Quantitative 31P-NMR for the Purity Determination of the Organophosphorus Compound Brigatinib and Its Method Validation.

The spectrum of 31P-NMR is fundamentally simpler than that of 1H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative 31P-NMR (31P-qNMR) than using quantitative 1H-NMR (1H-qNMR), which has been already established as an absolute determination method. We have previously reported a 31P-qNMR method for the absolute determination of cyclophosphamide hydrate and sofosbuvir as water-soluble and water-insoluble organophosphorus compounds, respectively. This study introduces the purity determination of brigatinib (BR), an organophosphorus compound with limited water solubility, using 31P-qNMR at multiple laboratories. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d4 were selected as the reference standards (RSs) for 31P-qNMR and 1H-qNMR, respectively. The qNMR solvents were chosen based on the solubilities of BR and the RSs for qNMR. CD3OH was selected as the solvent for 31P-qNMR measurements to prevent the influence of deuterium exchange caused by the presence of exchangeable intramolecular protons of BR and PAA on the quantitative values, while CD3OD was the solvent of choice for the 1H-qNMR measurements to prevent the influence of water signals and the exchangeable intramolecular protons of BR and PAA. The mean purity of BR determined by 31P-qNMR was 97.94 ± 0.69%, which was in agreement with that determined by 1H-qNMR (97.26 ± 0.71%), thus indicating the feasibility of purity determination of BR by 31P-qNMR. Therefore, the findings of this study may provide an effective method that is simpler than conventional 1H-qNMR for the determination of organophosphorus compounds.

Quantitative 31P-NMR for Purity Determination of Sofosbuvir and Method Validation.

Quantitative 1H-NMR (1H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the 31P nucleus because of the simplicity of its signals and previously reported 31P-qNMR in D2O. Here we report 31P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-bis(trimethylsilyl)benzene-d4 (1,4-BTMSB-d4) were used as reference standards for 31P-qNMR and 1H-qNMR, respectively, in methanol-d4. The purity of SOF determined by 31P-qNMR was 100.63 ± 0.95%, whereas that determined by 1H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the 31P signal of PAA and SOF were 3.38 ± 2.39 and 2.22 ± 0.19 Hz, respectively, suggesting that the T2 relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, dimethyl sulfoxide-d6 (DMSO-d6), was used as the dissolving solvent with PAA and sodium 4,4-dimethyl-4-silapentanesulfonate-d6 (DSS-d6) as reference standards for 31P-qNMR and 1H-qNMR, respectively. SOF purities determined by 31P-qNMR and 1H-qNMR were 99.10 ± 0.30 and 99.44 ± 0.29%, respectively. SOF purities determined by 31P-qNMR agreed with the established 1H-qNMR values, suggesting that an aprotic solvent is preferable for 31P-qNMR because it is unnecessary to consider the effect of deuterium exchange.

Preparation and Spectroscopic Characterization of Inclusion Complexes of 3D Ball-Milled Rifampicin with ß-cyclodextrin and γ-cyclodextrin : 3D Ball-Milled Rifampicin with ß-cyclodextrin and γ-cyclodextrin.

Rifampicin (RFP) solutions, intended to reduce incidence of prosthetic graft infection, were prepared as three-dimensional ground mixtures (3DGMs) using ß-cyclodextrin (ßCD) and γ-cyclodextrin (γCD) and characterized for their spectroscopic properties and solubility. Phase solubility diagrams revealed that 3DGMs (RFP/ßCD and RFP/γCD) produced a complex at 1:1 molar ratio. Pulsed field gradient nuclear magnetic resonance experiments indicated that the diffusion coefficients for RFP/ßCD and RFP/γCD were similar to the respective diffusion coefficients for ßCD and γCD. Rotating-frame Overhauser effect spectroscopy NMR spectra revealed the existence of a new exchanger peak for RFP/γCD, suggesting an intermolecular interaction different from that of RFP/ßCD. Differential scanning calorimetry confirmed the presence of endothermic peak at 191 °C indicating the manifestation of RFP in the inclusion complex. Interestingly, molecular interactions from the complexes, RFP/ßCD and RFP/γCD, revealed different patterns of inclusion in the 3DGMs. In RFP/ßCD, nuclear Overhauser effect spectroscopy NMR spectra indicated cross peaks for the protons of the methyl group of RFP and the protons (H-5 and H-6) in the ßCD cavity. The methyl group of RFP interacted with the narrow rim of ßCD. With RFP/γCD, cross peaks were due to the protons of the methyl group of RFP and the protons of the cavity of γCD suggesting multiple inclusion patterns. The observed multiple cross peaks affirm the inclusion of RFP into the CD cavity which enhanced its solubility by 1.6-2.0-fold when prepared as 3DGMs as RFP/ßCD and RFP/γCD, respectively.

Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 2).

As a new absolute quantitation method for low-molecular compounds, quantitative NMR (qNMR) has emerged. In the Japanese Pharmacopoeia (JP), 15 compounds evaluated by qNMR are listed as reagents used as the HPLC reference standards in the assay of crude drug section of the JP. In a previous study, we revealed that humidity affects purity values of hygroscopic reagents and that (i) humidity control before and during weighing is important for a reproducible preparation and (ii) indication of the absolute amount (not purity value), which is not affected by water content, is important for hygroscopic products determined by qNMR. In this study, typical and optimal conditions that affect the determination of the purity of ginsenoside Rb1 (GRB1), saikosaponin a (SSA), and barbaloin (BB) (i.e., hygroscopic reagents) by qNMR were examined. First, the effect of humidity before and during weighing on the purity of commercial GRB1, with a purity value determined by qNMR, was examined. The results showed the importance afore-mentioned. The results of SSA, which is relatively unstable in the dissolved state, suggested that the standardization of humidity control before and during weighing for a specific time provides a practical approach for hygroscopic products. In regard to BB, its humidity control for a specific time, only before weighing, is enough for a reproducible purity determination.

Unique Usage of a Classical Selective Homodecoupling Sequence for High-Resolution Quantitative 1H NMR.

Classical selective homodecoupling was used in a 1H NMR purity assay to improve accuracy by overcoming spectral overlaps due to 1H-1H spin coupling. Dummy irradiation at a specific frequency was used in addition to irradiation at a 1H resonance of the analyte to avoid irradiation bias. The method was validated in a 1H NMR purity assay of high-purity diethyl phthalate (National Metrology Institute of Japan Certified Reference Material (NMIJ CRM), purity: 99.98%). The obtained purity value biases were 0.27% or less. The utility of the method was demonstrated in another 1H NMR purity assay of dipropyl phthalate (NMIJ CRM, purity: 98.41%), which contained a tiny amount of the structurally similar compound methyl propyl phthalate as an impurity. An accurate assay was achieved with the method, giving a purity of 98.39%, whereas the conventional method gave a purity 99.13%.

TBL10 is required for O-acetylation of pectic rhamnogalacturonan-I in Arabidopsis thaliana.

O-Acetylated pectins are abundant in the primary cell wall of plants and growing evidence suggests they have important roles in plant cell growth and interaction with the environment. Despite their importance, genes required for O-acetylation of pectins are still largely unknown. In this study, we showed that TRICHOME BIREFRINGENCE LIKE 10 (AT3G06080) is involved in O-acetylation of pectins in Arabidopsis (Arabidopsis thaliana). The activity of the TBL10 promoter was strong in tissues where pectins are highly abundant (e.g. leaves). Two homozygous knock-out mutants of Arabidopsis, tbl10-1 and tbl10-2, were isolated and shown to exhibit reduced levels of wall-bound acetyl esters, equivalent of ~50% of the wild-type level in pectin-enriched fractions derived from leaves. Further fractionation revealed that the degree of acetylation of the pectin rhamnogalacturonan-I (RG-I) was reduced in the tbl10 mutant compared to the wild type, whereas the pectin homogalacturonan (HG) was unaffected. The degrees of acetylation in hemicelluloses (i.e. xyloglucan, xylan and mannan) were indistinguishable between the tbl10 mutants and the wild type. The mutant plants contained normal trichomes in leaves and exhibited a similar level of susceptibility to the phytopathogenic microorganisms Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea; while they displayed enhanced tolerance to drought. These results indicate that TBL10 is required for O-acetylation of RG-I, possibly as an acetyltransferase, and suggest that O-acetylated RG-I plays a role in abiotic stress responses in Arabidopsis.

Structure and Metabolic-Flow Analysis of Molecular Complexity in a (13) C-Labeled Tree by 2D and 3D NMR.

Improved signal identification for biological small molecules (BSMs) in a mixture was demonstrated by using multidimensional NMR on samples from (13) C-enriched Rhododendron japonicum (59.5 atom%) cultivated in air containing (13) C-labeled carbon dioxide for 14 weeks. The resonance assignment of 386 carbon atoms and 380 hydrogen atoms in the mixture was achieved. 42 BSMs, including eight that were unlisted in the spectral databases, were identified. Comparisons between the experimental values and the (13) C chemical shift values calculated by density functional theory supported the identifications of unlisted BSMs. Tracing the (13) C/(12) C ratio by multidimensional NMR spectra revealed faster and slower turnover ratios of BSMs involved in central metabolism and those categorized as secondary metabolites, respectively. The identification of BSMs and subsequent flow analysis provided insight into the metabolic systems of the plant.

Profiling planktonic biomass using element-specific, multicomponent nuclear magnetic resonance spectroscopy.

Planktonic metabolism plays crucial roles in Earth's elemental cycles. Chemical speciation as well as elemental stoichiometry is important for advancing our understanding of planktonic roles in biogeochemical cycles. In this study, a multicomponent solid-state nuclear magnetic resonance (NMR) approach is proposed for chemical speciation of cellular components, using several advanced NMR techniques. Measurements by ssNMR were performed on (13)C and (15)N-labeled Euglena gracilis, a flagellated protist. 3D dipolar-assisted rotational resonance, double-cross-polarization (1)H-(13)C correlation spectroscopy, and (1)H-(13)C solid-state heteronuclear single quantum correlation spectroscopy successively allowed characterization of cellular components. These techniques were then applied to E. gracilis cultured in high and low ammonium media to demonstrate the power of this method for profiling and comparing cellular components. Cellular NMR spectra indicated that ammonium induced both paramylon degradation and amination. Arginine was stored as a nitrogen reserve and ammonium replaced by arginine catabolism via the arginine dihydrolase pathway. (15)N and (31)P cellular ssNMR indicated arginine and polyphosphate accumulation in E. gracilis, respectively. This chemical speciation technique will contribute to environmental research by providing detailed information on environmental chemical properties.

How did COVID-19 pandemic affect the older adults' needs for robot technologies in Japan?: comparison of participatory design workshops during versus after the COVID-19 pandemic.

Social technology can improve the quality of social lives of older adults (OAs) and mitigate negative mental and physical health outcomes. When people engage with technology, they can do so to stimulate social interaction (stimulation hypothesis) or disengage from their real world (disengagement hypothesis), according to Nowland et al.'s model of the relationship between social Internet use and loneliness. External events, such as large periods of social isolation like during the COVID-19 pandemic, can also affect whether people use technology in line with the stimulation or disengagement hypothesis. We examined how the COVID-19 pandemic affected the social challenges OAs faced and their expectations for robot technology to solve their challenges. We conducted two participatory design (PD) workshops with OAs during and after the COVID-19 pandemic. During the pandemic, OAs' primary concern was distanced communication with family members, with a prevalent desire to assist them through technology. They also wanted to share experiences socially, as such OA's attitude toward technology could be explained mostly by the stimulation hypothesis. However, after COVID-19 the pandemic, their focus shifted towards their own wellbeing. Social isolation and loneliness were already significant issues for OAs, and these were exacerbated by the COVID-19 pandemic. Therefore, such OAs' attitudes toward technology after the pandemic could be explained mostly by the disengagement hypothesis. This clearly reflect the OA's current situation that they have been getting further digitally excluded due to rapid technological development during the pandemic. Both during and after the pandemic, OAs found it important to have technologies that were easy to use, which would reduce their digital exclusion. After the pandemic, we found this especially in relation to newly developed technologies meant to help people keep at a distance. To effectively integrate these technologies and avoid excluding large parts of the population, society must address the social challenges faced by OAs.

People's judgments of humans and robots in a classic moral dilemma.

How do ordinary people evaluate robots that make morally significant decisions? Previous work has found both equal and different evaluations, and different ones in either direction. In 13 studies (N = 7670), we asked people to evaluate humans and robots that make decisions in norm conflicts (variants of the classic trolley dilemma). We examined several conditions that may influence whether moral evaluations of human and robot agents are the same or different: the type of moral judgment (norms vs. blame); the structure of the dilemma (side effect vs. means-end); salience of particular information (victim, outcome); culture (Japan vs. US); and encouraged empathy. Norms for humans and robots are broadly similar, but blame judgments show a robust asymmetry under one condition: Humans are blamed less than robots specifically for inaction decisions-here, refraining from sacrificing one person for the good of many. This asymmetry may emerge because people appreciate that the human faces an impossible decision and deserves mitigated blame for inaction; when evaluating a robot, such appreciation appears to be lacking. However, our evidence for this explanation is mixed. We discuss alternative explanations and offer methodological guidance for future work into people's moral judgment of robots and humans.

Comprehensive signal assignment of 13C-labeled lignocellulose using multidimensional solution NMR and 13C chemical shift comparison with solid-state NMR.

A multidimensional solution NMR method has been developed using various pulse programs including HCCH-COSY and (13)C-HSQC-NOESY for the structural characterization of commercially available (13)C labeled lignocellulose from potatoes (Solanum tuberosum L.), chicory (Cichorium intybus), and corn (Zea mays). This new method allowed for 119 of the signals in the (13)C-HSQC spectrum of lignocelluloses to be assigned and was successfully used to characterize the structures of lignocellulose samples from three plants in terms of their xylan and xyloglucan structures, which are the major hemicelluloses in angiosperm. Furthermore, this new method provided greater insight into fine structures of lignin by providing a high resolution to the aromatic signals of the ß-aryl ether and resinol moieties, as well as the diastereomeric signals of the ß-aryl ether. Finally, the (13)C chemical shifts assigned in this study were compared with those from solid-state NMR and indicated the presence of heterogeneous dynamics in the polysaccharides where rigid cellulose and mobile hemicelluloses moieties existed together.

[Quantitative Determination Method of Aconitum Monoester Alkaloids Using Relative Molar Sensitivity (RMS) for the Assay in the Japanese Pharmacopoeia].

Recently, a novel quantitative method using relative molar sensitivity (RMS) was applied to quantify the ingredients of drugs and foods. An important development in this regard can be observed in the Japanese Pharmacopoeia (JP) 18, where the quantification of perillaldehyde, an unstable compound, in crude drug "Perilla Herb," was revised to incorporate the RMS method. In this study, the primary objective was to improve the tester safety and reduce the amount of reagents used in the JP test. To achieve this, the quantification of three toxic Aconitum monoester alkaloids (AMAs) was explored using the RMS method, employing a single reference compound for all three targets. These AMAs, namely benzoylmesaconine hydrochloride, benzoylhypaconine hydrochloride, and 14-anisoylaconine hydrochloride, which are the quantitative compounds of Kampo extracts containing Aconite Root (AR), were quantified using the reference compound benzoic acid (BA). Reliable RMS values were obtained using both 1H-quantitative NMR and HPLC/UV. Using the RMS of three AMAs relative to the BA, the AMA content (%) in commercial AMAs quantitative reagents were determined without analytical standards. Moreover, the quantitative values of AMAs using the RMS method and the calibration curve method using the three analytical standards were similar. Additionally, similar values were achieved for the three AMAs in the Kampo extracts containing AR using the RMS and the modified JP18 calibration curve methods. These results suggest that the RMS method is suitable for quantitative assays of the Kampo extracts containing AR and can serve as an alternative to the current method specified in the JP18.

Socially facilitative robots for older adults to alleviate social isolation: A participatory design workshop approach in the US and Japan.

Social technology can improve the quality of older adults' social lives and mitigate negative mental and physical health outcomes associated with loneliness, but it should be designed collaboratively with this population. In this paper, we used participatory design (PD) methods to investigate how robots might be used as social facilitators for middle-aged and older adults (age 50+) in both the US and Japan. We conducted PD workshops in the US and Japan because both countries are concerned about the social isolation of these older adults due to their rapidly aging populations. We developed a novel approach to participatory design of future technologies that spends 2/3 of the PD session asking participants about their own life experiences as a foundation. This grounds the conversation in reality, creates rapport among the participants, and engages them in creative critical thinking. Then, we build upon this foundation, pose an abstract topic, and ask participants to brainstorm on the topic based on their previous discussion. In both countries, participants were eager to actively discuss design ideas for socially facilitative robots and imagine how they might improve their social lives. US participants suggested design ideas for telepresence robots, social distancing robots, and social skills artificial intelligence programs, while Japanese participants suggested ideas for pet robots, robots for sharing experiences, and easy-to-operate instructor robots. Comparing these two countries, we found that US participants saw robots as tools to help facilitate their social connections, while Japanese participants envisioned robots to function as surrogate companions for their parents and distract them from loneliness when they were unavailable. With this paper, we contribute to the literature in two main ways, presenting: (1) A novel approach to participatory design of future technologies that grounds participants in their everyday experience, and (2) Results of the study indicating how middle-aged and older adults from the US and Japan wanted technologies to improve their social lives. Although we conducted the workshops during the COVID-19 pandemic, many findings generalized to other situations related to social isolation, such as older adults living alone.

1H-NMR metabolomics-based classification of Japanese sake and comparative metabolome analysis by gas chromatography-mass spectrometry.

Six categories of Japanese sake have been established by the National Tax Agency of Japan. In this system, the rice polishing ratio and the addition of alcohol are the main criteria for classification. The most common nuclear magnetic resonance (NMR) spectrometry method is 1H-NMR, and has higher throughput than gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) analysis due to its short measurement time, easy sample preparation, and high reproducibility. However, owing to the production of dominant ethanol signals, metabolome analyses have not been used for classifying Japanese sake using 1H-NMR. In this study, a technique to selectively suppress ethanol signals was used to classify Japanese sake by 1H-NMR, and a model was constructed to predict the rice polishing ratio. The results were compared to those obtained by GC-MS. The suppression of ethanol signals enabled the detection of trace components by 1H-NMR. In a principal component analysis (PCA) score plot of 1H-NMR spectra with ethanol signal suppression, PC1 was associated with both the addition of alcohol and the rice polishing ratio. Additionally, the separation of samples observed was similar when PCA score plots of 1H-NMR and GC-MS data were compared. Similarly, to predict the rice polishing ratio using partial least squares regression analysis, a model was constructed using 1H-NMR data, and showed nearly similar values for precision and predictive performance with the model constructed using GC-MS data. These results suggest that metabolomic analyses of Japanese sake based on 1H-NMR spectral patterns may be useful for classification.

Lessons Learned About Designing and Conducting Studies From HRI Experts.

The field of human-robot interaction (HRI) research is multidisciplinary and requires researchers to understand diverse fields including computer science, engineering, informatics, philosophy, psychology, and more disciplines. However, it is hard to be an expert in everything. To help HRI researchers develop methodological skills, especially in areas that are relatively new to them, we conducted a virtual workshop, Workshop Your Study Design (WYSD), at the 2021 International Conference on HRI. In this workshop, we grouped participants with mentors, who are experts in areas like real-world studies, empirical lab studies, questionnaire design, interview, participatory design, and statistics. During and after the workshop, participants discussed their proposed study methods, obtained feedback, and improved their work accordingly. In this paper, we present 1) Workshop attendees' feedback about the workshop and 2) Lessons that the participants learned during their discussions with mentors. Participants' responses about the workshop were positive, and future scholars who wish to run such a workshop can consider implementing their suggestions. The main contribution of this paper is the lessons learned section, where the workshop participants contributed to forming this section based on what participants discovered during the workshop. We organize lessons learned into themes of 1) Improving study design for HRI, 2) How to work with participants - especially children -, 3) Making the most of the study and robot's limitations, and 4) How to collaborate well across fields as they were the areas of the papers submitted to the workshop. These themes include practical tips and guidelines to assist researchers to learn about fields of HRI research with which they have limited experience. We include specific examples, and researchers can adapt the tips and guidelines to their own areas to avoid some common mistakes and pitfalls in their research.

Visual Perception of Moisture Is a Pathogen Detection Mechanism of the Behavioral Immune System.

The behavioral immune system (BIS) includes perceptual mechanisms for detecting cues of contamination. Former studies have indicated that moisture has a disgusting property. Therefore, moisture could be a target for detecting contamination cues by the BIS. We conducted two experiments to examine the psychophysical basis of moisture perception and clarify the relationship between the perception of moisture and the BIS. We assumed that the number of high luminance areas in a visual image provided optical information that would enable the visual perception of moisture. In two experiments, we presented eight images of dough that contained different amounts of moisture as experimental stimuli. The amount of moisture shown in the images was increased in eight steps, from 28.6 to 42.9% of the total weight of the dough. In Experiment 1, the images were randomly presented on a computer display, and the participants (n = 22) were asked to rank the images in the order of the visually perceived moisture content. In Experiment 2, the participants (n = 15) completed pairwise comparisons based on the perceived moistness of the images. Furthermore, to examine the BIS responses, the participants rated the strength of disgust evoked by the stimuli, their motivation to avoid touching the stimuli, and the estimated magnitude of the risk of contamination by physical contact with the stimuli. The results indicated that the moisture content and the numbers of high luminance areas in the images accurately predicted the perception of moisture, suggesting that the detection of visual moisture was highly accurate, and the optical information served as an essential perceptual cue for detecting moisture. On the other hand, the BIS responses peaked in response to stimuli having approximately 33 to 39% moisture content. These results show that objects containing a moderate amount of moisture could be the target of visually detecting pathogens by the BIS.

[Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 1)].

Quantitative NMR (qNMR) has been developed as an absolute quantitation method to determine the purity or content of organic compounds including marker compounds in crude drugs. The "qNMR test" has been introduced into the crude-drug section of the Japanese Pharmacopoeia (JP) for determining the purity of reagents used for the assay in the JP. In Supplement II to the JP 17th edition published in June 2019, fifteen compounds adopted qNMR test were listed as the reagents for the assay. To establish the "qNMR test" in the crude drug section of the JP, there were several problems to be solved. Previously, we reported that the handling impurity signals from reference substances and targeted marker compounds, chemical shifts of reference substances, and peak unity of signals of targeted marker compounds are important factors to conduct qNMR measurements with intended accuracy. In this study, we investigated that the hygroscopicity of reagents could cause the changes in the compounds' purity depending on increasing their water content. Twenty-one standard products used for the crude-drug test in JP were examined by water sorption-desorption analysis, and ginsenosides and saikosaponins were found to be hygroscopic. To prepare a sample solution of saikosaponin b2 for qNMR analysis, samples need to be maintained for 18 h at 25°C and 76% relative humidity; further, samples need to be weighed at the same humidity for the qNMR analysis.

SENSI: signal enhancement by spectral integration for the analysis of metabolic mixtures.

The method provided here can overcome the low S/N problem in (13)C NMR, by the integration of plural spectra to take advantage of high-resolution potential based on non-bucketing analysis without additional measurements. In addition, a new metabolite annotation approach using advanced STOCSY and quantum chemistry calculations was introduced in this study.

Modification of plant cell wall structure accompanied by enhancement of saccharification efficiency using a chemical, lasalocid sodium.

The cell wall is one major determinant of plant cell morphology, and is an attractive bioresource. Here, we report a novel strategy to modify plant cell wall property by small molecules. Lasalocid sodium (LS) was isolated by chemical screening to identify molecules that affect the cell morphology of tobacco BY-2 cells. LS treatment led to an increase in cell wall thickness, whilst the quantity and sugar composition of the cell wall remained unchanged in BY-2 cells. The chemical also disordered the cellular arrangement of hypocotyls of Arabidopsis plants, resulting in a decrease in hypocotyl length. LS treatment enhanced enzymatic saccharification efficiency in both BY-2 cells and Arabidopsis plants. Microarray analysis on Arabidopsis showed that exposure to LS upregulated type III peroxidase genes, of which some are involved in lignin biogenesis, and jasmonic acid response genes, and phloroglucinol staining supported the activation of lignification by the LS treatment. As jasmonic acid-mediated lignification is a typical reaction to cell wall damage, it is possible that LS induces cell wall loosening, which can trigger cell wall damage response. Thus, LS is a unique chemical for modification of cell wall and morphology through changes in cell wall architecture.

Multi-Spectroscopic Analysis of Seed Quality and 13C-Stable-Iotopologue Monitoring in Initial Growth Metabolism of Jatropha curcas L.

In the present study, we applied nuclear magnetic resonance (NMR), as well as near-infrared (NIR) spectroscopy, to Jatropha curcas to fulfill two objectives: (1) to qualitatively examine the seeds stored at different conditions, and (2) to monitor the metabolism of J. curcas during its initial growth stage under stable-isotope-labeling condition (until 15 days after seeding). NIR spectra could non-invasively distinguish differences in storage conditions. NMR metabolic analysis of water-soluble metabolites identified sucrose and raffinose family oligosaccharides as positive markers and gluconic acid as a negative marker of seed germination. Isotopic labeling patteren of metabolites in germinated seedlings cultured in agar-plate containg 13C-glucose and 15N-nitrate was analyzed by zero-quantum-filtered-total correlation spectroscopy (ZQF-TOCSY) and 13C-detected 1H-13C heteronuclear correlation spectroscopy (HETCOR). 13C-detected HETOCR with 13C-optimized cryogenic probe provided high-resolution 13C-NMR spectra of each metabolite in molecular crowd. The 13C-13C/12C bondmer estimated from 1H-13C HETCOR spectra indicated that glutamine and arginine were the major organic compounds for nitrogen and carbon transfer from roots to leaves.