Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Periodontal tissue susceptibility to glycaemic control in type 2 diabetes.

AIM: To assess the direct effect of intensive glycaemic control on periodontal tissues in patients with diabetes mellitus. MATERIALS AND METHODS: Twenty-nine patients with type 2 diabetes were enrolled and hospitalized to receive a 2-week intensive glycaemic control regimen. We observed and analysed the systemic and oral disease indicators before and after treatment and clarified the indicators related to periodontal inflammation. RESULTS: A significant reduction in glycaemic and periodontal parameters, including glycated albumin levels and periodontal inflamed surface area (PISA), was observed after treatment. The changes in PISA per tooth, indicative of periodontal healing, exhibited a bimodal distribution; the patients were divided into two groups on this basis. Correlations were observed between the changes in PISA per tooth and fasting plasma glucose, acetoacetic acid, and beta-hydroxybutyrate levels in the PISA-improved group. Significantly lower levels of C-peptide, coefficient of variation of R-R interval, and ankle-brachial pressure index were observed before treatment in the PISA non-improved group. CONCLUSIONS: Glycaemic control treatment can effectively improve periodontitis in patients with type 2 diabetes, even in the absence of specific periodontal treatments. However, the periodontal responsiveness to glycaemic control treatment depends on the systemic condition of the patient.

Novel mimetic tissue standards for precise quantitative mass spectrometry imaging of drug and neurotransmitter concentrations in rat brain tissues.

Understanding the relationship between the concentration of a drug and its therapeutic efficacy or side effects is crucial in drug development, especially to understand therapeutic efficacy in central nervous system drug, quantifying drug-induced site-specific changes in the levels of endogenous metabolites, such as neurotransmitters. In recent times, evaluation of quantitative distribution of drugs and endogenous metabolites using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) has attracted much attention in drug discovery research. However, MALDI-MSI quantification (quantitative mass spectrometry imaging, QMSI) is an emerging technique, and needs to be further developed for practicable and convenient use in drug discovery research. In this study, we developed a reliable QMSI method for quantification of clozapine (antipsychotic drug) and dopamine and its metabolites in the rat brain using MALDI-MSI. An improved mimetic tissue model using powdered frozen tissue for QMSI was established as an alternative method, enabling the accurate quantification of clozapine levels in the rat brain. Furthermore, we used the improved method to evaluate drug-induced fluctuations in the concentrations of dopamine and its metabolites. This method can quantitatively evaluate drug localization in the brain and drug-induced changes in the concentration of endogenous metabolites, demonstrating the usefulness of QMSI.

Rice grain structural characteristics of sake rice cultivar Hakutsurunishiki for daiginjo-shu brewing.

Hakutsurunishiki is a sake rice cultivar bred using Yamadabo (seed parent) and Wataribune 2 (pollen parent), equivalent to a Yamadanishiki sibling. This study evaluated the structural characteristics of the Hakutsurunishiki rice grain that contribute to the brewing characteristics of daiginjo-shu, via a comparison with Yamadanishiki. Hakutsurunishiki brown rice was a little heavy and had a large white core. Observing a cross-section of white rice after soaking revealed that the rice grain structure of Hakutsurunishiki was different from that of Yamadanishiki. Hakutsurunishiki white rice showed fewer voids than Yamadanishiki, promoting a slower water absorption rate. Glucose distribution in rice koji obtained by mass spectrometry imaging showed that Hakutsurunishiki rice koji, like Yamadanishiki, is tsuki-haze type, suggesting that its grain structure is suitable for making rice koji for daiginjo-shu. With these observations, we were able to clarify the structural characteristics of Hakutsurunishiki rice grain.

Correlation between plasma glutamate and adiponectin in patients with type 2 diabetes.

Visceral fat accumulation is a major determinant of type 2 diabetes mellitus and cardiovascular diseases. Recent studies have reported that glutamate is the most elevated amino acid in the plasma amino acid profile in patients with obesity and/or visceral fat accumulation. Here, we show the relationship between plasma glutamate and the clinical features of patients with type 2 diabetes. The study subjects were 62 (28 men and 34 women) Japanese patients with type 2 diabetes. Blood profiles, including glutamate and adiponectin (APN) levels and estimated visceral fat area (eVFA), were measured. We also evaluated the plasma amino acid levels in mice with or without obesity by GC/MS analysis. In patients with type 2 diabetes, plasma glutamate was positively correlated with BMI, eVFA, and fasting insulin but negatively correlated with APN and duration of diabetes. Additionally, multiple regression analysis revealed that plasma glutamate was a significant determinant of APN. The plasma glutamate level was most significantly increased in obese mice compared to control mice, and it was negatively correlated with APN. These results suggest that the level of plasma glutamate could be a strong indicator of adipocyte dysfunction in patients with type 2 diabetes.

The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4 receptor-dependent metabolism in myeloid cells.

Extracellular adenosine triphosphate (ATP) released by mucosal immune cells and by microbiota in the intestinal lumen elicits diverse immune responses that mediate the intestinal homeostasis via P2 purinergic receptors, while overactivation of ATP signaling leads to mucosal immune system disruption, which leads to pathogenesis of intestinal inflammation. In the small intestine, hydrolysis of luminal ATP by ectonucleoside triphosphate diphosphohydrolase (E-NTPD)7 in epithelial cells is essential for control of the number of T helper 17 (Th17) cells. However, the molecular mechanism by which microbiota-derived ATP in the colon is regulated remains poorly understood. Here, we show that E-NTPD8 is highly expressed in large-intestinal epithelial cells and hydrolyzes microbiota-derived luminal ATP. Compared with wild-type mice, Entpd8-/- mice develop more severe dextran sodium sulfate-induced colitis, which can be ameliorated by either the depletion of neutrophils and monocytes by injecting with anti-Gr-1 antibody or the introduction of P2rx4 deficiency into hematopoietic cells. An increased level of luminal ATP in the colon of Entpd8-/- mice promotes glycolysis in neutrophils through P2x4 receptor-dependent Ca2+ influx, which is linked to prolonged survival and elevated reactive oxygen species production in these cells. Thus, E-NTPD8 limits intestinal inflammation by controlling metabolic alteration toward glycolysis via the P2X4 receptor in myeloid cells.

Identification of potential quality markers in Indonesia's Arabica specialty coffee using GC/MS-based metabolomics approach.

INTRODUCTION: The cupping test is a widely used method for quality assessment of Arabica coffee. However, the cupping test is limited by the low number of certified panelists and the low throughput. Therefore, an analytical-based quality assessment may be a promising tool to complement the cupping test. A present, there is no report investigating quality marker candidates, focusing only on "specialty" grade Arabica coffee from Indonesia. OBJECTIVE: This study identified the potential quality marker(s) in Arabica Specialty coffee at different stages (green beans, roasted beans, and brewed coffee. METHODS: The metabolite profiles of ten different Arabica specialty-grade coffees were analyzed with different cup scores using gas chromatography-mass spectrometry (GC/MS). From the ten samples, green coffee beans, roasted coffee beans, and brewed coffee were selected. In addition, an orthogonal projection to latent structure (OPLS) regression analysis was conducted to obtain a potential quality marker based on the variable importance in projection (VIP). The potential quality marker(s) were validated by GC/MS metabolome profiling and OPLS analysis of different sets of samples consisting of 35 Arabica specialty-grade coffee samples. RESULTS: In Arabica coffee samples, the OPLS model of the three stages showed galactinol to have a high VIP score. Galactinol showed a consistent positive correlation with cup scores at all stages of coffee production (green beans, roasted beans, and brewed coffee). The correlation suggests galactinol is a potential quality marker after further validation using different samples. CONCLUSION: GC/MS combined with OPLS regression analysis suggested galactinol as a quality marker and provide an early screening method for Arabica coffee quality that complements the cupping test performed by certified panelists.

Metabolome analysis to investigate the effect of controlled fermentation on taste-related metabolites in terasi.

INTRODUCTION: Terasi is a fermented shrimp paste unique to Indonesia and is used in dishes to add umami and saltiness. In a previous study, the controlled fermentation of terasi was optimized using starters containing three bacterial isolates: Staphylococcus saprophyticus, Bacillus subtilis, and Lactobacillus murinus. However, the influence of controlled fermentation using these starters on the metabolites in terasi has not yet been studied. OBJECTIVES: Therefore, this study aimed to investigate the effect of controlled fermentation on taste-related metabolites in terasi using a metabolomics approach. RESULTS: Non-targeted analysis indicated that amino acids contributed to variations during fermentation. Subsequently, targeted analysis of amino acids revealed that terasi subjected to controlled fermentation using a starter with a 2:1:2 ratio of S. saprophyticus, B. subtilis, and L. murinus, respectively, resulted in a product containing D-amino acids, such as D-Asp, D-Gln, and D-Leu that was unique when compared to other terasi products prepared using controlled fermentation. Genetic analysis of isolates from the terasi produced using controlled fermentation was also carried out, and this is the first study to suggest that Staphylococcus spp. has the potential to produce D-amino acids. CONCLUSION: In conclusion, the ratio of bacterial species in starter cultures used in controlled fermentation influenced the amino acid profile of the product and starters with a higher ratio of Staphylococcus spp. may result in the production of D-amino acids.

Gas chromatography/mass spectrometry-based metabolite profiling of coffee beans obtained from different altitudes and origins with various postharvest processing.

INTRODUCTION: Coffee is a popular beverage because of its pleasant aroma and distinctive flavor. The flavor of coffee results from chemical transformations influenced by various intrinsic and extrinsic factors, including altitude, geographical origin, and postharvest processing. Despite is the importance of grading coffee quality, there is no report on the dominant factor that influences the metabolomic profile of green coffee beans and the correlated metabolites for each factor. OBJECTIVE: This study investigated the total metabolite profile of coffees from different altitudes and coffees subjected to different postharvest processing. METHOD: Arabica green coffee beans obtained from different geographical origins and different altitudes (400 and 800 m) and produced by different postharvest processes (dry, honey, and washed process) were used in this study. Coffee samples obtained from altitudes of 400-1600 m above sea level from various origins that were produced by the washed method were used for further study with regard to altitudes. Samples were subjected to gas chromatography/mass spectrometry (GC/MS) analysis and visualized using principal component analysis (PCA) and orthogonal partial least squares (OPLS) regression analysis. RESULTS: The PCA results showed sample separation based on postharvest processing in PC1 and sample separation based on altitude in PC2. A clear separation between samples from different altitudes was observed if the samples were subjected to the same postharvest processing method, and the samples were of the same origin. Based on this result, OPLS analysis was conducted using coffee samples obtained from various altitudes with the same postharvest processing. An OPLS model using altitude as a response variable and 79 metabolites annotated from the GC/MS analysis as an explanatory variable was constructed with good R2 and Q2 values. CONCLUSION: Postharvest processing was found to be the dominant factor affecting coffee metabolite composition; this was followed by geographical origin and altitude. The metabolites glutamic acid and galactinol were associated with the washed and honey process, while glycine, lysine, sorbose, fructose, glyceric acid, and glycolic acid were associated with the dry process. Two metabolites with high variable influence on projection scores in the OPLS model for altitude were inositol and serotonin, which showed positive and negative correlations, respectively. This is the first study to report characteristic coffee metabolites obtained from different altitudes.

Shrimp count size: GC/MS-based metabolomics approach and quantitative descriptive analysis (QDA) reveal the importance of size in white leg shrimp (Litopenaeus vannamei).

INTRODUCTION: "Count size" is a term used to represent the number of shrimps in one pound or kilogram that applies globally in the shrimp industry. Based on shrimp body weight, count sizes range over the smallest (> 70) up to the largest size (U15) of shrimp. Large shrimps are considered highly palatable; therefore, they are priced higher than the small shrimps. However, the pricing of shrimp has not been based on scientific findings since there have been no studies reporting the correlation between shrimp quality and shrimp size. OBJECTIVE: In this study, we aimed to investigate the importance of shrimp size in terms of metabolite profile and sensory properties. METHODS: Nine groups of Litopenaeus vannamei, categorized based on their body weight similarity, were collected from various sampling sites regardless of the difference in days of culture (count size 16/20, 21/25, 26/30, 41/50, and 51/60). Gas chromatography/mass spectrometry (GC/MS)-based metabolomics analysis was employed to characterize their metabolite profiles. Furthermore, a robust PLS regression model was constructed to predict the shrimp size using metabolome data. Following this, the difference in sensory attributes among commercial shrimp count sizes 21/25-41/50 was confirmed using quantitative descriptive analysis (QDA). RESULTS: Small shrimp (> 70-51/60) had higher accumulation of proteinogenic and non-proteinogenic amino acids, sugars, and organic acids compared to large shrimps (41/50-16/20). The QDA of commercial count sizes (21/25-41/50) performed by trained panelists showed that sweetness, juiciness, crispness, and red color attributes increased with an increase in shrimp size. Based on the PLS model, proline as a sweet-tasting metabolite also showed an increased level along with the shrimp size. CONCLUSIONS: These findings demonstrate the importance of shrimp count size with regard to shrimp quality.

Mass Spectrometric Enzyme Histochemistry Method Developed for Visualizing In Situ Cholinesterase Activity in Mus musculus and Drosophila melanogaster.

Enzyme histochemistry facilitates enzyme activity visualization in situ; however, as it is a color-based method, molecular quantification is prohibitive. This study aimed to develop a semiquantitative, mass spectrometry imaging (MSI)-based enzyme histochemistry method to determine endogenous cholinesterase (ChE) activity. Using deuterium-labeled acetylcholine (ACh-d9) as a substrate to distinguish ACh-d9 and choline-d9 from endogenous acetylcholine and choline, respectively, the heterogeneous localization of de novo ChE activity was visualized using MSI, devoid of interferences from in situ factors. Furthermore, a tissue inhibitor assay involving two ChE inhibitors in the mouse brain revealed specific ChE inhibition in the corpus callosum. To the best of our knowledge, this study is the first to report a visualization method for total ChE activity in the ganglia and abdomen in Drosophila melanogaster, indicating its applicability among different animals. The present results provide novel insights into the applicability of enzyme histochemistry via MSI to the metabolism of low-molecular-weight organic compounds (i.e., "small molecules") and semiquantitative capability, suggesting that MSI enzyme histochemistry may become a powerful tool for heterogeneous tissue studies.

Plasma metabolites associated with arterial stiffness in patients with type 2 diabetes.

BACKGROUND: Although an increased arterial stiffness has been associated with traditional coronary risk factors, the risk factors and pathology of arterial stiffness remain unclear. In this study, we aimed to identify the plasma metabolites associated with arterial stiffness in patients with type 2 diabetes mellitus. METHODS: We used the metabolomic data of 209 patients with type 2 diabetes as the first dataset for screening. To form the second dataset for validation, we enlisted an additional 31 individuals with type 2 diabetes. The non-targeted metabolome analysis of fasting plasma samples using gas chromatography coupled with mass spectrometry and the measurement of brachial-ankle pulse wave velocity (baPWV) were performed. RESULTS: A total of 65 annotated metabolites were detected. In the screening dataset, there were statistically significant associations between the baPWV and plasma levels of indoxyl sulfate (r = 0.226, p = 0.001), mannitol (r = 0.178, p = 0.010), mesoerythritol (r = 0.234, p = 0.001), and pyroglutamic acid (r = 0.182, p = 0.008). Multivariate regression analyses revealed that the plasma levels of mesoerythritol were significantly (ß = 0.163, p = 0.025) and that of indoxyl sulfate were marginally (ß = 0.124, p = 0.076) associated with baPWV, even after adjusting for traditional coronary risk factors. In the independent validation dataset, there was a statistically significant association between the baPWV and plasma levels of indoxyl sulfate (r = 0.430, p = 0.016). However, significant associations between the baPWV and plasma levels of the other three metabolites were not confirmed. CONCLUSIONS/INTERPRETATION: The plasma levels of indoxyl sulfate were associated with arterial stiffness in Japanese patients with type 2 diabetes. Although the plasma levels of mannitol, mesoerythritol, and pyroglutamic acid were also associated with arterial stiffness, further investigation is needed to verify the results.

GC/MS-based metabolic profiling for the evaluation of solid state fermentation to improve quality of Arabica coffee beans.

INTRODUCTION: Coffee fermentation has been reported as one key process in aroma and flavor development of coffee. However, natural fermentation often results in inconsistent quality of coffee. In this study, second fermentation using isolates from feces of civet (Luwak) and Cilembu sweet potato were used to improve the quality of Arabica green coffee beans. OBJECTIVES: The aim of this research was to improve the quality of various Arabica coffee from different origins in Indonesia by controlled-second fermentation. METHOD: The Arabica coffee beans used in this study were from three origins in Indonesia: Kintamani-Bali (I), Aceh-Gayo (II) and Nagarawangi-Sumedang (III). The second fermentation was done using three bacterial isolates coded as BF5C(2); UciSp14; and AF7E which belong to Bacillus genus. Quality assessment of fermented coffee was performed by cupping test following Specialty Coffee Association of America (SCAA) protocol by licensed Q graders, GC/MS metabolite profiling, and total polyphenol content measurement. RESULTS: The controlled-second fermentation for 4-8 h was successful to increase total polyphenol content well as to improve the complexity of coffee taste and coffee quality (cupping score > 84). Comparative GC/MS analysis showed that fermentation of coffee beans resulted in alteration of metabolite profiles of coffee beans compared with control, while still maintaining the characteristics of coffee based on each origin. CONCLUSION: The controlled-second fermentation was effective to increase the quality of coffee and alter metabolite composition of coffee that were associated with changes in taste profile of coffee. This report may serve as basis for producing coffee with better taste quality with higher polyphenols content through standardized fermentation production in industrial scale.

Metabolite profiling of whiteleg shrimp Litopenaeus vannamei from super-intensive culture in closed aquaculture systems: a recirculating aquaculture system and a hybrid zero water discharge-recirculating aquaculture system.

INTRODUCTION: The production of the whiteleg shrimp Litopenaeus vannamei now accounts for approximately 75% of the total shrimp production in Indonesia. The techniques used to produce whiteleg shrimp in Indonesia are still dominated by conventional rearing strategies using open-pond systems, which often contribute to unpredictable culture performance and weak sustainability. Alternative production strategies of closed aquaculture systems, including the recirculating aquaculture system (RAS) and hybrid zero water discharge-recirculating aquaculture system (hybrid system), have been developed and implemented for higher productivity, stability and sustainability of whiteleg shrimp grow-out production in Indonesia. Despite the positive aspects of the application of closed aquaculture systems in shrimp aquaculture, the differences in the characteristics of shrimp grown in closed RAS and hybrid systems compared to open-pond systems remain unclear. OBJECTIVE: This study aims to investigate the differences in the metabolite profiles of shrimp grown in intensive closed aquaculture systems, including an RAS and hybrid system, compared to those of shrimp grown in a semi-intensive, open, earthen pond system by means of non-targeted GC-MS metabolite profiling. METHODS: Shrimp cultured in the closed systems (RAS and hybrid system) and an open system (pond) were harvested and subjected to GC-MS non-targeted metabolomics analysis. A total of 112 metabolites were annotated from shrimp samples and subjected to principal component analysis (PCA). RESULTS: The metabolites annotated from GC-MS mainly included organic compounds, proteinogenic and non-proteinogenic amino acids, sugars, nucleosides and fatty acids. The results of principal component analysis showed several metabolites with high variable importance in projection (VIP) scores, including shikimic acid, ß-alanine, uric acid, hypoxanthine, inosine, homocysteine, methionine, phenylalanine, tryptophan and lysine, as the main metabolites differentiating the shrimp grown in the three production systems. CONCLUSION: Our findings showed that shrimp cultured in different aquaculture systems exhibited distinct metabolite profiles, and the metabolites showing high VIP scores, including shikimic acid, ß-alanine, uric acid, hypoxanthine, inosine, homocysteine, methionine, phenylalanine, tryptophan and lysine, may serve as candidate markers to indicate the differences in shrimp from different production systems.

Metabolic repair through emergence of new pathways in Escherichia coli.

Escherichia coli can derive all essential metabolites and cofactors through a highly evolved metabolic system. Damage of pathways may affect cell growth and physiology, but the strategies by which damaged metabolic pathways can be circumvented remain intriguing. Here, we use a ΔpanD (encoding for aspartate 1-decarboxylase) strain of E. coli that is unable to produce the ß-alanine required for CoA biosynthesis to demonstrate that metabolic systems can overcome pathway damage by extensively rerouting metabolic pathways and modifying existing enzymes for unnatural functions. Using directed cell evolution, rewiring and repurposing of uracil metabolism allowed formation of an alternative ß-alanine biosynthetic pathway. After this pathway was deleted, a second was evolved that used a gain-of-function mutation on ornithine decarboxylase (SpeC) to alter reaction and substrate specificity toward an oxidative decarboxylation-deamination reaction. After deletion of both pathways, yet another independent pathway emerged using polyamine biosynthesis, demonstrating the vast capacity of metabolic repair.

Potato tuber metabolomics-based prediction of chip color quality and application using gas chromatography/flame ionization detector.

Potato (Solanum tuberosum L.) tubers are usually harvested once a year; thus, long-term storage is required to supply quality-assured tubers throughout the year. Further, an applicable method to predict tuber quality during storage is needed. In this study, gas chromatography-mass spectrometry (GC/MS) metabolomics was applied to identify applicable biomarkers for prediction of potato chip color based on 3 years' field-grown tubers. The projections to latent structures (PLS) prediction model, calculated from a metabolome data set obtained before storage, was consistent with actual measured chip color values. Additionally, GC with frame ionization detector (GC/FID) metabolite fingerprinting simultaneously re-constructed more reliable and relevant prediction models for chip color quality compared to GC/MS. Moreover, nine metabolites detected by GC/MS analysis were further validated as applicable prediction markers. This strategy will provide a practical and cost-effective quality-control tool for potato processing manufacturers on an industrial scale.

Bulk RNA degradation by nitrogen starvation-induced autophagy in yeast.

Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is non-selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy-dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2-type ribonuclease, generating 3'-NMPs that were immediately converted to nucleosides by the vacuolar non-specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re-assimilated, but excreted from the cell. Bulk non-selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.

Tandem Mass Spectrometry Imaging Reveals Distinct Accumulation Patterns of Steroid Structural Isomers in Human Adrenal Glands.

Visualizing tissue distribution of steroid hormones is a promising application of MALDI mass spectrometry imaging (MSI). On-tissue chemical derivatization using Girard's T reagent has enhanced the ionization efficiency of steroids. However, discriminating between structural isomers with distinct bioactivities remains a challenge. Herein, we used ion trap MS/tandem MS (MS3) to distinguish a mineralcorticoid aldosterone (Aldo) and a glucocorticoid cortisol (F), from their structural isomers. Our method is also useful to detect hybrid steroids (18-hydroxycortisol [18-OHF] and 18-oxocortisol) with sufficient signal-to-noise ratio. The clinical applicability of the tandem MS method was evaluated by analyzing F, Aldo, and 18-OHF distributions in human adrenal glands. In such clinical specimens, small Aldo-producing cell clusters (APCCs) were identified and were first found to produce a high level of Aldo and not to contain F. Moreover, a part of APCCs produced 18-OHF, presumably converted from F by APCC-specific CYP11B2 activity. Catecholamine species were also visualized with another derivatization reagent (TAHS), and those profiling successfully discriminated pheochromocytoma species. These tandem MSI-methods, coupled with on-tissue chemical derivatization has proven to be useful for detecting low-abundance steroids, including Aldo and hybrid steroids and thus identifying steroid hormone-producing lesions.

Tailor-made poly-γ-glutamic acid production.

Poly-γ-glutamic acid (γ-PGA), which is produced by several Bacillus species, is a chiral biopolymer composed of D- and L-glutamate monomers and has various industrial applications. However, synthesized γ-PGA exhibits great structural diversity, and the structure must be controlled to broaden its industrial use. The biochemical pathways for γ-PGA production suggest that the polymer properties molecular weight (MW) and stereochemical composition are influenced by (1) the affinity of γ-PGA synthetase for the two alternative glutamate enantiomers and (2) glutamate racemase activity; hence, the availability of the monomers. In this study, we report tailor-made γ-PGA synthesis with B. subtilis by combining PGA synthetase and glutamate racemase genes from several Bacillus strains. The production of structurally diverse γ-PGA was thereby achieved. Depending on the PGA synthetase and glutamate racemase origins, the synthesized γ-PGA contained 3-60% D-glutamate. The exchange of PGA synthetase changed the MW from 40 to 8500 kDa. The results demonstrate the production of low-, medium-, and high-MW γ-PGA with the same microbial chassis.

GC/MS based metabolite profiling of Indonesian specialty coffee from different species and geographical origin.

INTRODUCTION: The consumption of high quality coffee such as specialty Arabica and fine Robusta coffee is increasing steadily in recent years. Development of single origin coffee is an important strategy to maintain coffee quality, grade and high cupping score. Indonesia is a top exporting country for Arabica coffee with high variety of specialty coffees from different origins. Despite its long standing reputation in global coffee market, very few is known about the variability among Indonesian specialty coffees. OBJECTIVES: This study aims to observe metabolite variability among Indonesian coffees from different species and geographical origins by means of non-targeted GC/MS metabolite profiling. METHODS: Sixty-four compounds were tentatively identified from 16 green and roasted coffee beans from different species and cultivation areas in Indonesia and were subjected to principal component analysis (PCA). Ten Specialty Arabica coffee and five Fine Robusta representing all important high quality coffees of Indonesia were also analyzed independently to further classify Indonesian coffee according to their origin. RESULTS: PCA results of 16 green and roasted coffee beans of different species and cultivation areas showed that samples were separated along PC1 based on different roasting condition (green and roasted) with 52.9% variance and were separated along PC2 based on different species with 19.3% variance. The result from this study showed the clustering of samples based on three major cultivation areas in Indonesia (western, central, eastern part). Metabolites showing higher concentration in Sulawesi, Papua, Flores and Sumatra samples were glycerol, glucuno-1,5-lactone, gluconic acid and sorbitol. A clear distinction in galactitol and galactinol concentration between all samples from eastern part of Indonesia and western and middle part of Indonesia was also observed. CONCLUSIONS: Our results showed that each region (western, central and eastern part of Indonesia) has signature compounds that may serve as discriminant markers for coffee authentication. This is the first report on the classification of Indonesian specialty coffee based on their metabolic profiles and can act as a basis for marker identification for routine procedures in industry.