A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Dietary supplementation of methionine, lysine, and tryptophan as possible modulators of growth, immune response, and disease resistance in striped catfish (Pangasius hypophthalmus).

The present study investigated the potential role of different essential amino acids (AA) in striped catfish (Pangasius hypophthalmus). Fish (initial weight = 17.91±0.27 g, n = 260) were fed with eight isonitrogenous (30%), and isolipidic diets (6%) formulated to include different combinations of tryptophan (Trp), methionine (Met), and lysine (Lys) (T0: Zero AA, T1: Trp, T2: Lys, T3: Met, T4: Trp+Met, T5: Lys+Trp, T6: Met+Lys, T7: Lys+Trp+Met) for eight weeks. The dose of amino acid supplementation, whether individually or in combination, was 5g of each amino acid per kg of diet. The trial comprised eight treatments, with each treatment consisted of three replicates (n = 10/replicate). At the end of the growth experiment, the highest total body weight, crude protein, digestive enzymatic activity, immune response, and amino acids level were observed in treatments supplemented with amino acids compared to T0. After the growth experiment, fish in all treatments were exposed to Staphylococcus aureus (5×105 CFU/ml). For bacterial challenge trial, the T0 treatment was designated as positive (+ve T0) and negative control (-ve T0). Following the S. aureus challenge, fish fed with amino acids showed a better response to reactive oxygen species and lipid peroxidation, as indicated by the increased levels of catalase and superoxide dismutase. Conversely, the concentration of malondialdehyde gradually decreased in all treatments compared to the +ve T0 treatment. It is concluded that supplementation of amino acids improved the growth, protein content, and immunocompetency against S. aureus in striped catfish. The most favorable outcomes in striped catfish were shown by fish supplemented with T7 diet. These essential amino acids hold potential as efficient supplements for use in the intensive aquaculture for striped catfish.

Relative biological efficacy of methionine hydroxy analogue-free acid compared to dl-methionine in the broiler chickens.

BACKGROUND: In the broiler's diets based on corn-soya bean meal, methionine (Met) and cystine (Cys), known as sulphur amino acids (SAAs), are the first limiting indispensable amino acids because of their limited presence, which are supplemented with different synthetic sources. Evaluation of the biological effectiveness of these sources can be important in their correct replacement, especially in the starter and growth diets. OBJECTIVES: The current study was done to assess the relative biological efficacy (RBE) of liquid Met hydroxy analogue-free acid (MHA-FA) in comparison with dl-Met (dl-Met) based on broiler performance traits at different levels of digestible SAA in the 1-11 (starter) and 11-25 (grower) days of age periods. METHODS: Two experiments were developed with treatments consisting of a basal diet without Met addition that met the nutrient and energy requirements of broilers with the exception of SAAs (Met + Cys) and five increasing Met doses for both sources (dl-Met and/or MHA-FA), resulting in digestible SAA concentrations from 0.62% to 1.02% of diet in the starter period (Trial 1) and 0.59% to 0.94% of diet in the grower period (Trial 2). The multi-linear regression model and slope ratio method were employed to calculate the RBE of MHA-FA compared with dl-Met for measured variables. RESULTS: In both experiments, the results obtained during the starter and grower periods with the different Met supplementations show significant growth responses to digestible SAAs levels. By increasing dietary dl-Met and/or MHA-FA levels, the growth performance traits and immune responses were improved (quadratic; p < 0.05). The RBE of MHA-FA compared to dl-Met on an equimolar basis was estimated 66%-89% (59%-79% on a weight-to-weight basis). CONCLUSIONS: It is concluded that the RBE of MHA-FA in comparison with dl-Met depends on broiler chicken age and what attribute is being evaluated.

A novel methionine nanoparticle in broiler chickens: Bioavailability and requirements.

This bioassay evaluated the bioavailability (RBV) of a novel nanoparticle of methionine (nano-Met) relative to DL-methionine (DL-Met), and estimated methionine requirements for both sources in starting broilers. Five supplemental levels (0.05, 0.10, 0.15, 0.20, and 0.25% of diet) of DL-Met or nano-Met were added to a basal diet containing 0.35% standardized ileal digestible (SID) methionine to create 11 experimental diets, including a basal diet and 10 experimental diets containing 0.40, 0.45, 0.50, 0.55, and 0.60% SID-Met, respectively. A total of 825 one-day-old male Ross 308 birds were randomly assigned to 11 treatments with 5 pen replicates and 15 birds each. Body weight gain (BWG), breast meat yield (BMY), and thigh meat yield (TMY) increased (P < 0.001) while feed conversion ratio (FCR) and malondialdehyde (MDA) concentration in meat samples decreased (P < 0.001) with increasing dietary methionine. Based on the slope-ratio method, the RBV of nano-Met relative to DL-Met for BWG, FCR, and TMY were 102 (48-155%; R2 = 0.71), 134 (68-201%; R2 = 0.77), and 110% (27-193%; R2 = 0.55), respectively. Considering the statistical accuracy of the spline models, the estimated values of DL-Met for maximum BWG and nano-Met for maximum TMY were 0.578% and 0.561%, respectively, which were statistically higher than those recommended for commercial settings. The highest effect size of supplemental methionine was on MDA (ƞ2p = 0.924), followed by FCR (ƞ2p = 0.578), BMY (ƞ2p = 0.575), BWG (ƞ2p = 0.430), and TMY (ƞ2p = 0.332), suggesting the potent antioxidant properties of methionine. Our findings suggest that reducing the particle size of DL-Met to nanoparticles could be a promising strategy to enhance the efficiency of methionine supplementation in broilers, an idea that requires further investigation in future research.

Dietary Methionine Restriction Improves Gut Health and Alters the Plasma Metabolomic Profile in Rats by Modulating the Composition of the Gut Microbiota.

Dietary methionine restriction (MetR) offers an integrated set of beneficial health effects, including delaying aging, extending health span, preventing fat accumulation, and reducing oxidative stress. This study aimed to investigate whether MetR exerts entero-protective effects by modulating intestinal flora, and the effect of MetR on plasma metabolites in rats. Rats were fed diets containing 0.86% methionine (CON group) and 0.17% methionine (MetR group) for 6 weeks. Several indicators of inflammation, gut microbiota, plasma metabolites, and intestinal barrier function were measured. 16S rRNA gene sequencing was used to analyze the cecal microbiota. The MetR diet reduced the plasma and colonic inflammatory factor levels. The MetR diet significantly improved intestinal barrier function by increasing the mRNA expression of tight junction proteins, such as zonula occludens (ZO)-1, claudin-3, and claudin-5. In addition, MetR significantly increased the levels of short-chain fatty acids (SCFAs) by increasing the abundance of SCFAs-producing Erysipclotxichaceae and Clostridium_sensu_stricto_1 and decreasing the abundance of pro-inflammatory bacteria Proteobacteria and Escherichia-Shigella. Furthermore, MetR reduced the plasma levels of taurochenodeoxycholate-7-sulfate, taurocholic acid, and tauro-ursodeoxycholic acid. Correlation analysis identified that colonic acetate, total colonic SCFAs, 8-acetylegelolide, collettiside I, 6-methyladenine, and cholic acid glucuronide showed a significant positive correlation with Clostridium_sensu_stricto_1 abundance but a significant negative correlation with Escherichia-Shigella and Enterococcus abundance. MetR improved gut health and altered the plasma metabolic profile by regulating the gut microbiota in rats.

Rescue of Methionine Dependence by Cobalamin in a Human Colorectal Cancer Cell Line.

Methionine dependence is a characteristic of most cancer cells where they are unable to proliferate when the essential amino acid methionine is replaced with its precursor homocysteine in the growing media. Normal cells, on the other hand, thrive under these conditions and are referred to as methionine-independent. The reaction that adds a methyl group from 5-methyltetrahydrofolate to homocysteine to regenerate methionine is catalyzed by the enzyme methionine synthase with the cofactor cobalamin (vitamin B12). However, decades of research have shown that methionine dependence in cancer is not due to a defect in the activity of methionine synthase. Cobalamin metabolism has been tied to the dependent phenotype in rare cell lines. We have identified a human colorectal cancer cell line in which the cells regain the ability to proliferation in methionine-free, L-homocystine-supplemented media when cyanocobalamin is supplemented at a level of 1 µg/mL. In human SW48 cells, methionine replacement with L-homocystine does not induce any measurable increase in apoptosis or reactive oxygen species production in this cell line. Rather, proliferation is halted, then restored in the presence of cyanocobalamin. Our data show that supplementation with cyanocobalamin prevents the activation of the integrated stress response (ISR) in methionine-deprived media in this cell line. The ISR-associated cell cycle arrest, characteristic of methionine-dependence in cancer, is also prevented, leading to the continuation of proliferation in methionine-deprived SW48 cells with cobalamin. Our results highlight differences between cancer cell lines in the response to cobalamin supplementation in the context of methionine dependence.

Methionine biosynthetic genes and methionine sulfoxide reductase A are required for Rhizoctonia solani AG1-IA to cause sheath blight disease in rice.

Rhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate-active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative-stress-enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double-stranded RNA-mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease-causing ability of Rs_MET13-silenced R. solani and facilitates its growth on 10 mM H2O2-containing minimal-media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H2O2 treatment and also during infection in rice. Rs_MsrA-silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray-induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice.

[Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice].

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1ß, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1ß in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1ß, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1ß in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.

Bifidobacterium alleviate metabolic disorders via converting methionine to 5'-methylthioadenosine.

Dietary patterns and corresponding gut microbiota profiles are associated with various health conditions. A diet rich in polyphenols, primarily plant-based, has been shown to promote the growth of probiotic bacteria in the gastrointestinal tract, subsequently reducing the risk of metabolic disorders in the host. The beneficial effects of these bacteria are largely due to the specific metabolites they produce, such as short-chain fatty acids and membrane proteins. In this study, we employed a metabolomics-guided bioactive metabolite identification platform that included bioactivity testing using in vitro and in vivo assays to discover a bioactive metabolite produced from probiotic bacteria. Through this approach, we identified 5'-methylthioadenosine (MTA) as a probiotic bacterial-derived metabolite with anti-obesity properties. Furthermore, our findings indicate that MTA administration has several regulatory impacts on liver functions, including modulating fatty acid synthesis and glucose metabolism. The present study elucidates the intricate interplay between dietary habits, gut microbiota, and their resultant metabolites.

Impact of [11C]methionine PET with Bayesian penalized likelihood reconstruction on glioma grades based on new WHO 2021 classification.

OBJECTIVE: The uptake of [11C]methionine in positron emission tomography (PET) imaging overlapped in earlier images of tumors. Bayesian penalized likelihood (BPL) reconstruction increases the quantitative values of tumors compared with conventional ordered subset-expectation maximization (OSEM). The present study aimed to grade glioma malignancy based on the new WHO 2021 classification using [11C]methionine PET images reconstructed using BPL. METHODS: We categorized 32 gliomas in 28 patients as grades 2/3 (n = 15) and 4 (n = 17) based on the WHO 2021 classification. All [11C]methionine images were reconstructed using OSEM + time-of-flight (TOF) and BPL + TOF (ß = 200). Maximum standardized uptake value (SUVmax) and tumor-to-normal tissue ratio (T/Nmax) were measured at each lesion. RESULTS: The mean SUVmax was 4.65 and 4.93 in grade 2/3 and 6.38 and 7.11 in grade 4, and the mean T/Nmax was 7.08 and 7.22 in grade 2/3 and 9.30 and 10.19 in grade 4 for OSEM and BPL, respectively. The BPL significantly increased these values in grade 4 gliomas. The area under the receiver operator characteristic (ROC) curve (AUC) for SUVmax was the highest (0.792) using BPL. CONCLUSIONS: The BPL increased mean SUVmax and mean T/Nmax in lesions with higher contrast such as grade 4 glioma. The discrimination power between grades 2/3 and 4 in SUVmax was also increased using [11C]methionine PET images reconstructed with BPL.

Methionyl-Methionine Dipeptide Enhances Mammogenesis and Lactogenesis by Suppressing the Expression of a Novel Long Noncoding RNA MGPNCR to Inhibit eIF4B Dephosphorylation.

Previous research has demonstrated that in pregnant mice deficient in l-methionine (Met), the mixture of the dipeptide l-methionyl-l-methionine (Met-Met) with Met was more effective than Met alone in promoting mammogenesis and lactogenesis. This study aimed to investigate the role of a novel long noncoding RNA (lncRNA), named mammary gland proliferation-associated lncRNA (MGPNCR), in these processes. Transcriptomic analysis of mammary tissues from Met-deficient mice, supplemented either with a Met-Met/Met mixture or with Met alone, revealed significantly higher MGPNCR expression in the Met group compared to the mixture group, a finding recapitulated in a mammary epithelial cell model. Our findings suggested that MGPNCR hindered mammogenesis and milk protein synthesis by binding to eukaryotic initiation factor 4B (eIF4B). This interaction promoted the dephosphorylation of eIF4B at serine-422 by enhancing its association with protein phosphatase 2A (PP2A). Our study sheds light on the regulatory mechanisms of lncRNA-mediated dipeptide effects on mammary cell proliferation and milk protein synthesis. These insights underscore the potential benefits of utilizing dipeptides to improve milk protein in animals and potentially in humans.

Systemic intracellular analysis for balancing complex biosynthesis in a transcriptionally deregulated Escherichia coli l-Methionine producer.

l-Methionine (l-Met) has gained remarkable interest due to its multifaceted and versatile applications in the fields of nutrition, pharmaceuticals and clinical practice. In this study, the fluxes of the challenging l-Met biosynthesis in the producer strain Escherichia coli (E. coli) DM2853 were fine-tuned to enable improved l-Met production. The potential bottlenecks identified in sulfur assimilation and l-Met synthesis downstream of O-succinyl-l-homoserine (OSHS) were addressed by overexpressing glutaredoxin 1 (grxA), thiosulfate sulfurtransferase (pspE) and O-succinylhomoserine lyase (metB). Although deemed as a straightforward target for improving glucose-to-Met conversion, the yields remained at approximately 12%-13% (g/g). Instead, intracellular l-Met pools increased by up to four-fold with accelerated kinetics. Overexpression of the Met exporter ygaZH may serve as a proper valve for releasing the rising internal Met pressure. Interestingly, the export kinetics revealed maximum saturated export rates already at low growth rates. This scenario is particularly advantageous for large-scale fermentation when product formation is ideally uncoupled from biomass formation to achieve maximum performance within the technical limits of large-scale bioreactors.

Tenascin-X is increased with decreased expression of miR-378a-5p and miR-486-5p in mice fed a methionine-choline-deficient diet that induces hepatic fibrosis.

We previously reported that tenascin-X (Tnxb) aggravates hepatic fibrosis in mice fed a high-fat and high-cholesterol diet with high levels of phosphorus and calcium (HFCD). In this study, we investigated Tnxb expression in livers with fibrosis caused by administration of a methionine-chorine-deficient (MCD) diet in mice. Whole transcriptome analysis showed that Tnxb was one of the genes with increased expression in livers of MCD diet-fed mice compared with that in livers of normal diet (ND)-fed mice. In microarray and subsequent microRNA (miRNA) network analyses, miR-378a-5p and miR-486-5p were identified in livers of MCD diet-fed mice as downregulated miRNAs, which have their predicted target sites in the 3' untranslated region of Tnxb mRNA and might suppress the translation of Tnxb mRNA. RT-qPCR analyses of livers of MCD diet-fed mice compared with livers of ND-fed mice verified the upregulation of Tnxb and fibrosis-triggering genes and conversely the downregulation of miR-378a-5p and miR-486-5p. Overexpression of miR-378a-5p and miR-486-5p resulted in decreased level not only of the FLAG-tagged fibrinogen-like domain of Tnxb protein (FLAG-mTNX-FG) but also of endogenous Tnxb protein in murine cultured cells. These results indicate that expression of Tnxb is regulated by miR-378a-5p and miR-486-5p in hepatic fibrosis following MCD diet feeding.

KLF10/CBS increases the sensitivity of gastric carcinoma cells to methionine restriction by promoting sulfur transfer pathway.

Gastric cancer metastasis is a major cause of poor prognosis. Our previous research showed that methionine restriction (MR) lowers the invasiveness and motility of gastric carcinoma. In this study, we investigated the particular mechanisms of MR on gastric carcinoma metastasis. In vitro, gastric carcinoma cells (AGS, SNU-5, MKN7, KATO III, SNU-1, and MKN45) were grown in an MR medium for 24 h. In vivo, BALB/c mice were given a methionine-free (Met-) diet. Transwell assays were used to investigate cell invasion and migration. The amounts of Krüppel like factor 10 (KLF10) and cystathionine ß-synthase (CBS) were determined using quantitative real-time PCR and Western blot. To determine the relationship between KLF10 and CBS, chromatin immunoprecipitation and a dual-luciferase reporter experiment were used. Hematoxylin-eosin staining was used to detect lung metastasis. Liquid chromatography-mass spectrometry was used to determine cystathionine content. MR therapy had varying effects on the invasion and migration of gastric carcinoma cells AGS, SNU-5, MKN7, KATO III, SNU-1, and MKN45. KLF10 was highly expressed in AGS cells but poorly expressed in KATO III cells. KLF10 improved MR's ability to prevent gastric carcinoma cell invasion and migration. In addition, KLF10 may interact with CBS, facilitating transcription. Further detection revealed that inhibiting the KLF10/CBS-mediated trans-sulfur pathway lowered Met-'s inhibitory effect on lung metastasis development. KLF10 transcription activated CBS, accelerated the trans-sulfur pathway, and increased gastric carcinoma cells' susceptibility to MR.

Reduction of Tumor Biomarkers from very High to Normal and Extensive Metastatic Lesions to Undetectability in a Patient With Stage IV HER2-positive Breast Cancer Treated With Low-dose Trastuzumab Deruxtecan in Combination With Oral Recombinant Methioninase and a Low-methionine Diet.

BACKGROUND/AIM: Breast cancer is the most common and the deadliest cancer among women in the world. Treatment options for HER2-positive metastatic breast cancer patients are limited. Trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate (ADC), has recently been introduced as second-line chemotherapy for HER2-positive metastatic breast cancer. The aim of the present study was to evaluate the efficacy of methionine restriction with oral recombinant methioninase (o-rMETase) and a low-methionine diet combined with T-DXd, on a patient with HER2-positive recurrent stage IV breast cancer. CASE REPORT: A 66-year-old female was diagnosed with HER2-positive metastatic breast cancer. Computed tomography (CT) indicated peritoneal dissemination, thickening of the sigmoid colon and splenic flexure and widespread bone metastases. The patient was previously treated with fulvestrant, trastuzumab, pertuzumab, paclitaxel and capecitabine which were ineffective. T-DXd was administered as a second-line chemotherapy. Since the patient experienced strong side effects, the dose of T-Dxd was decreased. The patient began methionine restriction using o-rMETase and a low-methionine diet along with T-DXd. After the start of the combined treatment, CA15-3 and CA27.29, tumor markers for breast cancer, decreased rapidly from a very high level. The levels of both tumor markers are currently normal. Additionally, peritoneal-dissemination nodules, ascites and the thickness of the sigmoid colon and splenic flexure are no longer detected on CT. The patient maintains a high performance status, without severe side effects of the combination treatment. CONCLUSION: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with T-DXd as second-line chemotherapy, was highly effective in a patient with HER2-positive stage IV breast cancer.

Radical S-Adenosyl-l-Methionine Enzyme PylB: A C-Centered Radical to Convert l-Lysine into (3R)-3-Methyl-d-Ornithine.

PylB is a radical S-adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3R)-3-methyl-d-ornithine, a precursor in the biosynthesis of the 22nd proteogenic amino acid pyrrolysine. This protein highly resembles that of the radical SAM tyrosine and tryptophan lyases, which activate their substrate by abstracting a H atom from the amino-nitrogen position. Here, combining in vitro assays, analytical methods, electron paramagnetic resonance spectroscopy, and theoretical methods, we demonstrated that instead, PylB activates its substrate by abstracting a H atom from the Cγ position of l-lysine to afford the radical-based ß-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3R)-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5'-deoxyadenosyl radical. Finally, we identified significant in vitro production of 5'-thioadenosine, an unexpected shunt product that we propose to result from the quenching of the 5'-deoxyadenosyl radical species by the nearby [Fe4S4] cluster.

High methionine diet mediated oxidative stress and proteasome impairment causes toxicity in liver.

Methionine (Met) rich diet inducing oxidative stress is reported to alter many organs. Proteasome as a regulator of oxidative stress can be targeted. This study was performed to investigate if excessive methionine supplementation causes hepatotoxicity related to proteasome dysfunction under endogenous oxidative stress in rats. Male Wistar albino rats (n = 16) were divided into controls and treated groups. The treated rats (n = 08) received orally L-methionine (1 g/kg/day) for 21 days. Total homocysteine (tHcy), total oxidant status (TOS), total antioxidant status (TAS), hepatic enzymes levels: aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), with total bilirubin (TBil), albumin (Alb), and C-reactive protein (CRP) were determined in plasma by biochemical assays. Liver supernatants were used for malondialdehyde (MDA), protein carbonyls (PC), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), 20S proteasome activities and their subunits expression, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) evaluation by appropriate methods and light microscopy for liver histological examination. Methionine treatment increased homocysteine, TOS, oxidative stress index (OSI), MDA and PC but decreased TAS, GSH, CAT, SOD, GPx with the 20S proteasome activities and their ß subunits expression. Liver proteins: AST, ALT, LDH, ALP, TBil and CRP were increased but Alb was decreased. Liver histology was also altered. An increase in liver TNF-α and IL-6 levels were observed. These findings indicated that methionine supplementation associated oxidative stress and proteasome dysfunction, caused hepatotoxicity and inflammation in rat. Further investigations should be to better understand the relation between methionine, oxidative stress, proteasome, and liver injuries.

Highly chromophoric fluorescent-labeled methionyl-initiator tRNAs applicable in living cells.

Fluorescent initiator tRNAs (tRNAi) play a crucial role in studying protein synthesis, yet generating highly fluorescent tRNAi complexes remains challenging. We present an optimized strategy to effectively generate highly fluorescent initiator-tRNA complexes in living cells. Our strategy allows the generation of Fluo-Met-tRNAiMet complexes. These complexes can have highly chromogenic N-terminal labeling. For generating such complexes, we use either purified fluorescent methionine (PFM) or non-purified fluorescently labeled methionine (NPFM). Furthermore, PFM promotes the active generation of endogenous tRNAi in cells, leading to highly efficient Fluo-Met-tRNAiMet complexes. Finally, PFM-tRNAiMet complexes also facilitate the visualization of native fluorescently labeled Tat binding to beads. This demonstrates the potential of our approach to advance precision protein engineering and biotechnology applications.

Simultaneous Determination of One-Carbon Folate Metabolites and One-Carbon-Related Amino Acids in Biological Samples Using a UHPLC-MS/MS Method.

One-carbon folate metabolites and one-carbon-related amino acids play an important role in human physiology, and their detection in biological samples is essential. However, poor stability as well as low concentrations and occurrence in different species in various biological samples make their quantification very challenging. The aim of this study was to develop a simple, fast, and sensitive ultra-high-performance liquid chromatography MS/MS (UHPLC-MS/MS) method for the simultaneous quantification of various one-carbon folate metabolites (folic acid (FA), tetrahydrofolic acid (THF), p-aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CH3THF), 10-formylfolic acid (10-CHOFA), 5,10-methenyl-5,6,7,8-tetrahydrofolic acid (5,10-CH+-THF), and 4-α-hydroxy-5-methyltetrahydrofolate (hmTHF)) and one-carbon-related amino acids (homocysteine (Hcy), methionine (Met), S-ade-L-homocysteine (SAH), and S-ade-L-methionine (SAM)). The method was standardized and validated by determining the selectivity, carryover, limits of detection, limits of quantitation, linearity, precision, accuracy, recovery, and matrix effects. The extraction methods were optimized with respect to several factors: protease-amylase treatment on embryos, deconjugation time, methanol precipitation, and proteins' isoelectric point precipitation on the folate recovery. Ten one-carbon folate metabolites and four one-carbon-related amino acids were detected using the UHPLC-MS/MS technique in various biological samples. The measured values of folate in human plasma, serum, and whole blood (WB) lay within the concentration range for normal donors. The contents of each analyte in mouse plasma were as follows: pABG (864.0 nmol/L), 5-CH3THF (202.2 nmol/L), hmTHF (122.2 nmol/L), Met (8.63 µmol/L), and SAH (0.06 µmol/L). The concentration of each analyte in mouse embryos were as follows: SAM (1.09 µg/g), SAH (0.13 µg/g), Met (16.5 µg/g), 5,10-CH+THF (74.3 ng/g), pABG (20.6 ng/g), and 5-CH3THF (185.4 ng/g). A simple and rapid sample preparation and UHPLC-MS/MS method was developed and validated for the simultaneous determination of the one-carbon-related folate metabolites and one-carbon-related amino acids in different biological samples.