Monofluorophore-based Two-Photon Ratiometric Fluorescent Probe for the Quantitative Imaging of Fatty Acid Amide Hydrolase in Live Neurons and Mouse Brain Tissues.

Fatty acid amide hydrolase (FAAH) plays a crucial role in the metabolism of the endocannabinoid system by hydrolyzing a series of bioactive amides, whose abnormal levels are associated with neuronal disorders including Alzheimer's disease (AD). However, due to the lack of suitable quantitative sensing tools, real-time and accurate monitoring of the activity of FAAH in living systems remains unresolved. Herein, a novel enzyme-activated near-infrared two-photon ratiometric fluorescent probe (CANP) based on a naphthylvinylpyridine monofluorophore is successfully developed, in which the electron-withdrawing amide moiety is prone to be hydrolyzed to an electron-donating amine group under the catalysis of FAAH, leading to the activation of the intramolecular charge transfer process and the emergence of a new 80 nm red-shifted emission, thereby achieving a ratiometric luminescence response. Benefiting from the high selectivity, high sensitivity, and ratiometric response to FAAH, the probe CANP is successfully used to quantitatively monitor and image the FAAH levels in living neurons, by which an amyloid ß (Aß)-induced upregulation of endogenous FAAH activity is observed. Similar increases in FAAH activity are found in various brain regions of AD model mice, indicating a potential fatty acid amide metabolite-involved pathway for the pathological deterioration of AD. Moreover, our quantitative FAAH inhibition experiments further demonstrate the great value of CANP as an efficient visual probe for in situ and precise assessment of FAAH inhibitors in complex living systems, assisting the discovery of FAAH-related therapeutic agents.

An Extremely Sensitive Ultra-High Throughput Growth Selection Assay for the Identification of Amidase Activity.

In the last decades, biocatalysis has offered new perspectives for the synthesis of (chiral) amines, which are essential building blocks for pharmaceuticals, fine and bulk chemicals. In this regard, amidases have been employed due to their broad substrate scope and their independence from expensive cofactors. To expand the repertoire of amidases, tools for their rapid identification and characterization are greatly demanded. In this work an ultra-high throughput growth selection assay based on the production of the folate precursor p-aminobenzoic acid (PABA) is introduced to identify amidase activity. PABA-derived amides structurally mimic the broad class of commonly used chromogenic substrates derived from p-nitroaniline. This suggests that the assay should be broadly applicable for the identification of amidases. Unlike conventional growth selection assays that rely on substrates as nitrogen or carbon source, our approach requires PABA in sub-nanomolar concentrations, making it exceptionally sensitive and ideal for engineering campaigns that aim at enhancing amidase activities from minimally active starting points, for example. The presented assay offers flexibility in the adjustment of sensitivity to suit project-specific needs using different expression systems and fine-tuning with the antimetabolite sulfathiazole. Application of this PABA-based assay facilitates the screening of millions of enzyme variants on a single agar plate within two days, without the need for laborious sample preparation or expensive instruments, with transformation efficiency being the only limiting factor. KEY POINTS: • Ultra-high throughput assay (tens of millions on one agar plate) for amidase screening • High sensitivity by coupling selection to folate instead of carbon or nitrogen source • Highly adjustable in terms of sensitivity and expression of the engineering target.

Connection between protein-tyrosine kinase inhibition and coping with oxidative stress in Bacillus subtilis.

In bacteria, attenuation of protein-tyrosine phosphorylation occurs during oxidative stress. The main described mechanism behind this effect is the H2O2-triggered conversion of bacterial phospho-tyrosines to protein-bound 3,4-dihydroxyphenylalanine. This disrupts the bacterial tyrosine phosphorylation-based signaling network, which alters the bacterial polysaccharide biosynthesis. Herein, we report an alternative mechanism, in which oxidative stress leads to a direct inhibition of bacterial protein-tyrosine kinases (BY-kinases). We show that DefA, a minor peptide deformylase, inhibits the activity of BY-kinase PtkA when Bacillus subtilis is exposed to oxidative stress. High levels of PtkA activity are known to destabilize B. subtilis pellicle formation, which leads to higher sensitivity to oxidative stress. Interaction with DefA inhibits both PtkA autophosphorylation and phosphorylation of its substrate Ugd, which is involved in exopolysaccharide formation. Inactivation of defA drastically reduces the capacity of B. subtilis to cope with oxidative stress, but it does not affect the major oxidative stress regulons PerR, OhrR, and Spx, indicating that PtkA inhibition is the main pathway for DefA involvement in this stress response. Structural analysis identified DefA residues Asn95, Tyr150, and Glu152 as essential for interaction with PtkA. Inhibition of PtkA depends also on the presence of a C-terminal α-helix of DefA, which resembles PtkA-interacting motifs from known PtkA activators, TkmA, SalA, and MinD. Loss of either the key interacting residues or the inhibitory helix of DefA abolishes inhibition of PtkA in vitro and impairs postoxidative stress recovery in vivo, confirming the involvement of these structural features in the proposed mechanism.

DDAH-1 maintains endoplasmic reticulum-mitochondria contacts and protects dopaminergic neurons in Parkinson's disease.

The loss of dopaminergic neurons in the substantia nigra is a hallmark of pathology in Parkinson's disease (PD). Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the critical enzyme responsible for the degradation of asymmetric dimethylarginine (ADMA) which inhibits nitric oxide (NO) synthase and has been implicated in neurodegeneration. Mitochondrial dysfunction, particularly in the mitochondria-associated endoplasmic reticulum membrane (MAM), plays a critical role in this process, although the specific molecular target has not yet been determined. This study aims to examine the involvement of DDAH-1 in the nigrostriatal dopaminergic pathway and PD pathogenesis. The distribution of DDAH-1 in the brain and its colocalization with dopaminergic neurons were observed. The loss of dopaminergic neurons and aggravated locomotor disability after rotenone (ROT) injection were showed in the DDAH-1 knockout rat. L-arginine (ARG) and NO donors were employed to elucidate the role of NO respectively. In vitro, we investigated the effects of DDAH-1 knockdown or overexpression on cell viability and mitochondrial functions, as well as modulation of ADMA/NO levels using ADMA or ARG. MAM formation was assessed by the Mitofusin2 oligomerization and the mitochondrial ubiquitin ligase (MITOL) phosphorylation. We found that DDAH-1 downregulation resulted in enhanced cell death and mitochondrial dysfunctions, accompanied by elevated ADMA and reduced NO levels. However, the recovered NO level after the ARG supplement failed to exhibit a protective effect on mitochondrial functions and partially restored cell viability. DDAH-1 overexpression prevented ROT toxicity, while ADMA treatment attenuated these protective effects. The declines of MAM formation in ROT-treated cells were exacerbated by DDAH-1 downregulation via reduced MITOL phosphorylation, which was reversed by DDAH-1 overexpression. Together, the abundant expression of DDAH-1 in nigral dopaminergic neurons may exert neuroprotective effects by maintaining MAM formation and mitochondrial function probably via ADMA, indicating the therapeutic potential of targeting DDAH-1 for PD.

Evaluating the Impact of Probiotic Therapy on the Endocannabinoid System, Pain, Sleep and Fatigue: A Randomized, Double-Blind, Placebo-Controlled Trial in Dancers.

Emerging research links the endocannabinoid system to gut microbiota, influencing nociception, mood, and immunity, yet the molecular interactions remain unclear. This study focused on the effects of probiotics on ECS markers-cannabinoid receptor type 2 (CB2) and fatty acid amide hydrolase (FAAH)-in dancers, a group selected due to their high exposure to physical and psychological stress. In a double-blind, placebo-controlled trial (ClinicalTrials.gov NCT05567653), 15 dancers were assigned to receive either a 12-week regimen of Lactobacillus helveticus Rosell-52 and Bifidobacterium longum Rosell-17 or a placebo (PLA: n = 10, PRO: n = 5). There were no significant changes in CB2 (probiotic: 0.55 to 0.29 ng/mL; placebo: 0.86 to 0.72 ng/mL) or FAAH levels (probiotic: 5.93 to 6.02 ng/mL; placebo: 6.46 to 6.94 ng/mL; p > 0.05). A trend toward improved sleep quality was observed in the probiotic group, while the placebo group showed a decline (PRO: from 1.4 to 1.0; PLA: from 0.8 to 1.2; p = 0.07841). No other differences were noted in assessed outcomes (pain and fatigue). Probiotic supplementation showed no significant impact on CB2 or FAAH levels, pain, or fatigue but suggested potential benefits for sleep quality, suggesting an area for further research.

Endocannabinoid Hydrolase Inhibitors: Potential Novel Anxiolytic Drugs.

Over the past decade, the idea of targeting the endocannabinoid system to treat anxiety disorders has received increasing attention. Previous studies focused more on developing cannabinoid receptor agonists or supplementing exogenous cannabinoids, which are prone to various adverse effects due to their strong pharmacological activity and poor receptor selectivity, limiting their application in clinical research. Endocannabinoid hydrolase inhibitors are considered to be the most promising development strategies for the treatment of anxiety disorders. More recent efforts have emphasized that inhibition of two major endogenous cannabinoid hydrolases, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), indirectly activates cannabinoid receptors by increasing endogenous cannabinoid levels in the synaptic gap, circumventing receptor desensitization resulting from direct enhancement of endogenous cannabinoid signaling. In this review, we comprehensively summarize the anxiolytic effects of MAGL and FAAH inhibitors and their potential pharmacological mechanisms, highlight reported novel inhibitors or natural products, and provide an outlook on future directions in this field.

Structural study for substrate recognition of human N-terminal glutamine amidohydrolase 1 in the arginine N-degron pathway.

The N-degron pathway determines the half-life of proteins by selectively destabilizing the proteins bearing N-degrons. N-terminal glutamine amidohydrolase 1 (NTAQ1) plays an essential role in the arginine N-degron (Arg/N-degron) pathway as an initializing enzyme via the deamidation of the N-terminal (Nt) glutamine (Gln). However, the Nt-serine-bound conformation of hNTAQ1 according to the previously identified crystal structure suggests the possibility of other factors influencing the recognition of Nt residues by hNTAQ1. Hence, in the current study, we aimed to further elucidate the substrate recognition of hNTAQ1; specifically, we explored 12 different substrate-binding conformations of hNTAQ1 depending on the subsequent residue of Nt-Gln. Results revealed that hNTAQ1 primarily interacts with the protein Nt backbone, instead of the side chain, for substrate recognition. Here, we report that the Nt backbone of proteins appears to be a key component of hNTAQ1 function and is the main determinant of substrate recognition. Moreover, not all second residues from Nt-Gln, but rather distinctive and charged residues, appeared to aid in detecting substrate recognition. These new findings define the substrate-recognition process of hNTAQ1 and emphasize the importance of the subsequent Gln residue in the Nt-Gln degradation system. Our extensive structural and biochemical analyses provide insights into the substrate specificity of the N-degron pathway and shed light on the mechanism underlying hNTAQ1 substrate recognition. An improved understanding of the protein degradation machinery could aid in developing therapies to promote overall health through enhanced protein regulation, such as targeted protein therapies.

Two different types of hydrolases co-degrade ochratoxin A in a highly efficient degradation strain Lysobacter sp. CW239.

Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 µg/L OTA was completely degraded by 1.0 µg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-ß-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest.

Cascade Mesophase Transitions of Multi-enzyme Responsive Polymeric Formulations.

Studying how synthetic polymer assemblies respond to sequential enzymatic stimuli can uncover intricate interactions in biological systems. Using amidase- and esterase-responsive PEG-based diblock (DBA) and triblock amphiphiles (TBAs), we created two distinct formulations: amidase-responsive DBA with esterase-responsive TBA and vice versa. We studied their cascade responses to the two enzymes and the sequence of their introduction. These formulations underwent cascade mesophase transitions upon the addition of the DBA-degrading enzyme, transitioning from (i) coassembled micelles to (ii) triblock-based hydrogel, and ultimately to (iii) dissolved polymers when exposed to the TBA hydrolyzing enzyme. The specific pathway of the two mesophase transitions depended on the compositions of the formulations and the enzyme introduction sequence. The results highlight the potential for designing polymeric formulations with programmable multistep enzymatic responses, mimicking the complex behavior of biological macromolecules.

Impact of miR-222-3p-mediated downregulation of arylacetamide deacetylase on drug hydrolysis and lipid accumulation.

Arylacetamide deacetylase (AADAC) is involved in drug hydrolysis and lipid metabolism. In 23 human liver samples, no significant correlation was observed between AADAC mRNA (19.7-fold variation) and protein levels (137.6-fold variation), suggesting a significant contribution of post-transcriptional regulation to AADAC expression. The present study investigated whether AADAC is regulated by microRNA in the human liver and elucidate its biological significance. Computational analysis predicted two potential miR-222-3p recognition elements in the 3'-untranslated region (UTR) of AADAC. Luciferase assay revealed that the miR-222-3p recognition element was functional in downregulating AADAC expression. In HEK293 cells transfected with an AADAC expression plasmid containing 3'-UTR, miR-222-3p overexpression decreased AADAC protein level and activity, whereas miR-222-3p inhibition increased them. Similar results were observed in human hepatoma-derived Huh-1 cells endogenously expressing AADAC and HepaSH cells that are hepatocytes from chimeric mice with humanized livers. In individual human liver samples, AADAC protein levels inversely correlated with miR-222-3p levels. Overexpression of miR-222-3p resulted in increased lipid accumulation in Huh-1 cells, which was reversed by AADAC overexpression. In contrast, miR-222-3p inhibition decreased lipid accumulation, which was reversed by AADAC knockdown. In conclusion, we found that hepatic AADAC was downregulated by miR-222-3p, resulting in decreased drug hydrolysis and increased lipid accumulation.

Preservation of conjugated primary bile acids by oxygenation of the small intestinal microbiota in vitro.

Bile acids play a critical role in the emulsification of dietary lipids, a critical step in the primary function of the small intestine, which is the digestion and absorption of food. Primary bile acids delivered into the small intestine are conjugated to enhance functionality, in part, by increasing aqueous solubility and preventing passive diffusion of bile acids out of the gut lumen. Bile acid function can be disrupted by the gut microbiota via the deconjugation of primary bile acids by bile salt hydrolases (BSHs), leading to their conversion into secondary bile acids through the expression of bacterial bile acid-inducible genes, a process often observed in malabsorption due to small intestinal bacterial overgrowth. By modeling the small intestinal microbiota in vitro using human small intestinal ileostomy effluent as the inocula, we show here that the infusion of physiologically relevant levels of oxygen, normally found in the proximal small intestine, reduced deconjugation of primary bile acids, in part, through the expansion of bacterial taxa known to have a low abundance of BSHs. Further recapitulating the small intestinal bile acid composition of the small intestine, limited conversion of primary into secondary bile acids was observed. Remarkably, these effects were preserved among four separate communities, each inoculated with a different small intestinal microbiota, despite a high degree of taxonomic variability under both anoxic and aerobic conditions. In total, these results provide evidence for a previously unrecognized role that the oxygenated environment of the small intestine plays in the maintenance of normal digestive physiology. IMPORTANCE: Conjugated primary bile acids are produced by the liver and exist at high concentrations in the proximal small intestine, where they are critical for proper digestion. Deconjugation of these bile acids with subsequent transformation via dehydroxylation into secondary bile acids is regulated by the colonic gut microbiota and reduces their digestive function. Using an in vitro platform modeling the small intestinal microbiota, we analyzed the ability of this community to transform primary bile acids and studied the effect of physiological levels of oxygen normally found in the proximal small intestine (5%) on this metabolic process. We found that oxygenation of the small intestinal microbiota inhibited the deconjugation of primary bile acids in vitro. These findings suggest that luminal oxygen levels normally found in the small intestine may maintain the optimal role of bile acids in the digestive process by regulating bile acid conversion by the gut microbiota.

Probing the enzymatic activity and maturation process of the EcAIII Ntn-amidohydrolase using local random mutagenesis.

This report describes a comprehensive approach to local random mutagenesis of the E. coli Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the randomization series RDM1. Here, random mutagenesis was applied in the center of the EcAIII molecule, i.e., in the region important for substrate binding and its immediate neighborhood (series RDM2, RDM3, RDM7), in the vicinity of the catalytic threonine triplet (series RDM4, RDM5, RDM6), in the linker region (series RDM8), and in the sodium-binding (stabilization) loop (series RDM9). The results revealed that the majority of the new EcAIII variants have abolished or significantly reduced rate of autoprocessing, even if the mutation was not in a highly conserved sequence and structure regions. AlphaFold-predicted structures of the mutants suggest the role of selected residues in the positioning of the linker and stabilization of the scissile bond in precisely correct orientation, enabling the nucleophilic attack during the maturation process. The presented data highlight the details of EcAIII geometry that are important for the autoproteolytic maturation and for the catalytic mechanism in general, and can be treated as a guide for protein engineering experiments with other Ntn-hydrolases.

The three-dimensional structure of DapE from Enterococcus faecium reveals new insights into DapE/ArgE subfamily ligand specificity.

DapE is a Zn2+-metallohydrolase recognized as a drug target for bacterial control. It is a homodimer that requires the exchange of interface strands by an induced fit essential for catalysis. Identifying novel anti-DapE agents requires greater structural details. Most of the characterized DapEs are from the Gram-negative group. Here, two high-resolution DapE crystal structures from Enterococcus faecium are presented for the first time with novel aspects. A loosened enzyme intermediate between the open and closed conformations is observed. Substrates may bind to loose state, subsequently it closes, where hydrolysis occurs, and finally, the change to the open state leads to the release of the products. Mutation of His352 suggests a role, along with His194, in the oxyanion stabilization in the mono-metalated Zn2+ isoform, while in the di-metalated isoform, the metal center 2 complements it function. An aromatic-π box potentially involved in the interaction of DapE with other proteins, and a peptide flip could determine the specificity in the Gram-positive ArgE/DapE group. Finally, details of two extra-catalytic cavities whose geometry changes depending on the conformational state of the enzyme are presented. These cavities could be a target for developing non-competitive agents that trap the enzyme in an inactive state.

Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants.

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.

In vitro and in vivo stability of a highly efficient long-acting cocaine hydrolase.

It is recognized as a promising therapeutic strategy for cocaine use disorder to develop an efficient enzyme which can rapidly convert cocaine to physiologically inactive metabolites. We have designed and discovered a series of highly efficient cocaine hydrolases, including CocH5-Fc(M6) which is the currently known as the most efficient cocaine hydrolase with both the highest catalytic activity against (-)-cocaine and the longest biological half-life in rats. In the present study, we characterized the time courses of protein appearance, pH, structural integrity, and catalytic activity against cocaine in vitro and in vivo of a CocH5-Fc(M6) bulk drug substance produced in a bioreactor for its in vitro and in vivo stability after long-time storage under various temperatures (- 80, - 20, 4, 25, or 37 °C). Specifically, all the tested properties of the CocH5-Fc(M6) protein did not significantly change after the protein was stored at any of four temperatures including - 80, - 20, 4, and 25 °C for ~ 18 months. In comparison, at 37 °C, the protein was less stable, with a half-life of ~ 82 days for cocaine hydrolysis activity. Additionally, the in vivo studies further confirmed the linear elimination PK profile of CocH5-Fc(M6) with an elimination half-life of ~ 9 days. All the in vitro and in vivo data on the efficacy and stability of CocH5-Fc(M6) have consistently demonstrated that CocH5-Fc(M6) has the desired in vitro and in vivo stability as a promising therapeutic candidate for treatment of cocaine use disorder.

Early and late-onset preeclampsia: effects of DDAH2 polymorphisms on ADMA levels and association with DDAH2 haplotypes.

Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.

A novel N-acetylglucosamine-6-phosphate deacetylase that is essential for chitin utilization in the chitinolytic bacterium, Chitiniphilus shinanonensis.

AIMS: We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified using transposon mutagenesis, followed by negatively selecting a mutant incapable of growing on chitin, a polysaccharide consisting of N-acetyl-d-glucosamine (GlcNAc). We focused on the physiological role of CsPLD protein in chitin utilization. METHODS AND RESULTS: Recombinant CsPLD expressed in Escherichia coli exhibited GlcNAc-6-phosphate deacetylase (GPD) activity, which is involved in the metabolism of amino sugars. However, SAY3 possesses two genes (csnagA1 and csnagA2) in its genome that code for proteins whose primary sequences are homologous to those of typical GPDs. Recombinant CsNagA1 and CsNagA2 also exhibited GPD activity with 23 and 1.6% of catalytic efficiency (kcat/Km), respectively, compared to CsPLD. The gene-disrupted mutant, Δcspld was unable to grow on chitin or GlcNAc, whereas the three mutants, ΔcsnagA1, ΔcsnagA2, and ΔcsnagA1ΔcsnagA2 grew similarly to SAY3. The determination of GPD activity in the crude extracts of each mutant revealed that CsPLD is a major enzyme that accounts for almost all cellular activities. CONCLUSIONS: Deacetylation of GlcNAc-6P catalyzed by CsPLD (but not by typical GPDs) is essential for the assimilation of chitin and its constituent monosaccharide, GlcNAc, as a carbon and energy source in C. shinanonensis.

Combined in vivo effect of N-acylethanolamine-hydrolyzing acid amidase and glycogen synthase kinase-3ß inhibition to treat multiple sclerosis.

The current pharmacological approaches to multiple sclerosis (MS) target its inflammatory and autoimmune components, but effective treatments to foster remyelination and axonal repair are still lacking. We therefore selected two targets known to be involved in MS pathogenesis: N-acylethanolamine-hydrolyzing acid amidase (NAAA) and glycogen synthase kinase-3ß (GSK-3ß). We tested whether inhibiting these targets exerted a therapeutic effect against experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The combined inhibition of NAAA and GSK-3ß by two selected small-molecule compounds, ARN16186 (an NAAA inhibitor) and AF3581 (a GSK-3ß inhibitor), effectively mitigated disease progression, rescuing the animals from paralysis and preventing a worsening of the pathology. The complementary activity of the two inhibitors reduced the infiltration of immune cells into the spinal cord and led to the formation of thin myelin sheaths around the axons post-demyelination. Specifically, the inhibition of NAAA and GSK-3ß modulated the over-activation of NF-kB and STAT3 transcription factors in the EAE-affected mice and induced the nuclear translocation of ß-catenin, reducing the inflammatory insult and promoting the remyelination process. Overall, this work demonstrates that the dual-targeting of key aspects responsible for MS progression could be an innovative pharmacological approach to tackle the pathology.

Quantification of N-acetyl-l-aspartate in dried blood spots: A simple and fast LC-MS/MS neonatal screening method for the diagnosis of Canavan disease.

BACKGROUND: Canavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene (ASPA). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective. METHODS: We therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/MS)-based method for quantification of N-acetylaspartate in dried blood spots and established reference ranges for neonates and older controls. With this test, we investigated 45 samples of 25 Canavan patients including 8 with a neonatal sample. RESULTS: Measuring N-acetylaspartate concentration in dried blood with this novel test, all Canavan patients (with variable severity) were well separated from the control group (median; range: 5.7; 1.6-13.6 µmol/L [n = 45] vs 0.44; 0.24-0.99 µmol/L [n = 59] (p < 0.05)). There was also no overlap when comparing neonatal samples of Canavan patients (7.3; 5.1-9.9 µmol/L [n = 8]) and neonatal controls (0.93; 0.4-1.8 µmol/L [n = 784]) (p < 0.05). CONCLUSIONS: We have developed a new LC-MS/MS-based screening test for early postnatal diagnosis of Canavan disease that should be further evaluated in a population-based study once a promising treatment becomes available. The method meets the general requirements of newborn screening and should be appropriate for multiplexing with other screening approaches that combine chromatographic and mass spectrometry techniques.

Biodegradation of ochratoxin A by Brevundimonas diminuta HAU429: Characterized performance, toxicity evaluation and functional enzymes.

Ochratoxin A (OTA) is a notorious mycotoxin commonly contaminating food products worldwide. In this study, an OTA-degrading strain Brevundimonas diminuta HAU429 was isolated by using hippuryl-L-phenylalanine as the sole carbon source. The biodegradation of OTA by strain HAU429 was a synergistic effect of intracellular and extracellular enzymes, which transformed OTA into ochratoxin α (OTα) through peptide bond cleavage. Cytotoxicity tests and cell metabolomics confirmed that the transformation of OTA into OTα resulted in the detoxification of its hepatotoxicity since OTA but not OTα disturbed redox homeostasis and induced oxidative damage to hepatocytes. Genome mining identified nine OTA hydrolase candidates in strain HAU429. They were heterologously expressed in Escherichia coli, and three novel amidohydrolase BT6, BT7 and BT9 were found to display OTA-hydrolyzing activity. BT6, BT7 and BT9 showed less than 45 % sequence identity with previously identified OTA-degrading amidohydrolases. BT6 and BT7 shared 60.9 % amino acid sequence identity, and exhibited much higher activity towards OTA than BT9. BT6 and BT7 could completely degrade 1 µg mL-1 of OTA within 1 h and 50 min, while BT9 hydrolyzed 100 % of OTA in the reaction mixture by 12 h. BT6 was the most thermostable retaining 38 % of activity after incubation at 70 °C for 10 min, while BT7 displayed the highest tolerance to ethanal remaining 76 % of activity in the presence of 6 % ethanol. This study could provide new insights towards microbial OTA degradation and promote the development of enzyme-catalyzed OTA detoxification during food processing.