Heterologous expression of the Glycine soja Kunitz-type protease inhibitor GsKTI improves resistance to drought stress and Helicoverpa armigera in transgenic Arabidopsis lines.

Kunitz-like protease inhibitors (KTIs) have been identified to play critical roles in insect defense, but evidence for their involvement in drought stress is sparse. The aim of this study was to identify and functionally characterize a Kunitz-like protease inhibitor, GsKTI, from the wild soybean (Glycine soja) variety ED059. Expression patterns suggest that drought stress and insect herbivory may induce GsKTI transcript levels. Transgenic Arabidopsis lines overexpressing GsKTI have been shown to exhibit enhanced drought tolerance by regulating the ABA signaling pathway and increasing xylem cell number. Transgenic Arabidopsis leaves overexpressing GsKTI interfered with insect digestion and thus had a negative effect on the growth of Helicoverpa armigera. It is concluded that GsKTI increases resistance to drought stress and insect attack in transgenic Arabidopsis lines.

The Role of Serine-Glyoxylate Aminotransferase and Malyl-CoA Lyase in the Metabolism of Methylococcus capsulatus Bath.

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin-Benson-Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation. The Δsga mutant obtained by double homologous recombination showed a prolonged lag phase, and after the lag period, the growth rate became similar to that of the wild type strain. The elevated intracellular levels of glutamate, serine, glycine, alanine, methionine, leucine, and succinate suggested significant metabolic changes in the mutant cells. Deletion of the mcl gene resulted in very poor growth and glycine only partially improved growth of the mutant strain. Cells of Δmcl mutant possess lower content of histidine, but enhanced level of alanine, leucine, and lysine than those of the wild type strain. Our data imply the importance of the serine cycle enzymes in metabolism of the methanotroph as well as relationships of the three C1 assimilation pathways in the gammaproteobacterial methanotrophs.

Differences in rectal amino acid levels determine bacteria-originated sex pheromone specificity in two closely related flies.

Sex pheromones are widely used by insects as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Although researchers have obtained extensive knowledge about sex pheromones, little is known about the differentiation mechanism of sex pheromones in closely related species. Using Bactrocera dorsalis and Bactrocera cucurbitae as the study model, we investigated how the male-borne sex pheromones are different. The results demonstrated that both 2,3,5-trimethylpyrazine (TMP) and 2,3,5,6-tetramethylpyrazine (TTMP) were sex pheromones produced by rectal Bacillus in the two flies. However, the TMP/TTMP ratios were reversed, indicating sex pheromone specificity in the two flies. Bacterial fermentation results showed that different threonine and glycine levels were responsible for the preference of rectal Bacillus to produce TMP or TTMP. Accordingly, threonine (glycine) levels and the expression of the threonine and glycine coding genes were significantly different between B. dorsalis and B. cucurbitae. In vivo assays confirmed that increased rectal glycine and threonine levels by amino acid feeding could significantly decrease the TMP/TTMP ratios and result in significantly decreased mating abilities in the studied flies. Meanwhile, decreased rectal glycine and threonine levels due to RNAi of the glycine and threonine coding genes was found to significantly increase the TMP/TTMP ratios and result in significantly decreased mating abilities. The study contributes to the new insight that insects and their symbionts can jointly regulate sex pheromone specificity in insects, and in turn, this helps us to better understand how the evolution of chemical communication affects speciation.

Salivary metabolomic profile associated with cariogenic risk in children.

OBJECTIVES: To identify the metabolomic differences in the saliva of healthy children versus children with active carious lesions and to estimate the predictive capacity of a model based on the salivary metabolomic profile. METHODS: A study of cases (n = 31) and controls (n = 37) was designed for children aged between 6 and 12 (mean age of the cases: 8.9; controls: 8.7). The said children attended public health centers in Valencia, Spain. Intraoral examinations were performed by a single examiner using ICDAS II diagnostic criteria. Unstimulated total saliva samples were analyzed by nuclear magnetic resonance (NMR) spectroscopy. RESULTS: The dft index for cases was 2.84 while it was 0.19 for the control group, the DMFT index was 1.13 and 0.11, respectively. The predictive model generated by the multivariate PLS-DA analysis projects a separation between the cases and the controls on the score chart with a predictive capacity and generating an area under the curve of 0.71. The metabolites: 3-methyl-2-oxovalerate, 3-hydroxybutyrate, lactate, acetone, citrate, ornithine, ethanolamine, taurine, proline, glycine, mannose, glucose, 1-6-Anhydro-ß-d-glucose and citraconate, are those that show greater significance in the model. In the controls, glycine (Cohen's d = 0.430) and glucose (Cohen's d = 0.560) present higher means compared to the cases. On the contrary, taurine (Cohen's d= -0.474) and mannose (Cohen's d= -0.456) show higher means in cases compared to controls. CONCLUSIONS: Our findings show a difference in the salivary metabolomic profiles, specifically in the groups of saccharides and amino acids, suggesting an association of these with the level of caries risk. CLINICAL SIGNIFICANCE: The results reported in the present study reinforce the use of salivary metabolomics as a research method for the search for salivary biomarkers that allow the evaluation of caries risk in patients. Furthermore, it brings us closer to a personalized medicine that will help in dental caries prevention strategies.

Novel Phenylene Lipids That Are Positive Allosteric Modulators of Glycine Receptors and Inhibitors of Glycine Transporter 2.

Chronic pain is a complex condition that remains resistant to current therapeutics. We previously synthesized a series of N-acyl amino acids (NAAAs) that inhibit the glycine transporter, GlyT2, some of which are also positive allosteric modulators of glycine receptors (GlyRs). In this study, we have synthesized a library of NAAAs that contain a phenylene ring within the acyl tail with the objective of improving efficacy at both GlyT2 and GlyRs and also identifying compounds that are efficacious as dual-acting modulators to enhance glycine neurotransmission. The most efficacious positive allosteric modulator of GlyRs was 2-[8-(2-octylphenyl)octanoylamino]acetic acid (8-8 OPGly) which potentiates the EC5 for glycine activation of GlyRα1 by 1500% with an EC50 of 664 nM. Phenylene-containing NAAAs with a lysine headgroup were the most potent inhibitors of GlyT2 with (2S)-6-amino-2-[8-(3-octylphenyl)octanoylamino]hexanoic acid (8-8 MPLys) inhibiting GlyT2 with an IC50 of 32 nM. The optimal modulator across both proteins was (2S)-6-amino-2-[8-(2-octylphenyl)octanoylamino]hexanoic acid (8-8 OPLys), which inhibits GlyT2 with an IC50 of 192 nM and potentiates GlyRs by up to 335% at 1 µM. When tested in a dual GlyT2/GlyRα1 expression system, 8-8 OPLys caused the greatest reductions in the EC50 for glycine. This suggests that the synergistic effects of a dual-acting modulator cause greater enhancements in glycinergic activity compared to single-target modulators and may provide an alternate approach to the development of new non-opioid analgesics for the treatment of chronic pain.

N-oleoyl glycine and N-oleoyl alanine attenuate alcohol self-administration and preference in mice.

The endocannabinoid system (ECS) plays a key modulatory role during synaptic plasticity and homeostatic processes in the brain and has an important role in the neurobiological processes underlying drug addiction. We have previously shown that an elevated ECS response to psychostimulant (cocaine) is involved in regulating the development and expression of cocaine-conditioned reward and sensitization. We therefore hypothesized that drug-induced elevation in endocannabinoids (eCBs) and/or eCB-like molecules (eCB-Ls) may represent a protective mechanism against drug insult, and boosting their levels exogenously may strengthen their neuroprotective effects. Here, we determine the involvement of ECS in alcohol addiction. We first measured the eCBs and eCB-Ls levels in different brain reward system regions following chronic alcohol self-administration using LC-MS. We have found that following chronic intermittent alcohol consumption, N-oleoyl glycine (OlGly) levels were significantly elevated in the prefrontal cortex (PFC), and N-oleoyl alanine (OlAla) was significantly elevated in the PFC, nucleus accumbens (NAc) and ventral tegmental area (VTA) in a region-specific manner. We next tested whether exogenous administration of OlGly or OlAla would attenuate alcohol consumption and preference. We found that systemic administration of OlGly or OlAla (60 mg/kg, intraperitoneal) during intermittent alcohol consumption significantly reduced alcohol intake and preference without affecting the hedonic state. These findings suggest that the ECS negatively regulates alcohol consumption and boosting selective eCBs exogenously has beneficial effects against alcohol consumption and potentially in preventing relapse.

Beyond photorespiration: the significance of glycine and serine in leaf metabolism.

The elucidation and removal of photorespiratory metabolic constraints will be necessary to improve crop yield in the next agricultural revolution. Fu et al. studied metabolic fluxes in the photorespiratory pathway and report that serine is the major export, whereas dynamic alterations in glycine pools orchestrate CO2 assimilation during the induction and relaxation of photorespiration.

Mammary gland development in male rats perinatally exposed to propiconazole, glyphosate, or their mixture.

This study aimed to assess whether perinatal exposure to propiconazole (PRO), glyphosate (GLY) or their mixture (PROGLY) alters key endocrine pathways and the development of the male rat mammary gland. To this end, pregnant rats were orally exposed to vehicle, PRO, GLY, or a mixture of PRO and GLY from gestation day 9 until weaning. Male offspring were euthanized on postnatal day (PND) 21 and PND60. On PND21, GLY-exposed rats showed reduced mammary epithelial cell proliferation, whereas PRO-exposed ones showed increased ductal p-Erk1/2 expression without histomorphological alterations. On PND60, GLY-exposed rats showed reduced mammary gland area and estrogen receptor alpha expression and increased aromatase expression, whereas PRO-exposed ones showed enhanced lobuloalveolar development and increased lobular hyperplasia. However, PROGLY did not modify any of the endpoints evaluated. In summary, PRO and GLY modified the expression of key molecules and the development of the male mammary gland individually but not together.

Amino Chemoassay Profiling of Aromatic Aldehydes-Unraveling Drivers of Their Skin Sensitization Potency.

Aromatic aldehydes are ubiquitous in humans' everyday life. As aldehydes, they can form imines (Schiff bases) with amino groups of skin proteins, leading to immune response-triggered allergic contact dermatitis. Many known aromatic aldehydes are considered as weak or nonsensitizers, but others like atranol and chloratranol, two components of the fragrance oak moss absolute, show strong sensitization potency. This large discrepancy in potency and, in particular, the underlying reaction mechanisms are only little understood so far. To reduce this knowledge gap, our chemoassay employing glycine-para-nitroanilide (Gly-pNA) as an amino model nucleophile was applied to 23 aromatic aldehydes. The determined Gly-pNA second-order rate constants for imine formation (k1 ≤ 2.85 L·mol-1·min-1) and the imine stability constant (K ≤ 333 L·mol-1) are on the lower end of the known amino reactivity scale for aldehydes, confirming many aromatic aldehydes as less potent sensitizers in line with animal and human data. The substantially higher sensitization potency of atranol and chloratranol, in turn, is reflected by their unique reaction chemistry profiles, inter alia, identifying them as cross-linkers able to form thermodynamically more stable epitopes with skin proteins (despite low formation kinetics, k1). The discussion further includes a comparison of experimentally determined k1 values with computed reactivity data (Taft σ*), the impact of the substitution pattern of the aryl ring on the reactivity with Gly-pNA, and analytically determined adduct patterns. Overall, this work provides new insights into the reaction of aromatic aldehydes with amino groups under aqueous conditions and fosters a better understanding of the chemistry underlying skin sensitization.

Astrocyte Calcium Signaling Shifts the Polarity of Presynaptic Plasticity.

Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic receptors expressed on their surface, astrocytes detect extracellular neurotransmitters, and in turn, release gliotransmitters to modify synaptic strength, while they can also alter neuronal membrane excitability by modulating extracellular ionic milieu. Given the seemingly large repertoire of synaptic modulation, when, where and how astrocytes interact with synapses remain to be fully understood. Previously, we have identified a role for astrocyte NMDA receptor and L-VGCCs signaling in heterosynaptic presynaptic plasticity and promoting the heterogeneity of presynaptic strengths at hippocampal synapses. Here, we have sought to further clarify the mode by which astrocytes regulate presynaptic plasticity by exploiting a reduced culture system to globally evoke NMDA receptor-dependent presynaptic plasticity. Recording from a postsynaptic neuron intracellularly loaded with BAPTA, briefly bath applying NMDA and glycine induces a stable decrease in the rate of spontaneous glutamate release, which requires the presence of astrocytes and the activation of A1 adenosine receptors. Upon preventing astrocyte calcium signaling or blocking L-VGCCs, NMDA + glycine application triggers an increase, rather than a decrease, in the rate of spontaneous glutamate release, thereby shifting the presynaptic plasticity to promote an increase in strength. Our findings point to a crucial and surprising role of astrocytes in controlling the polarity of NMDA receptor and adenosine-dependent presynaptic plasticity. Such a pivotal mechanism unveils the power of astrocytes in regulating computations performed by neural circuits and is expected to profoundly impact cognitive processes.

The glycine receptor alpha 3 subunit mRNA expression shows sex-dependent differences in the adult mouse brain.

BACKGROUND: The glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating neurons. Impairments of the glycine receptors can cause motor and sensory deficits. Glycine exerts inhibition through interaction with ligand-gated ion channels composed of alpha and beta subunits. We have investigated the mRNA expression of the glycine receptor alpha 3 (Glra3) subunit in the nervous system as well as in several peripheral organs of female and male mice. RESULTS: Single-cell RNA sequencing (scRNA-seq) data analysis on the Zeisel et al. (2018) dataset indicated widespread but low expression of Glra3 in vesicular glutamate transporter 2 (Vglut2, Slc17a6) positive and vesicular inhibitory amino acid transporter (Viaat, Slc32a1)positive neurons of the mouse central nervous system. Highest occurrence of Glra3 expression was identified in the cortex, amygdala, and striatal regions, as well as in the hypothalamus, brainstem and spinal cord. Bulk quantitative real-time-PCR (qRT-PCR) analysis demonstrated Glra3 expression in cortex, amygdala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Additionally, male mice expressed higher levels of Glra3 in all investigated brain areas compared with female mice. Lastly, RNAscope spatially validated Glra3 expression in the areas indicated by the single-cell and bulk analyses. Moreover, RNAscope analysis confirmed co-localization of Glra3 with Slc17a6 or Slc32a1 in the central nervous system areas suggested from the single-cell data. CONCLUSIONS: Glra3 expression is low but widespread in the mouse central nervous system. Clear sex-dependent differences have been identified, indicating higher levels of Glra3 in several telencephalic and diencephalic areas, as well as in cerebellum and brainstem, in male mice compared with female mice.

1 H, 15 N, and 13 C backbone and side chain resonance assignments of the cold shock domain of the Arabidopsis thaliana glycine-rich protein AtGRP2.

AtGRP2 (Arabidopsis thaliana glycine-rich protein 2) is a 19-kDa RNA-binding glycine-rich protein that regulates key processes in A. thaliana. AtGRP2 is a nucleo-cytoplasmic protein with preferential expression in developing tissues, such as meristems, carpels, anthers, and embryos. AtGRP2 knockdown leads to an early flowering phenotype. In addition, AtGRP2-silenced plants exhibit a reduced number of stamens and abnormal development of embryos and seeds, suggesting its involvement in plant development. AtGRP2 expression is highly induced by cold and abiotic stresses, such as high salinity. Moreover, AtGRP2 promotes double-stranded DNA/RNA denaturation, indicating its role as an RNA chaperone during cold acclimation. AtGRP2 is composed of an N-terminal cold shock domain (CSD) followed by a C-terminal flexible region containing two CCHC-type zinc fingers interspersed with glycine-rich sequences. Despite its functional relevance in flowering time regulation and cold adaptation, the molecular mechanisms employed by AtGRP2 are largely unknown. To date, there is no structural information regarding AtGRP2 in the literature. Here, we report the 1H, 15N, and 13C backbone and side chain resonance assignments, as well as the chemical shift-derived secondary structure propensities, of the N-terminal cold shock domain of AtGRP2, encompassing residues 1-90. These data provide a framework for AtGRP2-CSD three-dimensional structure, dynamics, and RNA binding specificity investigation, which will shed light on its mechanism of action.

Excitatory GluN1/GluN3A glycine receptors (eGlyRs) in brain signaling.

GluN3A is a glycine-binding subunit belonging to the NMDA receptor (NMDAR) family that can assemble with GluN1 subunits to form unconventional NMDARs insensitive to glutamate and activated by glycine only. The existence of such excitatory glycine receptors (eGlyRs) in the central nervous system (CNS) has long remained elusive. Recently, eGlyRs have been identified in specific brain regions, where they represent a novel neuronal signaling modality by which extracellular glycine tunes neuronal excitability, circuit function, and behavior. In this review, we summarize the emerging knowledge regarding these underappreciated receptors. The existence of eGlyRs reshapes current understanding of NMDAR diversity and of glycinergic signaling, previously thought to be primarily inhibitory. Given that GluN3A expression is concentrated in brain regions regulating emotional responses, eGlyRs are potential new targets of therapeutic interest in neuropsychiatry.

Efficient production of natural sunscreens shinorine, porphyra-334, and mycosporine-2-glycine in Saccharomyces cerevisiae.

Mycosporine-like amino acids (MAAs) are promising natural sunscreens mainly produced in marine organisms. Until now, metabolic engineering efforts to produce MAAs in heterologous hosts have mainly focused on shinorine production, and the low production levels are still not suitable for industrial applications. In this study, we successfully developed Saccharomyces cerevisiae strains that can efficiently produce various disubstituted MAAs, including shinorine, porphyra-334, and mycosporine-2-glycine (M2G), which are formed by conjugating serine, threonine, and glycine to mycosporine-glycine (MG), respectively. We first generated an MG-producing strain by multiple integration of the biosynthetic genes from cyanobacteria and applying metabolic engineering strategies to increase sedoheptulose-7-phosphate pool, a substrate for MG production. Next, five mysD genes from cyanobacteria, which encode D-Ala-D-Ala ligase homologues that conjugate an amino acid to MG, were introduced into the MG-producing strain to determine the substrate preference of each MysD enzyme. MysDs from Lyngbya sp., Nostoclinckia, and Euhalothece sp. showed high specificity toward serine, threonine, and glycine, resulting in efficient production of shinorine, porphyra-334, and M2G, respectively. This is the first report on the production of porphyra-334 and M2G in S. cerevisiae. Furthermore, we identified that the substrate specificity of MysD was determined by the omega loop region of 43-45 amino acids predicted based on its structural homology to a D-Ala-D-Ala ligase from Thermus thermophilus involved in peptidoglycan biosynthesis. The substrate specificities of two MysD enzymes were interchangeable by swapping the omega loop region. Using the engineered strain expressing mysD from Lyngbya sp. or N. linckia, up to 1.53 g/L shinorine or 1.21 g/L porphyra-334 was produced by fed-batch fermentation in a 5-L bioreactor, the highest titer reported so far. These results suggest that S. cerevisiae is a promising host for industrial production of different types of MAAs, providing a sustainable and eco-friendly alternative for the development of natural sunscreens.

Serine metabolism during differentiation of human iPSC-derived astrocytes.

Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. l-Serine is the precursor of the two main co-agonists of the N-methyl-d-aspartate receptor, glycine and d-serine. Strikingly, dysfunctions in both l- and d-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l-serine, glycine, threonine, l- and d-aspartate (which level is unexpectedly higher than that of d-serine) show the same biosynthetic time course. A significant utilization of l-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d-serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations.

Use of alkyne-tagged myristic acid to detect N-terminal myristoylation.

Protein N-terminal myristoylation is a lipidic modification typically occurring to the α-amino group of N-terminal glycine residues of proteins. It is catalyzed by the N-myristoyltransferase (NMT) enzyme family. Many studies in the past three decades have highlighted the importance of N-terminal glycine myristoylation as it affects protein localization, protein-protein interaction, and protein stability, thereby regulating multiple biological processes, including immune cell signaling, cancer progression, and infections. This book chapter will present protocols for using alkyne-tagged myristic acid to detect the N-myristoylation of targeted proteins in cell lines and compare global N-myristoylation levels. We then described a protocol of SILAC proteomics that compare the levels of N-myristoylation on a proteomic scale. These assays allow for the identification of potential NMT substrates and the development of novel NMT inhibitors.

Adaptive responses in uteroplacental metabolism and fetoplacental nutrient shuttling and sensing during placental insufficiency.

Glucose, lactate, and amino acids are major fetal nutrients. During placental insufficiency-induced intrauterine growth restriction (PI-IUGR), uteroplacental weight-specific oxygen consumption rates are maintained, yet fetal glucose and amino acid supply is decreased and fetal lactate concentrations are increased. We hypothesized that uteroplacental metabolism adapts to PI-IUGR by altering nutrient allocation to maintain oxidative metabolism. Here, we measured nutrient flux rates, with a focus on nutrients shuttled between the placenta and fetus (lactate-pyruvate, glutamine-glutamate, and glycine-serine) in a sheep model of PI-IUGR. PI-IUGR fetuses weighed 40% less and had decreased oxygen, glucose, and amino acid concentrations and increased lactate and pyruvate versus control (CON) fetuses. Uteroplacental weight-specific rates of oxygen, glucose, lactate, and pyruvate uptake were similar. In PI-IUGR, fetal glucose uptake was decreased and pyruvate output was increased. In PI-IUGR placental tissue, pyruvate dehydrogenase (PDH) phosphorylation was decreased and PDH activity was increased. Uteroplacental glutamine output to the fetus and expression of genes regulating glutamine-glutamate metabolism were lower in PI-IUGR. Fetal glycine uptake was lower in PI-IUGR, with no differences in uteroplacental glycine or serine flux. These results suggest increased placental utilization of pyruvate from the fetus, without higher maternal glucose utilization, and lower fetoplacental amino acid shuttling during PI-IUGR. Mechanistically, AMP-activated protein kinase (AMPK) activation was higher and associated with thiobarbituric acid-reactive substances (TBARS) content, a marker of oxidative stress, and PDH activity in the PI-IUGR placenta, supporting a potential link between oxidative stress, AMPK, and pyruvate utilization. These differences in fetoplacental nutrient sensing and shuttling may represent adaptive strategies enabling the placenta to maintain oxidative metabolism.NEW & NOTEWORTHY These results suggest increased placental utilization of pyruvate from the fetus, without higher maternal glucose uptake, and lower amino acid shuttling in the placental insufficiency-induced intrauterine growth restriction (PI-IUGR) placenta. AMPK activation was associated with oxidative stress and PDH activity, supporting a putative link between oxidative stress, AMPK, and pyruvate utilization. These differences in fetoplacental nutrient sensing and shuttling may represent adaptive strategies enabling the placenta to maintain oxidative metabolism at the expense of fetal growth.

Germline-enforced enrichment for charged amino acids in TCR beta chain (TCRß) complementarity determining region 3 (CDR-B3) alters T cell development, repertoire content, and antigen recognition.

T cell receptor beta chain (TCRß) diversity (Dß) gene segments are highly conserved across evolution, with trout Dß1 sequence identical to human and mouse Dß1. A key conserved feature is enrichment for glycine in all three Dß reading frames (RFs). Previously, we found that replacement of mouse Dß1 with a typical immunoglobulin DH sequence, which unlike Dß is enriched for tyrosine, leads to an increase in the use of tyrosine in TCRß complementarity determining region 3 (CDR-B3) after thymic selection, altering T cell numbers, CDR-B3 diversity, and T cell function. To test whether the incorporation of charged amino acids into the Dß sequence in place of glycine would also influence T cell biology, we targeted the TCRß locus with a novel glycine-deficient DßDKRQ allele that replaces Dß1 coding sequence with charged amino acids in all three reading frames. Developing T cells using DßDKRQ expressed TCR CDR-B3s depleted of tyrosine and glycine and enriched for germline-encoded lysine, arginine, and glutamine. Total thymocytes declined in number during the process of ß selection that occurs during the transition from the DN3bc to DN4 stage. Conventional thymocyte and T cell numbers remained reduced at all subsequent thymic stages and in the spleen. By contrast, regulatory T cell numbers were increased in Peyer's patches and the large intestine. In terms of functional consequences, T cell reactivity to an ovalbumin immunodominant epitope was reduced. These findings buttress the view that natural selection of Dß sequence is used to shape the pre-immune TCRß repertoire, affecting both conventional and regulatory T cell development and influencing epitope recognition.

Cellular Response and Molecular Mechanism of Glyphosate Degradation by Chryseobacterium sp. Y16C.

Glyphosate is one of the most widely used herbicides worldwide. Unfortunately, the continuous use of glyphosate has resulted in serious environmental contamination and raised public concern about its impact on human health. In our previous study, Chryseobacterium sp. Y16C was isolated and characterized as an efficient degrader that can completely degrade glyphosate. However, the biochemical and molecular mechanisms underlying its glyphosate biodegradation ability remain unclear. In this study, the physiological response of Y16C to glyphosate stimulation was characterized at the cellular level. The results indicated that, in the process of glyphosate degradation, Y16C induced a series of physiological responses in the membrane potential, reactive oxygen species levels, and apoptosis. The antioxidant system of Y16C was activated to alleviate the oxidative damage caused by glyphosate. Furthermore, a novel gene, goW, was expressed in response to glyphosate. The gene product, GOW, is an enzyme that catalyzes glyphosate degradation, with putative structural similarities to glycine oxidase. GOW encodes 508 amino acids, with an isoelectric point of 5.33 and a molecular weight of 57.2 kDa, which indicates that it is a glycine oxidase. GOW displays maximum enzyme activity at 30 °C and pH 7.0. Additionally, most of the metal ions exhibited little influence on the enzyme activity except for Cu2+. Finally, with glyphosate as the substrate, the catalytic efficiency of GOW was higher than that of glycine, although opposite results were observed for the affinity. Taken together, the current study provides new insights to deeply understand and reveal the mechanisms of glyphosate degradation in bacteria.

Glycine and aging: Evidence and mechanisms.

The restriction of calories, branched-chain amino acids, and methionine have all been shown to extend lifespan in model organisms. Recently, glycine was found to boost longevity in genetically heterogenous mice. This simple amino acid similarly extends lifespan in rats and improves health in mammalian models of age-related disease. While compelling data indicate that glycine is a pro-longevity molecule, divergent mechanisms may underlie its effects on aging. Glycine is abundant in collagen, a building block for glutathione, a precursor to creatine, and an acceptor for the enzyme glycine N-methyltransferase (GNMT). A review of the literature strongly implicates GNMT, which clears methionine from the body by taking a methyl group from S-adenosyl-L-methionine and methylating glycine to form sarcosine. In flies, Gnmt is required for reduced insulin/insulin-like growth factor 1 signaling and dietary restriction to fully extend lifespan. The geroprotector spermidine requires Gnmt to upregulate autophagy genes and boost longevity. Moreover, the overexpression of Gnmt is sufficient to extend lifespan and reduce methionine levels. Sarcosine, or methylglycine, declines with age in multiple species and is capable of inducing autophagy both in vitro and in vivo. Taken all together, existing evidence suggests that glycine prolongs life by mimicking methionine restriction and activating autophagy.