Tryptophan Metabolism in Alzheimer's Disease with the Involvement of Microglia and Astrocyte Crosstalk and Gut-Brain Axis.

Alzheimer's disease (AD) is an age-dependent neurodegenerative disease characterized by extracellular Amyloid Aß peptide (Aß) deposition and intracellular Tau protein aggregation. Glia, especially microglia and astrocytes are core participants during the progression of AD and these cells are the mediators of Aß clearance and degradation. The microbiota-gut-brain axis (MGBA) is a complex interactive network between the gut and brain involved in neurodegeneration. MGBA affects the function of glia in the central nervous system (CNS), and microbial metabolites regulate the communication between astrocytes and microglia; however, whether such communication is part of AD pathophysiology remains unknown. One of the potential links in bilateral gut-brain communication is tryptophan (Trp) metabolism. The microbiota-originated Trp and its metabolites enter the CNS to control microglial activation, and the activated microglia subsequently affect astrocyte functions. The present review highlights the role of MGBA in AD pathology, especially the roles of Trp per se and its metabolism as a part of the gut microbiota and brain communications. We (i) discuss the roles of Trp derivatives in microglia-astrocyte crosstalk from a bioinformatics perspective, (ii) describe the role of glia polarization in the microglia-astrocyte crosstalk and AD pathology, and (iii) summarize the potential of Trp metabolism as a therapeutic target. Finally, we review the role of Trp in AD from the perspective of the gut-brain axis and microglia, as well as astrocyte crosstalk, to inspire the discovery of novel AD therapeutics.

Ergothioneine Protects Against UV-Induced Oxidative Stress Through the PI3K/AKT/Nrf2 Signaling Pathway.

Background: Ergothioneine (EGT) is an antioxidant, which could be detected in human tissues, and human skin cells could utilize EGT and play an anti-oxidative role in keratinocytes. And in this study we are going to elucidate whether EGT could protect the skin from photoaging by Ultraviolet (UV) exposure in mice and its molecule pathway. Methods: Histological analysis was performed for evaluating the skin structure change. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured with biological assay for evaluating oxidative and antioxidative ability of skin exposed to UV light. And the level of marker molecules in mouse skin were detected by hydroxyproline (Hyp) assay, immunohistochemical analysis, Western blot, and quantitative real-time PCR (qRT-PCR). The markers of skin aging and cell death were tested by cell culture and treatment, Western blot and qRT-PCR. Results: EGT decreased the levels of inflammatory factors induced by UV exposure in mouse skin. MDA and SOD activity detection showed that EGT decreased MDA levels, increased SOD activity, and upregulated PI3K/Akt/Nrf2 signals in mouse skin exposed to UV, which further activated Nrf2 in the nucleus and enhanced the expression of Nrf2 target genes. In the cell model, we revealed that EGT could inhibit the increase in senescence-associated ß-galactosidase-positive cells and p16 and γ-H2A.X positive cells induced by etoposide and activate PI3K/Akt/Nrf2 signaling. Moreover, a PI3K inhibitor blocked EGT protection against etoposide-induced cell death. Conclusion: The study showed EGT may play an important protective role against cell damage or death through the PI3K/Akt/Nrf2 signaling pathway in skin.

New Frontiers in Nonheme Enzymatic Oxyferryl Species.

Non-heme mononuclear iron dependent (NHM-Fe) enzymes exhibit exceedingly diverse catalytic reactivities. Despite their catalytic versatilities, the mononuclear iron centers in these enzymes show a relatively simple architecture, in which an iron atom is ligated with 2-4 amino acid residues, including histidine, aspartic or glutamic acid. In the past two decades, a common high-valent reactive iron intermediate, the S = 2 oxoferryl (Fe(IV)-oxo or Fe(IV)=O) species, has been repeatedly discovered in NHM-Fe enzymes containing a 2-His-Fe or 2-His-1-carboxylate-Fe center. However, for 3-His/4-His-Fe enzymes, no common reactive intermediate has been identified. Recently, we have spectroscopically characterized the first S = 1 Fe(IV) intermediate in a 3-His-Fe containing enzyme, OvoA, which catalyzes a novel oxidative carbon-sulfur bond formation. In this review, we summarize the broad reactivities demonstrated by S = 2 Fe(IV)-oxo intermediates, the discovery of the first S = 1 Fe(IV) intermediate in OvoA and the mechanistic implication of such a discovery, and the intrinsic reactivity differences of the S = 2 and the S = 1 Fe(IV)-oxo species. Finally, we postulate the possible reasons to utilize an S = 1 Fe(IV) species in OvoA and their implications to other 3-His/4-His-Fe enzymes.

Biochemical and Structural Characterization of OvoATh2: A Mononuclear Nonheme Iron Enzyme from Hydrogenimonas thermophila for Ovothiol Biosynthesis.

Ovothiol A and ergothioneine are thiol-histidine derivatives with sulfur substitutions at the δ-carbon or ε-carbon of the l-histidine imidazole ring, respectively. Both ovothiol A and ergothioneine have protective effects on many aging-related diseases, and the sulfur substitution plays a key role in determining their chemical and biological properties, while factors governing sulfur incorporation regioselectivities in ovothiol and ergothioneine biosynthesis in the corresponding enzymes (OvoA, Egt1, or EgtB) are not yet known. In this study, we have successfully obtained the first OvoA crystal structure, which provides critical information to explain their C-S bond formation regioselectivity. Furthermore, OvoATh2 exhibits several additional activities: (1) ergothioneine sulfoxide synthase activity akin to Egt1 in ergothioneine biosynthesis; (2) cysteine dioxygenase activity using l-cysteine and l-histidine analogues as substrates; (3) cysteine dioxygenase activity upon mutation of an active site tyrosine residue (Y406). The structural insights and diverse chemistries demonstrated by OvoATh2 pave the way for future comprehensive structure-function correlation studies.

An S=1 Iron(IV) Intermediate Revealed in a Non-Heme Iron Enzyme-Catalyzed Oxidative C-S Bond Formation.

Ergothioneine (ESH) and ovothiol A (OSHA) are two natural thiol-histidine derivatives. ESH has been implicated as a longevity vitamin and OSHA inhibits the proliferation of hepatocarcinoma. The key biosynthetic step of ESH and OSHA in the aerobic pathways is the O2 -dependent C-S bond formation catalyzed by non-heme iron enzymes (e.g., OvoA in ovothiol biosynthesis), but due to the lack of identification of key reactive intermediate the mechanism of this novel reaction is unresolved. In this study, we report the identification and characterization of a kinetically competent S=1 iron(IV) intermediate supported by a four-histidine ligand environment (three from the protein residues and one from the substrate) in enabling C-S bond formation in OvoA from Methyloversatilis thermotoleran, which represents the first experimentally observed intermediate spin iron(IV) species in non-heme iron enzymes. Results reported in this study thus set the stage to further dissect the mechanism of enzymatic oxidative C-S bond formation in the OSHA biosynthesis pathway. They also afford new opportunities to study the structure-function relationship of high-valent iron intermediates supported by a histidine rich ligand environment.

Correspondence on "Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1".

In their recent Angewandte Chemie publication (doi: 10.1002/anie.202112063), Cen, Wang, Zhou et al. reported the crystal structure of a ternary complex of the non-heme iron endoperoxidase FtmOx1 (PDB entry 7ETK). The biochemical data assessed in this study were from a retracted study (doi: 10.1038/nature15519) by Zhang, Liu, Zhang et al.; no additional biochemical data were included, yet there was no discussion on the source of the biochemical data in the report by Cen, Wang, Zhou et al. Based on this new crystal structure and subsequent QM/MM-MD calculations, Cen, Wang, Zhou et al. concluded that their work provided evidence supporting the CarC-like mechanistic model for FtmOx1 catalysis. However, the authors did not accurately describe either the CarC-like model or the COX-like model, and they did not address the differences between them. Further, and contrary to their interpretations in the manuscript, the authors' data are consistent with the COX-like model once the details of the CarC-like and COX-like models have been carefully analyzed.

The whole-genome sequencing of prevalent DENV-1 strains during the largest dengue virus outbreak in Xishuangbanna Dai autonomous prefecture in 2019.

In 2019, a serious dengue virus (DENV) infection broke out in the Xishuangbanna Dai Autonomous Prefecture, China. Therefore, we conducted a molecular epidemiological analysis in people that contracted DENV serotype 1 (DENV-1) during this year. We analyzed the molecular epidemiology of six DENV-1 epidemic strains in 2019 by full-length genome sequencing, amino acid mutation site analysis, evolutionary tree analysis, and recombination site comparison analysis. Through the analysis of amino acid mutation sites, it was found that DENV-1 strain (MW386867) was different from the other five epidemic DENV-1 strains in Xishuangbanna in 2019. MW386867 had unique mutation sites at six loci. The six epidemic DENV-1 strains in Xishuangbanna in 2019 were divided into two clusters. MW386867 was highly similar to the MG679800 (Myanmar 2017), MG679801 (Myanmar 2017), and KC172834 (Laos 2008), and the other five strains were highly similar to JQ045660 (Vietnam 2011), FJ176780 (GuangDong 2006). Genetic recombination analysis revealed that there was no recombination signal in the six epidemic DENV-1 strains in Xishuangbanna in 2019. We speculate that the DENV-1 epidemic in 2019 has a co-epidemic of local strains and cross-border strains.

Dissecting the Mechanism of the Nonheme Iron Endoperoxidase FtmOx1 Using Substrate Analogues.

FtmOx1 is a nonheme iron (NHFe) endoperoxidase, catalyzing three disparate reactions, endoperoxidation, alcohol dehydrogenation, and dealkylation, under in vitro conditions; the diversity complicates its mechanistic studies. In this study, we use two substrate analogues to simplify the FtmOx1-catalyzed reaction to either a dealkylation or an alcohol dehydrogenation reaction for structure-function relationship analysis to address two key FtmOx1 mechanistic questions: (1) Y224 flipping in the proposed COX-like model vs α-ketoglutarate (αKG) rotation proposed in the CarC-like mechanistic model and (2) the involvement of a Y224 radical (COX-like model) or a Y68 radical (CarC-like model) in FtmOx1-catalysis. When 13-oxo-fumitremorgin B (7) is used as the substrate, FtmOx1-catalysis changes from the endoperoxidation to a hydroxylation reaction and leads to dealkylation. In addition, consistent with the dealkylation side-reaction in the COX-like model prediction, the X-ray structure of the FtmOx1•CoII•αKG•7 ternary complex reveals a flip of Y224 to an alternative conformation relative to the FtmOx1•FeII•αKG binary complex. Verruculogen (2) was used as a second substrate analogue to study the alcohol dehydrogenation reaction to examine the involvement of the Y224 radical or Y68 radical in FtmOx1-catalysis, and again, the results from the verruculogen reaction are more consistent with the COX-like model.

Corrigendum: Clinical Characteristics and Risk Factors for Severe Dengue Fever in Xishuangbanna, During the Dengue Outbreak in 2019.

[This corrects the article DOI: 10.3389/fmicb.2022.739970.].

OvoAMtht from Methyloversatilis thermotolerans ovothiol biosynthesis is a bifunction enzyme: thiol oxygenase and sulfoxide synthase activities.

Mononuclear non-heme iron enzymes are a large class of enzymes catalyzing a wide-range of reactions. In this work, we report that a non-heme iron enzyme in Methyloversatilis thermotolerans, OvoAMtht, has two different activities, as a thiol oxygenase and a sulfoxide synthase. When cysteine is presented as the only substrate, OvoAMtht is a thiol oxygenase. In the presence of both histidine and cysteine as substrates, OvoAMtht catalyzes the oxidative coupling between histidine and cysteine (a sulfoxide synthase). Additionally, we demonstrate that both substrates and the active site iron's secondary coordination shell residues exert exquisite control over the dual activities of OvoAMtht (sulfoxide synthase vs. thiol oxygenase activities). OvoAMtht is an excellent system for future detailed mechanistic investigation on how metal ligands and secondary coordination shell residues fine-tune the iron-center electronic properties to achieve different reactivities.

Clinical Characteristics and Risk Factors for Severe Dengue Fever in Xishuangbanna, During the Dengue Outbreak in 2019.

Background: Dengue poses a large burden on the public health systems worldwide. severe dengue (SD) could lead to more serious clinical symptoms and even death. This study aimed to identify the cause of SD in a clinical trial during the dengue outbreak in Xishuangbanna in 2019, and could provide new insights into the pathogenic mechanisms of SD. Methods: Mosquito-borne viral (DENV, JEV, and CHIKV) infections were identified. The epidemiological factors and clinical symptoms of inpatients in Xishuangbanna were recorded. The IgG and IgM levels in the serum of dengue inpatients were evaluated, and secondary infections were identified. Then, the structural proteins (C/PrM/E) were sequenced and compared with those of the same type of DENV in the same area as before, and their structures were predicted by the SWISS-MODEL (expasy.org). The full-length viral genomes were sequenced and aligned with representative strains by BioEidt or MEGA 5.0. Results: In this outbreak, the clinical symptoms were more serious in SD. The proportion of SD inpatients of male and Han nationality was larger than that of dengue fever (DF) inpatients (p < 0.05). DENV-2 infection was the majority in DF, with 45 inpatients. However, DENV-1 infection was the most common SD, with 54 inpatients. There were 3 DENV-3-positive inpatients in the DF group and 6 ZIKV-positive inpatients in the SD group. A secondary infection accounted for 76.47% (78 cases) of SD inpatients, but secondary infections were only in 20% (17 cases) of DF inpatients. In the three-dimensional structure of protein analysis, the C/PrM/E of DENV-1 and DENV-2 showed more stability than previous epidemic strains, while DENV-3 in 2019 showed a looser spatial structure. After a complete genome sequencing and analysis, all six DENV-2 strains belonged to cosmopolitan, five of which clustered into one branch. The GC/AT of the five strains decreased from 2014 to 2018. Compared with DF strains, SD strains had no mutations of commonness. Conclusions: SD may related to secondary heteromorphic dengue in Xishuangbanna in 2019. The coinfection of ZIKV could be another related factor for SD. The currently datas were very limited and only suggestive.

Plasmonic photoreactors-coated plastic tubing as combined-active-and-passive antimicrobial flow sterilizer.

Plastic materials are ubiquitous in medical devices and consumer goods. As bacterial contamination of plastic surfaces can pose significant health risks, there is a need for effective approaches both to inactivate bacteria on plastic surfaces and to prevent colonization of plastic surfaces. In this study, we evaluate a plasmonic photoreactor coating for plastic surfaces that provides both active and passive antimicrobial effects and implement a visible light-driven antibacterial flow sterilizer. We demonstrate that this approach inactivates bacteria in an aqueous suspension passed through a photoreactor-coated polyethylene tubing, achieving log reduction values (LRVs) > 5 for both Gram-positive and -negative bacteria under resonant LED illumination. Importantly, the antimicrobial flow sterilizers do not cause a detectable loss of functionality for monoclonal antibodies that were included in this work as an example of high-value biologics that require sterilization. Under ambient light illumination, the plasmonic photoreactor coating exhibits a significant inhibitory effect on bacterial colonization and biofilm formation. The inhibitory effect was substantially weaker for mammalian cells, indicating some selectivity in the protection provided by the coating.

Light-driven Oxidative Demethylation Reaction Catalyzed by a Rieske-type Non-heme Iron Enzyme Stc2.

Rieske-type non-heme iron oxygenases/oxidases catalyze a wide range of transformations. Their applications in bioremediation or biocatalysis face two key barriers: the need of expensive NAD(P)H as a reductant and a proper reductase to mediate the electron transfer from NAD(P)H to the oxygenases. To bypass the need of both the reductase and NAD(P)H, using Rieske-type oxygenase (Stc2) catalyzed oxidative demethylation as the model system, we report Stc2 photocatalysis using eosin Y/sulfite as the photosensitizer/sacrificial reagent pair. In a flow-chemistry setting to separate the photo-reduction half-reaction and oxidation half-reaction, Stc2 photo-biocatalysis outperforms the Stc2-NAD(P)H-reductase (GbcB) system. In addition, in a few other selected Rieske enzymes (NdmA, CntA, and GbcA), and a flavin-dependent enzyme (iodotyrosine deiodinase, IYD), the eosin Y/sodium sulfite photo-reduction pair could also serve as the NAD(P)H-reductase surrogate to support catalysis, which implies the potential applicability of this photo-reduction system to other redox enzymes.

Implications for an imidazol-2-yl carbene intermediate in the rhodanase-catalyzed C-S bond formation reaction of anaerobic ergothioneine biosynthesis.

In the anaerobic ergothioneine biosynthetic pathway, a rhodanese domain containing enzyme (EanB) activates tne hercynine's sp2 ε-C-H Dona ana replaces it with a C-S bond to produce ergothioneine. The key intermediate for this trans-sulfuration reaction is the Cys412 persulfide. Substitution of the EanB-Cys412 persulfide with a Cys412 perselenide does not yield the selenium analog of ergothioneine, selenoneine. However, in deuterated buffer, the perselenide-modified EanB catalyzes the deuterium exchange between hercynine's sp2 ε-C-H bond and D2O. Results from QM/MM calculations suggest that the reaction involves a carbene intermediate and that Tyr353 plays a key role. We hypothesize that modulating the pKa of Tyr353 will affect the deuterium-exchange rate. Indeed, the 3,5-difluoro tyrosine containing EanB catalyzes the deuterium exchange reaction with k ex of ~10-fold greater than the wild-type EanB (EanBWT). With regards to potential mechanisms, these results support the involvement of a carbene intermediate in EanB-catalysis, rendering EanB as one of the few carbene-intermediate involving enzymatic systems.

Co-circulation of three dengue virus serotypes led to a severe dengue outbreak in Xishuangbanna, a border area of China, Myanmar, and Laos, in 2019.

Dengue fever was included in the top 10 global health threats announced by the World Health Organization (WHO) in early 2019. In some southern provinces of China, autochthonous outbreaks have also been reported over the last decade. An unexpected large outbreak of dengue fever was reported in Xishuangbanna, a border area of China, Myanmar, and Laos, in 2019. Among the 226 hospitalized cases, 90 were diagnosed as severe dengue according to the 2009 WHO guidelines. Serotyping and phylogenetic analyses of envelope gene sequences from 246 randomly selected samples showed that three serotypes of dengue virus were co-circulating in this outbreak, which is very rare in this area. Dengue virus serotype 1 (DENV-1, genotype I) and serotype 2 (DENV-2, Cosmopolitan genotype and Asian genotype) were the main pathogenic agents of this outbreak. Dengue virus serotype 3 (DENV-3) epidemic strains were classified as genotype III and formed a close cluster with the Thailand 2015 epidemic strain. The co-circulation may have led to more serious clinical symptoms and a larger scale epidemic. This finding is of great importance in understanding the circulation of DENV and to strengthen the detection and management of dengue fever in border areas.

Chemical modifications of proteins and their applications in metalloenzyme studies.

Protein chemical modifications are important tools for elucidating chemical and biological functions of proteins. Several strategies have been developed to implement these modifications, including enzymatic tailoring reactions, unnatural amino acid incorporation using the expanded genetic codes, and recognition-driven transformations. These technologies have been applied in metalloenzyme studies, specifically in dissecting their mechanisms, improving their enzymatic activities, and creating artificial enzymes with non-natural activities. Herein, we summarize some of the recent efforts in these areas with an emphasis on a few metalloenzyme case studies.

Molecular characterization of structural protein genes of dengue virus serotype 1 epidemic in Yunnan, Southwest China, in 2018.

A dengue virus serotype 1 (DENV-1) epidemic occurred from October to December 2018 in Xishuangbanna, Yunnan, Southwest China, neighboring Myanmar, Laos, and Vietnam. In this study, we investigated the molecular characteristics, evolution, and potential source of DENV from Xishuangbanna. The C (capsid), prM (premembrane), and E (envelope) genes of DENV isolated from 87 serum samples obtained from local patients were amplified and sequenced, and the sequences were evaluated by identification of mutations, phylogenetic and homologous recombination analysis, and secondary structure prediction. Phylogenetic analysis showed that all of the epidemic DENV strains from Xishuangbanna could be grouped in a branch with DENV-1 isolates, and were most similar to the Fujian 2005 (China, DQ193572) and Singapore 2016 (MF314188) strains. When compared with DENV-1SS (the standard strain), there were 31 non-synonymous mutations, but no obvious homologous recombination signal was found. Secondary structure prediction showed that some changes had occurred in a helical region in proteins of the MN123849 and MN123854 strains, but there were few changes in the disordered region. This study reveals the molecular characteristics of the structural genes of the Xishuangbanna epidemic strains in 2018 and provides a reference for molecular epidemiology, infection, and pathogenicity research and vaccine development.

Single-step Replacement of an Unreactive C-H Bond by a C-S Bond Using Polysulfide as the Direct Sulfur Source in Anaerobic Ergothioneine Biosynthesis.

Ergothioneine, a natural longevity vitamin and antioxidant, is a thiol-histidine derivative. Recently, two types of biosynthetic pathways were reported. In the aerobic ergothioneine biosynthesis, a non-heme iron enzyme incorporates a sulfoxide to an sp2 C-H bond in trimethyl-histidine (hercynine) through oxidation reactions. In contrast, in the anaerobic ergothioneine biosynthetic pathway in a green sulfur bacterium, Chlorobium limicola, a rhodanese domain containing protein (EanB) directly replaces this unreactive hercynine C-H bond with a C-S bond. Herein, we demonstrate that polysulfide (HSSnSR) is the direct sulfur-source in EanB-catalysis. After identifying EanB's substrates, X-ray crystallography of several intermediate states along with mass spectrometry results provide additional mechanistic details for this reaction. Further, quantum mechanics/molecular mechanics (QM/MM) calculations reveal that protonation of Nπ of hercynine by Tyr353 with the assistance of Thr414 is a key activation step for the hercynine sp2 C-H bond in this trans-sulfuration reaction.

Hybrid Plasmonic Photoreactors as Visible Light-Mediated Bactericides.

Photocatalytic compounds and complexes, such as tris(bipyridine)ruthenium(II), [Ru(bpy)3]2+, have recently attracted attention as light-mediated bactericides that can help to address the need for new antibacterial strategies. We demonstrate in this work that the bactericidal efficacy of [Ru(bpy)3]2+ and the control of its antibacterial function can be significantly enhanced through combination with a plasmonic nanoantenna. We report strong, visible light-controlled bacterial inactivation with a nanocomposite design that incorporates [Ru(bpy)3]2+ as a photocatalyst and a Ag nanoparticle (NP) core as a light-concentrating nanoantenna into a plasmonic hybrid photoreactor. The hybrid photoreactor platform is facilitated by a self-assembled lipid membrane that encapsulates the Ag NP and binds the photocatalyst. The lipid membrane renders the nanocomposite biocompatible in the absence of resonant illumination. Upon illumination, the plasmon-enhanced photoexcitation of the metal-to-ligand charge-transfer band of [Ru(bpy)3]2+ prepares the reactive excited state of the complex that oxidizes the nanocomposite membrane and increases its permeability. The photooxidation induces the release of [Ru(bpy)3]2+, Ag+, and peroxidized lipids into the ambient medium, where they interact synergistically to inactivate bacteria. We measured a 7 order of magnitude decrease in Gram-positive Arthrobacter sp. and a 4 order of magnitude decrease in Gram-negative Escherichia coli colony forming units with the photoreactor bactericides after visible light illumination for 1 h. In both cases, the photoreactor exceeds the bactericidal standard of a log reduction value of 3 and surpasses the antibacterial effect of free Ag NPs or [Ru(bpy)3]2+ by >4 orders of magnitude. We also implement the inactivation of a bacterial thin film in a proof-of-concept study.