Iron-Doped Polymer Dots with Enhanced Fluorescence and Dual Enzyme Activity for Versatile Bioassays.

Nanozymes with multiple functionalities endow biochemical sensing with more sensitive and efficient analytical performance by widening the sensing modes. Meanwhile, the target-oriented design of multifunctional nanozymes for certain biosensing remains challenging. Herein, a constructive strategy of doping iron into polymer dots (PDs) to achieve nanozymes with excellent oxidase-mimicking and peroxidase-mimicking activity is proposed. Compared with the Fe-free PDs prepared under the same mild condition, the Fe-doped PDs (Fe-PDs) exhibit greatly boosted fluorescence at 500 nm. While applying 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate, the fluorescence of the Fe-PDs can be further quenched by oxTMB due to the inner filter effect (IFE). Inspired by this, a simple but efficient colorimetric and fluorometric dual-mode sensing platform is developed for monitoring the reducing substances ascorbic acid (AA), α-glucosidase (α-Glu), and its inhibitors (AGIs). We believe that such multifunctional enzyme-mimic materials will provoke the exploration of multimode sensing strategy with strong practicality to serve as a versatile tool in biochemical sensing.

Bidirectional Associations between Parental Feeding Practices and Child Eating Behaviors in a Chinese Sample.

BACKGROUND: Child eating behaviors (CEBs) and parental feeding practices (PFPs) play critical roles in childhood obesity. However, the bidirectional relationships between CEBs and PFPs remain equivocal. This longitudinal study aimed to explore their bidirectional relationships. METHODS: A convenience sample of 870 parents with preschoolers was recruited in this longitudinal study (Shanghai, China). Three non-responsive feeding practices (NFPs), three responsive feeding practices (RFPs), five CEBs, and covariates were collected using validated questionnaires at baseline and the 6-month follow-up. Cross-lagged analyses using structural equation modeling (SEM) were performed to examine their bidirectional relationships. RESULTS: Eight hundred and fifty-three parents completed questionnaires, with a response rate of 98%. The mean age of their children at baseline was 4.39 years (standard deviation = 0.72 years). Eighteen out of sixty longitudinal cross-lagged paths were statistically significant. Parental encouragement of healthy eating and content-restricted feeding were found to be bidirectionally associated with child food fussiness. Four parent-driven associations and one child-driven association were identified between RFPs and CEBs. For example, monitoring was negatively associated with children's unhealthy eating habits (ß = -0.066, standard error (SE) = 0.025, p < 0.01). Eight child-driven associations and one parent-driven association were observed between NFPs and CEBs. For example, higher child satiety responsiveness predicted a higher pressure to eat (ß = 0.057, SE = 0.029, p < 0.01) and the use of food as a reward (ß = 0.083, SE = 0.031, p < 0.01). CONCLUSIONS: There were bidirectional, parent-driven, and child-driven associations. Parents should be encouraged to adopt RFPs to shape CEBs. Increasing parents' understanding of CEBs and providing them with reasonable coping strategies would help optimize PFPs.

N-oxide reduction of quinoxaline-1,4-dioxides catalyzed by porcine aldehyde oxidase SsAOX1.

Quinoxaline-1,4-dioxides (QdNOs) are a class of quinoxaline derivatives that are widely used in humans or animals as drugs or feed additives. However, the metabolic mechanism, especially the involved enzymes, has not been reported in detail. In this study, the N-oxide reduction enzyme, porcine aldehyde oxidase SsAOX1 was identified and characterized. The SsAOX1 gene was cloned from pig liver through reverse-transcription polymerase chain reaction using degenerate primers, which encode a 147-kDa protein with typical aldehyde oxidase motifs, two [2Fe-2S] centers, a flavin adenine dinucleotide (FAD) binding domain, and a molybdenum cofactor domain. After heterologous expression in a prokaryote, purified SsAOX1 formed a functional homodimer under native conditions. Importantly, the SsAOX1 catalyzed the N-oxide reduction at the N1 position of three representative QdNOs (quinocetone, mequindox, and cyadox), which are commonly used as animal feed additives. SsAOX1 has the highest activity toward quinocetone, followed by mequindox and cyadox, with kcat/K(m) values of 1.94 ± 0.04, 1.27 ± 0.15, and 0.43 ± 0.09 minute(-1) µM(-1), respectively. However, SsAOX1 has the lowest substrate affinity for quinocetone, followed by the cyadox and mequindox, with K(m) values of 4.36 ± 0.56, 3.16 ± 0.48, and 2.96 ± 0.51 µM, respectively. In addition, using site-directed mutagenesis, we found that substitution of glycine 1019 with threonine endows SsAOX1 with N-oxide reductive activity at the N4 position. The goal of this study was to identify and characterize the N-oxide reduction enzyme for a class of veterinary drugs, QdNOs, which will aid in the elucidation of the metabolic pathways of QdNOs and will provide a theoretical basis for their administration and new veterinary drug design.

Proteomic changes in chicken primary hepatocytes exposed to T-2 toxin are associated with oxidative stress and mitochondrial enhancement.

T-2 toxin is a mycotoxin that is toxic to plants, animals, and humans. However, its molecular mechanism remains unclear, especially in chickens. In this study, using 2D electrophoresis with MALDI-TOF/TOF-MS, 53 proteins were identified as up- or downregulated by T-2 toxin in chicken primary hepatocytes. Functional network analysis by ingenuity pathway analysis showed that the top network altered by T-2 toxin is associated with neurological disease, cancer, organismal injury, and abnormalities. Most of the identified proteins were associated with one of eight functional classes, including cell redox homeostasis, transcriptional or translational regulation, cell cycle or cell proliferation, stress response, lipid metabolism, transport, carbohydrate metabolism, and protein degradation. Subcellular location categorization showed that the identified proteins were predominantly located in the mitochondrion (34%) and interestingly, the expression of all the identified mitochondrial proteins was increased. Further cellular analysis showed that T-2 toxin was able to induce the ROS accumulation and could lead to an increase in mitochondrial mass and adenosine 5'-triphosphate content, which indicated that oxidative stress and mitochondrial enhancement occurred in T-2 toxin-treated cells. Overall, these results characterize the global proteomic response of chicken primary hepatocytes to T-2 toxin, which may lead to a better understanding of the molecular mechanisms underlying its toxicity.

Trp266 determines the binding specificity of a porcine aflatoxin B1 aldehyde reductase for aflatoxin B1-dialdehyde.

Aflatoxin B1 (AFB1) is a severe threat to human and animal health. The aflatoxin B1 aldehyde reductase (AFAR) family specifically catalyzes AFB1-dialdehyde, a toxic metabolic intermediate of AFB1, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB1-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB1-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB1-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB1-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π-π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB1-dialdehyde.

Carbonyl reduction of mequindox by chicken and porcine cytosol and cloned carbonyl reductase 1.

Mequindox (MEQ) is a novel synthetic quinoxaline 1,4-dioxides derivative, which is widely used as a veterinary drug and animal feed additive. However, the metabolic mechanism of MEQ is rarely reported. The N-oxide reduction mechanism of MEQ was reported in our previous work. In this article, the toxicity and the reduction of the carbonyl of MEQ were studied. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays demonstrated that the carbonyl-reduced MEQ, 2-isoethanol MEQ was much less toxic than MEQ. High-performance liquid chromatography analysis showed that the cytosol extracts of chicken and pig livers were able to reduce MEQ to 2-isoethanol MEQ and the reaction was NADPH-dependent. Further study via enzyme-inhibitory experiment revealed that carbonyl reductase 1 (CBR1) participated in this metabolism. The enzyme activity analysis showed that both chicken CBR1 (cCBR1) and porcine CBR1 (pCBR1) were capable of catalyzing the carbonyl reduction of MEQ and its N-oxide reductive metabolite, 1-deoxymequindox. By comparison of the kinetic constants, we observed that the activity of cCBR1 was higher than pCBR1 to MEQ and the standard substrate of CBR1, menadione. On the other hand, both CBR1s exhibited higher activity to 1-deoxymequindox than MEQ. Mutation analysis suggested that the difference of amino acid at position 141/142 may be one possible reason that caused the activity difference between cCBR1 and pCBR1. Thus far, CBR1 was first reported to participate in the carbonyl reduction of MEQ. Our results will be helpful to recognize the metabolic pathways of quinoxaline drugs deeply and to provide a theoretical basis for controlling the negative effects of these drugs.

The mechanism of enzymatic and non-enzymatic N-oxide reductive metabolism of cyadox in pig liver.

Cyadox is a novel quinoxaline-1,4-dioxide with the potential for development as a substitute for the banned veterinary drugs carbadox and olaquindox. In this paper, using pigs as the test subjects, the metabolic mechanism of cyadox N-oxide reduction in liver is demonstrated. There exist two metabolic mechanisms for the N-oxide reduction of cyadox, the enzymatic and non-enzymatic routes. It is found that cyadox can be enzymatically reduced to 4-cyadox monoxide and 1-cyadox monoxide; this process is catalyzed by aldehyde oxidase and xanthine oxidase in the cytosol and by cytochrome b5 reductase in the microsomes. On the other hand, cyadox is only reduced to 4-cyadox monoxide in the non-enzymatic reduction mediated by heme groups of catalase and cytochrome P450s. We supposed that, owing to the position of the side chain in cyadox, the 1-N-oxide and 4-N-oxide bonds in the quinoxaline ring had different biochemical activities, which caused cyadox to be shunted to the distinct metabolic mechanisms. Additionally, this research gives the first evidence of FAD- and NAD(P)H-dependent non-enzymatic catalase reduction of a heterocyclic N-oxide. The research provides a basic foundation for the formulation of safety controls for animal products and the properties and metabolism of heterocyclic N-oxides.