The effects of rumen protected methionine supplementation on the performance of primiparous dairy cows using the Presynch-Ovsynch protocol.

The purpose of this study was to examine the effects of rumen-protected methionine (RPM) supplementation on the reproductive and productive performance of primiparous dairy cows fed two levels of protein. The Presynch-Ovsynch protocol was used to synchronize 36 lactating Holstein cows that were assigned randomly to one of six dietary treatments: (1) 14% CP and without RPM diet (14CP-0RPM; n = 6), (2) 14% CP and 15 g/head/day RPM (14CP-15RPM; n = 6), (3) 14% CP and 25 g/head/day RPM (14CP-25RPM; n = 6), (4) 16% CP and without RPM diet (16CP-0RPM; n = 6), (5) 16% CP and 15 g/head/day RPM (16CP-15RPM; n = 6), and (6) 16% CP and 25 g/head/day RPM (16CP-25RPM; n = 6). Independent of CP levels, feeding RPM had reduced the calving interval (P < 0.01). Feeding RPM increased (P < 0.01) overall plasma progesterone (P4). Feeding 16CP-15RPM increased (P < 0.01) overall plasma P4. Feeding 16% CP increased (P < 0.01) 4% fat corrected milk, energy corrected milk, milk fat and protein yield, and milk casein. Moreover, feeding the 25RPM has increased (P < 0.01) 4% fat corrected milk, energy corrected milk, milk fat, and protein yield. Compared with other treatments, feeding 16CP-25RPM or 16CP-15RPM enhanced (P < 0.01) milk yield and milk fat yield. In conclusion, feeding 16% CP with RPM boosted the productivity and reduced the calving interval in primiparous lactating dairy cows.

Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.

Mitochondria retain their own genome and translational apparatus that is highly specialized in the synthesis of a handful of proteins, essential components of the oxidative phosphorylation system. During evolution, the players and mechanisms involved in mitochondrial translation have acquired some unique features, which we have only partially disclosed. The study of the mitochondrial translation process has been historically hampered by the lack of an in vitro translational system and has largely relied on the analysis of the incorporation rate of radiolabeled amino acids into mitochondrial proteins in cellulo or in organello. In this chapter, we describe methods to monitor mitochondrial translation by labeling newly synthesized mitochondrial polypeptides with [S35]-methionine in either yeast or mammalian whole cells or isolated mitochondria.

Concentrations of vitamin B12 and folate in maternal serum and fetal fluids, metabolite interrelationships, and hepatic transcript abundance of key folate and methionine cycle genes: the impacts of maternal nutrition during the first 50 d of gestation.

Adequate maternal nutrition is key for proper fetal development and epigenetic programming. One-carbon metabolites (OCM), including vitamin B12, folate, choline, and methionine, play a role in epigenetic mechanisms associated with developmental programming. This study investigated the presence of B12 and folate in maternal serum, allantoic fluid (ALF), and amniotic fluid (AMF), as well as how those concentrations in all three fluids correlate to the concentrations of methionine-folate cycle intermediates in heifers receiving either a control (CON) or restricted (RES) diet for the first 50 d of gestation and fetal hepatic gene expression for methionine-folate cycle enzymes. Angus cross heifers (n = 43) were estrus synchronized, bred via artificial insemination with semen from a single sire, and randomly assigned to one of two nutrition treatments (CON = 20, RES = 23). Heifers were ovariohysterectomized on either day 16 (n = 14), 34 (n = 15), or 50 of gestation (n = 14), where samples of maternal serum (n = 42), ALF (n = 29), and AMF (n = 11) were collected and analyzed for concentrations of folate and B12. Concentrations of B12 and folate in ALF were greater (P < 0.05) in RES compared to CON. For ALF, folate concentrations were also greater (P < 0.01) on day 34 compared to day 50. There was a significant (P = 0.04) nutrition × fluid interaction for B12 concentrations where concentrations were greatest in restricted ALF, intermediate in control ALF, and lowest in CON and RES serum and AMF. Folate concentrations were greatest (P < 0.01) in ALF, intermediate in serum, and lowest in AMF. Additionally, positive correlations (P < 0.05) were found between ALF and AMF folate concentrations and AMF concentrations of methionine, serine, and glycine. Negative correlations (P < 0.05) between AMF folate and serum homocysteine were also observed. Both positive and negative correlations (P < 0.05) depending on the fluid evaluated were found between B12 and methionine, serine, and glycine concentrations. There was a downregulation (P = 0.05) of dihydrofolate reductase and upregulation (P = 0.03) of arginine methyltransferase 7 gene expression in RES fetal liver samples compared with CON fetal liver on day 50. Combined, these data show restricted maternal nutrition results in increased B12 and folate concentrations present in fetal fluids, and increased expression of genes for enzymes within one-carbon metabolism.

When pregnant cattle have restricted access to feed or specific nutrients, calf development can be affected, and the degree of impairment depends, at least partially, on timing, duration, and severity of the limitations. A biochemical pathway present in cells that can be affected by limited nutrition is one-carbon metabolism. This pathway is related to epigenetics, which regulates gene expression or the turning on and off of genes. Two important vitamins in one-carbon metabolism are vitamins B12 and folate. By understanding the amounts of those vitamins available to the developing calf, we can gain better insight into the regulation and potential avenues of improvement of calf growth and development. In this study, we found a nutrient restricted maternal diet increased the amount of B12 and folate in calf allantoic and amniotic fluids. We also found that folate and B12 were correlated to the presence of other nutrients in serum, allantoic fluid, and amniotic fluid. In addition, we found that a protein methylating gene in one-carbon metabolism had increased expression in calves from heifers receiving limited nutrition. This study is an important step in understanding how the nutrients available to a pregnant heifer during gestation affects nutrients available to the conceptus.

Methionine consumption by cancer cells drives a progressive upregulation of PD-1 expression in CD4 T cells.

Programmed cell death protein 1 (PD-1), expressed on tumor-infiltrating T cells, is a T cell exhaustion marker. The mechanisms underlying PD-1 upregulation in CD4 T cells remain unknown. Here we develop nutrient-deprived media and a conditional knockout female mouse model to study the mechanism underlying PD-1 upregulation. Reduced methionine increases PD-1 expression on CD4 T cells. The genetic ablation of SLC43A2 in cancer cells restores methionine metabolism in CD4 T cells, increasing the intracellular levels of S-adenosylmethionine and yielding H3K79me2. Reduced H3K79me2 due to methionine deprivation downregulates AMPK, upregulates PD-1 expression and impairs antitumor immunity in CD4 T cells. Methionine supplementation restores H3K79 methylation and AMPK expression, lowering PD-1 levels. AMPK-deficient CD4 T cells exhibit increased endoplasmic reticulum stress and Xbp1s transcript levels. Our results demonstrate that AMPK is a methionine-dependent regulator of the epigenetic control of PD-1 expression in CD4 T cells, a metabolic checkpoint for CD4 T cell exhaustion.

Effect of increasing dietary methionine-to-lysine ratio during early gestation on fetal development and piglet birth weight.

It was hypothesized that increasing dietary methionine (Met) for sows in early gestation would have a positive effect on fetal and placental growth and development, thereby also increasing the birth weight of piglets. The objective of the study was to investigate the effect of increasing the total dietary methionine-to-lysine ratio (Met:Lys) from 0.29 (Control diet) to 0.41 (Met diet) from mating to day 50 of gestation. A total of 349 multiparous sows were allocated to either the Control or Met diet group. The sows' backfat thickness was measured pre-farrowing, post-farrowing, and at weaning in the previous cycle and on days 14, 50 and 112 of gestation in the current cycle. On day 50, three Control and six Met sows were slaughtered. In 116 litters, piglets were weighed and measured individually at farrowing. The dietary treatment did not affect the sows' backfat thickness before or during gestation (P > 0.05). The number of liveborn and stillborn piglets at farrowing were similar in both groups (P > 0.05) and no differences in average piglet birth weight, total litter weight at birth or within-litter variation in birth weight (P > 0.05) were observed. In conclusion, increasing the dietary Met:Lys ratio for sows in early gestation had no effect on piglet birth weight.

The Assessment of Glioblastoma Metabolic Activity via 11C-Methionine PET and Radiomics.

Nowadays, the quantitative analysis of PET/CT data in patients with glioblastoma is not strictly standardized in the clinic and does not exclude the human factor. This study aimed to evaluate the relationship between the radiomic features of glioblastoma 11C-methionine PET images and the tumor-to-normal brain (T/N) ratio determined by radiologists in clinical routine. PET/CT data were obtained for 40 patients (mean age 55 ± 12 years; 77.5% men) with a histologically confirmed diagnosis of glioblastoma. Radiomic features were calculated for the whole brain and tumor-containing regions of interest using the RIA package for R. We redesigned the original RIA functions for GLCM and GLRLM calculation to reduce computation time significantly. Machine learning over radiomic features was applied to predict T/N with the best median correlation between the true and predicted values of 0.73 (p = 0.01). The present study showed a reproducible linear relationship between 11C-methionine PET radiomic features and a T/N indicator routinely assessed in brain tumors. Radiomics enabled utilizing texture properties of PET/CT neuroimaging that may reflect the biological activity of glioblastoma and can potentially augment the radiological assessment.

BHMT polymorphism and susceptibility to PTE in Chinese patients.

OBJECTIVE: Pulmonary thromboembolism (PTE) is as a common form of venous thrombosis and a potentially fatal cardiovascular disorder, which has become a severe clinical problem with high incidence and mortality. The PTE has a strong genetic basis, which contributes up to half of the variance in PTE incidence and susceptibility single-nucleotide polymorphisms (SNPs) is associated with PTE. Betaine homocysteine methyltransferase (BHMT) is an essential enzyme that catalyzes the remethylating reaction from homocysteine to methionine and participates in conserving methionine and detoxifying homocysteine. In this work, we aimed to explore BHMT polymorphism and susceptibility to PTE in Chinese patients. PATIENTS AND METHODS: Variant loci of the BHMT gene were screened in serum samples of PTE patients, followed by verification using Sanger sequencing. These polymorphic loci were validated in 16 PTE patients and 16 matched normal patients. The frequency differences between the allele and genotypes were compared using the Hardy-Weinberg equilibrium test and Chi-square test. RESULTS: A SNP was identified in PTE patients and a heterozygous transition of G>A (Arg239Gln) in rs3733890 was found. The variance difference at rs3733890 between normal patients (2/16, 0.125) and PTE patients (9/16, 0.5625) was significant (p<0.01). CONCLUSIONS: Therefore, we concluded that the BHMT polymorphism, rs3733890 may be a susceptibility SNP for PTE.

Synthesis of L-methionine-loaded chitosan nanoparticles for controlled release and their in vitro and in vivo evaluation.

Therapeutically popular controlled release-enabling technology has forayed into the nutrition sector. Polymer coated forms of L-methionine used in soy protein diets, and its intermediate metabolite, S-adenosyl-L-methionine, used in myriad of medical conditions have proved more efficacious over (highly catabolized) free forms. In this premier study, L-methionine-loaded chitosan nanoparticles (M-NPs) were synthesized using ionic gelation method and their efficacy was evaluated. Biophysical characterization of the NPs was done using a Nanopartica SZ 100 analyser, transmission electron microscopy, and Fourier transform infrared spectroscopy. The M-NPs were spherical and smooth and 218.9 ± 7.4 nm in size and in vitro testing confirmed the controlled release of methionine. A 60-days feeding trial in L. rohita fish fingerlings was conducted. A basal diet suboptimal (0.85%) in methionine was provided with one of the supplements as under: none (control), 0.8% chitosan NPs (0.8% NPs), 1.2% L-methionine (1.2% M) (crystalline free form), 0.6% M-NPs and 1.2% M-NPs. While the addition of 0.6% M-NPs to the basal diet complemented towards meeting the established dietary requirement and resulted in significantly highest (P < 0.05) growth and protein efficiency and sero-immunological test scores (serum total protein, serum globulin, serum albumin: globulin ratio, phagocytic respiratory burst/NBT reduction and lysozyme activity), 1.2% supplementation in either form (free or nano), for being 0.85% excess, was counterproductive. Liver transaminases and dehydrogenases corroborated enhanced growth. It was inferred that part of the methionine requirement in nano form (M-NPs) can confer intended performance and health benefits in animals relying on plant proteins-based diets limiting in this essential amino acid. The study also paves the way for exploring chitosan NPs-based sustained delivery of amino acids in human medical conditions.

Methionine, cysteine, and butylated hydroxytoluene enhance cryosurvival of ram semen on post-thaw and post-incubation time points.

Despite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders is yet to be found. This study was designed to investigate the different doses of methionine (2.5 and 5 mM), cysteine (1 and 2 mM), and butylated hydroxytoluene (BHT) (1 and 2 mM) for ram semen cryopreservation on post-thaw and post-incubation (6 h) time points over spermatological parameters. Semen samples were collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5 mM methionine, 5 mM methionine, 1 mM cysteine, 2 mM cysteine, 1 mM BHT, and 2 mM BHT). Semen samples were put into French straws (0.25 mL), and freezing procedure (two-step) was conducted via a programmable gamete freezer. At both time points, motility, HOST, PSA-FITC, and TUNEL assays were made to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results compared to the control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6 h of time. The study demonstrated that supplementing sperm freezing extenders with previous antioxidants may create new approaches to cryopreservation procedures, and through increasing success rate of freezing, fertility results may increase to better results in near future.

Increased dietary Trp, Thr, and Met supplementation improves growth performance and protein deposition of salmonella-challenged growing pigs under poor housing conditions.

Highly intensified rearing conditions and precarious sanitary management predispose pigs to immune system activation, altered amino acid (AA) metabolism, and decreased growth performance. Thus, the main objective of this study was to evaluate the effects of increased dietary tryptophan (Trp), threonine (Thr), and methionine + cysteine (Met + Cys) supplementation on performance, body composition, metabolism, and immune responses of group-housed growing pigs under challenging sanitary conditions. A hundred and twenty pigs (25.4 ± 3.7 kg) were randomly assigned to a 2 × 2 factorial arrangement, consisting of two sanitary conditions (SC, good [GOOD] or salmonella-challenge and poor housing condition [Salmonella Typhimurium (ST) + POOR]) and two diets, control (CN) or supplemented with AA (Trp, Thr, and Met + Cys:Lys ratios 20% higher than those of the CN diet [AA>+]). Pigs were followed during the growing phase (25-50 kg) and the trial lasted 28 d. The ST + POOR SC pigs were challenged with Salmonella Typhimurium and raised in a poor housing condition. The ST + POOR SC increased rectal temperature, fecal score, serum haptoglobin, and urea concentration (P < 0.05) and decreased serum albumin concentration (P < 0.05) compared with GOOD SC. Body weight, average daily feed intake, average daily gain (ADG), feed efficiency (G:F), and protein deposition (PD) were greater in GOOD SC than in ST + POOR SC (P < 0.01). However, pigs housed in ST + POOR SC fed with AA+ diet had lower body temperature (P < 0.05), increased ADG (P < 0.05) and nitrogen efficiency (P < 0.05), and a tendency for improved PD and G:F (P < 0.10) compared with CN diet fed pigs. Regardless of the SC, pigs fed AA+ diet had lower serum albumin (P < 0.05) and tended to decrease serum urea levels (P < 0.10) compared with CN diet. The results of this study suggest that the ratio of Trp, Thr, and Met + Cys to Lys for pigs are modified by sanitary conditions. Furthermore, supplementation of diets with a blend of Trp, Thr, and Met + Cys improves performance, especially under salmonella-challenge and poor housing conditions. Dietary tryptophan, threonine, and methionine supplementation can modulate immune status and influence resilience to sanitary challenges.

Immune system activation alters pigs' physiology and metabolism, increasing maintenance requirements and reducing voluntary feed intake and weight gain. Dietary functional amino acid supplementation (tryptophan, threonine, and methionine) is a strategy to support the immune system activation for immune components production, maintenance of the gut barrier integrity, and reduction of the oxidative status. Additionally, amino acid supplementation may mitigate growth performance losses. In this context, this study was conducted to investigate the effect of diets with or without tryptophan, threonine, and methionine supplementation on the performance and immune system activation of growing pigs under a sanitary challenge. The amino acid supplementation mitigated the immune system activation of challenged growing pigs and improved growth performance when compared to pigs fed diets with no supplementation. The functional amino acid supplementation may be an efficient nutritional strategy to optimize health and growth performance of immune-challenged pigs.

Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20.

BACKGROUND: Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. METHODS: Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsRM, was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25-6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. RESULTS: The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. CONCLUSIONS: We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster.

Montelukast prevents mice against carbon tetrachloride- and methionine-choline deficient diet-induced liver fibrosis: Reducing hepatic stellate cell activation and inflammation.

AIMS: Montelukast is an antagonist of cysteinyl leukotriene receptor 1 (CysLTR1) that protects against inflammation and oxidative stress. However, the function of montelukast in liver fibrosis remains unknown. In this study, we examined whether the pharmacological inhibition of CysLTR1 could protect mice against hepatic fibrosis. MATERIALS AND METHODS: Carbon tetrachloride (CCl4) and methionine-choline deficient (MCD) diet models were used in this study. The expression of CysLTR1 in liver were detected by RT-qPCR and Western blot analysis. Liver hydroxyproline levels, fibrotic genes expression, serum biochemical indexes and inflammatory factors were used to evaluate the effect of montelukast on liver fibrosis, injury, and inflammation. In vitro, we used the RT-qPCR and Western blot analysis to assess CysLTR1 in mouse primary hepatic stellate cell (HSC) and human LX-2 cell line. The role of montelukast on HSC activation and the underlying mechaisms were determined using RT-qPCR analysis, Western blot and immunostaining assays. KEY FINDINGS: Chronic stimulation from CCl4 and MCD diet upregulated the mRNA and protein levels of CysLTR1 in the liver. Pharmacological inhibition of CysLTR1 by montelukast ameliorated liver inflammation and fibrosis in both models. Mechanistically, montelukast suppressed HSC activation by targeting the TGFß/Smad pathway in vitro. The hepatoprotective effect of montelukast was also associated with reduced liver injury and inflammation. SIGNIFICANCE: Montelukast suppressed CCl4- and MCD-induced chronic hepatic inflammation and liver fibrosis. CysLTR1 might be a therapeutic target for treating liver fibrosis.

Characterization by LC-MS/MS of oxidized products identified in synthetic peptide somatostatin and cetrorelix submitted to forced oxidative stress by hydrogen peroxide: Two case studies.

In a broader scenario, the forced degradation studies provided by the ICH guidelines for Q1A, Q1B, and Q2B degradation studies allow to know the CQA of the molecule used as a drug product, to determine the appropriate analytical methods, excipients, and storage conditions ensuring the quality of the drug, its efficacy, and patient safety. In this study, we focused our attention on understanding how oxidative stress is performed by H2 O2 -impacted small synthetic peptides that do not contain residues susceptible to oxidation such as methionine. Among the amino acids susceptible to oxidation, methionine is the most reactive and depending on the structure of the protein where it is exposed, it tends to oxidize by converting into methionine sulfone or methionine sulfoxide by oxidation of its sulfur atom. Scouting experiments obtained by forced oxidative stress conditions are presented on two small synthetic peptides that do not contain any methionine residues spiked with different amounts of H2 O2 , and they are analyzed by LC-MS/MS. Less frequent oxidation products than those commonly observed on proteins/peptides-containing methionine have been characterized on both peptides. The study demonstrated that somatostatin, by means of one residue of tryptophan on the molecule, can generate traces of several oxidized products detected by UPLC-MS. Furthermore, even at a negligible level, oxidation on tyrosine and proline in cetrorelix that does not contain methionine nor tryptophan has been detected by UHPLC-MS/MS. Identification and quantification of oxidized species were achieved by high-resolution MS and MS/MS experiments. Thus, FDSs undoubtedly aid the evaluation of the CQAs as an important component of the characterization package as recommended by HAs and ICH, facilitating the understanding of unforeseen features of the studied molecule used as drugs.

Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids.

Methionine restriction (MR) provides metabolic benefits in many organisms. However, mechanisms underlying the MR-induced effect remain incompletely understood. Here, we show in the budding yeast S. cerevisiae that MR relays a signal of S-adenosylmethionine (SAM) deprivation to adapt bioenergetic mitochondria to nitrogenic anabolism. In particular, decreases in cellular SAM constrain lipoate metabolism and protein lipoylation required for the operation of the tricarboxylic acid (TCA) cycle in the mitochondria, leading to incomplete glucose oxidation with an exit of acetyl-CoA and α-ketoglutarate from the TCA cycle to the syntheses of amino acids, such as arginine and leucine. This mitochondrial response achieves a trade-off between energy metabolism and nitrogenic anabolism, which serves as an effector mechanism promoting cell survival under MR.

Physiological and genomic analyses of cobalamin (vitamin B12)-auxotrophy of Lysobacter auxotrophicus sp. nov., a methionine-auxotrophic chitinolytic bacterium isolated from chitin-treated soil.

A novel bacterium, designated 5-21aT, isolated from chitin-treated upland soil, exhibits methionine (Met) auxotrophy and chitinolytic activity. A physiological experiment revealed the cobalamin (synonym, vitamin B12)(Cbl)-auxotrophic property of strain 5-21aT. The newly determined complete genomic sequence indicated that strain 5-21aT possesses only the putative gene for Cbl-dependent Met synthase (MetH) and lacks that for the Cbl-independent one (MetE), which implies the requirement of Cbl for Met-synthesis in strain 5-21aT. The set of genes for the upstream (corrin ring synthesis) pathway of Cbl synthesis is absent in the genome of strain 5-21aT, which explains the Cbl-auxotrophy of 5-21aT. This strain was characterized via a polyphasic approach to determine its taxonomic position. The nucleotide sequences of two copies of the 16S rRNA gene of strain 5-21aT indicated the highest similarities to Lysobacter soli DCY21T(99.8 and 99.9 %) and Lysobacter panacisoli CJ29T(98.7 and 98.8 %, respectively), whose Cbl-auxotrophic properties were revealed in this study. The principal respiratory quinone was Q-8. The predominant cellular fatty acids were iso-C15:0, iso-C16:0 and iso-C17:1 ω9c. The complete genome sequence of strain 5-21aT revealed that the genome size was 4 155 451 bp long and the G+C content was 67.87 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain 5-21aT and its most closely phylogenetic relative L. soli DCY21T were 88.8 and 36.5%, respectively. Based on genomic, chemotaxonomic, phenotypic and phylogenetic data, strain 5-21aT represents a novel species in the genus Lysobacter, for which the name Lyobacter auxotrophicus sp. nov. is proposed. The type strain is 5-21aT (=NBRC 115507T=LMG 32660T).

A novel methionine metabolism-related signature predicts prognosis and immunotherapy response in lung adenocarcinoma.

Recent research revealed methionine metabolism as a key mediator of tumor initiation and immune evasion. However, the relationship between methionine metabolism and tumor microenvironment (TME) in lung adenocarcinoma (LUAD) remains unknown. Here, we comprehensively analyzed the genomic alterations, expression patterns, and prognostic values of 68 methionine-related regulators (MRGs) in LUAD. We found that most MRGs were highly prognostic based on 30 datasets including 5024 LUAD patients. Three distinct MRG modification patterns were identified, which showed significant differences in clinical outcomes and TME characteristics: The C2 subtype was characterized by higher immune score, while the C3 subtype had more malignant cells and worse survival. We developed a MethScore to measure the level of methionine metabolism in LUAD. MethScore was positively correlated with T-cell dysfunction and tumor-associated macrophages (TAMs), indicating a dysfunctional TME phenotype in the high MethScore group. In addition, two immunotherapy cohorts confirmed that patients with a lower MethScore exhibited significant clinical benefits. Our study highlights the important role of methionine metabolism in modeling the TME. Evaluating methionine modification patterns will enhance our understanding of TME characteristics and can guide more effective immunotherapy strategies.

Effect of Ethanol-Induced Methyl Donors Consumption on the State of Hypomethylation in Cervical Cancer.

Cervical cancer causes malignant tumors in females and threatens the physical and mental health of women. Current research shows that persistent infection of high-risk HPV is the main cause of cervical cancer. However, not all cervical cancer is caused by HPV infection, which may also be related to other factors, such as nutritional status and lifestyle. This study focuses on the effect of alcohol consumption on the methylation status of cervical cancer from the perspective of methyl donors. We established a mouse tumor-bearing model with cervical cancer SiHa cells, and at the same time, we cultured SiHa cells in vitro. Different concentrations of ethanol were administered to the model mice and SiHa cells. Then, we detected the levels of the methyl-donor folate and methionine and their metabolite homocysteine levels in mice serum, tumor tissues, and SiHa cells. Furthermore, we determined the expression of the members of the DNA methyltransferase family (DNMT1, DNMT3a, and DNMT3b) in tumor tissue by immunohistochemistry. qRT-PCR and Western blotting analysis were used to detect the mRNA and protein levels of members of the DNA methyltransferase family in cervical cancer SiHa cells. Our results show that the levels of the methyl donor (folate and methionine) decreased with the increase of ethanol concentration (p < 0.05), and the homocysteine level increased significantly (p < 0.05). In SiHa cells, the mRNA and protein levels of the DNMT family members and their receptors were significantly higher than those in the control group (p < 0.05). Collectively, these results suggest that ethanol could influence DNMT expression by inducing methyl donor consumption, thereby causing cervical cancer cells to exhibit genome-wide hypomethylation.

Effects of a 28-day feeding trial of grain-containing versus pulse-based diets on cardiac function, taurine levels and digestibility in domestic dogs.

In 2018, the US Food and Drug Administration reported a link between canine dilated cardiomyopathy (DCM) and grain-free diets. Evidence to support a link has emerged, but the specific ingredients responsible and the role of taurine or other causative factors remain unclear. We hypothesized dogs fed pulse-based, grain-free diets for 28 days will show decreased macronutrient digestibility, increased fecal bile acid excretion, and reduced plasma cystine, cysteine, methionine and taurine, causing sub-clinical cardiac or blood changes indicative of early DCM. Three diets were formulated using white rice flour (grain), whole lentil (grain-free), or wrinkled pea (grain-free) and compared to the pre-trial phase on a commercial grain-based diet. After 28 days of feeding each diet, the wrinkled pea diet impaired stroke volume and cardiac output, increased end-systolic ventricular diameter and increased plasma N-Terminal Pro-B-type Natriuretic Peptide (NT-ProBNP), albeit in a sub-clinical manner. Digestibility of some macronutrients and sulphur-containing amino acids, excluding taurine, also decreased with pulse-based compared to grain-based diets, likely due to higher fiber levels. Plasma taurine levels were unchanged; however, plasma methionine was significantly lower after feeding all test diets compared to the commercial diet. Overall, DCM-like changes observed with the wrinkled pea diet, but not lentil diet, after only 4 weeks in a breed not known to be susceptible support a link between pea-based diets and canine nutritionally-mediated DCM.

Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis.

BACKGROUND: Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes. RESULTS: We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl ß-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system. CONCLUSION: Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.

The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains.

Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetylation. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit. Yet, whereas well-resolved structures for ribosome-bound RAC, SRP and Sec61, are available, structural information on the mode of ribosome interaction of eukaryotic MetAPs or of the five cotranslationally active NATs is only available for NatA. Here, we present cryo-EM structures of yeast Map1 and NatB bound to ribosome-nascent chain complexes. Map1 is mainly associated with the dynamic rRNA expansion segment ES27a, thereby kept at an ideal position below the tunnel exit to act on the emerging substrate nascent chain. For NatB, we observe two copies of the NatB complex. NatB-1 binds directly below the tunnel exit, again involving ES27a, and NatB-2 is located below the second universal adapter site (eL31 and uL22). The binding mode of the two NatB complexes on the ribosome differs but overlaps with that of NatA and Map1, implying that NatB binds exclusively to the tunnel exit. We further observe that ES27a adopts distinct conformations when bound to NatA, NatB, or Map1, together suggesting a contribution to the coordination of a sequential activity of these factors on the emerging nascent chain at the ribosomal exit tunnel.