A High-Throughput Absolute Quantification of Protein-Bound Sulfur Amino Acids from Model and Crop Plant Seeds.

In this procedure, we describe a high-throughput absolute quantification protocol for the protein-bound sulfur amino acids, cysteine (Cys) and methionine (Met), from plant seeds. This procedure consists of performic acid oxidation that transforms bound Cys into cysteic acid (CysA) and bound Met into methionine sulfone (MetS) followed by acid hydrolysis. The absolute quantification step is performed by multiple reaction monitoring tandem mass spectrometry (LC-MS/MS). The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common acid hydrolysis protocol, followed by water extraction that includes DL-Ser-d3 and L-Met-d3 as internal standards to enable the quantification of the absolute levels of the protein-bound Cys and Met with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples from Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound cysteine and methionine from seeds.

Association of Long-term Exposure to Air Pollution and Dementia Risk: The Role of Homocysteine, Methionine, and Cardiovascular Burden.

BACKGROUND AND OBJECTIVES: Growing evidence links air pollution with dementia risk, but the biological mechanisms are largely unknown. We investigated the role played by homocysteine (tHcy) and methionine in this association and explored whether this could be explained by cardiovascular diseases (CVDs). METHODS: Data were extracted from the ongoing Swedish National study on Aging and Care in Kungsholmen (SNAC-K), a longitudinal population-based study. At baseline, 2,512 dementia-free participants were examined up to 2013 (mean follow-up: 5.18 ± 2.96 years). Two air pollutants (particulate matter ≤2.5 µm [PM2.5] and nitrogen oxides [NOx]) were assessed yearly from 1990 until 2013 using dispersion models at residential addresses. The hazard ratio of dementia over air pollution levels was estimated using Cox models adjusted for age, sex, education, smoking, socioeconomic status, physical activity, retirement age, creatinine, year of assessment, and the use of supplements. The total effect of air pollutants on dementia was decomposed into 4 pathways involving tHcy/methionine: (1) direct effect; (2) indirect effect (mediation); (3) effect due to interaction; and (4) effect due to both mediation and interaction. To test whether the association was independent from CVDs (ischemic heart disease, atrial fibrillation, heart failure, and stroke), we repeated the analyses excluding those individuals who developed CVDs. RESULTS: The mean age of the study participants was 73.4 years (SD: 10.4), and 62.1% were female individuals. During an average period of 5 years (mean: 5.18; SD: 2.96 years), 376 cases with incident dementia were identified. There was a 70% increased hazard of dementia per unit increase of PM2.5 during the 5 years before baseline (hazard ratio [HR]: 1.71; 95% CI 1.33-2.09). Overall, 50% (51.6%; 95% CI 9.0-94.1) of the total effect of PM2.5 on dementia was due to mediation of tHcy (6.6%; 95% CI 1.6-11.6) and/or interaction (47.8%; 95% CI 4.9-91.7) with tHcy and 48.4% (p = 0.03) to the direct effect of PM2.5 on dementia. High levels of methionine reduced the dementia hazard linked to PM2.5 by 31% (HR: 0.69; 95% CI 0.56-0.85) with 24.8% attributable to the interaction with methionine and 25.9% (p = 0.001) to the direct effect of PM2.5. No mediation effect was found through methionine. Attenuated results were obtained for NOx. Findings for tHcy were attenuated after excluding those who developed CVDs, while remained similar for methionine. DISCUSSION: High levels of homocysteine enhanced the dementia risk attributed to air pollution, while high methionine concentrations reduced this risk. The impact of homocysteine on cardiovascular conditions partly explains this association. Alternative pathways other than cardiovascular mechanisms may be at play between methionine and dementia.

Extraction, characterization, and anti-nonalcoholic steatohepatitis activity of a (1,3) (1,6)-ß-D-glucan from the Polyporus umbellatus (Pers.) Fries.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by inflammation and hepatic steatosis that may coincide with fibrotic activity. To date, no pharmacological agents have been approved for NASH treatment. Here, a homogeneous (1,3),(1,6)-ß-D-glucan (PUP-W-1, Mw: 41.07 kDa) was successfully purified from Polyporus umbellatus (Pers.) Fries sclerotia and characterized. The analysis showed that the PUP-W-1 backbone consisted of a repeating chain of eight →3)-ß-D-Glcp-(1 â†’ units, with branched chains of four ß-D-Glcp residues, joined by repeating 1,6-linkage units at the O-6 position of the backbone. The pharmacological effects of PUP-W-1 treatment in the context of NASH pathogenesis were explored using a methionine choline-deficient (MCD) diet-induced murine steatohepatitis model. The MCD model mice exhibited pronounced steatohepatitis, inflammatory activity, steatosis, stellate cell activation, and mild fibrotic activity. Treatment of the mice for three weeks with PUP-W-1 prevented the development of NASH due to the suppression of inflammation, lipid accumulation, and fibrosis. As suggested by these findings, PUP-W-1 may hold promise as a natural drug candidate or precursor for the treatment of NASH.

Quantitative fusion omics reveals that refrigeration drives methionine degradation through perturbing 5-methyltetrahydropteroyltriglutamate-homocysteine activity.

Postharvest senescence and quality deterioration of fresh tea leaves occurred due to the limitation of processing capacity. Refrigerated storage prolongs the shelf life of fresh tea. In this study, quantitative fusion omics delineated the translational landscape of metabolites and proteins in time-series (0-12 days) refrigerated tea by UHPLC-Q-Orbitrap HRMS. Accurate quantification results showed the content of amino acids, especially l-theanine, decreased with the lengthening of the storage duration (15.57 mg g-1 to 7.65 mg g-1) driven by theanine synthetase. Downregulation of enzyme 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase expression led to methionine degradation (6.29 µg g-1 to 1.78 µg g-1). Refrigerated storage inhibited serine carboxypeptidase-like acyltransferases activity (59.49 % reduction in 12 days) and induced the polymerization of epicatechin and epigallocatechin and generation of procyanidin dimer and δ-type dehydrodicatechin, causing the manifestation of color deterioration. A predictive model incorporating zero-order reaction and Arrhenius equation was constructed to forecast the storage time of green tea.

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation.

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin 'egg-box' structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

Melatonin Attenuates Inflammation, Oxidative Stress, and DNA Damage in Mice with Nonalcoholic Steatohepatitis Induced by a Methionine- and Choline-Deficient Diet.

Nonalcoholic steatohepatitis (NASH) is a disease with a high incidence worldwide, but its diagnosis and treatment are poorly managed. In this study, NASH pathophysiology and DNA damage biomarkers were investigated in mice with NASH treated and untreated with melatonin (MLT). C57BL/6 mice were fed a methionine- and choline-deficient (MCD) diet for 4 weeks to develop NASH. Melatonin was administered at 20 mg/kg during the last 2 weeks. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured, and hepatic tissue was dissected for histological analysis, evaluation of lipoperoxidation, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as nuclear factor-erythroid 2 (Nrf2), tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), and transforming growth factor beta (TGF-ß) expression by immunohistochemistry. DNA damage was evaluated using Comet assay, while a micronucleus test in bone marrow was performed to assess the genomic instability associated with the disease. Melatonin decreased AST and ALT, liver inflammatory processes, balloonization, and fibrosis in mice with NASH, decreasing TNF-α, iNOS, and TGF-ß, as well as oxidative stress, shown by reducing lipoperoxidation and intensifying Nrf2 expression. The SOD and GPx activities were increased, while CAT was decreased by treatment with MLT. Although the micronucleus frequency was not increased in mice with NASH, a protective effect on DNA was observed with MLT treatment in blood and liver tissues using Comet assay. As conclusions, MLT slows down the progression of NASH, reducing hepatic oxidative stress and inflammatory processes, inhibiting DNA damage via anti-inflammatory and antioxidant actions.

Quantifying methionine sulfoxide in therapeutic protein formulation excipients as sensitive oxidation marker.

Methionine is a common excipient used in therapeutic protein liquid formulations as stabilizer and antioxidant. The oxidation of methionine to methionine sulfoxide can be regarded as a sensitive marker of oxidative stress for drug product storage conditions. In this study, a sensitive HPLC method for the quantification of methionine sulfoxide in formulated protein product was developed and qualified according to regulatory requirements using a SIELC® Primesep 100 column with UV detection. The separation involves a mixed-mode mechanism including reversed phase and cationic exchange modalities. The operating range of the method was established between 1 µM and 35 µM of methionine sulfoxide. In this testing range, the method was shown to be linear (R2 > 0.99), accurate (Recovery 92.9 - 103.6%, average recovery = 99.8 ± 1.4%) and precise (intermediate precision at LoQ, CV = 2.9%). The developed test system was successfully applied to study the effects of temperature and storage conditions on methionine sulfoxide formation in complex therapeutic antibody formulations.

Improving crude protein and methionine production, selective lignin degradation and digestibility of wheat straw by Inonotus obliquus using response surface methodology.

BACKGROUND: To date, fungus-assisted pretreatment of agricultural residue has not become the preferred method to produce protein-enriched and ruminally digestible animal feed because of low time efficiency of fungal delignification and protein production, i.e. the long solid-state fermentation period, and because of laccase as a potential inhibitor of cellulose activity. In this study, response surface methodology was employed to optimize the parameters in the process of producing nutritious animal feed from wheat straw with Inonotus obliquus pretreatment. RESULTS: The mineral salt solution containing (w/v) (NH4 )2 SO4 1%, MgSO4 ·7H2 O 0.03%, KH2 PO4 0.011%, Tween-80 0.4%, and corn starch 10% with pH of 7.4 was optimized. Inonotus obliquus rapidly and completely colonized on wheat straw with an ergosterol content of 280 µg g-1 dry matter, consuming 45% of lignin after 15 days of fermentation, producing maximums of lignin peroxidase (1729 IU g-1 ), manganese peroxidase (610 IU g-1 ) and laccase (98 IU g-1 ) on days 5, 15, and 25, respectively. The crude protein (102.4 g kg-1 ) of 15-day fermented wheat straw increased by ~132%. After hydrolysis, the essential protein-bound amino acids (15.3 g kg-1 ) increased by ~47%, within which Met and Lys measured ~1070% and ~60% higher. The treatment with I. obliquus also improved the in vitro gas production after 72 h (IVGP72 ) of wheat straw to 178.8 mL g-1 organic matter (~43% increase). CONCLUSION: For the first time, we found that I. obliquus is an effective white rot fungus turning wheat straw into ruminally digestible animal feed without laccase inhibitor.

The Influence of UV Varnishes on the Content of Cysteine and Methionine in Women Nail Plates-Chromatographic Studies.

The main purpose of this work was to determine if the use of hybrid nail polishes causes changes in concentration of the most important sulfur amino acids that build nail plate structures, cysteine and methionine. We found that the average contents of cysteine and methionine in studied samples before the use of hybrid manicure were 1275.3 ± 145.9 nmol mg-1 and 111.7 ± 23.8 nmol mg-1, respectively. After six months of hybrid manicure use, the average amount of these sulfur amino acids in studied samples were 22.1% and 36.5% lower in the case of cysteine and methionine, respectively. The average amounts of cysteine and methionine in nail plate samples after the use of hybrid manicures were 992.4 ± 96.2 nmol mg-1 and 70.9 ± 14.8 nmol mg-1, respectively. We also confirmed that in studied women the application of UV light varnishes reduced the thickness of the nail plate, from 0.50 ± 0.12 mm before to 0.46 ± 0.12 mm after the use of the hybrid manicure.

pH-Regulating Nanoplatform for the "Double Channel Chase " of Tumor Cells by the Synergistic Cascade between Chlorine Treatment and Methionine-Depletion Starvation Therapy.

During rapid proliferation and metabolism, tumor cells show a high dependence on methionine. The deficiency of methionine exhibits significant inhibition on tumor growth, which provides a potential therapeutic target in tumor therapy. Herein, ClO2-loaded nanoparticles (fluvastatin sodium&metformin&bupivacaine&ClO2@CaSiO3@MnO2-arginine-glycine-aspatic acid (RGD) (MFBC@CMR) NPs) were prepared for synergistic chlorine treatment and methionine-depletion starvation therapy. After outer layer MnO2 was degraded in the high glutathione (GSH) tumor microenvironment (TME), MFBC@CMR NPs released metformin (Me) to target the mitochondria, thus interfering with the tricarboxylic acid (TCA) cycle and promoting the production of lactate. In addition, released fluvastatin sodium (Flu) by the NPs acted on monocarboxylic acid transporter 4 (MCT4) in the cell membrane to inhibit lactate leakage and induce a decrease of intracellular pH, further prompting the NPs to release chlorine dioxide (ClO2), which then oxidized methionine, inhibited tumor growth, and produced large numbers of Cl- in the cytoplasm. Cl- could enter mitochondria through the voltage-dependent anion channel (VDAC) channel, which was opened by bupivacaine (Bup). The disruption of Cl- homeostasis promotes mitochondrial damage and membrane potential decline, leading to the release of cytochrome C (Cyt-C) and apoptosis inducing factor (AIF) and further inducing cell apoptosis. To sum up, the pH-regulating and ClO2-loaded MFBC@CMR nanoplatform can achieve cascade chlorine treatment and methionine-depletion starvation therapy toward tumor cells, which is of great significance for improving the clinical tumor treatment effect.

Dietary Methyl-Group Donor Intake and Breast Cancer Risk in the European Prospective Investigation into Cancer and Nutrition (EPIC).

(1) Background: Methyl-group donors (MGDs), including folate, choline, betaine, and methionine, may influence breast cancer (BC) risk through their role in one-carbon metabolism; (2) Methods: We studied the relationship between dietary intakes of MGDs and BC risk, adopting data from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort; (3) Results: 318,686 pre- and postmenopausal women were followed between enrolment in 1992-2000 and December 2013-December 2015. Dietary MGD intakes were estimated at baseline through food-frequency questionnaires. Multivariable Cox proportional hazards regression models were used to quantify the association between dietary intake of MGDs, measured both as a calculated score based on their sum and individually, and BC risk. Subgroup analyses were performed by hormone receptor status, menopausal status, and level of alcohol intake. During a mean follow-up time of 14.1 years, 13,320 women with malignant BC were identified. No associations were found between dietary intakes of the MGD score or individual MGDs and BC risk. However, a potential U-shaped relationship was observed between dietary folate intake and overall BC risk, suggesting an inverse association for intakes up to 350 µg/day compared to a reference intake of 205 µg/day. No statistically significant differences in the associations were observed by hormone receptor status, menopausal status, or level of alcohol intake; (4) Conclusions: There was no strong evidence for an association between MGDs involved in one-carbon metabolism and BC risk. However, a potential U-shaped trend was suggested for dietary folate intake and BC risk. Further research is needed to clarify this association.

Highly sensitive determination of methionine by solvent-based de-emulsification dispersive liquid-liquid microextraction using bio-stabilized silver nanoparticles.

In this study, a solvent-based de-emulsification dispersive liquid-liquid microextraction method coupled with surface plasmon resonance of silver nanoparticles was developed for determination of trace levels of methionine. The stable and dispersed silver nanoparticles were synthesized by applying ascorbic acid as reducer and Stenotrophomonas sp. bacterial suspension as bio-stabilizer and then preconcentrated in organic phase according to a facile dispersive liquid-liquid microextraction procedure based on 1-octanol as extraction solvent, methyltrioctylammonium chloride (aliquat 336) as disperser and acetone as de-emulsifier. The presence of methionine influenced the intensity of plasmon resonance absorbance of silver nanoparticles, which was employed as a colorimetric probe for the determination of this amino acid. Under the optimal conditions, the linear analytical range of 5.6 to 234.5 nmol/L and a detection limit of 3.4 nmol/L were achieved for methionine. The relative standard deviation for seven replicate measurements of 33.5 and 107.2 nmol/L of methionine was 4.3 and 2.1%, respectively. The suggested method was successfully applied for the determination of methionine in biological samples.

Sulfur fertility management to enhance methionine and cysteine in soybeans.

BACKGROUND: Soybeans (Glycine max) are a major protein source both for humans and non-ruminant livestock; however, the usability of soybean protein is limited by the concentration of the essential sulfur (S)-containing amino acids methionine and cysteine (MET+CYS). Traditional efforts to improve protein quality in soybeans have largely been focused on plant breeding but soil S fertility may also influence seed MET+CYS concentration. Crop S deficiencies are increasingly common due to soil depletion by high yields and reduced atmospheric deposition. We report on a survey of commercial soybean fields and two replicated split-plot field experiments in the mid-Atlantic region, USA. The experimental treatments were two levels (0 or 100 kg S ha-1 ) of broadcast gypsum (CaSO4 ) and two levels (0 or 11 kg-S ha-1 ) of foliar Epsom salt (MgSO4 ) applied to two soybean cultivars. The objective was to assess the variability of, and effect of, S fertilization on S and MET+CYS concentrations in soybean seeds. RESULTS: Sulfur ranged from 2.35 to 3.54 mg g-1 and MET+CYS ranged from 5.5 to 9.2 mg g-1 protein in seeds from commercial fields surveyed. Sulfur application increased seed MET+CYS concentration 1.3 to twofold in two replicated field experiments. Overall, MET+CYS concentration in protein ranged from 3.9 to 12.8 mg g-1 and was linearly predicted (R2  = 0.65) by seed S. CONCLUSIONS: Soybean seed S and MET+CYS concentrations vary widely. We show that field-scale S application can greatly enhance soybean MET+CYS content and therefore protein quality. © 2021 Society of Chemical Industry.

Effects of rumen-protected methionine supplementation on the performance of high production dairy cows in the tropics.

Increasing methionine availability in dairy cow diets during the first third of lactation may enhance their performance and health. The aim of this study was to determine the effect of supplementing rumen-protected methionine (Smartamine® M, SM) in a lactation diet with protein and energy levels calculated according to the literature. Seventy-six multiparous Holstein cows (39.1 ± 6.8 kg of milk/d and 65 ± 28 DIM) were assigned to 1 of 2 dietary treatments (38/treatment) according to a randomized complete block design with a 2-wk (covariate) and 10-wk experimental period. Treatments were a basal diet (CON; 3.77 Lys:1Met); and CON + 23 g SM (2.97 Lys:1 Met). Individual milk samples were taken every 2 weeks to determine milk composition. Blood was collected from 24 cows on d+30 d to measure plasma AA levels. Body weight and body condition score (BCS) were measured at the beginning and the end of the experiment. The SM diet promoted higher milk yield (41.7 vs. 40.1 kg/d; P = 0.03). Energy-corrected milk yield (41.0 vs. 38.0 kg/d), milk protein yield (1.30 vs. 1.18 kg/d), milk protein (3.14% vs. 2.97%) and casein (2.39% vs. 2.28%) were also different (P < 0.01) as well as milk fat yield (1.42 vs. 1.29 kg/d; P = 0.02). A trend (P = 0.06) for higher milk fat % (3.41% vs. 3.21%) was observed. Both diets resulted in similar body weight, but CON-fed cows tended (P = 0.08) to have higher BCS. Higher plasma methionine levels were determined with SM compared with CON (29.6 vs. 18.4 µM; P < 0.01), but lysine and histidine were not different. Dietary supplementation of RPM improved productive performance by increasing milk yield and milk components yields, suggesting better dietary AA utilization when Met levels are adjusted in Lys-adequate lactation diets.

Use of alternative protein sources for fishmeal replacement in the diet of largemouth bass (Micropterus salmoides). Part II: effects of supplementation with methionine or taurine on growth, feed utilization, and health.

Fishmeal has long been a staple protein feedstuff for fish, but its global shortage and high price have prompted its replacement with alternative sustainable sources. In this experiment involving largemouth bass (a carnivorous fish), a new mixture of feedstuffs (45% poultry byproduct meal, 30% soybean meal, 15% blood meal, and 10% krill shrimp meal) was added to low (14.5%) fishmeal diets along with 0.0%, 0.5% taurine, 0.5% methionine, or 0.5% taurine plus 0.5% methionine (dry matter basis). The positive control diet [65.3% fishmeal (46% crude protein on dry matter basis)] and all low-fishmeal diets contained 40% true protein and 10% lipids. There were 3 tanks per treatment group (20 fish/tank). Fish with the mean initial body weight of 16.6 g were fed to satiety twice daily. Compared with the unsupplemented low-fishmeal group, supplementing either 0.5% methionine or 0.5% methionine plus 0.5% taurine to the low-fishmeal diet improved (P < 0.05) the growth, feed utilization, retention of dietary protein and lipids, and health of largemouth bass, reduced (P < 0.05) the occurrence of black skin syndrome from ~ 40 to ~ 10%. Histological sections of tissues from the fish with black skin syndrome showed retina degeneration, liver damage, and enteritis in the intestine. Compared with methionine supplementation, supplementing 0.5% taurine alone to the low-fishmeal diet did not affect the growth or feed efficiency of fish and had less beneficial effects (P < 0.05) on ameliorating the black skin syndrome. These results indicated that: (a) the basal low-fishmeal diet was inadequate in methionine or taurine; and (b) dietary supplementation with methionine was an effective method to improve the growth performance, feed efficiency, and health of largemouth bass. Further studies are warranted to understand the pathogenesis of the black skin syndrome in largemouth bass.

Construction of a synthetic microbial community for the biosynthesis of volatile sulfur compound by multi-module division of labor.

3-(Methylthio)-1-propanol, reminiscent of cauliflower and cooked vegetable aroma, is an important sulfur compound in Baijiu. It is important to develop a method to increase 3-(methylthio)-1-propanol content to improve flavor quality of products. In this study, a synthetic microbial community was employed to enhance the content of 3-(methylthio)-1-propanol by multi-module division of labor approach. Firstly, the synthetic pathway of 3-(methylthio)-1-propanol was reconstructed and classified into three modules. Later, the hyper producers in each module were isolated and negative interaction between the members was relieved. Finally, a synthetic microbial community was constructed using three species containing one hyper producer from each module. Furthermore, the transcription characteristics of the species in each module were validated by metatranscriptomic analysis. The constructed synthetic microbial community can be used to biosynthesize 3-(methylthio)-1-propanol for Baijiu. This work provided a novel and workable strategy to design synthetic microbial community to enhance the flavor feature of other fermented foods.

Yellow loosestrife (Lysimachia vulgaris var. davurica) ameliorates liver fibrosis in db/db mice with methionine- and choline-deficient diet-induced nonalcoholic steatohepatitis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH), a liver disease caused by a nonalcoholic fatty liver, is increasing in incidence worldwide. Owing to the complexity of its pathogenic mechanisms, there are no therapeutic agents for this disease yet. The ideal drug for NASH needs to concurrently decrease hepatic lipid accumulation and exert anti-inflammatory, antifibrotic, and antioxidative effects in the liver. Because of their multipurpose therapeutic effects, we considered that medicinal herbs are suitable for treating patients with NASH. METHODS: We determined the efficacy of the alcoholic extract of Lysimachia vulgaris var. davurica (LV), an edible medicinal herb, for NASH treatment. For inducing NASH, C57BLKS/J lar-Leprdb/Leprdb (db/db) male mice were fed with a methionine-choline deficient (MCD) diet ad libitum. After 3 weeks, the LV extract and a positive control (GFT505) were administered to mice by oral gavage for 3 weeks with a continued MCD diet as needed. RESULTS: In mice with diet-induced NASH, the LV extract could relieve the disease symptoms; that is, the extract ameliorated hepatic lipid accumulation and also showed antioxidative and anti-inflammatory effects. The LV extract also activated nuclear factor E2-related factor 2 (Nrf2) expression, leading to the upregulation of antioxidants and detoxification signaling. Moreover, the extract presented remarkable efficacy in alleviating liver fibrosis compared with GFT505. This difference was caused by significant LV extract-mediated reduction in the mRNA expression of fibrotic genes like the alpha-smooth muscle actin and collagen type 3 alpha 1. Reduction of fibrotic genes may thus relate with the downregulation of transforming growth factor beta (TGFß)/Smad signaling by LV extract administration. CONCLUSIONS: Lipid accumulation and inflammatory responses in the liver were alleviated by feeding LV extract to NASH-induced mice. Moreover, the LV extract strongly prevented liver fibrosis by blocking TGFß/Smad signaling. Hence, LV showed sufficient potency for use as a therapeutic agent against NASH.

Identification of methionine -rich insoluble proteins in the shell of the pearl oyster, Pinctada fucata.

The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.

A Biochemical Lanthanide-Encoding Approach Enables Quantitative Monitoring of the Bacterial Response to Vancomycin Treatment.

A pathogenic bacterium has its own mechanisms for not only pathogenic attack but also exogenous invasion defense, in which the bacterial cell wall is the front line of attack and defense. We developed a biochemical lanthanide-encoding approach to quantify the uncanonical d-amino acid (d-X) that was edited in a small proportion into the terminal acyl-d-Ala-d-X of nascent peptidoglycan UDP-MurNAc-pentapeptides in the bacterial cell wall. This approach overcomes the difficulties regarding quantification and accuracy issues encountered by the popular optical imaging and traditional high-performance liquid chromatography-based methods. Newly synthesized azide-d-Leu and ketone-d-Met were used together with alkynyl-d-Ala for their metabolic assembly and then bioorthogonally encoded by the correspondingly fabricated DBCO-DOTA-Gd, H2NO-DOTA-Eu, and azide-DOTA-Sm tags. This approach allows direct quantification of the d-X in situ in the cell wall using 158Gd, 153Eu, and 154Sm species-unspecific isotope dilution inductively coupled plasma mass spectrometry, avoiding any tedious and complex "cell-broken" pretreatment procedures that might induce racemization of the d-X. The obtained site-specific and accurate in situ information about the d-X enables quantitative monitoring of the bacterial response when Staphylococcus aureus meets vancomycin, showing that the amounts of azide-d-Leu and ketone-d-Met assembled are more important after determining the structure- and composition-dependent bacterial antibiotic resistance mechanisms. In addition, we found that the combined use of vancomycin and d-Ala restores the efficacy of vancomycin and might be a wise and simple way to combat vancomycin intermediate-resistant S. aureus.

Citrus reticulata Blanco peel extract ameliorates hepatic steatosis, oxidative stress and inflammation in HF and MCD diet-induced NASH C57BL/6 J mice.

Excessive lipid deposition, oxidative stress and inflammation in liver tissues are regarded as crucial inducers of nonalcoholic steatohepatitis (NASH), which is the most frequent chronic liver disease and closely related to obesity and insulin resistance. In this work, the preventive and therapeutic effects of Citrus reticulata Blanco (Jizigan) peel extract (JZE) on NASH induced by high fat (HF) diet and methionine choline-deficient (MCD) diet in C57BL/6 mice were investigated. We found that daily supplementation of JZE with an HF diet effectively ameliorated glucose tolerance and insulin resistance. In addition, the key indexes of lipid profiles, oxidative stress, hepatic steatosis and inflammatory factors were also ameliorated in both NASH mouse models. Furthermore, JZE treatment activated nuclear factor erythroid-2-related factor 2 (Nrf2) in the livers of diet- induced NASH mice. Our study suggests that JZE might alleviate NASH via the activation of Nrf2 signaling and that citrus Jizigan could be used as a dietary therapy for NASH and related metabolic syndrome.