The association of protein-bound methionine sulfoxide with proteomic basis for aging in beech seeds.

BACKGROUND: European beech (Fagus sylvatica L.) trees produce seeds irregularly; therefore, it is necessary to store beech seeds for forestation. Despite the acquisition of desiccation tolerance during development, beech seeds are classified as intermediate because they lose viability during long-term storage faster than typical orthodox seeds. In this study, beech seeds stored for short (3 years) or long (20 years) periods under optimal conditions and displaying 92 and 30% germination capacity, respectively, were compared. RESULTS: Aged seeds displayed increased membrane damage, manifested as electrolyte leakage and lipid peroxidation levels. Analyses have been based on embryonic axes, which contained higher levels of reactive oxygen species (ROS) and higher levels of protein-bound methionine sulfoxide (MetO) in aged seeds. Using label-free quantitative proteomics, 3,949 proteins were identified, of which 2,442 were reliably quantified pointing to 24 more abundant proteins and 35 less abundant proteins in beech seeds under long-term storage conditions. Functional analyses based on gene ontology annotations revealed that nucleic acid binding activity (molecular function), ribosome organization or biogenesis and transmembrane transport (cellular processes), translational proteins (protein class) and membranous anatomical entities (cellular compartment) were affected in aged seeds. To verify whether MetO, the oxidative posttranslational modification of proteins that can be reversed via the action of methionine sulfoxide reductase (Msr) enzymes, is involved in the aging of beech seeds, we identified and quantified 226 MetO-containing proteins, among which 9 and 19 exhibited significantly up- and downregulated MetO levels, respectively, in beech seeds under long-term storage conditions. Several Msr isoforms were identified and recognized as MsrA1-like, MsrA4, MsrB5 and MsrB5-like in beech seeds. Only MsrA1-like displayed decreased abundance in aged seeds. CONCLUSIONS: We demonstrated that the loss of membrane integrity reflected in the elevated abundance of membrane proteins had a higher impact on seed aging progress than the MetO/Msr system. Proteome analyses enabled us to propose protein Sec61 and glyceraldehyde-3-phosphate dehydrogenase as potential longevity modulators in beech seeds.

8-Cl-Ado and 8-NH2-Ado synergize with venetoclax to target the methionine-MAT2A-SAM axis in acute myeloid leukemia.

Targeting the metabolic dependencies of acute myeloid leukemia (AML) cells is a promising therapeutical strategy. In particular, the cysteine and methionine metabolism pathway (C/M) is significantly altered in AML cells compared to healthy blood cells. Moreover, methionine has been identified as one of the dominant amino acid dependencies of AML cells. Through RNA-seq, we found that the two nucleoside analogs 8-chloro-adenosine (8CA) and 8-amino-adenosine (8AA) significantly suppress the C/M pathway in AML cells, and methionine-adenosyltransferase-2A (MAT2A) is one of most significantly downregulated genes. Additionally, mass spectrometry analysis revealed that Venetoclax (VEN), a BCL-2 inhibitor recently approved by the FDA for AML treatment, significantly decreases the intracellular level of methionine in AML cells. Based on these findings, we hypothesized that combining 8CA or 8AA with VEN can efficiently target the Methionine-MAT2A-S-adenosyl-methionine (SAM) axis in AML. Our results demonstrate that VEN and 8CA/8AA synergistically decrease the SAM biosynthesis and effectively target AML cells both in vivo and in vitro. These findings suggest the promising potential of combining 8CA/8AA and VEN for AML treatment by inhibiting Methionine-MAT2A-SAM axis and provide a strong rationale for our recently activated clinical trial.

Characterization of unique EDTA-insensitive methylthioalkylmalate synthase from Eutrema japonicum and its potential application in synthetic biology.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum, commonly referred to as Japanese wasabi. Eutremajaponicum MAMs (EjMAMs) were expressed in an Escherichiacoli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l-methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform.

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.

Modes of Interactions with DNA/HSA Biomolecules and Comparative Cytotoxic Studies of Newly Synthesized Mononuclear Zinc(II) and Heteronuclear Platinum(II)/Zinc(II) Complexes toward Colorectal Cancer Cells.

A series of mono- and heteronuclear platinum(II) and zinc(II) complexes with 4,4',4″-tri-tert-butyl-2,2':6',2″-terpyridine ligand were synthesized and characterized. The DNA and protein binding properties of [ZnCl2(terpytBu)] (C1), [{cis-PtCl(NH3)2(µ-pyrazine)ZnCl(terpytBu)}](ClO4)2 (C2), [{trans-PtCl(NH3)2(µ-pyrazine)ZnCl(terpytBu)}](ClO4)2 (C3), [{cis-PtCl(NH3)2(µ-4,4'-bipyridyl)ZnCl(terpytBu)}](CIO4)2 (C4) and [{trans-PtCl(NH3)2(µ-4,4'-bipyridyl)ZnCl(terpytBu)}](CIO4)2 (C5) (where terpytBu = 4,4',4″-tri-tert-butyl-2,2':6',2″-terpyridine), were investigated by electronic absorption, fluorescence spectroscopic, and molecular docking methods. Complexes featuring transplatin exhibited lower Kb and Ksv constant values compared to cisplatin analogs. The lowest Ksv value belonged to complex C1, while C4 exhibited the highest. Molecular docking studies reveal that the binding of complex C1 to DNA is due to van der Waals forces, while that of C2-C5 is due to conventional hydrogen bonds and van der Waals forces. The tested complexes exhibited variable cytotoxicity toward mouse colorectal carcinoma (CT26), human colorectal carcinoma (HCT116 and SW480), and non-cancerous mouse mesenchymal stem cells (mMSC). Particularly, the mononuclear C1 complex showed pronounced selectivity toward cancer cells over non-cancerous mMSC. The C1 complex notably induced apoptosis in CT26 cells, effectively arrested the cell cycle in the G0/G1 phase, and selectively down-regulated Cyclin D.

Different methionine to cysteine supplementation ratios altered bone quality of broilers with or without Eimeria challenge assessed by dual energy X-ray absorptiometry and microtomography.

Despite the acknowledged significance of nutrition in bone development, effects of methionine (Met) and cysteine (Cys) on bone quality remain under-researched, particularly during Eimeria challenge. We investigated the effects of different supplemental Met to Cys ratios (MCR) on bone quality of broilers under Eimeria challenge. A total of 720 fourteen-day old Cobb500 broilers were allocated into a 5 × 2 factorial arrangement. Five diets with Met and Cys supplemented at MCR of 100:0, 75:25, 50:50, 25:75, and 0:100 were fed to the birds with or without Eimeria challenge. Body composition was measured by dual energy x-ray absorptiometry, and the femur bone characteristics were assessed by microtomography. Data were analyzed by two-way ANOVA and orthogonal polynomial contrast. The results reaffirmed the detrimental effects of Eimeria challenge on bone quality. On 9 d post inoculation (DPI), significant interaction effects were found for whole body bone mineral content (BMC), lean tissue weight, and body weight (P < 0.05); in the nonchallenged group (NCG), these parameters linearly decreased as MCR decreased (P < 0.05). In the challenged group (CG), body weight and lean tissue weight were unaffected by MCR, and BMC linearly increased as MCR decreased (P < 0.05). For the cortical bone of femoral metaphysis on 6 DPI, bone mineral density (BMD) linearly increased as MCR decreased (P < 0.05). Bone volume to tissue volume ratio (BV/TV) in the CG linearly increased as MCR decreased (P < 0.05). On 9 DPI, BMC and TV linearly increased as MCR decreased (P < 0.05) in the NCG. BMD and BV/TV changed quadratically as MCR decreased (P < 0.05). For the trabecular bone of femoral metaphysis on 9 DPI, BV/TV, and trabecular number linearly increased as MCR decreased (P < 0.05) in the NCG. For the femoral diaphysis, BV, TV, BMC on 6 DPI, and BMD on 9 DPI linearly increased as MCR decreased (P < 0.05). In conclusion, this study showed that both Eimeria challenge and varying supplemental MCR could influence bone quality of broilers.

Resolution of N-acetyl-DL-methionine methyl ester by the lipase from Brucella thiophenivorans.

In this study, lipase-catalyzed resolution of N-acetyl-DL-methionine methyl ester (N-Ac-DL-MetOMe) was evaluated. A lipase from Brucella thiophenivorans was prone to exhibit high activity and excellent enantioselectivity toward N-Ac-DL-MetOMe to produce the key chiral intermediate N-acetyl-L-methionine methyl ester (N-Ac-L-MetOMe). The results showed that the enzymatic reaction was carried out in 100 g/L racemic substrate for 2 h, the conversion reached 51.3%, the enantiomeric excess value N-Ac-L-MetOMe exceeded 99%, and the enantiomeric ratio value >200. Therefore, the lipase from B. thiophenivorans has potential prospects for the resolution of N-Ac-DL-MetOMe to produce the important intermediate N-Ac-L-MetOMe.

Anaerobic respiration of host-derived methionine sulfoxide protects intracellular Salmonella from the phagocyte NADPH oxidase.

Intracellular Salmonella experiencing oxidative stress downregulates aerobic respiration. To maintain cellular energetics during periods of oxidative stress, intracellular Salmonella must utilize terminal electron acceptors of lower energetic value than molecular oxygen. We show here that intracellular Salmonella undergoes anaerobic respiration during adaptation to the respiratory burst of the phagocyte NADPH oxidase in macrophages and in mice. Reactive oxygen species generated by phagocytes oxidize methionine, generating methionine sulfoxide. Anaerobic Salmonella uses the molybdenum cofactor-containing DmsABC enzymatic complex to reduce methionine sulfoxide. The enzymatic activity of the methionine sulfoxide reductase DmsABC helps Salmonella maintain an alkaline cytoplasm that supports the synthesis of the antioxidant hydrogen sulfide via cysteine desulfuration while providing a source of methionine and fostering redox balancing by associated dehydrogenases. Our investigations demonstrate that nontyphoidal Salmonella responding to oxidative stress exploits the anaerobic metabolism associated with dmsABC gene products, a pathway that has accrued inactivating mutations in human-adapted typhoidal serovars.

Synthesis and biological evaluation of selective Pepstatin based trifluoromethylated inhibitors of Cathepsin D.

Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two non-proteinogenic statin residues, is among the most potent inhibitor of CD but lacks selectivity and suffers from poor bioavailability. Eight analogues of Pepstatin A, were synthesized, replacing residues in P3 or P1 position by non-canonical (S)- and (R)-α-Trifluoromethyl Alanine (TfmAla), (S)- and (R)-Trifluoromethionine (TFM) or non-natural d-Valine. The biological activities of those analogues were quantified on isolated CD and Pepsin by fluorescence-based assay (FRET) and cytotoxicity of the best fluorinated inhibitors was evaluated on SKOV3 ovarian cancer cell line. (R)-TFM based analog of Pepstatin A (compound 6) returned a sub-nanomolar IC50 against CD and an increased selectivity. Molecular Docking experiments could partially rationalize these results. Stabilized inhibitor 6 in the catalytic pocket of CD showed strong hydrophobic interactions of the long and flexible TFM side chain with lipophilic residues of S1 and S3 sub-pockets of the catalytic pocket. The newly synthesized inhibitors returned no cytotoxicity at IC50 concentrations on SKOV3 cancer cells, however the compounds derived from (S)-TfmAla and (R)-TFM led to modifications of cells morphologies, associated with altered organization of F-actin and extracellular Fibronectin.

Maternal Mediterranean-Style Diet Adherence during Pregnancy and Metabolomic Signature in Postpartum Plasma: Findings from the Boston Birth Cohort.

BACKGROUND: The health benefits of a Mediterranean-style diet (MSD) are well observed, but the underlying mechanisms are unclear. Metabolomic profiling offers a systematic approach for identifying which metabolic biomarkers and pathways might be affected by an MSD. OBJECTIVES: This study aimed to identify postpartum plasma metabolites that are associated with MSD adherence during pregnancy and to further test whether these identified metabolites may vary by maternal characteristics. METHODS: We analyzed data from 1410 mothers enrolled in the Boston Birth Cohort (BBC). A maternal food frequency questionnaire (FFQ) was administered and epidemiologic information was obtained via an in-person standard questionnaire interview within 24-72 h postpartum. Maternal clinical information was extracted from electronic medical records. A Mediterranean-style diet score (MSDS) was calculated using responses to the FFQ. Metabolomic profiling in postpartum plasma was conducted by liquid chromatography-MS. Linear regression models were used to assess the associations of each metabolite with an MSDS, adjusting for covariates. RESULTS: Among the 380 postpartum plasma metabolites analyzed, 24 were associated with MSDS during pregnancy (false discovery rate < 0.05). Of 24 MSDS-associated metabolites, 19 were lipids [for example, triacylglycerols, phosphatidylcholines (PCs), PC plasmalogen, phosphatidylserine, and phosphatidylethanolamine]; others were amino acids (methionine sulfoxide and threonine), tropane (nor-psi-tropine), vitamin (vitamin A), and nucleotide (adenosine). The association of adenosine and methionine sulfoxide with MSDS differed by race (P-interaction = 0.033) and maternal overweight or obesity status (P-interaction = 0.021), respectively. CONCLUSIONS: In the BBC, we identified 24 postpartum plasma metabolites associated with MSDS during pregnancy. The associations of the 2 metabolites varied by maternal race and BMI. This study provides a new insight into dietary effects on health under the skin. More studies are needed to better understand the metabolic pathways underlying the short- and long-term health benefits of an MSD during pregnancy.

Dose-responses of zinc as zinc-methionine supplements on antioxidant status, hematological parameters, immune response and the expression of IL-4 and IL-6 genes of ewes in the hot season.

This study was implemented to evaluate the effects of different zinc doses as Zinc-Met supplement (Zinpro®) on the antioxidant status, blood immune cells, antibody titers, and the expression of IL-4 and IL-6 genes of ewes in the hot season. In a completely randomized design, 24 ewes were assigned to treatments as follow: 0, 15, 30 and 45 mg/kg zinc as Zinc-Met supplementation for 40 days in region with 40 °C and vaccinated against food-and-mouth disease as an immune challenge at day 30, and then blood samples were collected on day 40. Ewes were fed a basal diet containing 29.9 mg zinc/kg. The highest activity of the antioxidant enzyme and the lowest lipid peroxidation values were found in ewes receiving 30 and 45 mg/kg zinc following a linear trend. The highest lymphocytes count and antibody titers were found in ewes received 30 mg zinc/kg. There were no significant differences among treatments for the relative expression of genes. In overall, zinc supplementation non-significantly up-regulate interleukin-4 and down-regulate interleukin-6. It was concluded that zinc supplementation as Zinc-Met could enhance the antioxidant status and immune response of ewes under heat stress; supplementation of diet with 30 mg zinc/kg (300 mg/kg Zinpro®) appeared to be the most effective dose.

Angioprotective and Follicle-Stimulating Effects of Administration of Bone Marrow Mesenchymal Stromal Cells in Chronic Inflammation of Internal Reproductive Organs.

Induced inflammation of reproductive organs in female Wistar rats was associated with an increase in the diameters of arteries and veins and number of blood vessels in the ovary medulla in combination with an increase in the number of lymphatic vessels; these changes were accompanied by reduction of the ovarian reserve and number of yellow bodies. Intravenous and submucosal injection of bone marrow multipotent mesenchymal stromal cells (BМ-ММSC) led to further increase in the diameters of arteries and veins and number of primordial and primary follicles. The injection of conditioned medium of BМ-ММSC cultures generally produced the same effects, which could demonstrate the secretory mechanisms of their influence on local angiogenesis and folliculogenesis.

Redox-Responsive Polymer Nanoreactors Based on Methionine Sulfoxide for Monitoring Cell Adhesion.

Expanding the category of redox-responsive monomers suitable for enzymolysis efficiency regulation and application to living biosystems is a prerequisite to complementing the fabrication of stimuli-responsive polymer nanoreactors. However, the development of redox-responsive monomers is severely limited by chemical oxidation and low biocompatibility. This work presents a protocol for overcoming this problem by the self-assembly of redox-responsive polymer nanoreactors containing segments of water-soluble methionine sulfoxide residues and poly(styrene-co-maleic anhydride-l-methionine), and by immobilizing α-l-fucosidase into the nanoreactors. These nanoreactors demonstrate highly selective responses to a mild redox triggered by H2O2 from the initial state (VO) to an oxidation state (VO1), and are reduced by methionine sulfoxide reductase A to mold the VO' state. It resulted in significantly enhanced enzymolysis efficiency and maximal reaction rates 8.1-fold (VO) and 23.3-fold (VO1) higher than those of the free enzyme. Moreover, cell adhesion was evaluated by the highly selective determination of l-fucose on cell surfaces. Using a combination of chemical oxidation and enzymatic reduction, this work achieves reiterative enzymolysis efficiency regulation of polymer nanoreactors, which has great potential for the construction of redox-responsive nanoreactors and for monitoring cell adhesion.

Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on milk fat, rumen environment and biohydrogenation, and rumen protozoa in lactating cows fed diets with increased risk for milk fat depression.

Biohydrogenation-induced milk fat depression (MFD) is a reduction in milk fat synthesis caused by bioactive fatty acids (FA) produced during altered ruminal microbial metabolism of unsaturated FA. The methionine analog 2-hydroxy-4-(methylthio)butanoate (HMTBa) has been shown to reduce the shift to the alternate biohydrogenation pathway and maintain higher milk fat yield in high-producing cows fed diets lower in fiber and higher in unsaturated FA. The objective of this experiment was to verify the effect of HMTBa on biohydrogenation-induced MFD and investigate associated changes in rumen environment and fermentation. Twenty-two rumen cannulated high-producing Holstein cows [168 ± 66 d in milk; 42 ± 7 kg of milk/d (mean ± standard deviation)] were used in a randomized design performed in 2 blocks (1 = 14 cows, 2 = 8 cows). Treatments were control (corn carrier) and HMTBa (0.1% of diet dry matter). The experiment included a 7-d covariate period followed by 3 phases that fed diets with increasing risk of MFD. The diet during the covariate and low-risk phase (7 d) was 32% neutral detergent fiber with no additional oil. The diet during the moderate-risk phase (17 d) was 29% neutral detergent fiber with 0.75% soybean oil. Soybean oil was increased to 1.5% for the last 4 d. The statistical model included the random effect of block and time course data were analyzed with repeated measures including the random effect of cow and tested the interaction of treatment and time. There was no effect of block or interaction of block and treatment or time. There was no overall effect of treatment or treatment by time interaction for dry matter intake, milk yield, and milk protein concentration and yield. Overall, HMTBa increased milk fat percent (3.2 vs. 3.6%) and yield (1,342 vs. 1,543 g/d) and there was no interaction of treatment and dietary phase. Additionally, HMTBa decreased the concentration of trans-10 18:1 in milk fat and rumen digesta. Average total ruminal concentration of volatile FA across the day and total-tract dry matter and fiber digestibility were not affected by HMTBa, but HMTBa increased average rumen butyrate and decreased propionate concentration and increased total protozoa abundance. Additionally, HMTBa increased the fractional rate of α-linoleic acid clearance from the rumen following a bolus predominantly driven by a difference in the first 30 min. Plasma insulin was decreased by HMTBa. In conclusion, HMTBa prevented the increase in trans FA in milk fat associated with MFD through a mechanism that is independent of total volatile FA concentration, but involves modification of rumen biohydrogenation. Decreased propionate and increased butyrate and ruminal protozoa may also have functional roles in the mechanism.

Oxidative brain and cerebellum injury induced by d-galactosamine: Protective effect of S-methyl methionine sulfonium chloride.

The objective of this study was to examine the protective effects of S-methyl methionine sulfonium chloride (MMSC) against galactosamine (GalN)-induced brain and cerebellum injury in rats. A total of 22 female Sprague-Dawley rats were randomly divided into four groups as follows: Group I (n = 5), intact animals; Group II (n = 6), animals received 50 mg/kg/day of MMSC by gavage technique for 3 consecutive days; Group III (n = 5), animals injected with a single dose of 500 mg/kg of GalN intraperitoneally (ip); and Group IV (n = 6), animals injected with the same dose of GalN 1 h after MMSC treatment. After 6 h of the last GalN treatment (at the end of the experiments), all animals were killed under anesthesia, brain and cerebellum tissues were dissected out. Reduced glutathione, total antioxidant status levels, and antioxidant enzymes (catalase, superoxide dismutase, and glutathione-related enzymes), aryl esterase, and carbonic anhydrase activities remarkably declined whereas advanced oxidized protein products, reactive oxygen species, total oxidant status, oxidative stress index levels, and myeloperoxidase, acetylcholinesterase, lactate dehydrogenase, and xanthine oxidase activities were significantly elevated in the GalN group compared with intact rats. In contrast, the administration of MMSC to GalN groups reversed these alterations. In conclusion, we may suggest that MMSC has protective effects against GalN-induced brain and cerebellar toxicity in rats.

Are the Main Methionine Sources Equivalent? A Focus on DL-Methionine and DL-Methionine Hydroxy Analog Reveals Differences on Rainbow Trout Hepatic Cell Lines Functions.

The replacement of fishmeal by plant proteins in aquafeeds imposes the use of synthetic methionine (MET) sources to balance the amino acid composition of alternative diets and so to meet the metabolic needs of fish of agronomic interest such as rainbow trout (RT-Oncorhynchus mykiss). Nonetheless, debates still exist to determine if one MET source is more efficiently used than another by fish. To address this question, the use of fish cell lines appeared a convenient strategy, since it allowed to perfectly control cell growing conditions notably by fully depleting MET from the media and studying which MET source is capable to restore cell growth/proliferation and metabolism when supplemented back. Thus, results of cell proliferation assays, Western blots, RT-qPCR and liquid chromatography analyses from two RT liver-derived cell lines revealed a better absorption and metabolization of DL-MET than DL-Methionine Hydroxy Analog (MHA) with the activation of the mechanistic Target Of Rapamycin (mTOR) pathway for DL-MET and the activation of integrated stress response (ISR) pathway for MHA. Altogether, the results clearly allow to conclude that both synthetic MET sources are not biologically equivalent, suggesting similar in vivo effects in RT liver and, therefore, questioning the MHA efficiencies in other RT tissues.

Beneficial impact of dietary methyl methionine sulfonium chloride and/or L-carnitine supplementation on growth performance, feed efficiency, and serum biochemical parameters in broiler chicken: role of IGF-1 and MSTN genes.

The purpose of this study was to examine the effect of dietary supplementation with methyl methionine sulfonium chloride (MMSC), and L-carnitine (L-CAR) alone or in combination on the growth performance of broilers through their impact on the expression of IGF-1 and MSTN genes associated with growth in broilers. One-day-old female Ross 308 broiler chicks were allocated into four groups, each of which received a broiler starter diet and water daily ad libitum. The control group (group 1) was given drinking water without any additives. Group 2 received 0.25 g L-carnitine per liter of drinking water, group 3 received 0.25 g MMSC per liter of drinking water, and group 4 received 0.25 g of both L-carnitine and MMSC per liter of drinking water. Birds were given a starter diet to 21 days after which they received a broiler grower diet to 35 days when the experiment ended. There were five replicate groups of 12 birds per treatment. Body weights and feed intake were recorded weekly. Compared to the control group of birds, supplementation with MMSC either alone or in combination with L-carnitine resulted in an increase in growth rate or feed utilization efficiency; L-carnitine by itself had no effect. MMSC supplementation, again either alone or in combination with L-carnitine, increased jejunal and ileal villi height, increased serum total proteins and globulins, downregulated myostatin (MSTN) mRNA, and upregulated insulin growth factor-1 (IGF-1) mRNA expression. Supplementation with L-carnitine alone showed none of these effects. We conclude that MMSC supplementation improved growth performance through the upregulation of IGF-1 mRNA expression and downregulation of MSTN mRNA expression.

Photo- and Radiation-Induced One-Electron Oxidation of Methionine in Various Structural Environments Studied by Time-Resolved Techniques.

Oxidation of methionine (Met) is an important reaction that plays a key role in protein modifications during oxidative stress and aging. The first steps of Met oxidation involve the creation of very reactive and short-lived transients. Application of complementary time-resolved radiation and photochemical techniques (pulse radiolysis and laser flash photolysis together with time-resolved CIDNP and ESR techniques) allowed comparing in detail the one-electron oxidation mechanisms initiated either by ●OH radicals and other one-electron oxidants or the excited triplet state of the sensitizers e.g., 4-,3-carboxybenzophenones. The main purpose of this review is to present various factors that influence the character of the forming intermediates. They are divided into two parts: those inextricably related to the structures of molecules containing Met and those related to external factors. The former include (i) the protection of terminal amine and carboxyl groups, (ii) the location of Met in the peptide molecule, (iii) the character of neighboring amino acid other than Met, (iv) the character of the peptide chain (open vs cyclic), (v) the number of Met residues in peptide and protein, and (vi) the optical isomerism of Met residues. External factors include the type of the oxidant, pH, and concentration of Met-containing compounds in the reaction environment. Particular attention is given to the neighboring group participation, which is an essential parameter controlling one-electron oxidation of Met. Mechanistic aspects of oxidation processes by various one-electron oxidants in various structural and pH environments are summarized and discussed. The importance of these studies for understanding oxidation of Met in real biological systems is also addressed.

Impact of Dietary Zinc Chloride Hydroxide and Zinc Methionine on the Faecal Microbiota of Healthy Adult Horses and Ponies.

Zinc supplements are often used in equine nutrition to support skin and hoof quality or the immune function. However, no data on the effects of dietary zinc on the intestinal microbiota of horses and ponies are available so far. In the present study, varying dietary zinc concentrations (maintenance (4 mg/kg BW0.75/day), 120 mg/kg dry matter (DM)/day and 240 mg/kg DM/day) were achieved by the supplementation of either zinc chloride hydroxide or zinc methionine (six treatment periods of 4 weeks each). Eight healthy adult ponies and two healthy adult horses were included, and faecal samples were collected at the end of each treatment period to analyse the microbiota (16S rDNA sequencing) and microbial metabolites. With increasing dietary zinc concentrations, the richness of the faecal microbiota decreased, independently of the zinc compound used. In addition, a decrease of the relative abundance of Bacteroidetes and Fibrobacteres as well as of acetate and total short-chain fatty acids in the faeces of the animals was observed at high zinc intakes. Effects on the bacterial order, family and genus level were also detected, which were partly more pronounced for zinc chloride hydroxide. It can be concluded that high dietary zinc levels decrease the richness and fermentative activity of the faecal microbiota of horses and ponies. Although all animals were healthy throughout the study, the effects could be critical for gut health, and deserve more research. The detected differences between the zinc compounds used indicate differences in the bioavailability of organic and inorganic zinc sources in equines.

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.