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Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the functions of these TF condensates in 3-Dimentional (3D) genome organization and gene regulation remain elusive. In response to methionine (met) starvation, budding yeast TF Met4 and a few co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form co-localized puncta-like structures in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes is clustered in 3D in a Met4-dependent manner. A MET3pr -GFP reporter inserted near several native Met4-binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4-binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to methionine depletion., Competing Interests: JL, LS, YL, SB, FZ, XZ, LB No competing interests declared, (© 2024, Lee et al.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels 1 are essential for pacemaking activity and neural signalling 2,3 . Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain 4 and epileptic seizures 5 . The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor 6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs.  7,8 ). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Serine β-lactamase TEM-1 is the first β-lactamase discovered and is still common in Gram-negative pathogens resistant to β-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a "global suppressor", has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of β-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the β-lactamase to be reactivated.

Congenital heart disease is one of the most common congenital malformations and thus represents a considerable public health burden. Hence, the identification of individuals and families with an increased genetic predisposition to congenital heart disease (CHD) and its possible prevention is important. Even though CHD is associated with the lack of folate during early pregnancy, the genetic background of folate and methionine metabolism perturbations and their influence on CHD risk is not clear. While some genes, such as those coding for cytosolic enzymes of folate/methionine cycles, have been extensively studied, genetic studies of folate transporters (de)glutamation enzymes and mitochondrial enzymes of the folate cycle are lacking. Among genes coding for cytoplasmic enzymes of the folate cycle, MTHFR , MTHFD1 , MTR , and MTRR have the strongest association with CHD, while among genes for enzymes of the methionine cycle BHMT and BHMT2 are the most prominent. Among mitochondrial folate cycle enzymes, MTHFD2 plays the most important role in CHD formation, while FPGS was identified as important in the group of (de)glutamation enzymes. Among transporters, the strongest association with CHD was demonstrated for SLC19A1.

Histone modifications are associated with regulation of gene expression that controls a vast array of biological processes. Often, these associations are drawn by correlating the genomic location of a particular histone modification with gene expression or phenotype; however, establishing a causal relationship between histone marks and biological processes remains challenging. Consequently, there is a strong need for experimental approaches to directly manipulate histone modifications. A class of mutations on the N-terminal tail of histone H3, lysine-to-methionine (K-to-M) mutations, was identified as dominant-negative inhibitors of histone methylation at their respective and specific residues. The dominant-negative nature of K-to-M mutants makes them a valuable tool for studying the function of specific methylation marks on histone H3. Here, we review recent applications of K-to-M mutations to understand the role of histone methylation during development and homeostasis. We highlight important advantages and limitations that require consideration when using K-to-M mutants, particularly in a developmental context., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The emergence of fungicide resistance severely threatens crop production by limiting the availability and application of established fungicides. Therefore, it is urgent to identify new fungicidal targets for controlling plant diseases. Here, we characterized the function of a conserved homoserine O-acetyltransferase (HOA) from the rice blast fungus Magnaporthe oryzae that could serve as the candidate antifungal target. Deletion of the MoMET2 and MoCYS2 genes encoding HOAs perturbed the biosynthesis of methionine and S-adenyl methionine, a methyl group donor for epigenetic modifications, and severely attenuated the development and virulence of M. oryzae. The ∆Momet2 mutant is significantly increased in 5-methylcytosine (5mC) modification that represses the expression of genes required for pathogenicity, including MoGLIK and MoCDH-CYT. We further showed that host-induced gene silencing (HIGS) targeting MoMET2 and MoCYS2 effectively controls rice blasts. Our studies revealed the importance of HOA in the development and virulence of M. oryzae, which suggests the potential feasibility of HOA as new targets for novel anti-rice blast measurements., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The protein brain-derived neurotrophic factor (BDNF) promotes neural plasticity of the central nervous system and plays an important role for learning and memory. A single nucleotide polymorphism (rs6265) at position 66 in the pro-region of the human BDNF gene, resulting in a substitution of the amino acid valine (val) with methionine (met), leads to attenuated BDNF secretion and has been associated with reduced neurocognitive function. Inhomogeneous results have been found regarding the effect of the BDNF genotype on behavior. We determined the BDNF genotype and performance on the Compound Remote Associate (CRA) task as a common measure of creativity in 76 healthy university students. In our main analyses, we did not find significant differences between met-carriers (n = 30) and non-met carriers (n = 46). In a secondary analysis, we found that met-carriers had a slower solution time (medium effect size) for items of medium difficulty. Our results suggest that met-carriers and non-met-carriers do not generally differ regarding their creativity, but non-met-carriers may have a certain advantage when it comes to moderately difficult problems. The wider literature suggests that both genetic variants come with advantages and disadvantages. Future research needs to sharpen our understanding of the disadvantages and, potentially, advantages met allele carriers may have., Competing Interests: The authors report there are no competing interests to declare., (Copyright: © 2023 Hertenstein et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The natural variation in mammalian longevity and its underlying mechanisms remain an active area of aging research. In the latest issue of The EMBO Journal, Liu et al (2023) analyze gene expression levels in 103 mammalian species across three tissues, revealing tissue-specific associations between gene expression patterns and longevity. Remarkably, the study suggests that methionine restriction, a strategy shown to increase lifespan, may extend beyond artificial interventions and is similarly employed by natural selection., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Competing Interests: Conflict of interest statement. None declared.

Cyclic GMP-AMP synthase (cGAS) is the major sensor for cytosolic DNA and activates type I interferon signaling and plays an essential role in antitumor immunity. However, it remains unclear whether the cGAS-mediated antitumor activity is affected by nutrient status. Here, our study reports that methionine deprivation enhances cGAS activity by blocking its methylation, which is catalyzed by methyltransferase SUV39H1. We further show that methylation enhances the chromatin sequestration of cGAS in a UHRF1-dependent manner. Blocking cGAS methylation enhances cGAS-mediated antitumor immunity and suppresses colorectal tumorigenesis. Clinically, cGAS methylation in human cancers correlates with poor prognosis. Thus, our results indicate that nutrient stress promotes cGAS activation via reversible methylation, and suggest a potential therapeutic strategy for targeting cGAS methylation in cancer treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fibromyalgia (FM) is a disorder characterized by chronic musculoskeletal pain, fatigue, and cognitive problems. Neurotransmitters, mainly catecholamines, appear to be involved in regulating the etiology of FM. Catechol-O-methyltransferase ( COMT ) is involved in catabolizing catecholamines such as norepinephrine. The most common variant studied in the COMT gene is the valine (Val) to methionine (Met) substitution at codon 158. This is the first study in Sudan addressing FM cases and genetic susceptibility to the disease. We aimed in this study to investigate the frequency of COMT Val 158 Met polymorphism among patients with FM, rheumatoid arthritis, and in healthy individuals. Genomic DNA from forty female volunteers was analyzed: twenty were from primary and secondary FM patients, ten were from rheumatoid arthritis patients, and ten were from healthy control. FM patients' age was ranging from 25 years to 55 with a mean of 41.14 ± 8.90. The mean age of the rheumatoid arthritis patients and healthy individuals was 31.3 ± 7.5 and 38.6 ± 11.2, respectively. Samples were genotyped for COMT single nucleotide polymorphism rs4680 (Val158Met), using the amplification-refractory mutation system (ARMS-PCR). Genotyping data have been analyzed using the Chi-square and Fisher exact test. The most common genotype among the study participants was the heterozygous Val/Met found in all participants. It was the only genotype found in the healthy participants. The genotype Met/Met was found only in FM patients. The genotype Val/Val was found only in rheumatoid patients. Analyses have shown no association between the Met/Met genotype and FM, and this could be due to a small sample size. In a larger sample size, a significant association could be found as this genotype was shown only by FM patients. Moreover, the Val/Val genotype, which is shown only among rheumatoid patients, might protect them from developing FM symptoms., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2023 Safaa Mamoun Abdelmageid et al.)

Candida albicans is a predominant species causing candidemia in hospitalized patients. This study aimed to investigate the association of culture medium metabolomic profiles with biofilm formation and invasion properties of clinical bloodstream-isolated C. albicans. A total of twelve isolates and two reference strains were identified by virulent phenotypes. Their susceptibility was determined by the microdilution method, following EUCAST guidelines. Biofilm formation was evaluated with metabolic activity, morphology and agglutinin-like sequence 3 (ALS3) mRNA expression. Invasion into the vascular endothelial EA.hy926 cells was determined by lactate dehydrogenase release and internalization assay. Their metabolomic profiles were assessed by high-resolution accurate-mass spectrometry (HRAMS). The results showed four different phenotypes of C. albicans: high-biofilm/invasive (50%), high-biofilm/non-invasive (7%), low-biofilm/invasive (36%) and low-biofilm/non-invasive (7%). The metabolomic profiles of the culture medium determined strong correlation of the virulent phenotypes and the alteration of metabolites in the methionine metabolism pathway, such as homocysteine, 5-methyltetrahydrofolate and S-adenosylmethioninamine. Moreover, thiamine and biotin levels were significantly increased in Isolate03, representative of a high-biofilm/invasive phenotype. These results suggest that methionine and vitamin B metabolism pathways might be influenced by their virulent phenotypes and pathogenic traits. Therefore, their metabolism pathways might be a potential target for reducing virulence of C. albicans bloodstream infections., Competing Interests: Declaration of competing interest We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcomes., (Copyright © 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Brain-derived neurotrophic factor (BDNF) has been implicated in a multitude of neurodevelopmental processes including neuronal differentiation, axonal outgrowth, synaptic plasticity, or survival. One human-specific single nucleotide polymorphism (rs6265) in the BDNF gene causes a substitution of valine (Val) to methionine (Met) at codon 66 in the pro domain of the protein (Val66Met). This substitution is associated to reduced hippocampal volumes, poor performance on hippocampal-dependent memory tasks, and some mental disorders such as schizophrenia, depression or Alzheimer's disease. Here we generated three iPSC lines from healthy donors, either homozygous (Val/Val and Met/Met) or heterozygous (Val/Met) for the polymorphism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The ethylene insensitive 3/ethylene insensitive 3-like (EIN3/EIL) plays an indispensable role in fruit ripening. However, the regulatory mechanism that links post-translational modification of EIN3/EIL to fruit ripening is largely unknown. Here, we studied the expression of 13 MaEIL genes during banana fruit ripening, among which MaEIL9 displayed higher enhancement particularly in the ripening stage. Consistent with its transcript pattern, abundance of MaEIL9 protein gradually increased during the ripening process, with maximal enhancement in the ripening. DNA affinity purification (DAP)-seq analysis revealed that MaEIL9 directly targets a subset of genes related to fruit ripening, such as the starch hydrolytic genes MaAMY3D and MaBAM1. Stably overexpressing MaEIL9 in tomato fruit hastened fruit ripening, whereas transiently silencing this gene in banana fruit retarded the ripening process, supporting a positive role of MaEIL9 in fruit ripening. Moreover, oxidation of methionines (Met-129, Met-130, and Met-282) in MaEIL9 resulted in the loss of its DNA-binding capacity and transcriptional activation activity. Importantly, we identified MaEIL9 as a potential substrate protein of methionine sulfoxide reductase A MaMsrA4, and oxidation of Met-129, Met-130, and Met-282 in MaEIL9 could be restored by MaMsrA4. Collectively, our findings reveal a novel regulatory network controlling banana fruit ripening, which involves MaMsrA4-mediated redox regulation of the ethylene signaling component MaEIL9., (© 2022 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

In Acidithiobacillus ferrooxidans, proteins such as CcO are present in the electron transport pathway. They cause ferrous iron oxidation to ferric leading to the electron release. CcO has two copper atoms (CuA, CuB). CuA plays an important role in electron transfer. According to previous studies, the conversion of histidine to methionine in a similar protein increased the redox potential and was directly related to the number of electrons received. Also, the binding of methionine 233 to CuA and CuB in the wild protein structure is the reason for the selection of the H230 M mutation in the CuA site. Then, wild-type and H230 M mutant were simulated in the presence of a bilayer membrane POPC using the gromacs version 5.1.4. The changes performed in the H230 M mutant were evaluated by MD simulations analyzes. CcO and CoxA proteins are the last two proteins in the chain and were docked by the PatchDock server. By H230 M mutation, the connection between CuA and M230 weakens. The M230 moves further away from CuA, resulting become more flexible. Therefore, the Methionine gets closer to E149 of the CoxA leading to the higher stability of the CcO/CoxA complex. The results of RMSF analysis at the mutation point showed a significant increase. This indicates more flexibility in the active site. And leads to an increase in E 0 in the mutation point, an increase in the rate of electron reception, and an improved bioleaching process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gerstmann-Sträussler-Scheinker disease (GSS) is a rare genetic prion disease. A large GSS kindred linked to the serine-for-phenylalanine substitution at codon 198 of the prion protein gene (GSS-F198S) is characterized by conspicuous accumulation of prion protein (PrP)-amyloid deposits and neurofibrillary tangles. Recently, we demonstrated the transmissibility of GSS-F198S prions to bank vole carrying isoleucine at 109 PrP codon (BvI). Here we investigated: (i) the transmissibility of GSS-F198S prions to voles carrying methionine at codon 109 (BvM); (ii) the induction of hyperphosphorylated Tau (pTau) in two vole lines, and (iii) compared the phenotype of GSS-F198S-induced pTau with pTau induced in BvM following intracerebral inoculation of a familial Alzheimer's disease case carrying Presenilin 1 mutation (fAD-PS1). We did not detect prion transmission to BvM, despite the high susceptibility of BvI previously observed. Immunohistochemistry established the presence of induced pTau depositions in vole brains that were not affected by prions. Furthermore, the phenotype of pTau deposits in vole brains was similar in GSS-F198S and fAD-PS1. Overall, results suggest that, regardless of the cause of pTau deposition and its relationship with PrP Sc in GSS-F198S human-affected brains, the two components possess their own seeding properties, and that pTau deposition is similarly induced by GSS-F198S and fAD-PS1.

Methionine/valine polymorphism at position 129 of the human prion protein, huPrP, is tightly associated with the pathogenic phenotype, disease progress, and age of onset of neurodegenerative diseases such as Creutzfeldt-Jakob disease or Fatal Familial Insomnia. This raises the question of whether and how the amino acid type at position 129 influences the structural properties of huPrP, affecting its folding, stability, and amyloid formation behavior. Here, our detailed biophysical characterization of the 129M and 129V variants of recombinant full-length huPrP(23-230) by amyloid formation kinetics, CD spectroscopy, molecular dynamics simulations, and sedimentation velocity analysis reveals differences in their aggregation propensity and oligomer content, leading to deviating pathways for the conversion into amyloid at acidic pH. We determined that the 129M variant exhibits less secondary structure content before amyloid formation and higher resistance to thermal denaturation compared to the 129V variant, whereas the amyloid conformation of both variants shows similar thermal stability. Additionally, our molecular dynamics simulations and rigidity analyses at the atomistic level identify intramolecular interactions responsible for the enhanced monomer stability of the 129M variant, involving more frequent minimum distances between E196 and R156, forming a salt bridge. Removal of the N-terminal half of the 129M full-length variant diminishes its differences compared to the 129V full-length variant and highlights the relevance of the flexible N terminus in huPrP. Taken together, our findings provide insight into structural properties of huPrP and the effects of the amino acid identity at position 129 on amyloid formation behavior., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction： Metabolism methionine and of folate play a vital function in cellular methylation reactions, DNA synthesis and epigenetic process.However, polymorphisms of methionine have received much attention in recent medical genetics research. Objectives: To ascertain whether the common polymorphisms of the MTRR (Methionine Synthase Reductase) A66G gene could play a role in affecting susceptibility to Chronic Myeloid Leukemia (CML) in Sudanese individuals. Methods: In a case-controlled study, we extracted and analyzed DNA from 200 CML patients and 100 healthy control subjects by the PCR-RFLP method. Results: We found no significant difference in age orgender between the patient group and controls. The MTRR A66G genotypes were distributed based on the Hardy-Weinberg equilibrium (p > 0.05). The variation of MTRR A66G was less significantly frequent in cases with CML (68.35%) than in controls (87%) (OR = 0.146, 95% CI = 0.162−0.662, p < 0.002). Additionally, AG and GG genotypes and G allele were reducing the CML risk (Odds ratio [OR] = 0.365; 95% CI [0.179−0.746]; p = 0.006; OR = 0.292; 95% CI [0.145−0.590]; p = 0.001 and OR = 0.146; 95% CI [0.162−0.662]; p = 0.002 and OR = 2.0; 95% CI [1.3853−2.817]; respectively, (p = 0.000)). Conclusions: Our data demonstrated that heterozygous and homozygous mutant genotypes of MTRR polymorphisms were associated with decreased risk of developing CML in the Sudanese population.

The serine hydroxymethyltransferase (SHMT; E.C. 2.1.2.1) is involved in the interconversion of serine/glycine and tetrahydrofolate (THF)/5,10-methylene THF, playing a key role in one-carbon metabolism, the de novo purine pathway, cellular methylation reactions, redox homeostasis maintenance, and methionine and thymidylate synthesis. GmSHMT08 is the soybean gene underlying soybean cyst nematode (SCN) resistance at the Rhg4 locus. GmSHMT08 protein contains four tetrahydrofolate (THF) cofactor binding sites (L129, L135, F284, N374) and six pyridoxal phosphate (PLP) cofactor binding/catalysis sites (Y59, G106, G107, H134, S190A, H218). In the current study, proteomic analysis of a data set of protein complex immunoprecipitated using GmSHMT08 antibodies under SCN infected soybean roots reveals the presence of enriched pathways that mainly use glycine/serine as a substrate (glyoxylate cycle, redox homeostasis, glycolysis, and heme biosynthesis). Root and leaf transcriptomic analysis of differentially expressed genes under SCN infection supported the proteomic data, pointing directly to the involvement of the interconversion reaction carried out by the serine hydroxymethyltransferase enzyme. Direct site mutagenesis revealed that all mutated THF and PLP sites at the GmSHMT08 resulted in increased SCN resistance. We have shown the involvement of PLP sites in SCN resistance. Specially, the effect of the two Y59 and S190 PLP sites was more drastic than the tested THF sites. This unprecedented finding will help us to identify the biological outcomes of THF and PLP residues at the GmSHMT08 and to understand SCN resistance mechanisms.

The biological improvement of triticale, a cereal of increasing importance in agriculture, may be accelerated via the production of doubled haploid lines using in vitro culture. Among the relevant factors affecting the culture efficiency are Cu(II) or Ag(I) acting, e.g., as cofactors of enzymes. The copper ions are known to positively affect green plant regeneration efficiency. However, the biochemical basis, mainly its role in the generation of in vitro-induced genetic and epigenetic variation and green plant regeneration efficiency, is not well understood. Here, we employed structural equation modeling to evaluate the relationship between de novo DNA methylation affecting the asymmetric context of CHH sequences, the methylation-sensitive Amplified Fragment Length Polymorphism related sequence variation, and the concentration of Cu(II) and Ag(I) ions in induction media, as well as their effect on S-adenosyl-L-methionine perturbations, observed using FTIR spectroscopy, and the green plant regeneration efficiency. Our results allowed the construction of a theory-based model reflecting the biological phenomena associated with green plant regeneration efficiency. Furthermore, it is shown that Cu(II) ions in induction media affect plant regeneration, and by manipulating their concentration, the regeneration efficiency can be altered. Additionally, S-adenosyl-L-methionine is involved in the efficiency of green plant regeneration through methylation of the asymmetric CHH sequence related to de novo methylation. This shows that the Yang cycle may impact the production of green regenerants.

Background: Improved understanding of the stemness regulation mechanism in intrahepatic cholangiocarcinoma (ICC) could identify targets and guidance for adjuvant transarterial chemoembolization (TACE)., Methods: TCGA database was excavated to identify the ICC stemness-associated genes. The pro-stemness effect of target genes was further analyzed by sphere formation assay, qRT-PCR, western blot, flow cytometric analysis, IHC, CCK8 assay and metabolomic analysis. Based on multivariate analysis, a nomogram for ICC patients with adjuvant TACE was established and our result was further confirmed by a validation cohort. Finally, the effect of dietary methionine intervention on chemotherapy was estimated by in vivo experiment and clinical data., Results: In this study, we identified four ICC stemness-associated genes (SDHAF2, MRPS34, MRPL11, and COX8A) that are significantly upregulated in ICC tissues and negatively associated with clinical outcome. Functional studies indicated that these 4-key-genes are associated with self-renewal ability of ICC and transgenic expression of these 4-key-genes could enhance chemoresistance of cholangiocarcinoma cells. Mechanistically, the 4-key-genes-mediated pro-stemness requires the activation of methionine cycle, and their promotion on ICC stemness characteristic is dependent on MAT2A. Importantly, we established a novel nomogram to evaluate the effectiveness of TACE for ICC patients. Further dietary methionine intervene studies indicated that patients with adjuvant TACE might benefit from dietary methionine restriction if they have a relatively high nomogram score (≥ 135)., Conclusions: Our results show that four ICC stemness-associated genes could serve as novel biomarkers in predicting ICC patient's response to adjuvant TACE and their pro-stemness ability may be attributed to the activation of the methionine cycle., (© 2022. The Author(s).)

Abstract: The catechol-O-methyltransferase (COMT) gene has been noted to play an important role in individual variations in the aging process. We investigated whether COMT polymorphism could influence cognition related to white matter networks. More specifically, we examined whether methionine (Met) allele loading is associated with better individual cognitive performance. Thirty-four healthy elderly participants were recruited; each participant's COMT genotype was determined, and Korean version of Montreal Cognitive Assessment scores and a diffusion tensor image were obtained for all participants. The Met carrier group showed significantly lower mean diffusivity, axial diffusivity, and radial diffusivity values for the right hippocampus, thalamus, uncinate fasciculus, and left caudate nucleus than the valine homozygote group. The Met carrier group also scored higher for executive function and attention on the Korean version of Montreal Cognitive Assessment. Based on these results, we can assume that the COMT Met allele has a protective effect on cognitive decline contributing to individual differences in cognitive function in late life period., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

In recent years, morbidity and mortality of prostate cancer (PCa) have increased dramatically, while mechanistic understanding of its onset and progression remains unmet. LncRNA SNHG3 has been proved to stimulate malignant progression of multiple cancers, whereas its functional mechanism in PCa needs to be deciphered. In this study, our analysis in the TCGA database revealed high SNHG3 expression in PCa tissue. Further analysis in starBase, TargetScan, and mirDIP databases identified the SNHG3/miR-152-3p/SLC7A11 regulatory axis. FISH was conducted to assess the distribution of SNHG3 in PCa tissue. Dual-luciferase reporter gene and RIP assays confirmed the relationship among the three objects. Next, qRT-PCR and western blot were conducted to measure expression levels of SNHG3, miR-152-3p, and SLC7A11. CCK-8, colony formation, Transwell, and flow cytometry were carried out to assess proliferation, migration, invasion, methionine dependence, apoptosis, and the cell cycle. It was noted that SNHG3 as a molecular sponge of miR-152-3p stimulated proliferation, migration, and invasion, restrained methionine dependence and apoptosis, and affected the cell cycle of PCa cells via targeting SLC7A11. Additionally, we constructed xenograft tumor models in nude mice and confirmed that knockdown of SNHG3 could restrain PCa tumor growth and elevate methionine dependence in vivo. In conclusion, our investigation improved understanding of the molecular mechanism of SNHG3 modulating PCa progression, thereby generating novel insights into clinical therapy for PCa., (© 2022. The Author(s).)

Background: Metabolic reprogramming and redox homeostasis contribute to esophageal squamous cell carcinoma (ESCC). CDC-like kinase 4 (CLK4) is a dual-specificity kinase that can phosphorylate substrates' tyrosine or serine/threonine residue. However, the role and mechanism of CLK4 in ESCC remain unknown., Methods: CLK4 expression was analysed using publicly available datasets and confirmed in ESCC tissues and cell lines. The biological roles of CLK4 were studied with gain and loss-of-function experiments. Mass spectrometry was employed to examine the effects of CLK4 on metabolic profiling. In vitro kinase assay, co-immunoprecipitation, glutathione S-transferase pulldown, chromatin immunoprecipitation and luciferase reporter were used to elucidate the relationship among CLK4, microphthalmia-associated transcription factor (MITF), COP1 and ZRANB1., Results: CLK4 down-regulation was observed in ESCC cell lines and clinical samples and associated with the methylation of its promoter. Low levels of CLK4 promoted ESCC development by affecting the purine synthesis pathway and nicotinamide adenine dinucleotide phosphate (NADPH)/nicotinamide adenine dinucleotide phosphate (NADP + ) ratio. Interestingly, CLK4 inhibited ESCC development by blocking MITF-enhanced de novo purine synthesis and redox balance. Mechanistically, wild type CLK4 (WT-CLK4) but not kinase-dead CLK4-K189R mutant phosphorylated MITF at Y360. This modification promoted its interaction with E3 ligase COP1 and its K63-linked ubiquitination at K308/K372, leading to sequestosome 1 recognition and autophagic degradation. However, the deubiquitinase ZRANB1 rescued MITF ubiquitination and degradation. In turn, MITF bound to E- rather than M-boxes in CLK4 promoter and transcriptionally down-regulated its expression in ESCC. Clinically, the negative correlations were observed between CLK4, MITF, and purine metabolic markers, which predicts a poor clinical outcome of ESCC patients. Notably, CLK4 itself was a redox-sensitive kinase, and its methionine oxidation at M307 impaired kinase activity, enhanced mitochondria length and inhibited lipid peroxidation, contributing to ESCC., Conclusions: Our data highlight the potential role of CLK4 in modulating redox status and nucleotide metabolism, suggesting potential therapeutic targets in ESCC treatment., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Short-range, non-covalent interactions between amino acid residues determine protein structures and contribute to protein functions in diverse ways. The interactions of the thioether of methionine with the aromatic rings of tyrosine, tryptophan, and/or phenylalanine has long been discussed and such interactions are favorable on the order of 1-3 kcal mol -1 . Here, we carry out a new bioinformatics survey of known protein structures where we assay the propensity of three aromatic residues to localize around the [-CH 2 -S-CH 3 ] of methionine. We term these groups "3-bridge clusters". A dataset consisting of 33,819 proteins with less than 90% sequence identity was analyzed and such clusters were found in 4093 structures (or 12% of the non-redundant dataset). All sub-classes of enzymes were represented. A 3D coordinate analysis shows that most aromatic groups localize near the CH 2 and CH 3 of methionine. Quantum chemical calculations support that the 3-bridge clusters involve a network of interactions that involve the Met-S, Met-CH 2 , Met-CH 3 , and the π systems of nearby aromatic amino acid residues. Selected examples of proposed functions of 3-bridge clusters are discussed.

Activating variants in the receptor tyrosine kinase RE arranged during T ransfection (RET) cause multiple endocrine neoplasia type 2 (MEN 2), an autosomal dominantly inherited cancer-susceptibility syndrome. The variant c.166C>A, p.Leu56Met in RET was recently reported in two patients with medullary thyroid cancer (MTC). The presence of a pheochromocytoma in one of the patients, suggested a possible pathogenic role of the variant in MEN 2A. Here, we present clinical follow up of a Danish RET Leu56Met cohort. Patients were evaluated for signs of MEN 2 according to a set of predefined criteria. None of the seven patients in our cohort exhibited evidence of MEN 2. Furthermore, we found the Leu56Met variant in our in-house diagnostic cohort with an allele frequency of 0.59%, suggesting that it is a common variant in the population. Additionally, none of the patients who harbored the allele were listed in the Danish MTC and MEN 2 registries. In conclusion, our findings do not support a pathogenic role of the Leu56Met variant in MEN 2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hansen, Borgwardt, Rasmussen, Godballe, Poulsen, Vieira, Mathiesen and Rossing.)

Loss of metabolic homeostasis is a hallmark of aging and is characterized by dramatic metabolic reprogramming. To analyze how the fate of labeled methionine is altered during aging, we applied 13 C5-Methionine labeling to Drosophila and demonstrated significant changes in the activity of different branches of the methionine metabolism as flies age. We further tested whether targeted degradation of methionine metabolism components would "reset" methionine metabolism flux and extend the fly lifespan. Specifically, we created transgenic flies with inducible expression of Methioninase , a bacterial enzyme capable of degrading methionine and revealed methionine requirements for normal maintenance of lifespan. We also demonstrated that microbiota-derived methionine is an alternative and important source in addition to food-derived methionine. In this genetic model of methionine restriction (MetR), we also demonstrate that either whole-body or tissue-specific Methioninase expression can dramatically extend Drosophila health- and lifespan and exerts physiological effects associated with MetR. Interestingly, while previous dietary MetR extended lifespan in flies only in low amino acid conditions, MetR from Methioninase expression extends lifespan independently of amino acid levels in the food. Finally, because impairment of the methionine metabolism has been previously associated with the development of Alzheimer's disease, we compared methionine metabolism reprogramming between aging flies and a Drosophila model relevant to Alzheimer's disease, and found that overexpression of human Tau caused methionine metabolism flux reprogramming similar to the changes found in aged flies. Altogether, our study highlights Methioninase as a potential agent for health- and lifespan extension., Competing Interests: The authors declare no competing interest.

Hypoxia is the most prominent feature in human solid tumors and induces activation of hypoxia-inducible factors and their downstream genes to promote cancer progression. However, whether and how hypoxia regulates overall mRNA homeostasis is unclear. Here we show that hypoxia inhibits global-mRNA decay in cancer cells. Mechanistically, hypoxia induces the interaction of AGO2 with LUBAC, the linear ubiquitin chain assembly complex, which co-localizes with miRNA-induced silencing complex and in turn catalyzes AGO2 occurring Met 1 -linked linear ubiquitination (M1-Ubi). A series of biochemical experiments reveal that M1-Ubi of AGO2 restrains miRNA-mediated gene silencing. Moreover, combination analyses of the AGO2-associated mRNA transcriptome by RIP-Seq and the mRNA transcriptome by RNA-Seq confirm that AGO2 M1-Ubi interferes miRNA-targeted mRNA recruiting to AGO2, and thereby facilitates accumulation of global mRNAs. By this mechanism, short-term hypoxia may protect overall mRNAs and enhances stress tolerance, whereas long-term hypoxia in tumor cells results in seriously changing the entire gene expression profile to drive cell malignant evolution., (© 2021. The Author(s).)

Genetic prion disease accounts for 10-15% of prion disease. While insertion of four or more octapeptide repeats are clearly pathogenic, smaller repeat insertions have an unclear pathogenicity. The goal of this case series was to provide an insight into the characteristics of the 2-octapeptide repeat genetic variant and to provide insight into the risk for Creutzfeldt-Jakob disease in asymptomatic carriers. 2-octapeptide repeat insertion prion disease cases were collected from the National Prion Disease Pathology Surveillance Center (US), the National Prion Clinic (UK), and the National Creutzfeldt-Jakob Disease Registry (Australia). Three largescale population genetic databases were queried for the 2-octapeptide repeat insertion allele. Eight cases of 2-octapeptide repeat insertion were identified. The cases were indistinguishable from the sporadic Creutzfeldt-Jakob cases of the same molecular subtype. Western blot characterization of the prion protein in the absence of enzymatic digestion with proteinase K revealed that 2-octapeptide repeat insertion and sporadic Creutzfeldt-Jakob disease have distinct prion protein profiles. Interrogation of large-scale population datasets suggested the variant is of very low penetrance. The 2-octapeptide repeat insertion is at most a low-risk genetic variant. Predictive genetic testing for asymptomatic blood relatives is not likely to be justified given the low risk.

Brain-derived neurotrophic factor (BDNF) is a pleiotropic neuronal growth and survival factor that is indispensable in the brain, as well as in multiple other tissues and organs, including the cardiovascular system. In approximately 30% of the general population, BDNF harbors a nonsynonymous single nucleotide polymorphism that may be associated with cardiometabolic disorders, coronary artery disease, and Duchenne muscular dystrophy cardiomyopathy. We recently showed that transgenic mice with the human BDNF rs6265 polymorphism (Val66Met) exhibit altered cardiac function, and that cardiomyocytes isolated from these mice are also less contractile. To identify the underlying mechanisms involved, we compared cardiac function by echocardiography and performed deep sequencing of RNA extracted from whole hearts of all three genotypes (Val/Val, Val/Met, and Met/Met) of both male and female Val66Met mice. We found female-specific cardiac alterations in both heterozygous and homozygous carriers, including increased systolic (26.8%, p = 0.047) and diastolic diameters (14.9%, p = 0.022), increased systolic (57.9%, p = 0.039) and diastolic volumes (32.7%, p = 0.026), and increased stroke volume (25.9%, p = 0.033), with preserved ejection fraction and fractional shortening. Both males and females exhibited lower heart rates, but this change was more pronounced in female mice than in males. Consistent with phenotypic observations, the gene encoding SERCA2 ( Atp2a2 ) was reduced in homozygous Met/Met mice but more profoundly in females compared to males. Enriched functions in females with the Met allele included cardiac hypertrophy in response to stress, with down-regulation of the gene encoding titin ( Tcap ) and upregulation of BNP ( Nppb ), in line with altered cardiac functional parameters. Homozygous male mice on the other hand exhibited an inflammatory profile characterized by interferon-γ (IFN-γ)-mediated Th1 immune responses. These results provide evidence for sex-based differences in how the BDNF polymorphism modifies cardiac physiology, including female-specific alterations of cardiac-specific transcripts and male-specific activation of inflammatory targets.

Predicting events in the ever-changing environment is a fundamental survival function intrinsic to the physiology of sensory systems, whose efficiency varies among the population. Even though it is established that a major source of such variations is genetic heritage, there are no studies tracking down auditory predicting processes to genetic mutations. Thus, we examined the neurophysiological responses to deviant stimuli recorded with magnetoencephalography (MEG) in 108 healthy participants carrying different variants of Val158Met single-nucleotide polymorphism (SNP) within the catechol-O-methyltransferase (COMT) gene, responsible for the majority of catecholamines degradation in the prefrontal cortex. Our results showed significant amplitude enhancement of prediction error responses originating from the inferior frontal gyrus, superior and middle temporal cortices in heterozygous genotype carriers (Val/Met) vs homozygous (Val/Val and Met/Met) carriers. Integrating neurophysiology and genetics, this study shows how the neural mechanisms underlying optimal deviant detection vary according to the gene-determined cathecolamine levels in the brain., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition affecting behavior and communication, presenting with extremely different clinical phenotypes and features. ASD etiology is composite and multifaceted with several causes and risk factors responsible for different individual disease pathophysiological processes and clinical phenotypes. From a genetic and epigenetic side, several candidate genes have been reported as potentially linked to ASD, which can be detected in about 10-25% of patients. Folate gene polymorphisms have been previously associated with other psychiatric and neurodegenerative diseases, mainly focused on gene variants in the DHFR gene (5q14.1; rs70991108, 19bp ins/del), MTHFR gene (1p36.22; rs1801133, C677T and rs1801131, A1298C), and CBS gene (21q22.3; rs876657421, 844ins68). Of note, their roles have been scarcely investigated from a sex/gender viewpoint, though ASD is characterized by a strong sex gap in onset-risk and progression. The aim of the present review is to point out the molecular mechanisms related to intracellular folate recycling affecting in turn remethylation and transsulfuration pathways having potential effects on ASD. Brain epigenome during fetal life necessarily reflects the sex-dependent different imprint of the genome-environment interactions which effects are difficult to decrypt. We here will focus on the DHFR , MTHFR and CBS gene-triad by dissecting their roles in a sex-oriented view, primarily to bring new perspectives in ASD epigenetics.

Alaska Native people are under-represented in genetic research but have unique gene variation that may critically impact their response to pharmacotherapy. Full resequencing of CYP2C9 in a cross-section of this population identified CYP2C9 Met1Leu ( M1L ) , a novel, relatively common single nucleotide polymorphism hypothesized to confer CYP2C9 poor metabolizer phenotype by disrupting the start codon. M1L is present at a minor allele frequency of 6.3% in Yup'ik Alaska Native people and thus can contribute to the risk of an adverse drug response from narrow-therapeutic-index CYP2C9 substrates such as ( S ) - warfarin. This study's objective was to characterize the catalytic efficiency of the Leu1 variant enzyme in vivo by evaluating the pharmacokinetic behavior of naproxen, a probe substrate for CYP2C9 activity, in genotyped Yup'ik participants. We first confirmed the selectivity of ( S )-naproxen O -demethylation by CYP2C9 using activity-phenotyped human liver microsomes and selective cytochrome P450 inhibitors and then developed and validated a novel liquid chromatography mass spectrometry method for simultaneous quantification of ( S ) - naproxen, ( S ) -O -desmethylnaproxen, and naproxen acyl glucuronide in human urine. The average ratio of ( S ) -O -desmethylnaproxen to unchanged ( S ) - naproxen in urine was 18.0 ± 8.0 ( n = 11) for the homozygous CYP2C9 Met1 reference group and 10.3 ± 6.6 ( n = 11) for the Leu1 variant carrier group ( P = 0.011). The effect of M1L variation on CYP2C9 function and its potential to alter the pharmacokinetics of drugs metabolized by the enzyme has clinical implications and should be included in a variant screening panel when pharmacogenetic testing in the Alaska Native population is warranted. SIGNIFICANCE STATEMENT: The novel CYP2C9 Met1Leu variant in Alaska Native people was recently identified. This study validated ( S ) - naproxen as a CYP2C9 probe substrate to characterize the in vivo functional activity of the CYP2C9 Leu1 variant. The results of this pharmacogenetic-pharmacokinetic study suggest that the CYP2C9 Leu1 variant exhibits loss of enzyme activity. This finding may be important to consider when administering narrow-therapeutic-index medications metabolized by CYP2C9 and also compels further investigation to characterize novel genetic variation in understudied populations., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 by The Author(s).)

Previous research has shown a consistent association among genetic factors, psychological symptoms and pain associated with fibromyalgia. However, how these symptoms interact to moderate genetic factors in fibromyalgia has rarely been studied to date. The present research investigates whether psychological symptoms can moderate the effects of catechol-O-methyltransferase on pain and fatigue. A total of 108 women diagnosed with fibromyalgia and 77 healthy control participants took part in the study. Pain, fatigue, and psychological symptoms (anxiety, depression, pain catastrophizing, fear of pain and fear of movement) were measured by self-report questionnaires. Two types of statistical analyses were performed; the first was undertaken to explore the influences of COMT genotypes on clinical symptoms by comparing patients with fibromyalgia and healthy controls. In the second analysis, moderation analyses to explore the role of psychological symptoms as potential factors that moderate the relationship between pain/fatigue and COMT genotypes were performed. The main results indicated that patients carrying the Met/Met genotype reported significantly higher levels of fatigue than heterozygote carriers (i.e., Met/Val genotype) and higher levels of fatigue, but not significantly different, than Val homozygote carriers. Among patients with fibromyalgia carrying methionine alleles (i.e., Met/Met + Met/Val carriers), only those who scored high on medical fear of pain, experienced an intensified feeling of fatigue. Thus, the present research suggests that fear of pain, as a psychological symptom frequently described in fibromyalgia may act as a moderating factor in the relationship between the Met allele of the COMT gene and the increase or decrease in self-reported fatigue. Although further research with wider patient samples is needed to confirm the present findings, these results point out that the use of psychological interventions focused on affective symptomatology might be a useful tool to reduce the severity of fibromyalgia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Background: The S-adenosyl-methionine (SAM) availability is crucial for DNA methylation, an epigenetic mechanism involved in nonsyndromic cleft lip with or without cleft palate (NSCL/P) expression. The aim of this study was to assess the association between single-nucleotide polymorphisms (SNPs) of genes involved in SAM synthesis and NSCL/P in a Chilean population., Methods: In 234 cases and 309 controls, 18 SNPs in AHCY, MTR, MTRR, and MAT2A were genotyped, and the association between them and the phenotype was evaluated based on additive (allele), dominant, recessive and haplotype models, by odds ratio (OR) computing., Results: Three deep intronic SNPs of MTR showed a protective effect on NSCL/P expression: rs10925239 (OR 0.68; p = 0.0032; q = 0.0192), rs10925254 (OR 0.66; p = 0.0018; q = 0.0162), and rs3768142 (OR 0.66; p = 0.0015; q = 0.0162). Annotations in expression database demonstrate that the protective allele of the three SNPs is associated with a reduction of MTR expression summed to the prediction by bioinformatic tools of its potentiality to modify splicing sites., Conclusions: The protective effect against NSCL/P of these intronic MTR SNPs seems to be related to a decrease in MTR enzyme expression, modulating the SAM availability for proper substrate methylation. However, functional analyses are necessary to confirm our findings., Impact: SAM synthesis pathway genetic variants are factors associated to NSCL/P. This article adds new evidence for folate related genes in NSCL/P in Chile. Its impact is to contribute with potential new markers for genetic counseling.

The assimilation of inorganic sulfate and the synthesis of the sulfur-containing amino acids methionine and cysteine is mediated by a multibranched biosynthetic pathway. We have investigated this circuitry in the fungal pathogen Candida albicans, which is phylogenetically intermediate between the filamentous fungi and Saccharomyces cerevisiae. In S. cerevisiae, this pathway is regulated by a collection of five transcription factors (Met4, Cbf1, Met28, and Met31/Met32), while in the filamentous fungi the pathway is controlled by a single Met4-like factor. We found that in C. albicans, the Met4 ortholog is also a core regulator of methionine biosynthesis, where it functions together with Cbf1. While C. albicans encodes this Met4 protein, a Met4 paralog designated Met28 (Orf19.7046), and a Met31 protein, deletion, and activation constructs suggest that of these proteins only Met4 is actually involved in the regulation of methionine biosynthesis. Both Met28 and Met31 are linked to other functions; Met28 appears essential, and Met32 appears implicated in the regulation of genes of central metabolism. Therefore, while S. cerevisiae and C. albicans share Cbf1 and Met4 as central elements of the methionine biosynthesis control, the other proteins that make up the circuit in S. cerevisiae are not members of the C. albicans control network, and so the S. cerevisiae circuit likely represents a recently evolved arrangement., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Context: The I148M (rs738409-G) variant in PNPLA3 increases liver fat content but may be protective against cardiovascular disease. Insulin resistance (IR) amplifies the effect of PNPLA3-I148M on liver fat., Objective: To study whether PNPLA3-I148M confers an antihyperlipidemic effect in insulin-resistant patients., Design: Cross-sectional study comparing the impact of PNPLA3-I148M on plasma lipids and lipoproteins in 2 cohorts, both divided into groups based on rs738409-G allele carrier status and median HOMA-IR., Setting: Tertiary referral center., Patients: A total of 298 obese patients who underwent a liver biopsy during bariatric surgery (bariatric cohort: age 49 ± 9 years, body mass index [BMI] 43.2 ± 6.8 kg/m2), and 345 less obese volunteers in whom liver fat was measured by proton magnetic resonance spectroscopy (nonbariatric cohort: age 45 ± 14 years, BMI 29.7 ± 5.7 kg/m2)., Main Outcome Measures: Nuclear magnetic resonance profiling of plasma lipids, lipoprotein particle subclasses and their composition., Results: In both cohorts, individuals carrying the PNPLA3-I148M variant had significantly higher liver fat content than noncarriers. In insulin-resistant and homozygous carriers, PNPLA3-I148M exerted a distinct antihyperlipidemic effect with decreased very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) particles and their constituents, and increased high-density lipoprotein particles and their constituents, compared with noncarriers. VLDL particles were smaller and LDL particles larger in PNPLA3-I148M carriers. These changes were geometrically opposite to those due to IR. PNPLA3-I148M did not have a measurable effect in patients with lower IR, and its effect was smaller albeit still significant in the less obese than in the obese cohort., Conclusions: PNPLA3-I148M confers an antiatherogenic plasma lipid profile particularly in insulin-resistant individuals., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Soybean is an important source of protein, oil and carbohydrates, as well as other beneficial nutrients. A major function of proteins in nutrition is to supply adequate amounts of amino acids. Although they are essential for human nutrition, the sulfur-containing amino acids cysteine (Cys) and methionine (Met) are often limited and the genetic control of their content in soybean seeds is poorly characterized. This study aimed to characterize the phenotypic variation and identify quantitative trait loci (QTL) associated with Cys and Met content in a core set of 137 soybean lines, representative of the genetic diversity among Canadian short-season soybean, spanning maturity groups 000-II (MG000-II). Significant phenotypic differences were found among these lines for Cys, Met and Cys + Met content. Using both a mixed linear model and six multi-locus methods with a catalogue of 2.18 M SNPs, we report a total of nine QTLs and seventeen QTNs of which seven comprise promising candidate genes. This work allowed us to reproducibly detect multiple novel loci associated with sulfur-containing amino acid content. The markers and genes identified in this study may be useful for soybean genetic improvement aiming to increase Cys and Met content.

Background: Alteration in brain-derived neurotrophic factor (BDNF) production is a marker of neuropathological conditions, which has led to the investigation of Val66Met polymorphism occurring in the human BDNF gene ( BDNF ). Presently, there are no reported methods available for the analysis of Val66Met impact on human BDNF functioning. Purpose: To develop a qRT-PCR protocol for the allele-specific expression evaluation of the Val66Met polymorphism in BDNF . Methods: Using RNA extracted from muscle samples of 9 healthy volunteers (32.9 ± 10.3 y) at rest and following a maximal effort aerobic capacity exercise test, a protocol was developed for the detection of Val66/Met66 allele-specific BDNF expression in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) - relative to housekeeping genes - and validated by absolute quantification in Droplet Digital Polymerase Chain Reaction (ddPCR). Results: Differences in the relative values of BDNF mRNA were confirmed by ddPCR analysis. HPRT1 and B2M were the most stable genes expressed in muscle tissue among different metabolic conditions, while GAPDH revealed to be metabolic responsive. Conclusion : Our qRT-PCR protocol successfully determines the allele-specific detection and changes in BDNF expression regarding the Val66Met polymorphism., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site., Competing Interests: The authors have declared that no competing interests exist.

Methionine restrictive diet may alleviate ischaemia/reperfusion (I/R)-induced myocardial injury, but its underlying mechanism remains unclear. HE staining was performed to evaluate the myocardial injury caused by I/R and the effect of methionine-restricted diet (MRD) in I/R mice. IHC and Western blot were carried out to analyse the expression of CSE, CHOP and active caspase3 in I/R mice and hypoxia/reoxygenation (H/R) cells. TUNEL assay and flow cytometry were used to assess the apoptotic status of I/R mice and H/R cells. MTT was performed to analyse the proliferation of H/R cells. H2S assay was used to evaluate the concentration of H2S in the myocardial tissues and peripheral blood of I/R mice. I/R-induced mediated myocardial injury and apoptosis were partially reversed by methionine-restricted diet (MRD) via the down-regulation of CSE expression and up-regulation of CHOP and active caspase3 expression. The decreased H2S concentration in myocardial tissues and peripheral blood of I/R mice was increased by MRD. Accordingly, in a cellular model of I/R injury established with H9C2 cells, cell proliferation was inhibited, cell apoptosis was increased, and the expressions of CSE, CHOP and active caspase3 were dysregulated, whereas NaHS treatment alleviated the effect of I/R injury in H9C2 cells in a dose-dependent manner. This study provided a deep insight into the mechanism underlying the role of MRD in I/R-induced myocardial injury., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The imbalance of prenatal micronutrients may perturb one-carbon (C1) metabolism and increase the risk for neuropsychiatric disorders. Prenatal excessive methionine (MET) produces in mice behavioral phenotypes reminiscent of human schizophrenia. Whether in-utero programming or early life caregiving mediate these effects is, however, unknown. Here, we show that the behavioral deficits of MET are independent of the early life mother-infant interaction. We also show that MET produces in early life profound changes in the brain C1 pathway components as well as glutamate transmission, mitochondrial function, and lipid metabolism. Bioinformatics analysis integrating metabolomics and transcriptomic data reveal dysregulations of glutamate transmission and lipid metabolism, and identify perturbed pathways of methylation and redox reactions. Our transcriptomics Linkage analysis of MET mice and schizophrenia subjects reveals master genes involved in inflammation and myelination. Finally, we identify potential metabolites as early biomarkers for neurodevelopmental defects and suggest therapeutic targets for schizophrenia.

Several soybean ( Glycine max ) germplasms, such as Nishiyamahitashi 98-5 (NH), have an intense seaweed-like flavor after cooking because of their high seed S -methylmethionine (SMM) content. In this study, we compared the amounts of amino acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed a comparably higher SMM content alongside a higher free Met content in NH seeds, suggesting that the SMM-hyperaccumulation phenotype of NH soybean was related to Met metabolism in seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, we examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci in segregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus on chromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon insertion into the intron of a gene, Glyma.10g172700 , is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700- encoded recombinant protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NH efficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of Met to sections of developing soybean seeds resulted in transient increases in Met levels, followed by continuous increases in SMM concentrations, which was likely caused by Met methyltransferase activity in the seeds. Accordingly, we propose that the SMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting in transient accumulation of Met, which is converted into SMM to avoid the harmful effects caused by excess free Met., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently classified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K-digested abnormal prion protein (PrP res ) isoform identified by Western blotting (type 1 or type 2). Converging evidence led to the view that MM/MV1, VV/MV2, and VV1 and MM2 sCJD cases are caused by distinct prion strains. However, in a significant proportion of sCJD patients, both type 1 and type 2 PrP res were reported to accumulate in the brain, which raised questions about the diversity of sCJD prion strains and the coexistence of two prion strains in the same patient. In this study, a panel of sCJD brain isolates ( n  = 29) that displayed either a single or mixed type 1/type 2 PrP res were transmitted into human-PrP-expressing mice (tgHu). These bioassays demonstrated that two distinct prion strains (M1 CJD and V2 CJD ) were associated with the development of sCJD in MM1/MV1 and VV2/MV2 patients. However, in about 35% of the investigated VV and MV cases, transmission results were consistent with the presence of both M1 CJD and V2 CJD strains, including in patients who displayed a "pure" type 1 or type 2 PrP res The use of a highly sensitive prion in vitro amplification technique that specifically probes the V2 CJD strain revealed the presence of the V2 CJD prion in more than 80% of the investigated isolates, including isolates that propagated as a pure M1 CJD strain in tgHu. These results demonstrate that at least two sCJD prion strains can be present in a single patient. IMPORTANCE sCJD occurrence is currently assumed to result from spontaneous and stochastic formation of a misfolded PrP nucleus in the brains of affected patients. This original nucleus then recruits and converts nascent PrP C into PrP Sc , leading to the propagation of prions in the patient's brain. Our study demonstrates the coexistence of two prion strains in the brains of a majority of the 23 sCJD patients investigated. The relative proportion of these sCJD strains varied both between patients and between brain areas in a single patient. These findings strongly support the view that the replication of an sCJD prion strain in the brain of a patient can result in the propagation of different prion strain subpopulations. Beyond its conceptual importance for our understanding of prion strain properties and evolution, the sCJD strain mixture phenomenon and its frequency among patients have important implications for the development of therapeutic strategies for prion diseases., (Copyright © 2020 Cassard et al.)

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress., Competing Interests: Declaration of Interests J.M.D. is a consultant for FORGE Life Sciences and co-founder of Galilei BioSciences. D.W.L has received funding from, and is a scientific advisory board member of, Aeovian Pharmaceuticals, which seeks to develop novel, selective mTOR inhibitors for the treatment of various diseases. The remaining authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Neonatal diabetes has been shown to be associated with high neuropsychiatric morbidity in a genotype-phenotype-dependent manner. However, the specific impact of different mutations on intellectual functioning is still insufficiently characterized. Specifically, only a small number of subjects with developmental delay have been comprehensively assessed, creating a knowledge gap about patients carrying the heaviest burden., Research Design and Methods: We assessed the intellectual functioning and mental health of the complete Norwegian population with K ATP channel neonatal diabetes. Eight sulfonylurea-treated children (five with the p.V59M genotype [ KCNJ11 ]) were assessed using age-matched control subjects with type 1 diabetes. The investigations included a physical and motor developmental examination, cerebral MRI, psychometrical examination, and questionnaires assessing intellectual capabilities and psychiatric morbidity., Results: A strong genotype-phenotype correlation was found, revealing the p.V59M genotype as highly associated with substantial intellectual disability, with no significant correlation with the time of sulfonylurea initiation. Consistent with previous studies, other genotypes were associated with minor cognitive impairment. Cerebral MRI verified normal brain anatomy in all but one child., Conclusions: We here presented a comprehensive assessment of intellectual functioning in the largest cohort of p.V59M subjects to date. The level of intellectual disability revealed not only changes the interpretation of other psychological measures but downplays a strong protective effect of sulfonylurea. Within the scope of this study, we could not find evidence supporting an early treatment start to be beneficial, although a weaker effect cannot be ruled out., (© 2020 by the American Diabetes Association.)

Objective: L-methionine is an important sulfur-containing amino acid essential for humans and animals. Its biosynthesis pathway is complex and highly regulated. This study aims to explore the bottleneck limiting the improvement of L-methionine productivity and apply efficient strategies to increase L-methionine production in engineered E. coli., Results: The enzyme O-succinylhomoserine sulfhydrylase involved in thiolation of OSH to form homocysteine was overexpressed in the engineered strain E. coli W3110 IJAHFEBC/PAm, resulting in L-methionine production increased from 2.8 to 3.22 g/L in shake flask cultivation. By exogenous addition of L-glycine as the precursor of one carbon unit, the titer of L-methionine was increased to 3.68 g/L. The glycine cleavage system was further strengthened for the efficient one carbon unit supply and a L-methionine titer of 3.96 g/L was obtained, which was increased by 42% compared with that of the original strain., Conclusions: Insufficient supply of one carbon unit was found to be the issue limiting the improvement of L-methionine productivity and its up-regulation significantly promoted the L-methionine production in the engineered E. coli.

Memory abnormalities are considered a core feature of posttraumatic stress disorder (PTSD). Studies attempting to quantify such memory dysfunction in PTSD have reported that individuals with this disorder exhibit selective memory bias toward negative material. The low expression Met allele of brain-derived neurotrophic factor (BDNF) Val66Met polymorphism has been associated with the aetiology of PTSD and with memory abnormalities. It is therefore possible that the BDNF Val66Met polymorphism can moderate the relationship between PTSD and memory bias. Here we examined this association in 50 civilian women with PTSD and 70 non-trauma-exposed healthy control women. All subjects were genotyped for the BDNF Val66Met (rs6265) polymorphism. Negative memory bias was assessed using a recognition memory task. Patients showed significantly greater negative memory bias compared to controls. In patients, negative memory bias significantly increased with increasing numbers of Met alleles; while no significant relationship was seen in controls. Further pairwise analyses revealed that patients with the Met allele had significantly greater negative memory bias than controls. These results suggest that the relationship between PTSD and negative memory bias can be moderated by the BDNF Val66Met polymorphism. More studies are needed to further clarify the relationship between this polymorphism and memory abnormalities in PTSD.

Human prion diseases are classified into sporadic, genetic, and acquired forms. Within this last group, iatrogenic Creutzfeldt-Jakob disease (iCJD) is caused by human-to-human transmission through surgical and medical procedures. After reaching an incidence peak in the 1990s, it is believed that the iCJD historical period is probably coming to an end, thanks to lessons learnt from past infection sources that promoted new prion prevention and decontamination protocols. At this point, we sought to characterise the biomarker profile of iCJD and compare it to that of sporadic CJD (sCJD) for determining the value of available diagnostic tools in promptly recognising iCJD cases. To that end, we collected 23 iCJD samples from seven national CJD surveillance centres and analysed the electroencephalogram and neuroimaging data together with a panel of seven CSF biomarkers: 14-3-3, total tau, phosphorylated/total tau ratio, alpha-synuclein, neurofilament light, YKL-40, and real-time quaking induced conversion of prion protein. Using the cut-off values established for sCJD, we found the sensitivities of these biomarkers for iCJD to be similar to those described for sCJD. Given the limited relevant information on this issue to date, the present study validates the use of current sCJD biomarkers for the diagnosis of future iCJD cases., Competing Interests: The authors declare no conflict of interest.