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L -Amino acids are the building blocks for proteins synthesized in ribosomes in all kingdoms of life, but d -amino acids ( d -aa) have important non-ribosome-based functions 1 . Mammals synthesize d -Ser and d -Asp, primarily in the central nervous system, where d -Ser is critical for neurotransmission 2 . Bacteria synthesize a largely distinct set of d -aa, which become integral components of the cell wall and are also released as free d -aa 3 , 4 . However, the impact of free microbial d -aa on host physiology at the host–microbial interface has not been explored. Here, we show that the mouse intestine is rich in free d -aa that are derived from the microbiota. Furthermore, the microbiota induces production of d -amino acid oxidase (DAO) by intestinal epithelial cells, including goblet cells, which secrete the enzyme into the lumen. Oxidative deamination of intestinal d -aa by DAO, which yields the antimicrobial product H 2 O 2 , protects the mucosal surface in the small intestine from the cholera pathogen. DAO also modifies the composition of the microbiota and is associated with microbial induction of intestinal sIgA. Collectively, these results identify d -aa and DAO as previously unrecognized mediators of microbe–host interplay and homeostasis on the epithelial surface of the small intestine. The mouse gut microbiota produce free d -amino acids and induce the production of d -amino acid oxidase by intestinal epithelial cells. Oxidative deamination of d -amino acids yields H 2 O 2 , which protects the mucosa from Vibrio cholera e.

Background Patient-derived organoids provide a powerful platform for elucidating mechanisms of drug resistance and tumor evolution in hepatocellular carcinoma (HCC) and identifying novel therapeutic targets. Methods Transcriptomic sequencing was used to compare gene expression patterns between organoid-forming and non-forming HCC tissues, as well as between sorafenib-resistant organoids and sorafenib-sensitive counterparts. The TCGA-liver hepatocellular carcinoma (LIHC) cohort was used to screen for the key molecular drivers of HCC evolution from the overlapping differentially expressed genes. The effects of D-amino acid oxidase (DAO) on the growth, migration, invasion, apoptosis, and generation of H 2 O 2 were evaluated in HepG2 and SK-Hep-1 cell lines and human HCC organoids. The therapeutic efficacy of DAO against HCC growth and drug resistance was validated with xenograft mouse model and organoids, respectively. Results Expression level of DAO was significantly downregulated in HCC tissues that successfully formed organoids compared to those that failed, as well as in sorafenib-resistant organoids versus their parental counterparts. In the TCGA-LIHC cohort, DAO expression was significantly reduced in advanced-stage HCC tissues and was inversely correlated with stemness- and epithelial-mesenchymal transition (EMT)-related molecules. Lower DAO expression was associated with poor overall survival in patients with HCC. In HepG2 cells, DAO knockdown significantly enhanced cell proliferation. Ectopic DAO expression suppressed proliferation, migration, and invasion in HepG2 and SK-Hep-1 cells. D-alanine (D-Ala) supplementation further enhanced the anti-proliferative effect of overexpressed DAO, but did not significantly alter the DAO-mediated suppression of migration or invasion. Ectopic expression of DAO induced apoptosis via the generation of H 2 O 2 upon simultaneous supplementation of D-Ala into the culture medium; the addition of catalase, an H₂O₂-degrading enzyme, significantly reversed the D-Ala-induced effects. In BALB/c nude mouse models, HCC cells overexpressing DAO formed significantly smaller tumors than the control cells ( P  = 0.010), and this tumor-suppressive effect was further enhanced by D-alanine supplementation. Ectopic DAO expression restored sorafenib sensitivity in resistant organoids. Conclusion DAO appears to be a novel endogenous stemness repressor. The reduction in DAO is a critical molecular event in the evolution of HCC. Therapeutically, combined DAO and D-amino acid supplementation is a promising strategy for HCC treatment, particularly for reversing sorafenib resistance.

D-Amino acid oxidase (DAAO) is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-isomers of neutral and polar amino acids. This enzymatic activity has been identified in most eukaryotic organisms, the only exception being plants. In the various organisms in which it does occur, DAAO fulfills distinct physiological functions: from a catabolic role in yeast cells, which allows them to grow on D-amino acids as carbon and energy sources, to a regulatory role in the human brain, where it controls the levels of the neuromodulator D-serine. Since 1935, DAAO has been the object of an astonishing number of investigations and has become a model for the dehydrogenase-oxidase class of flavoproteins. Structural and functional studies have suggested that specific physiological functions are implemented through the use of different structural elements that control access to the active site and substrate/product exchange. Current research is attempting to delineate the regulation of DAAO functions in the contest of complex biochemical and physiological networks.

Histamine intolerance, also referred to as enteral histaminosis or sensitivity to dietary histamine, is a disorder associated with an impaired ability to metabolize ingested histamine that was described at the beginning of the 21st century. Although interest in histamine intolerance has considerably grown in recent years, more scientific evidence is still required to help define, diagnose and clinically manage this condition. This article will provide an updated review on histamine intolerance, mainly focusing on its etiology and the existing diagnostic and treatment strategies. In this work, a glance on histamine intoxication will also be provided, as well as the analysis of some uncertainties historically associated to histamine intoxication outbreaks that may be better explained by the existence of interindividual susceptibility to ingested histamine. Keywords: diamine oxidase (DAO); food intolerance; food supplement; histamine; histamine intolerance; histamine intoxication; histaminosis; low-histamine diet.

Arsenic exposure model of offspring mice was established and intervened with 6-chlorobenzo[d]isoxazol-3-ol (CBIO), a D-amino acid oxidase (DAAO) inhibitor, to explore the role of DAAO in cognitive impairment of offspring mice induced by arsenic during early developmental stage. Female mice and their pups treated with 0 or 60 mg/L sodium arsenite (NaAsO 2 ) via drinkable water from the first day of gestation till the end of lactation. On the 28th day after birth, the offspring mice in the drinking distilled water group were randomly divided into control and 1 mg/mL CBIO group. The offspring mice in the arsenic group were divided into 60 mg/L NaAsO 2 group and 60 mg/L NaAsO 2  + 1 mg/mL CBIO group, CBIO was administered to the lateral ventricle for one week. Additionally, D-serine and L-serine concentrations were detected by UHPLC-MS/MS, Real-time RT-PCR and Western blot were applied to measure DAAO, serine racemase (SR), N-methyl-D-aspartate receptor (NMDAR), synaptophysin (SYP) and postsynaptic density (PSD95) levels in the hippocampus. Results disclosed that arsenic could reduce the levels of D-serine, L-serine, SR and NMDAR, while upregulate DAAO levels, however, inhibiting DAAO levels could increase D-serine and NR1 levels. These findings indicated that DAAO might be involved in cognitive impairment of offspring mice induced by arsenic during early developmental stage by affecting D-serine metabolism.

Aims/hypothesisDiabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy.MethodsRats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood–retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid–Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection.ResultsAt the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood–retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood–retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented.Conclusions/interpretationAltogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy.

D -amino acid oxidase (DAO) is a flavoenzyme that metabolizes certain D -amino acids, notably the endogenous N -methyl D -aspartate receptor (NMDAR) co-agonist, D -serine. As such, it has the potential to modulate the function of NMDAR and to contribute to the widely hypothesized involvement of NMDAR signalling in schizophrenia. Three lines of evidence now provide support for this possibility: DAO shows genetic associations with the disorder in several, although not all, studies; the expression and activity of DAO are increased in schizophrenia; and DAO inactivation in rodents produces behavioural and biochemical effects, suggestive of potential therapeutic benefits. However, several key issues remain unclear. These include the regional, cellular and subcellular localization of DAO, the physiological importance of DAO and its substrates other than D -serine, as well as the causes and consequences of elevated DAO in schizophrenia. Herein, we critically review the neurobiology of DAO, its involvement in schizophrenia, and the therapeutic value of DAO inhibition. This review also highlights issues that have a broader relevance beyond DAO itself: how should we weigh up convergent and cumulatively impressive, but individually inconclusive, pieces of evidence regarding the role that a given gene may have in the aetiology, pathophysiology and pharmacotherapy of schizophrenia?

d- Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining d -amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several d -amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic d- amino acids, in particular hydrophobic d -amino acids. In addition, the enzyme also significantly acted on cephalosporin C, a starting material for semisynthetic antibiotics, and d- Glu, a general substrate for d- aspartate oxidase but not for DAAO, showing its unique and practically useful substrate specificity. The apparent k cat and K m values of the enzyme toward good substrates were comparable to those of higher catalytic fungal DAAOs, and the thermal stability ( T 50 value of ~60 °C) was comparable to that of a thermophilic bacterial DAAO and significantly higher than that of other eukaryotic DAAOs. These results highlight the great potential of ReDAAO for use in practical applications.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. D-Serine, an endogenous coagonist of N-methyl-D-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the D-amino acid oxidase (DAO) gene, encoding a D-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and D-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the D-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through D-serine regulation and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing D-serine or controlling DAO activity in ALS should be tested in future studies.

The flavoenzyme d-amino acid oxidase (DAAO) is deputed to the degradation of d-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal d-amino acids to generate hydrogen peroxide (H2O2), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1–16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.