Untargeted metabolomics in the aqueous humor reveals the involvement of TAAR pathway in glaucoma.

Understanding the metabolic dysfunctions and underlying complex pathological mechanisms of neurodegeneration in glaucoma could help discover disease pathways, identify novel biomarkers, and rationalize newer therapeutics. Therefore, we aimed to investigate the local metabolomic alterations in the aqueous humor and plasma of primary glaucomatous patients. This study cohort comprised primary open-angle glaucoma (POAG), primary angle-closure glaucoma (PACG), and cataract control groups. Aqueous humor and plasma samples were collected from patients undergoing trabeculectomy or cataract surgery and subjected to high-resolution mass spectrometry (HRMS) analysis. Spectral information was processed, and the acquired data were subjected to uni-variate as well as multi-variate statistical analyses using MetaboAnalyst ver5.0. To further understand the localized metabolic abnormalities in glaucoma, metabolites affected in aqueous humor were distinguished from metabolites altered in plasma in this study. Nine and twelve metabolites were found to be significantly altered (p < 0.05, variable importance of projection >1 and log2 fold change ≥0.58/≤ -0.58) in the aqueous humor of PACG and POAG patients, respectively. The galactose and amino acid metabolic pathways were locally affected in the PACG and POAG groups, respectively. Based on the observation of the previous findings, gene expression profiles of trace amine-associated receptor-1 (TAAR-1) were studied in rat ocular tissues. The pharmacodynamics of TAAR-1 were explored in rabbits using topical administration of its agonist, ß-phenyl-ethylamine (ß-PEA). TAAR-1 was expressed in the rat's iris-ciliary body, optic nerve, lens, and cornea. ß-PEA elicited a mydriatic response in rabbit eyes, without altering intraocular pressure. Targeted analysis of ß-PEA levels in the aqueous humor of POAG patients showed an insignificant elevation. This study provides new insights regarding alterations in both localized and systemic metabolites in primary glaucomatous patients. This study also demonstrated the propensity of ß-PEA to cause an adrenergic response through the TAAR-1 pathway.

Design and Synthesis of Novel Indole Ethylamine Derivatives as a Lipid Metabolism Regulator Targeting PPARα/CPT1 in AML12 Cells.

Peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1 (CPT1) are important targets of lipid metabolism regulation for nonalcoholic fatty liver disease (NAFLD) therapy. In the present study, a set of novel indole ethylamine derivatives (4, 5, 8, 9) were designed and synthesized. The target product (compound 9) can effectively activate PPARα and CPT1a. Consistently, in vitro assays demonstrated its impact on the lipid accumulation of oleic acid (OA)-induced AML12 cells. Compared with AML12 cells treated only with OA, supplementation with 5, 10, and 20 µM of compound 9 reduced the levels of intracellular triglyceride (by 28.07%, 37.55%, and 51.33%) with greater inhibitory activity relative to the commercial PPARα agonist fenofibrate. Moreover, the compound 9 supplementations upregulated the expression of hormone-sensitive triglyceride lipase (HSL) and adipose triglyceride lipase (ATGL) and upregulated the phosphorylation of acetyl-CoA carboxylase (ACC) related to fatty acid oxidation and lipogenesis. This dual-target compound with lipid metabolism regulatory efficacy may represent a promising type of drug lead for NAFLD therapy.

Electrochemical Reversible Reforming between Ethylamine and Acetonitrile on Heterostructured Pd-Ni(OH)2 Nanosheets.

Rational design of electrocatalysts is essential to achieve desirable performance of electrochemical synthesis process. Heterostructured catalysts have thus attracted widespread attention due to their multifunctional intrinsic properties, and diverse catalytic applications with corresponding outstanding activities. Here, we report an in situ restoration strategy for the synthesis of ultrathin Pd-Ni(OH)2 nanosheets. Such Pd-Ni(OH)2 nanosheets exhibit excellent activity and selectivity towards reversible electrochemical reforming of ethylamine and acetonitrile. In the acetonitrile reduction process, Pd acts as reaction center, while Ni(OH)2 provide proton hydrogen through promoting the dissociation of water. Also ethylamine oxidation process can be achieved on the surface of the heterostructured nanosheets with abundant Ni(II) defects. More importantly, an electrolytic cell driven by solar cells was successfully constructed to realize ethylamine-acetonitrile reversible reforming. This work demonstrates the importance of heterostructure engineering in the rational synthesis of multifunctional catalysts towards electrochemical synthesis of fine chemicals.

Nonaqueous fractionation and overexpression of fluorescent-tagged enzymes reveals the subcellular sites of L-theanine biosynthesis in tea.

l-Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l-theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l-theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l-theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l-theanine synthetase and also to determine the subcellular localization of fluorescent-tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l-theanine biosynthesis, and cytosol-located CsTSI was the key l-theanine synthase. In tea shoot tissue, l-theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l-theanine synthases. In addition, l-theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.

[Ion chromatography for the determination of ethylamine in the workplace air].

Objective: To establish a method for the determination of ethylamine in the air of the workplace by ion chromatography. Methods: In August 2020, ethylamine in the air of the workplace was adsorbed by a basic silica gel tube, and ultrasonically desorbed with a 1 mmol/L sulfuric acid solution, and then qualitatively and quantitatively determined by ion chromatography. Results: The linear range of the method was 0.014-50 µg/ml, and the linear equation of the standard curve was y=0.1243x+0.0429, the correlation coefficient was r=0.9997. The detection limit of the method was 4.29 µg/L, and the lower limit of quantification was 14.29 µg/L. The lowest quantitative concentration was 0.012 mg/m(3) (in term of sampling 6.0 L) . The average desorption efficiency of the method was 97.31%, the precision was 1.00%-1.68%, and the standard recovery rate was 96.33%-99.61%. Conclusion: This method is fast, sensitive and accurate, and can be used for the determination of ethylamine in the air of workplace.

Efficient fermentative production of L-theanine by Corynebacterium glutamicum.

L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. L-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in L-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of L-theanine in Corynebacterium glutamicum, an industrially safe host. For L-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of L-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an L-glutamate overproducing strain, to produce L-theanine upon ethylamine addition to the hosts. Second, the L-glutamate exporter in host cells was disrupted, which markedly increased the L-theanine titer in GDK-9 cells and almost eliminated the accumulation of L-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated L-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived L-serine/L-alanine decarboxylases; however, these enzymes showed no L-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L L-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of L-theanine achieved using ethylamine supplementation.

Identification of a Novel Gene Encoding the Specialized Alanine Decarboxylase in Tea (Camellia sinensis) Plants.

Theanine, a unique amino acid in Camellia sinensis, accounts for more than 50% of total free amino acids in tea and has a significant contribution to the quality of green tea. Previous research indicated that theanine is synthesized from glutamic acid (Glu) and ethylamine mainly in roots, and that theanine accumulation depends on the availability of ethylamine which is derived from alanine (Ala) decarboxylation catalyzed by alanine decarboxylase (AlaDC). However, the specific gene encoding AlaDC protein remains to be discovered in tea plants or in other species. To explore the gene of AlaDC in tea plants, the differences in theanine contents and gene expressions between pretreatment and posttreatment of long-time nitrogen starvation were analyzed in young roots of two tea cultivars. A novel gene annotated as serine decarboxylase (SDC) was noted for its expression levels, which showed high consistency with theanine content, and the expression was remarkably high in young roots under sufficient nitrogen condition. To verify its function, full-length complementary DNA (cDNA) of this candidate gene was cloned from young roots of tea seedlings, and the target protein was expressed and purified from Escherichia coli (E. coli). The enzymatic activity of the protein for Ala and Ser was measured in vitro using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). The results illustrated that the target protein could catalyze the decarboxylation of Ala despite of its high similarity with SDC from other species. Therefore, this novel gene was identified as AlaDC and named CsAlaDC. Furthermore, the gene expression levels of CsAlaDC in different tissues of tea plants were also quantified with quantitative real-time PCR (qRT-PCR). The results suggest that transcription levels of CsAlaDC in root tissues are significantly higher than those in leaf tissues. That may explain why theanine biosynthesis preferentially occurs in the roots of tea plants. The expression of the gene was upregulated when nitrogen was present, suggesting that theanine biosynthesis is regulated by nitrogen supply and closely related to nitrogen metabolism for C. sinensis. The results of this study are significant supplements to the theanine biosynthetic pathway and provide evidence for the differential accumulation of theanine between C. sinensis and other species.

Serotonin-1A receptor dependent modulation of pain and reward for improving therapy of chronic pain.

Chronic pain conditions such as low back pain and osteoarthritis are the most prominent causes of disability worldwide. Morphine and other opioid drugs are the gold standard treatment for severe pain, including surgical pain, but the use of these drugs for chronic pain is limited largely because long term use of these drugs is associated with drug abuse and hyperalgesia which produces a negative impact on the treatment. Non-addictive treatments for chronic pain are, therefore, highly needed. Commonly used opioid drugs activate mu opioid receptors, resulting in an inhibition of tonic activity of nociceptive neurons. The rewarding effects of opioid drugs are also mediated via activation of mu opioid receptors and inhibition of GABA mediated control of the activity of dopamineregic neurons. Enhanced glutamate release and greater activity of NMDA glutamate receptors is linked to the hyperalgesic effects of opioid drugs. Evidence suggests that activation of serotonin (5-hydroxytryptamine; 5-HT)-1 A receptors modulates dopamine neurotransmission to inhibit rewarding effects of drugs of abuse. Activation of these receptors inhibits glutamate release from the sensory neurons to reduce pain transmission. To help develop strategies for improving therapeutics in chronic pain, and draw research interest in the synthesis of non-addictive opioid drugs which do not predispose to hyperalgesia, the present article concerns the potential mechanism involved in 5-HT-1 A receptor mediated inhibition of pain and reward.

The enzymatic resolution of 1-(4-chlorophenyl)ethylamine by Novozym 435 to prepare a novel triazolopyrimidine herbicide.

The kinetic resolution of (R,S)-1-(4-chlorophenyl)ethylamine was accomplished using a commercial lipase from Candida antarctica (Novozym 435). The performance of this lipase was investigated for the enantioselective amidation of (R,S)-1-(4-chlorophenyl)ethylamine, leaving the target product (S)-1-(4-chlorophenyl)ethylamine in its unreacted form. The effects of various types of solvents and an acyl donor, the molar ratio of the substrate to the acyl donor, and the reaction temperature were studied. The optimum reaction conditions were found to result in amidation with methyl 2-tetrahydrofuroate at 40°C in methyl tert-butyl ether, with a substrate/acyl donor molar ratio of 1:2.4. The conversion rate of (R,S)-1-(4-chlorophenyl)ethylamine was 52%, with an enantiomeric excess of 99% towards the unreacted substrate in a reaction time of 22 hours. Finally, using optically pure (S)-1-(4-chlorophenyl)ethylamine as the raw material, the chemical synthesis of (S)-N-(1-(4-chlorphenyl)ethyl)-2-(5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)acetamide, a novel triazolopyrimidine herbicide, was achieved, and the total yield and purity were 83.5% and 95.3%, respectively.

Application of the Asymmetric Pictet-Spengler Reaction in the Total Synthesis of Natural Products and Relevant Biologically Active Compounds.

Tetrahydroisoquinolines are the framework of numerous natural products predominantly alkaloids, an important and one of the most wide spread families of naturally occurring compounds in the plant kingdom. Tetrahydroisoquinolines are commonly constructed through an old reaction, the so-called Pictet−Spengler Reaction (PSR). In this reaction, a ß-aryl ethylamine undergoes an acid mediated condensation with a suitable aldehyde or ketone, followed by ring closure. In this review, we aim to highlight the applications of the asymmetric variant of this old name reaction in the total synthesis of natural products, chiefly, alkaloids, which exhibit significant biological properties.

Spinal sigma-1 receptor activation increases the production of D-serine in astrocytes which contributes to the development of mechanical allodynia in a mouse model of neuropathic pain.

We have previously demonstrated that activation of the spinal sigma-1 receptor (Sig-1R) plays an important role in the development of mechanical allodynia (MA) via secondary activation of the N-methyl-d-aspartate (NMDA) receptor. Sig-1Rs have been shown to localize to astrocytes, and blockade of Sig-1Rs inhibits the pathologic activation of astrocytes in neuropathic mice. However, the mechanism by which Sig-1R activation in astrocytes modulates NMDA receptors in neurons is currently unknown. d-serine, synthesized from l-serine by serine racemase (Srr) in astrocytes, is an endogenous co-agonist for the NMDA receptor glycine site and can control NMDA receptor activity. Here, we investigated the role of d-serine in the development of MA induced by spinal Sig-1R activation in chronic constriction injury (CCI) mice. The production of d-serine and Srr expression were both significantly increased in the spinal cord dorsal horn post-CCI surgery. Srr and d-serine were only localized to astrocytes in the superficial dorsal horn, while d-serine was also localized to neurons in the deep dorsal horn. Moreover, we found that Srr exists in astrocytes that express Sig-1Rs. The CCI-induced increase in the levels of d-serine and Srr was attenuated by sustained intrathecal treatment with the Sig-1R antagonist, BD-1047 during the induction phase of neuropathic pain. In behavioral experiments, degradation of endogenous d-serine with DAAO, or selective blockade of Srr by LSOS, effectively reduced the development of MA, but not thermal hyperalgesia in CCI mice. Finally, BD-1047 administration inhibited the development of MA and this inhibition was reversed by intrathecal treatment with exogenous d-serine. These findings demonstrate for the first time that the activation of Sig-1Rs increases the expression of Srr and d-serine in astrocytes. The increased production of d-serine induced by CCI ultimately affects dorsal horn neurons that are involved in the development of MA in neuropathic mice.

The coordination chemistry and magnetism of some 3d-4f and 4f amino-polyalcohol compounds.

Triethanolamine, teaH3, and diethanolamine, RdeaH2, 3d-4f and 4f compounds demonstrate an enormous variety in their structure and bonding. This review examines the synthetic strategies to these molecules and their magnetic properties, whilst trying to assess these ligands' suitability towards new SMMs and magnetic refrigerants.

Simplified microchip electrophoresis for rapid separation of urine proteins.

BACKGROUND: Urine protein test has been widely used in clinics, but to determine the type of proteinuria is usually difficult due to technical limitations. METHODS: In the current study, a rapid and simple method to separate and determine urine proteins by a microchip electrophoresis (ME) system has been developed in which only 4 min are required. RESULTS: Optimal separation conditions have been established by using 15 s injection time at 500 and 1,500 V separation voltage in 75 mmol/l borate buffer containing 0.8 mmol/l calcium lactate and 1% ϕ ethylamine (pH 10.55). Relative standard deviation (RSD) of migration time with purified human albumin and human transferring was 2.68% and 2.24%, and RSD of the peak area was 5.85% and 4.96%, respectively. The linear detection range was 1.0-15.0 g/l for purified human albumin and 1.0-10.0 g/l for human transferrin, with the same detection limit (S/N = 3) of 0.4 g/l. Finally, comparing to conventional agarose gel electrophoresis, the same results were obtained by using ME by testing clinical samples including 60 selective proteinuria, 105 nonselective proteinuria, and 6 overflow proteinuria. CONCLUSION: This newly established ME could have broad applications to determine the type of proteinuria in clinics.

Nitrogen fertilisation increases biogenic amines and amino acid concentrations in Vitis vinifera var. Riesling musts and wines.

BACKGROUND: Wines rich in biogenic amines can cause adverse health effects to the consumer. Being nitrogen-containing substances, the amount of amines in wines might be strongly influenced by the rate of nitrogen fertiliser application during grape production. The aim of this work was to evaluate the effect of nitrogen fertilisation in the vineyard on the formation of biogenic amines in musts and wines. RESULTS: In a field experiment which compared unfertilised and fertilised (60 and 150 kg N ha(-1)) vines over two separate years, the total amine concentrations in must and wine increased. The latter was due to an increase of individual amines such as ethylamine, histamine, isopentylamine, phenylethylamine and spermidine in the musts and wines with the nitrogen application. Furthermore, the fermentation process increased the concentration of histamine and ethylamine in most of the treatments, while spermidine, spermine and isopentylamine concentrations generally decreased. Throughout both vintages, the concentrations of tyramine and histamine of the investigated musts and wines never reached detrimental levels to the health of non-allergenic people. CONCLUSIONS: Nitrogen fertilisation has a significant effect on amines formation in musts and wines. Furthermore, during fermentation, ethylamine and histamine increased while other amines were presumably serving as N sources during fermentation.

[Site-specific mutation of ω-transaminase and the biocatalytic preparation of (S)-1-(2-fluorophenyl) ethylamine].

(S)-1-(2-fluorophenyl) ethylamine plays a crucial role as a chiral building block in pharmaceutical synthesis. ω-transaminases are widely recognized as environmentally friendly and efficient catalysts for the preparation of chiral amines. In this study, we isolated a novel ω-transaminase, PfTA, from Pseudogulbenkiania ferrooxidans through gene mining in the NCBI database. By employing semi-rational design, we obtained a Y168R/R416Q variant with enhanced enzyme activity. This variant exhibited the ability to catalyze the synthesis of (S)-1-(2-fluorophenyl) ethylamine from 2-fluorophenone, achieving a yield of 83.58% and an enantioselectivity exceeding 99% after a 10 h reaction. Compared to the wild type, the specific enzyme activity of the Y168R/R416Q variant reached 47.04 U/mg, which represents an increase of 11.65 times. Additionally, the catalytic efficiency, as measured by kcat/Km, was increased by 20.9 times. Molecular docking and structural simulation analysis revealed that the primary factor contributing to the improved catalytic efficiency is the expansion of the enzyme's active pocket and the alleviation of steric hindrance.

Engineered the Active Site of ω-Transaminase for Enhanced Asymmetric Synthesis Towards (S)-1-[4-(Trifluoromethyl)phenyl]ethylamine.

ω-Transaminase (ω-TA) is a promising biocatalyst for the synthesis of chiral amines. In this study, a ω-TA derived from Vitreoscilla stercoraria DSM 513 (VsTA) was heterologous expressed in recombinant E. coli cells and applied to reduce 4'-(trifluoromethyl)acetophenone (TAP) to (S)-1-[4-(trifluoromethyl)phenyl]ethylamine ((S)-TPE), a pharmaceutical intermediate of chiral amine. Aimed to a more efficient synthesis of (S)-TPE, VsTA was further engineered via a semi-rational strategy. Compared to wild-type VsTA, the obtained R411A variant exhibited 2.39 times higher activity towards TAP and enhanced catalytic activities towards other prochiral aromatic ketones. Additionally, better thermal stability for R411A variant was observed with 25.4% and 16.3% increase in half-life at 30 °C and 40 °C, respectively. Structure-guided analysis revealed that the activity improvement of R411A variant was attributed to the introduction of residue A411, which is responsible for the increase in the hydrophobicity of substrate tunnel and the alleviation of steric hindrance, thereby facilitating the accessibility of hydrophobic substrate TAP to the active center of VsTA. This study provides an efficient strategy for the engineering of ω-TA based on semi-rational approach and has the potential for the molecular modification of other biocatalysts.

Crystal-Phase-Engineered High-Entropy Alloy Aerogels for Enhanced Ethylamine Electrosynthesis from Acetonitrile.

Crystal-phase engineering that promotes the rearrangement of active atoms to form new structural frameworks achieves excellent result in the field of electrocatalysis and optimizes the performance of various electrochemical reactions. Herein, for the first time, it is found that the different components in metallic aerogels will affect the crystal-phase transformation, especially in high-entropy alloy aerogels (HEAAs), whose crystal-phase transformation during annealing is more difficult than medium-entropy alloy aerogels (MEAAs), but they still show better electrochemical performance. Specifically, PdPtCuCoNi HEAAs with the parent phase of face-centered cubic (FCC) PdCu possess excellent 89.24% of selectivity, 746.82 mmol h-1 g-1 cat. of yield rate, and 90.75% of Faraday efficiency for ethylamine during acetonitrile reduction reaction (ARR); while, maintaining stability under 50 h of long-term testing and ten consecutive electrolysis cycles. The structure-activity relationship indicates that crystal-phase regulation from amorphous state to FCC phase promotes the atomic rearrangement in HEAAs, thereby optimizing the electronic structure and enhancing the adsorption strength of reaction intermediates, improving the catalytic performance. This study provides a new paradigm for developing novel ARR electrocatalysts and also expands the potential of crystal-phase engineering in other application areas.

Stereoselective synthesis of (R)-(+)-1-(1-naphthyl)ethylamine by ω-amine transaminase immobilized on amino modified multi-walled carbon nanotubes and biocatalyst recycling.

Immobilized enzymes exhibit favorable advantages in biocatalysis, such as high operation stability, feasible reusability, and improved organic solvents tolerance. Herein, an immobilized ω-amine transaminase AtATA@MWCNTs-NH2 is successfully prepared using amino modified multi-walled carbon nanotubes as carrier and glutaraldehyde as crosslinker. Under the optimum immobilization conditions, the activity recovery is 78.7%. Compared with purified enzyme AtATA, AtATA@MWCNTs-NH2 possesses superior stability, even in harsh conditions (e.g., high temperature, acidic or alkali environment, and different kind of organic solvents). To simplify the separation and extraction of products, we choose methanol (10%, v/v) as the cosolvent, replacing DMSO (20%, v/v) in our previous work, for the catalytic reaction of AtATA@MWCNTs-NH2. AtATA@MWCNTs-NH2 can be used for stereoselective synthesis (R)-(+)- 1(1-naphthyl)ethylamine ((R)-NEA) for 15 cycles, with the e.e.p (enantiomeric excess) > 99.5%. The catalytic process of AtATA@MWCNTs-NH2 achieves cycle production of (R)-NEA using methanol as cosolvent.

Do the biogenic amines ethylamine, ethanolamine and methylamine reach toxic concentrations in foods?

Ethylamine, ethanolamine and methylamine are biogenic amines (BA) - active metabolites that, despite having important biological functions, may accumulate at toxic concentrations in certain foods. Very little information exists on the toxicity of these BA in this context. This study provides new insights into their cytotoxicity with respect to a human intestinal epithelial cell line, as assessed using real-time cell analyzer technology. A preliminary evaluation of the cytotoxic mode of action was also performed. The present results show that only ethylamine was cytotoxic for these cells at food concentrations. These new data should help establish legal limits for these BA in foods.