Microbial innovations in chromium remediation: mechanistic insights and diverse applications.

The ubiquity of hexavalent chromium (Cr(VI)) from industrial activities poses a critical environmental threat due to its persistence, toxicity and mutagenic potential. Traditional physico-chemical methods for its removal often entail significant environmental drawbacks. Recent advancements in remediation strategies have emphasized nano and bioremediation techniques as promising avenues for cost-effective and efficient Cr(VI) mitigation. Bioremediation harnesses the capabilities of biological agents like microorganisms, and algae to mitigate heavy metal contamination, while nano-remediation employs nanoparticles for adsorption purposes. Various microorganisms, including E. coli, Byssochlamys sp., Pannonibacter phragmitetus, Bacillus, Aspergillus, Trichoderma, Fusarium, and Chlorella utilize bioreduction, biotransformation, biosorption and bioaccumulation mechanisms to convert Cr(VI) to Cr(III). Their adaptability to different environments and integration with nanomaterials enhance microbial activity, offering eco-friendly solutions. The study provides a brief overview of metabolic pathways involved in Cr(VI) bioreduction facilitated by diverse microbial species. Nitroreductase and chromate reductase enzymes play key roles in nitrogen and chromium removal, with nitroreductase requiring nitrate and NADPH/NADH, while the chromium reductase pathway relies solely on NADPH/NADH. This review investigates the various anthropogenic activities contributing to Cr(VI) emissions and evaluates the efficacy of conventional, nano-remediation, and bioremediation approaches in curbing Cr(VI) concentrations. Additionally, it scrutinizes the mechanisms underlying nano-remediation techniques for a deeper understanding of the remediation process. It identifies research gaps and offers insights into future directions aimed at enhancing the real-time applicability of bioremediation methods for mitigating with Cr(VI) pollution and pave the way for sustainable remediation solutions.

Visualization of an Endogenous Mitochondrial Azoreductase Activity under Normoxic Conditions Using a Naphthalimide Azo-Based Fluorogenic Probe.

In this paper, we demonstrate the existence of an endogenous mitochondrial azoreductase (AzoR) activity that can induce the cleavage of N═N double bonds of azobenzene compounds under normoxic conditions. To this end, 100% OFF-ON azo-based fluorogenic probes derived from 4-amino-1,8-naphthalimide fluorophores were synthesized and evaluated. The in vitro study conducted with other endogenous reducing agents of the cell, including reductases, demonstrated both the efficacy and the selectivity of the probe for AzoR. Confocal experiments with the probe revealed an AzoR activity in the mitochondria of living cells under normal oxygenation conditions, and we were able to demonstrate that this endogenous AzoR activity appears to be expressed at different levels across different cell lines. This discovery provides crucial information for our understanding of the biochemical processes occurring within the mitochondria. It thus contributes to a better understanding of its function, which is implicated in numerous pathologies.

Revealing the degrading-possibility of methyl red by two azoreductases of Anoxybacillus sp. PDR2 based on molecular docking.

Azo dyes, as the most widely used synthetic dyes, are considered to be one of the culprits of water resources and environmental pollution. Anoxybacillus sp. PDR2 is a thermophilic bacterium with the ability to degrade azo dyes, whose genome contains two genes encoding azoreductases (named AzoPDR2-1 and AzoPDR2-2). In this study, through response surface methodology (RSM), when the initial pH, inoculation volume and Mg2+ addition amount were 7.18, 10.72% and 0.1 g/L respectively, the decolorization rate of methyl red (MR) (200 mg/L) could reach its maximum (98.8%). The metabolites after biodegradation were detected by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and liquid chromatography mass spectrometry (LC-MS/MS), indicating that MR was successfully decomposed into 4-aminobenzoic acid and other small substrates. In homologous modeling, it was found that both azoreductases were flavin-dependent azoreductases, and belonged to the α/ß structure, using the Rossmann fold. In their docking results with the cofactor flavin mononucleotide (FMN), FMN bound to the surface of the protein dimer. Nicotinamide adenine dinucleotide (NADH) was superimposed on the plane of the pyrazine ring between FMN and the activity pocket of protein. Besides, both azoreductase complexes (azoreductase-FMN-NADH) exhibited a substrate preference for MR. Asn104 and Tyr74 played an important role in the combination of the azoreductase AzoPDR2-1 complex and the azoreductase AzoPDR2-2 complex with MR, respectively. This provided assistance for studying the mechanism of azoreductase biodegradation of azo dyes in thermophilic bacteria.

Core-genome-mediated promising alternative drug and multi-epitope vaccine targets prioritization against infectious Clostridium difficile.

Prevention of Clostridium difficile infection is challenging worldwide owing to its high morbidity and mortality rates. C. difficile is currently being classified as an urgent threat by the CDC. Devising a new therapeutic strategy become indispensable against C. difficile infection due to its high rates of reinfection and increasing antimicrobial resistance. The current study is based on core proteome data of C. difficile to identify promising vaccine and drug candidates. Immunoinformatics and vaccinomics approaches were employed to construct multi-epitope-based chimeric vaccine constructs from top-ranked T- and B-cell epitopes. The efficacy of the designed vaccine was assessed by immunological analysis, immune receptor binding potential and immune simulation analyses. Additionally, subtractive proteomics and druggability analyses prioritized several promising and alternative drug targets against C. difficile. These include FMN-dependent nitroreductase which was prioritized for pharmacophore-based virtual screening of druggable molecule databases to predict potent inhibitors. A MolPort-001-785-965 druggable molecule was found to exhibit significant binding affinity with the conserved residues of FMN-dependent nitroreductase. The experimental validation of the therapeutic targets prioritized in the current study may worthy to identify new strategies to combat the drug-resistant C. difficile infection.

Antigiardial and antiamebic activities of fexinidazole and its metabolites: new drug leads for giardiasis and amebiasis.

The intestinal parasites Giardia lamblia and Entamoeba histolytica are major causes of morbidity and mortality associated with diarrheal diseases. Metronidazole is the most common drug used to treat giardiasis and amebiasis. Despite its efficacy, treatment failures in giardiasis occur in up to 5%-40% of cases. Potential resistance of E. histolytica to metronidazole is an increasing concern. Therefore, it is critical to search for more effective drugs to treat giardiasis and amebiasis. We identified antigiardial and antiamebic activities of the rediscovered nitroimidazole compound, fexinidazole, and its sulfone and sulfoxide metabolites. Fexinidazole is equally active against E. histolytica and G. lamblia trophozoites, and both metabolites were 3- to 18-fold more active than the parent drug. Fexinidazole and its metabolites were also active against a metronidazole-resistant strain of G. lamblia. G. lamblia and E. histolytica cell extracts exhibited decreased residual nitroreductase activity when metabolites were used as substrates, indicating nitroreductase may be central to the mechanism of action of fexinidazole. In a cell invasion model, fexinidazole and its metabolites significantly reduced the invasiveness of E. histolytica trophozoites through basement membrane matrix. A q.d. oral dose of fexinidazole and its metabolites at 10 mg/kg for 3 days reduced G. lamblia infection significantly in mice compared to control. The newly discovered antigiardial and antiamebic activities of fexinidazole, combined with its FDA-approval and inclusion in the WHO Model List of Essential Medicines for the treatment of human African trypanosomiasis, offer decreased risk and a shortened development timeline toward clinical use of fexinidazole for treatment of giardiasis or amebiasis.

High-level nitrofurantoin resistance in a clinical isolate of Klebsiella pneumoniae: a comparative genomics and metabolomics analysis.

Nitrofurantoin is a commonly used chemotherapeutic agent in the treatment of uncomplicated urinary tract infections caused by the problematic multidrug resistant Gram-negative pathogen Klebsiella pneumoniae. The present study aims to elucidate the mechanism of nitrofurantoin action and high-level resistance in K. pneumoniae using whole-genome sequencing (WGS), qPCR analysis, mutation structural modeling and untargeted metabolomic analysis. WGS profiling of evolved highly resistant mutants (nitrofurantoin minimum inhibitory concentrations > 256 mg/L) revealed modified expression of several genes related to membrane transport (porin ompK36 and efflux pump regulator oqxR) and nitroreductase activity (ribC and nfsB, involved in nitrofurantoin reduction). Untargeted metabolomics analysis of total metabolites extracted at 1 and 4 h post-nitrofurantoin treatment revealed that exposure to the drug caused a delayed effect on the metabolome which was most pronounced after 4 h. Pathway enrichment analysis illustrated that several complex interrelated metabolic pathways related to nitrofurantoin bacterial killing (aminoacyl-tRNA biosynthesis, purine metabolism, central carbohydrate metabolism, and pantothenate and CoA biosynthesis) and the development of nitrofurantoin resistance (riboflavin metabolism) were significantly perturbed. This study highlights for the first time the key role of efflux pump regulator oqxR in nitrofurantoin resistance and reveals global metabolome perturbations in response to nitrofurantoin, in K. pneumoniae.IMPORTANCEA quest for novel antibiotics and revitalizing older ones (such as nitrofurantoin) for treatment of difficult-to-treat Gram-negative bacterial infections has become increasingly popular. The precise antibacterial activity of nitrofurantoin is still not fully understood. Furthermore, although the prevalence of nitrofurantoin resistance remains low currently, the drug's fast-growing consumption worldwide highlights the need to comprehend the emerging resistance mechanisms. Here, we used multidisciplinary techniques to discern the exact mechanism of nitrofurantoin action and high-level resistance in Klebsiella pneumoniae, a common cause of urinary tract infections for which nitrofurantoin is the recommended treatment. We found that the expression of multiple genes related to membrane transport (including active efflux and passive diffusion of drug molecules) and nitroreductase activity was modified in nitrofurantoin-resistant strains, including oqxR, the transcriptional regulator of the oqxAB efflux pump. Furthermore, complex interconnected metabolic pathways that potentially govern the nitrofurantoin-killing mechanisms (e.g., aminoacyl-tRNA biosynthesis) and nitrofurantoin resistance (riboflavin metabolism) were significantly inhibited following nitrofurantoin treatment. Our study could help inform the improvement of nitrofuran derivatives, the development of new pharmacophores, or drug combinations to support the resurgence of nitrofurantoin in the management of multidrug resistant K. pneumouniae infection.

Both Nitro Groups Are Essential for High Antitubercular Activity of 3,5-Dinitrobenzylsulfanyl Tetrazoles and 1,3,4-Oxadiazoles through the Deazaflavin-Dependent Nitroreductase Activation Pathway.

3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.

Oxygen-insensitive nitroreductase bacteria-mediated degradation of TNT and proteomic analysis.

2,4,6-trinitrotoluene (TNT) is a nitroaromatic compound that causes soil and groundwater pollution during manufacture, transportation, and use, posing significant environmental and safety hazards. In this study, a TNT-degrading strain, Bacillus cereus strain T4, was screened and isolated from TNT-contaminated soil to explore its degradation characteristics and proteomic response to TNT. The results showed that after inoculation with the bacteria for 4 h, the TNT degradation rate reached 100% and was transformed into 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT), and 2,6-diamino-4-nitrotoluene (2,6-DANT), accompanied by the accumulation of nitrite and ammonium ions. Through proteomic sequencing, we identified 999 differentially expressed proteins (482 upregulated, 517 downregulated), mainly enriched in the pentose phosphate, glycolysis/gluconeogenesis, and amino acid metabolism pathways. In addition, the significant upregulation of nitroreductase and N-ethylmaleimide reductase was closely related to TNT denitration and confirmed that the strain T4 converted TNT into intermediate metabolites such as 2-ADNT and 4-ADNT. Therefore, Bacillus cereus strain T4 has the potential to degrade TNT and has a high tolerance to intermediate products, which may effectively degrade nitroaromatic pollutants such as TNT in situ remediation in combination with other bacterial communities.

Anti-Trypanosoma cruzi Activity, Mutagenicity, Hepatocytotoxicity and Nitroreductase Enzyme Evaluation of 3-Nitrotriazole, 2-Nitroimidazole and Triazole Derivatives.

Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential.

A highly selective ratio-metric fluorescent sensor for visualizing nitroreductase in hypoxic cells.

Herein, we have developed a novel single-molecular probe (NORP) for selective and accurate determination of NTR in living cells. It was discovered that up-regulation of endogenous NTR occurred in response to hypoxic stimulation, and there was a dependence between the NTR levels and the degree of hypoxia.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.

Roxarsone biotransformation by a nitroreductase and an acetyltransferase in Pseudomonas chlororaphis, a bacterium isolated from soil.

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox), a widely used organoarsenical feed additive, can enter soils and be further biotransformed into various arsenic species that pose human health and ecological risks. However, the pathway and molecular mechanism of Rox biotransformation by soil microbes are not well studied. Therefore, in this study, we isolated a Rox-transforming bacterium from manure-fertilized soil and identified it as Pseudomonas chlororaphis through morphological analysis and 16S rRNA gene sequencing. Pseudomonas chlororaphis was able to biotransform Rox to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), arsenate [As(V)], arsenite [As(III)], and dimethylarsenate [DMAs(V)]. The complete genome of Pseudomonas chlororaphis was sequenced. PcmdaB, encoding a nitroreductase, and PcnhoA, encoding an acetyltransferase, were identified in the genome of Pseudomonas chlororaphis. Expression of PcmdaB and PcnhoA in E. coli Rosetta was shown to confer Rox(III) and 3-AHPAA(III) resistance through Rox nitroreduction and 3-AHPAA acetylation, respectively. The PcMdaB and PcNhoA enzymes were further purified and functionally characterized in vitro. The kinetic data of both PcMdaB and PcNhoA were well fit to the Michaelis-Menten equation, and nitroreduction catalyzed by PcMdaB is the rate-limiting step for Rox transformation. Our results provide new insights into the environmental risk assessment and bioremediation of Rox(V)-contaminated soils.

[Comparison of Conventional Methods with Molecular Methods in the Diagnosis of Trichomonas vaginalis and Investigation of Metronidazole Resistance]. / Trichomonas vaginalis'in Tanisinda Konvansiyonel Yöntemlerin Moleküler Yöntemlerle Karsilastirilmasi ve Metronidazol Direncinin Arastirilmasi.

Trichomoniasis is a sexually transmitted parasitic infection caused by Trichomonas vaginalis. In the diagnosis of trichomoniasis, direct microscopy (DM) is preferred, which is a cheap and fast method, although it has low sensitivity. Culture methods, which are accepted as the gold standard, can only be applied in certain centers due to the need for experienced personnel and the ability to get results within 2-7 days, despite their high sensitivity. In this study, it was aimed to compare conventional microscopic and culture methods used in the routine diagnosis of T.vaginalis with polymerase chain reaction (PCR) method and to investigate ntr4 and/or ntr6 gene polymorphism in the nitroreductase gene region, which are thought to be associated with metronidazole resistance in T.vaginalis strains isolated from clinical specimens. Vaginal swab specimens were collected from the posterior fornix of the vagina with two sterile ecuvion sticks during the gynecological examinations of 200 patients who applied to the Balikesir University Health Practice and Research Hospital, Obstetrics and Gynecology Polyclinic between March 2019 and August 2021. The first swab sample was used for direct microscopic examination, Giemsa staining and conventional PCR analysis, while the second swab specimen was taken into trypticase-yeast-extract-maltose (TYM) medium for T.vaginalis culture and followed for eight days at 37 °C. All specimens were screened for the presence of T.vaginalis using primers specific to the ß-tubulin (btub1) gene region and clinical isolates grown in TYM medium were examined for metronidazole resistance using primers specific for the nitroreductase gene region by using conventional PCR. Drug resistance test was also performed for the isolates in which polymorphism associated with metronidazole resistance was detected. Eight (4%) of 200 patient specimens were found positive by both culture/staining and PCR methods. The mean age of the patients included in the study was 39.9, while the mean age of the patients with positive T.vaginalis was 41.8. The most common clinical findings in the patients were foul-smelling vaginal discharge (36%), groin pain (21%), vaginal itching (19%), and burning sensation during urination (18%). In three out of eight T.vaginalis strains isolated from clinical samples, the presence of polymorphism in the ntr6 gene, which is thought to be associated with metronidazole resistance, was demonstrated by PCR. It was observed that three isolates with ntr6 gene polymorphism were phenotypically resistant to metronidazole (MLK= 390 µM). In this study, the fact that three of eight clinical isolates that were resistant to metronidazole by the broth microdilution method and as well as showing ntr6 gene polymorphism supported the thesis that there might be a close relationship between metronidazole resistance and ntr6 gene polymorphism. As a result, the use of culture and molecular methods in the diagnosis of T.vaginalis, in addition to the microscopy method, may contribute to a more accurate laboratory diagnosis of the agent, to detect metronidazole resistance molecularly and phenotypically, to determine metronidazole resistance rates in our country and to update treatment protocols within the framework of these data.

An inducible germ cell ablation chicken model for high-grade germline chimeras.

Chicken embryos are a powerful and widely used animal model in developmental biology studies. Since the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) into recipients after genetic modifications. However, low inheritance caused by competition between host germ cells and the transferred cells is a common complication and greatly reduces production efficiency. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which nitroreductase produces cytotoxic alkylating agents from administered metronidazole, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus was used to drive the expression of the nitroreductase gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system 'germ cell-specific autonomous removal induction' (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.

IS1-related large-scale deletion of chromosomal regions harbouring the oxygen-insensitive nitroreductase gene nfsB causes nitrofurantoin heteroresistance in Escherichia coli.

Nitrofurantoin is a broad-spectrum first-line antimicrobial used for managing uncomplicated urinary tract infection (UTI). Loss-of-function mutations in chromosomal genes nfsA, nfsB and ribE of Escherichia coli are known to reduce nitrofurantoin susceptibility. Here, we report the discovery of nitrofurantoin heteroresistance in E. coli clinical isolates and a novel genetic mechanism associated with this phenomenon. Subpopulations with lower nitrofurantoin susceptibility than major populations (hereafter, nitrofurantoin-resistant subpopulations) in two E. coli blood isolates (previously whole-genome sequenced) were identified using population analysis profiling. Each isolate was known to have a loss-of-function mutation in nfsA. From each isolate, four nitrofurantoin-resistant isolates were derived at a nitrofurantoin concentration of 32 mg l-1, and a comparator isolate was obtained without any nitrofurantoin exposure. Genomes of derived isolates were sequenced on Illumina and Nanopore MinION systems. Genetic variation between isolates was determined based on genome assemblies and read mapping. Nitrofurantoin minimum inhibitory concentrations (MICs) of both blood isolates were 64 mg l-1, with MICs of major nitrofurantoin-susceptible populations varying from 4 to 8 mg l-1. Two to 99 c.f.u. per million demonstrated growth at the nitrofurantoin concentration of 32 mg l-1, which is distinct from that of a homogeneously susceptible or resistant isolate. Derived nitrofurantoin-resistant isolates had 11-66 kb deletions in chromosomal regions harbouring nfsB, and all deletions were immediately adjacent to IS1-family insertion sequences. Our findings demonstrate that the IS1-associated large-scale genetic deletion is a hitherto unrecognized mechanism of nitrofurantoin heteroresistance and could compromise UTI management. Further, frequencies of resistant subpopulations from nitrofurantoin-heteroresistant isolates may challenge conventional nitrofurantoin susceptibility testing in clinical settings.

[12]aneN3-modified camptothecin and PEGylated AIEgens co-assembly into core-shell nanoparticles with ROS/NTR dual-response for enhanced cancer therapy.

A novel dual-responsive nanoparticle (NP) system was aimed to be developed for the co-delivery of camptothecin (CPT) and plasmid encoding TNF-related apoptosis-inducing ligand (pTRAIL) DNA in cancer therapy. The combination of the prodrug CPT and the nucleic acid condensing di-(triazole-[12]aneN3) unit with 4-nitrobenzyl ester through alkyl chains resulted in three nitroreductase (NTR) responsive amphiphiles, CNN1-CNN3 (with 5, 8, and 11 carbon chains, respectively). Among them, CNN2 was the most effective in inhibiting the proliferation of HeLa cells in the presence of fusogenic lipid DOPE. The NPs composed of CNN2, pDNA, and DOPE were further co-assembled with ROS-responsive thioketal-linked amphiphilic polymer (TTP) to afford the core-shell NPs (CNN2-DT/pDNA) with an average size of 118 nm, which exhibited high drug-loading capacity, excellent serum tolerance, and good biocompatibility. In the presence of ROS, NTR, and NADH, the core-shell NPs were decomposed, leading to the efficient release of 80% CPT and abundant pDNA. The self-assembly and delivery process of CNN2-DT NPs and DNA were clearly observed through the AIE fluorescent imaging. In vitro and in vivo results demonstrated that the CNN2-DT/pTRAIL NPs synergistically promoted 68% apoptosis of tumor cells and inhibited tumor growth with negligible toxic side effects. This study showed that the combination of prodrug and nucleic acid through dual-responsive core-shell NPs provide a spatially and temporally-controlled strategy for cancer therapy.

A fluorescent probe for imaging nitroreductase with signal amplification in high-viscosity environments.

Herein, we developed a fluorescent probe ENBT for in vitro detection of nitroreductase (NTR) as well as imaging intracellular NTR. ENBT itself is non-fluorescent and it could be catalyzed by NTR to generate a viscosity-sensitive fluorophore EBT. The fluorescence intensity of EBT could be further enhanced in cancer cells with relatively high viscosity due to the inhibition of the twisted intramolecular charge transfer effect. The probe ENBT has a good response to NTR with a detection limit of 36.8 ng mL-1, and EBT has a good response to viscosity. Furthermore, different concentrations of NTR (0-1.4 µg mL-1) were used to react with the probe and the reaction systems were subjected to different viscosity solutions, and the fluorescence signals of the products in the viscosity range of 45.86-163.60 cP were increased up to 1.69-fold. ENBT was successfully used to image NTR in cells under different hypoxic conditions as well as in Staphylococcus aureus. Finally, lipopolysaccharide was added to stimulate an increase in cellular viscosity after ENBT was catalyzed by intracellular NTR into EBT, and the fluorescence signals were observed to increase by 1.72-fold. The signal amplification capability gives ENBT higher sensitivity and immunity to interference. Moreover, it has the advantages of mitochondrial targeting, large Stokes shift (190 nm), high selectivity, and can be easily synthesized.

Diagnosing Orthotopic Lung Tumor Using a NTR-Activatable Near-Infrared Fluorescent Probe by Tracheal Inhalation.

Nitroreductase (NTR) is an enzyme that is upregulated under tumor-depleted oxygen conditions. The majority of studies have been conducted on NTR, but many existing fluorescent imaging tools for monitoring NTR inevitably suffer from weak targeting, low sensitivity, and simple tumor models. Research on diagnosing lung tumors has been very popular in recent years, but targeting assays in orthotopic lung tumors is still of great research value, as such models better mimic the reality of cancer in the organism. Here, we developed a novel near-infrared (NIR) fluorescent probe IR-ABS that jointly targets NTR and carbonic anhydrase IX (CAIX). IR-ABS has excellent sensitivity and selectivity and shows exceptional NTR response in spectroscopic tests. The measurements ensured that this probe has good biosafety in both cells and mice. A better NTR response was found in hypoxic tumor cells at the cellular level, distinguishing tumor cells from normal cells. In vivo experiments demonstrated that IR-ABS achieves a hypoxic response at the zebrafish level and enables rapid and accurate tumor margin distinguishment in different mouse tumor models. More importantly, we successfully applied IR-ABS for NTR detection in orthotopic lung tumor models, further combined with tracheal inhalation drug delivery to improve targeting. To the best of our knowledge, we present for the first time a near-infrared imaging method for targeting lung cancerous tumor in situ via tracheal inhalation drug delivery, in contrast to the reported literature. This NIR fluorescence diagnostic strategy for targeting orthotopic lung cancer holds exciting potential for clinical aid in cancer diagnosis.

A nitroreductase DnrA catalyzes the biotransformation of several diphenyl ether herbicides in Bacillus sp. Za.

Diphenyl ether herbicides, typical globally used herbicides, threaten the agricultural environment and the sensitive crops. The microbial degradation pathways of diphenyl ether herbicides are well studied, but the nitroreduction of diphenyl ether herbicides by purified enzymes is still unclear. Here, the gene dnrA, encoding a nitroreductase DnrA responsible for the reduction of nitro to amino groups, was identified from the strain Bacillus sp. Za. DnrA had a broad substrate spectrum, and the Km values of DnrA for different diphenyl ether herbicides were 20.67 µM (fomesafen), 23.64 µM (bifenox), 26.19 µM (fluoroglycofen), 28.24 µM (acifluorfen), and 36.32 µM (lactofen). DnrA also mitigated the growth inhibition effect on cucumber and sorghum through nitroreduction. Molecular docking revealed the mechanisms of the compounds fomesafen, bifenox, fluoroglycofen, lactofen, and acifluorfen with DnrA. Fomesafen showed higher affinities and lower binding energy values for DnrA, and residue Arg244 affected the affinity between diphenyl ether herbicides and DnrA. This research provides new genetic resources and insights into the microbial remediation of diphenyl ether herbicide-contaminated environments. KEY POINTS: • Nitroreductase DnrA transforms the nitro group of diphenyl ether herbicides. • Nitroreductase DnrA reduces the toxicity of diphenyl ether herbicides. • The distance between Arg244 and the herbicides is related to catalytic efficiency.

CpxA/R-Controlled Nitroreductase Expression as Target for Combinatorial Therapy against Uropathogens by Promoting Reactive Oxygen Species Generation.

The antibiotic resistances emerged in uropathogens lead to accumulative treatment failure and recurrent episodes of urinary tract infection (UTI), necessitating more innovative therapeutics to curb UTI before systematic infection. In the current study, the combination of amikacin and nitrofurantoin is found to synergistically eradicate Gram-negative uropathogens in vitro and in vivo. The mechanistic analysis demonstrates that the amikacin, as an aminoglycoside, induced bacterial envelope stress by introducing mistranslated proteins, thereby constitutively activating the cpxA/R two-component system (Cpx signaling). The activation of Cpx signaling stimulates the expression of bacterial major nitroreductases (nfsA/nfsB) through soxS/marA regulons. As a result, the CpxA/R-dependent nitroreductases overexpression generates considerable quantity of lethal reactive intermediates via nitroreduction and promotes the prodrug activation of nitrofurantoin. As such, these actions together disrupt the bacterial cellular redox balance with excessively-produced reactive oxygen species (ROS) as "Domino effect", accelerating the clearance of uropathogens. Although aminoglycosides are used as proof-of-principle to elucidate the mechanism, the synergy between nitrofurantoin is generally applicable to other Cpx stimuli. To summarize, this study highlights the potential of aminoglycoside-nitrofurantoin combination to replenish the arsenal against recurrent Gram-negative uropathogens and shed light on the Cpx signaling-controlled nitroreductase as a potential target to manipulate the antibiotic susceptibility.