Plasmonic nanostructures acting as a light-driven O_(2)-sensitive nitroreductase mimic for enhanced photochemical oxidation of para-aminothiophenol

Nanozymes,as a novel form of enzyme mimics,have garnered considerable interest.Despite overcoming the main disadvantages of their natural analogs,they still face challenges such as restricted mimic types and low substrate specificity.Herein,we introduce a reactive ligand modification strategy to diversify enzyme mimic types.Specifically,we have utilized helical plasmonic nanorods(HPNRs)modified with para-nitrothiophenol(4-NTP)to create an oxygen-sensitive nitroreductase(NTR)with light-controllability.HPNRs act as a light-adjustable source of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate(NAD(P)H),providing photon-generated energetic electrons to adsorbed 4-NTP molecules.In the presence of O_(2),the activated 4-NTP transfers the captured electron to the adsorbed O_(2),mimicking the electron transfer process in its natural counterpart.This enhanced O_(2)activation notably boosts the oxidative coupling of para-aminothiophenol(4-ATP).Density functional theory(DFT)calculations reveal that hot electrons injected into the lowest unoccupied molecular orbital(LUMO)energy level of 4-NTP can be transferred to that of molecular oxygen.In conclusion,our findings underline the potential of the reactive ligand modification strategy in developing new types of enzyme reactions,which opens up promising avenues for the enhancement and diversification of nanozyme functionalities.

Recent progress in small molecule fluorescent probes for nitroreductase

Nitroreductase（NTR） is a member of flavin-containing enzymes that exists widely in bacteria. Hypoxia,which is a characteristic of locally advanced solid tumors, resulting from an imbalance between oxygen consumption and supply, can result in NTR overexpression. Using either nicotinamide adenine dinucleotide（NADH） or nicotinamide adenine dinucleotide phosphate（NADPH） as a source of reducing equivalents, NTR can catalyze the reduction of nitroaromatic compounds to the corresponding amines.Based on this reduction mechanism, NTR can be applied not only in the bioremediation and degradation of organic nitrogen compounds, but also in the development of NTR-targeted fluorescent probes to detect the hypoxic status of cancer cells. This review aims to provide a summary of the progress in fluorescent probes for NTR in recent years and elucidate the main fluorescent mechanisms that have been applied to design probes.

A novel nitroreductase-enhanced MRI contrast agent and its potential application in bacterial imaging

Nitroreductases(NTRs) are known to be able to metabolize nitro-substituted compounds in the presence of reduced nicotinamide adenine dinucleotide(NADH) as an electron donor. NTRs are present in a wide range of bacterial genera and, to a lesser extent, in eukaryotes hypoxic tumour cells and tumorous tissues, which makes it an appropriate biomarker for an imaging target to detect the hypoxic status of cancer cells and potential bacterial infections. To evaluate the specific activation level of NTR, great efforts have been devoted to the development of fluorescent probes to detect NTR activities using fluorogenic methods to probe its behaviour in a cellular context; however, NTR-responsive MRI contrast agents are still by far underexplored. In this study, para-nitrobenzyl substituted T_1-weighted magnetic resonance imaging(MRI)contrast agent Gd-DOTA-PNB(probe 1) has been designed and explored for the possible detection of NTR.Our experimental results show that probe 1 could serve as an MRI-enhanced contrast agent for monitoring NTR activity. The in vitro response and mechanism of the NTR catalysed reduction of probe 1 have been investigated through LC–MS and MRI. Para-nitrobenzyl substituted probe 1 was catalytically reduced by NTR to the intermediate para-aminobenzyl substituted probe which then underwent a rearrangement elimination reaction to Gd-DOTA, generating the enhanced T1-weighted MR imaging. Further, LC–MS and MRI studies of living Escherichia coli have confirmed the NTR activity detection ability of probe 1 at a cellular level. This method may potentially be used for the diagnosis of bacterial infections.

Fine-tailoring the linker of near-infrared fluorescence probes for nitroreductase imaging in hypoxic tumor cells

Imaging hypoxia using fluorescence probes for nitroreductase（NTR） have attracted much attention in last decade. At least three different linkers have been commonly used to connect the recognition unit and reporting unit in reported probes for NTR. Meanwhile, the linker is known to be a key factor for achieving best sensing performance. In this work, three near-infrared fluorescence probes CyNP-1, CyNP-2 and CyNP-3 were designed and synthesized from an aminocyanine dye CyNP. The three probes have the same recognition unit and same fluorescence reporting unit, but different linkers. CyNP-1 was found to have the best sensing performance for NTR with 40-fold of fluorescence enhancement. It is well investigated how the difference of the linkers brings out the different sensing performance by HPLC, MS and docking calculations. In the end, CyNP-1 was found to have good selectivity for NTR and used to imaging hypoxia in Hela cells.

Inhibition of Escherichia coli nitroreductase by the constituents in Syzygium aromaticum

Gut bacterial nitroreductases play an important role in reduction of various nitroaromatic compounds to the corresponding N-nitroso compounds,hydroxylamines or aromatic amines,most of which are carcinogenic and mutagenic agents.Inhibition of gut nitroreductases has been recognized as an attractive approach for reducing mutagen metabolites in the colon,so as to prevent colon diseases.In this study,the inhibitory effects of 55 herbal medicines against Escherichia coli(E.coli)nitroreductase(EcNfsA)were examined.Compared with other herbal extracts,Syzygium aromaticum extract showed superior inhibitory potency toward EcNf-sA mediated nitrofurazone reduction.Then,the inhibitory effects of 22 major constituents in Syzygium aromaticum against EcNfsA were evaluted.Compared with other tested natural compounds,ellagic acid,corilagin,betulinic acid,oleanic acid,ursolic acid,urolith-in M5 and isorhamnetin were found with strong to moderate inhibitory effect against EcNfsA,with IC50 values ranging from 0.67 to 28.98 mol·L^(−1).Furthermore,the inhibition kinetic analysis and docking simulation demonstrated that ellagic acid and betulinic acid potently inhibited EcNfsA(Ki<2μmol·L−1)in a competitively inhibitory manner,which created strong interactions with the catalytic triad of EcNfsA.In summary,our findings provide new scientific basis for explaining the anti-mutagenic activity of Syzygium aromaticum,where some newly identified EcNfsA inhibitors can be used for developing novel agents to reduce the toxicity induced by bacteri-al nitroreductase.

Introducing nitazoxanide as a promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in clinical isolates

Objective:To identify the frequencies(F) of ferredoxin and nitroreductase mutations were identified on Iranian clinical isolates of Giardia lamblia in order to predict whether the nitazoxanide can be prescribed as suitable drug for symptomatic to metronidazoleresistant giardiasis.Methods:Forty Giardia lamblia isolates as of 38 symptomatic and two metronidazole-resistant patients were collected from Iran.DNAs were extracted and amplified by targeting ferredoxin and Gl NR genes.The amplicons were directly sequenced to determine gene mutations.Results:The various amino acid substitutions(F:20%,Haplotype diversity:0.891,Tajima's D:-0.44013) were identified by analyzing ferredoxin gene in four symptomatic and two resistant isolates.Only,two haplotypes(F:5%,HD:0.345; Tajima's D:0.77815) characterized in metronidazole-resistant isolates of Gl NR,however,no point mutations was found in symptomatic isolates.Conclusions:Non-synonymous mutations of ferredoxin oxidoreductase gene reduce translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression and its activity.This leads to decrease in metronidazole drug delivery into the cells.Mutations in these isolates may lead to their resistance to metronidazole.No to low synonymous mutations of Gl NR demonstrates that nitazoxanide can be prescribed as promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in Iranian clinical isolates.

Evolving a novel red-emitting two-photon dye with optically tunable amino group for monitoring the degree of hypoxia during liver fibrosis

Two-photon imaging has attracted increasing attention owing to its deep tissue imaging capabilities.Therefore,many fluorophores have been developed to satisfy its requirements.However,long-wavelength emission fluorophores with an optically tunable group are rarely developed.In this study,two longwavelength emission fluorophores with an optically tunable amino group were successfully developed by introducing strong electron acceptor and large conjugated group to the TPQL dye.TPCO_(2)displayed a bright red emission(λem=638 nm,Φ=0.15)together with high two-photon action cross section and good water solubility,which enabled higher signal-to-background ratios and deep tissue imaging.The proof-of-concept probe(TPCO-NO_(2))was successfully applied to the high signal-to-background ratio imaging of nitroreductase in liver fibrosis,further realizing diagnosis of the degree of hypoxia during liver fibrosis.

Biodegradation of pendimethalin by Bacillus subtilis Y3

A bacterium strain Y3,capable of efficiently degrading pendimethalin,was isolated from activated sludge and identified as Bacillus subtilis according to its phenotypic features and 16 S rRNA phylogenetic analysis.This strain could grow on pendimethalin as a sole carbon source and degrade 99.5%of 100 mg/L pendimethalin within 2.5 days in batch liquid culture,demonstrating a greater efficiency than any other reported strains.Three metabolic products,6-aminopendimethalin,5-amino-2-methyl-3-nitroso-4-（pentan-3-ylamino） benzoic acid,and 8-amino-2-ethyl-5-（hydroxymethyl）-1,2-dihydroquinoxaline-6-carboxylic acid,were identified by HPLC-MS/MS,and a new microbial degradation pathway was proposed.A nitroreductase catalyzing nitroreduction of pendimethalin to 6-aminopendimethalin was detected in the cell lysate of strain Y3.The cofactor was nicotinamide adenine dinucleotide phosphate（NADPH） or more preferably nicotinamide adenine dinucleotide（NADH）.The optimal temperature and pH for the nitroreductase were 30℃ and 7.5,respectively.Hg^（2＋）,Ni^（2＋）,Pb^（2＋）,Co^（2＋）,Mn^（2＋） Cu^（2＋）,Ag~＋,and EDTA severely inhibited the nitroreductase activity,whereas Fe^（2＋）,Mg^（2＋）,and Ca^（2＋） enhanced it.This study provides an efficient pendimethalin-degrading microorganism and broadens the knowledge of the microbial degradation pathway of pendimethalin.