolar quantities of molecular hydrogen (H(2)) in an air sample. This method improves previous methods to attain simpler and lower-cost analyses, especially by avoiding the use of expensive or special devices, such as a Toepler pump, a cryogenic refrigerator, and a special evacuation system to keep the temperature of a coolant under reduced pressure. Instead, the system allows H(2) purification from the air matrix via automatic multi-step gas chromatographic separation using the coolants of both liquid nitrogen (77 K) and liquid nitrogen + ethanol (158 K) under 1 atm pressure. The analytical precision of the δD determination using the developed method was better than 4‰ for >5 nmol injections (250 mL STP for 500 ppbv air sample) and better than 15‰ for 1 nmol injections, regardless of the δD value, within 1 h for one sample analysis. Using the developed system, the δD values of H(2) can be quantified for atmospheric samples as well as samples of representative sources and sinks including those containing small quantities of H(2) , such as H(2) in soil pores or aqueous environments, for which there is currently little δD data available. As an example of such trace H(2) analyses, we report here the isotope fractionations during H(2) uptake by soils in a static chamber. The δD values of H(2) in these H(2)-depleted environments can be useful in constraining the budgets of atmospheric H(2) by applying an isotope mass balance model.

Expression and function of Sox21 during mouse cochlea development.

The development of the inner ear is an orchestrated process of morphogenesis with spatiotemporally controlled generations of individual cell types. Recent studies have revealed that the Sox gene family, a family of evolutionarily conserved HMG-type transcriptional factors, is differentially expressed in each cell type of the mammalian inner ear and plays critical roles in cell-fate determination during development. In this study, we examined the expression pattern of Sox21 in the developing and adult murine cochlea. Sox21 was expressed throughout the sensory epithelium in the early otocyst stage but became restricted to supporting cells during adulthood. Interestingly, the expression in adults was restricted to the inner phalangeal, inner border, and Deiters' cells: all of these cells are in direct contact with hair cells. Evaluations of the auditory brainstem-response revealed that Sox21(-/-) mice suffered mild hearing impairments, with an increase in hair cells that miss their appropriate planar cell polarity. Taken together with the previously reported critical roles of SoxB1 families in the morphogenesis of inner ear sensory and neuronal cells, our results suggest that Sox21, a counteracting partner of the SoxB1 family, controls fine-tuned cell fate decisions. Also, the characteristic expression pattern may be useful for labelling a particular subset of supporting cells.

Grain-scale heterogeneities in the stable carbon and oxygen isotopic compositions of the international standard calcite materials (NBS 19, NBS 18, IAEA-CO-1, and IAEA-CO-8).

We determined grain-scale heterogeneities (from 6 to 88 microg) in the stable carbon and oxygen isotopic compositions (delta(13)C and delta(18)O) of the international standard calcite materials (NBS 19, NBS 18, IAEA-CO-1, and IAEA-CO-8) using a continuous-flow isotope ratio mass spectrometry (CF-IRMS) system that realizes a simultaneous determination of the delta(13)C and the delta(18)O values with standard deviations (S.D.) of less than 0.05 per thousand for CO(2) gas. Based on the S.D. of the delta(13)C and delta(18)O values determined for CO(2) gases evolved from the different grains of the same calcite material, we found that NBS 19, IAEA-CO-1, and IEAE-CO-8 were homogeneous for delta(13)C (less than 0.10 per thousand S.D.), and that only NBS 19 was homogeneous for delta(18)O (less than 0.14 per thousand S.D.). On the level of single grains, we found that both IAEA-CO-1 and IAEA-CO-8 were heterogeneous for delta(18)O (1.46 per thousand and 0.76 per thousand S.D., respectively), and that NBS 18 was heterogeneous for both delta(13)C and delta(18)O (0.34 per thousand and 0.54 per thousand S.D., respectively). Closer inspection of NBS 18 grains revealed that the highly deviated isotopic compositions were limited to the colored grains. By excluding such colored grains, we could also obtain the homogeneous delta(13)C and delta(18)O values (less than 0.18 per thousand and less than 0.16 per thousand S.D., respectively) for NBS 18. We conclude that NBS 19, IAEA-CO-1, or pure grains in NBS 18 are suitable to be used as the standard reference material for delta(13)C, and that either NBS 19 or pure grains in NBS 18 are suitable to be used as the reference material for delta(18)O during the grain-scale isotopic analyses of calcite.

Determination of the 15N/14N, 17O/16O, and 18O/16O ratios of nitrous oxide by using continuous-flow isotope-ratio mass spectrometry.

We developed a rapid, sensitive, and automated analytical system to determine the delta15N, delta18O, and Delta17O values of nitrous oxide (N2O) simultaneously in nanomolar quantities for a single batch of samples by continuous-flow isotope-ratio mass spectrometry (CF-IRMS) without any cumbersome and time-consuming pretreatments. The analytical system consisted of a vacuum line to extract and purify N2O, a gas chromatograph for further purification of N2O, an optional thermal furnace to decompose N2O to O2, and a CF-IRMS system. We also used pneumatic valves and pneumatic actuators in the system so that we could operate it automatically with timing software on a personal computer. The analytical precision was better than 0.12 per thousand for delta15N with >4 nmol N2O injections, 0.25 per thousand for delta18O with >4 nmol N2O injections, and 0.20 per thousand for Delta17O with >20 nmol N2O injections for a single measurement. We were also easily able to improve the precision (standard errors) to better than 0.05 per thousand for delta15N, 0.10 per thousand for delta18O, and 0.10 per thousand for Delta17O through multiple analyses with more than four repetitions with 190 nmol samples using the automated analytical system. Using the system, the delta15N, delta18O, and Delta17O values of N2O can be quantified not only for atmospheric samples, but also for other gas or liquid samples with low N2O content, such as soil gas or natural water. Here, we showed the first ever Delta17O measurements of soil N2O.

Sensitive determinations of stable nitrogen isotopic composition of organic nitrogen through chemical conversion into N2O.

We present a method for high-sensitivity nitrogen isotopic analysis of particulate organic nitrogen (PON) in seawater and freshwater, for the purpose of determining the aquatic nitrogen fixation rate through the 15N2 tracer technique for samples that contain a low abundance of organisms. The method is composed of the traditional oxidation/reduction methods, such as the oxidation of PON to nitrate (NO3*) using persulfate, the reduction of NO3* to nitrite (NO2*) using spongy cadmium, and further reduction of NO2* to nitrous oxide (N2O) using sodium azide. Then, N2O is purged from the water and trapped cryogenically with subsequent release into a gas chromatography column to analyze the stable nitrogen isotopic composition using continuous-flow isotope ratio mass spectrometry (CF-IRMS) by simultaneously monitoring the NO+ ion currents at masses 30, 31, and 32. The nitrogen isotopic fractionation was consistent within each batch of analysis. The standard deviation of sample measurements was less than 0.3 per thousand for samples containing PON of more than 50 nmolN, and 0.5 per thousand for those of more than 20 nmolN, by subtracting the contribution of blank nitrogen, 8 +/- 2 nmol at final N2O. By using this method, we can determine delta15N for lower quantities of PON better than by other methods, so we can reduce the quantities of water samples needed for incubation to determine the nitrogen fixation rate. In addition, we can expand the method to determine the nitrogen isotopic composition of organic nitrogen in general, such as that of total dissolved nitrogen (TDN; sum of NO3*, NO2*, ammonium, and DON), by applying the method to filtrates.

Development of two-dimensional gas chromatography/isotope ratio mass spectrometry for the stable carbon isotopic analysis of C(2)-C(5) non-methane hydrocarbons emitted from biomass burning.

A two-dimensional gas chromatography/combustion/isotope ratio mass spectrometry (2D-GC/C/IRMS) system was developed for stable carbon isotopic measurements of C(2)-C(5) non-methane hydrocarbons (NMHCs) in biomass burning smoke. The 2D-GC/C/IRMS system successfully improved the accuracy and precision for the measurements of C(4) and C(5) saturated compounds in a smoke sample by selective injection of target compounds into a combustion furnace and consequently allowed us to provide complete baseline separation for all individual NMHCs. The analytical precision of the delta(13)C of each compound was better than 0.5 per thousand for more than 500 pmolC injections and 2.1 per thousand for 30 pmolC injections, which was estimated from replicate analysis of standard gases. This system was applied to the analysis of NMHCs in smoke samples collected from laboratory biomass burning experiments. From the combustion of three fuel materials (rice straw, pine wood, and maize), we found that the isotopic fractionation between fuel material and individual NMHCs is almost independent of the fuel material and thus the delta(13)C values of the fuel materials are reflected in delta(13)C values of most of NMHCs. However, only i-butane emitted from maize combustion showed anomalous (13)C-depletion of -11.6 per thousand relative to the delta(13)C value of maize. Such a large (13)C depletion suggests the specific isotopic fractionation process which is attributed to the maize combustion itself or the chemical properties of i-butane during production from a radical recombination reaction.

An analytical system for determining delta17O in CO2 using continuous flow-isotope ratio MS.

We developed a simple measurement system for delta17O in nanomole quantities of CO2 using continuous flow isotope ratio mass spectrometry (CF-IRMS). The analytical system consisted of a sample injection system, a helium-purged CO2 purification line, a capillary GC, a combustion unit, and CF-IRMS. A unique feature of the system is that we use molecular CO2 to determine the isotopic compositions including delta17O. The delta17O of CO2 in a sample is calculated from the mass ratios of both 45/44 and 46/44 of two different kinds of CO2, which have been purified quantitatively from different aliquots of a sample. While one aliquot (rCO2) flows into IRMS directly, the other (eCO2) flows through a CuO unit (900 degrees C) prior to injection into IRMS, to exchange oxygen atoms in the sample CO2 molecules with those in CuO for which we can assume Delta17O = 0. In our system, we introduce both rCO2 and eCO2 alternately to IRMS repeatedly by using an automatic multianalytical system to improve analytical precision statistically. The standard deviation of 0.35 per thousand for Delta17O can be realized using as little as 8.7 nmol CO2 in a approximately 3-h analysis. Based on this system, we have quantified delta17O in the stratospheric CO2 over Japan.

A selective unsaturated hydrocarbon subtraction technique for stable carbon isotopic analysis of atmospheric methyl chloride, methyl bromide, and C2-C5 saturated hydrocarbons using continuous-flow isotope ratio mass spectrometry.

Using continuous-flow isotope ratio mass spectrometry, we have developed a new analytical system which enables us to determine the stable carbon isotopic composition of CH3Cl, CH3Br, and C2-C5 saturated hydrocarbons in gas samples even if they contain substantial amounts of unsaturated hydrocarbons, using an I2O5 reagent for their selective subtraction. The analytical precision of the delta13C determinations is better than 0.5 per thousand for >300 pmolC injections and better than 5 per thousand for 20 pmolC injections. Using the system, delta13C values for CH3Cl and CH3Br were found in burning exhaust that contain a substantial quantity of unsaturated hydrocarbons. CH3Cl and CH3Br measured in exhaust from burning rice plants exhibit highly 13C-depleted values of -56.6 +/- 1.3 per thousand and -48.6 +/- 3.9 per thousand, respectively, while saturated hydrocarbons exhibit delta13C values (-26.4 to -28.9 per thousand) that are comparable with the total delta13C value of the parent material (rice plant; -28.0 per thousand). Using the system, we can determine the delta13C values of methyl halides and hydrocarbons in many kinds of gas samples.

Stable carbon and oxygen isotopic analysis of atmospheric carbon monoxide using continuous-flow isotope ratio MS by isotope ratio monitoring of CO.

We have developed a rapid and simple measurement system for both content and stable isotopic compositions (13C and 18O) of atmospheric CO, using continuous-flow isotope ratio mass spectrometry by simultaneously monitoring the CO+ ion currents at masses 28, 29, and 30. The analytical system consisted sequentially of a sample trapping port (liquid nitrogen temperature silica gel and molecular sieve 5A), a gas dryer, a CO purification column (molecular sieve 5A), a cryofocusing unit, and a final purification column using a GC capillary. Analytical precision of 0.2 per thousand for 13C and 0.4 per thousand for 18O can be realized for samples that contain as little as 300 pmol of CO within 40 min for one sample analysis. Analytical blanks associated with the method are less than 1 pmol. The extent of analytical error in delta13C due to mass-independent fractionation of oxygen in natural CO is estimated to be less than 0.3 per thousand. Based on this system, we report herein a kinetic isotopic effect during CO consumption in soil.

On-line measurement of intramolecular carbon isotope distribution of acetic acid by continuous-flow isotope ratio mass spectrometry.

Molecular and intramolecular carbon isotope measurements of acetic acid present in natural environments have been performed by off-line procedures. The off-line method is complicated and time-consuming and requires micromolar to millimolar amounts of sample. This limits geochemical isotopic studies, especially at the intramolecular level, on acetic acid present in natural samples. Here, we examine an on-line measurement of intramolecular carbon isotope distribution of acetic acid using continuous-flow isotope ratio mass spectrometry (CF-IRMS) coupled with an on-line pyrolysis system. This is achieved by measurement of the respective carbon isotope ratios of CH4 and CO2 produced by on-line pyrolysis of acetic acid. Results for authentic standards of pure acetic acid demonstrated the practicality of this on-line method, although the carbon isotope ratio of the methyl group could not be determined directly. The precision of the carbon isotope measurements was 0.4 per thousand (1sigma). The carbon isotope distribution determined by the on-line method was identical to that determined by the conventional off-line method within analytical error. The advantages of the on-line method compared with the conventional off-line method are that it is less laborious, requires less analytical time (less than one hour per sample) and, most importantly, uses smaller sample sizes (ca. 10 nanomole). An application of this on-line method to natural geochemical samples will provide an insight into the geochemical cycle of acetic acid.