Isolation of an α-glucosidase Inhibitor from Houttuynia cordata Thunb. and Its In vitro and In vivo Hypoglycemic Bioactivity.

The Houttuynia cordata Thunb. belongs to the Saururaceae family and is a well-known medicine and food homologous plant. Herein, the isolation of an α-glucosidase inhibitor from Houttuynia cordata Thunb. and characterization of its in vitro and in vivo hypoglycemic bioactivities are reported. We optimized the extraction conditions and isolated neochlorogenic acid (nCGA), which has α-glucosidase inhibitory activity from Houttuynia cordata Thunb. for the first time. nCGA competed with glucose for the α-glucosidase binding site, with a 50% inhibitory concentration (IC50) of 0.711 mg/mL. In vivo experiments in zebrafish showed that effects of nCGA on blood glucose varied by its concentrations. In particular, 4 mg/L nCGA significantly decreased the blood glucose level and inhibited effects of α-glucosidase in zebrafish. This work provides a theoretical basis for the extraction of hypoglycemic active ingredients from Houttuynia cordata Thunb. and a foundation for the development of natural and effective α-glucosidase inhibitors.

Research on the Prediction Model of Engine Output Torque and Real-Time Estimation of the Road Rolling Resistance Coefficient in Tracked Vehicles.

Road parameter identification is of great significance for the active safety control of tracked vehicles and the improvement of vehicle driving safety. In this study, a method for establishing a prediction model of the engine output torques in tracked vehicles based on vehicle driving data was proposed, and the road rolling resistance coefficient f was further estimated using the model. First, the driving data from the tracked vehicle were collected and then screened by setting the driving conditions of the tracked vehicle. Then, the mapping relationship between the engine torque Te, the engine speed ne, and the accelerator pedal position ß was obtained by a genetic algorithm-backpropagation (GA-BP) neural network algorithm, and an engine output torque prediction model was established. Finally, based on the vehicle longitudinal dynamics model, the recursive least squares (RLS) algorithm was used to estimate the f. The experimental results showed that when the driving state of the tracked vehicle satisfied the set driving conditions, the engine output torque prediction model could predict the engine output torque T^e in real time based on the changes in the ne and ß, and then the RLS algorithm was used to estimate the road rolling resistance coefficient f^. The average coefficient of determination R of the T^e was 0.91, and the estimation accuracy of the f^ was 98.421%. This method could adequately meet the requirements for engine output torque prediction and real-time estimation of the road rolling resistance coefficient during tracked vehicle driving.

Zebrafish as model organisms for toxicological evaluations in the field of food science.

Food safety has long been an area of concern. The selection of stable and efficient model organisms is particularly important for food toxicology studies. Zebrafish (Danio rerio) are small model vertebrates, and 70% of human genes have at least one zebrafish ortholog. Zebrafish have advantages as model organisms due to their short life cycle, strong reproductive ability, easy rearing, and low cost. Zebrafish embryos have the advantage of being sensitive to the breeding environment and thus have been used as biosensors. Zebrafish and their embryos have been widely used for food toxicology assessments. This review provides a systematic and comprehensive summary of food toxicology studies using zebrafish as model organisms. First, we briefly introduce the multidimensional mechanisms and structure-activity relationship studies of food toxicological assessment. Second, we categorize these studies according to eight types of hazards in foods, including mycotoxins, pesticides, antibiotics, heavy metals, endocrine disruptors, food additives, nanoparticles, and other food-related ingredients. Finally, we list the applications of zebrafish in food toxicology studies in line with future research prospects, aiming to provide a valuable reference for researchers in the field of food science.

Comparison of Two DNA Aptamers for Dopamine Using Homogeneous Binding Assays.

Dopamine is an essential neurotransmitter and its detection is important for bioanalytical chemistry. Two very different DNA aptamers have been reported for dopamine, one derived from an RNA aptamer (named Apt1) and other obtained via direct aptamer selection (named Apt2). In this study, we used four homogeneous binding assays to compare these two DNA dopamine aptamers. Thiazole orange (TO) fluorescence assay indicated that the Apt2 specifically bound with dopamine with a Kd of 2.37 µM, which was consistent with that from the isothermal titration calorimetry (ITC) assay. However, Apt1 had much less TO fluorescence change and also no signal from ITC. By labeling the two ends of the two aptamers by a fluorophore and a quencher, the aptamer beacons showed binding of dopamine only for Apt2. Finally, the label-free AuNP-based colorimetric assay showed no difference between these two aptamer sequences, and even non-binding random DNA showed the same response, indicating that AuNPs were not a good probe for detecting dopamine. According to the data, Apt1 does not appear to be able to bind dopamine specifically, while Apt2 showed specific binding and could be used for developing related biosensors.

USP31 acetylation at Lys1264 is essential for its activity and cervical cancer cell growth.

Ubiquitin-specific protease 31 (USP31) is a member of deubiquitinase family that is involved in nuclear factor-κB activation and sarcomagenesis. However, little is known about posttranslational modification in the regulation of its activity and cervical cancer cell growth. In our study, we found that the Lys1264 residue of USP31 can be modified with an acetyl group by high-resolution mass spectrometry in HeLa cell line, and site-specific mutagenesis can significantly increase USP31 ubiquitin hydrolase activity and decrease the expression of p65. When being transfected with a plasmid expressing mutated USP31, the number of cancer cells was significantly decreased. We also observed that mutated USP31 could promote apoptosis but not cell cycle by flow cytometer analysis. Overexpression of mutated USP31 could reverse the effect in USP31 knockdown cell line. To further investigate its activity in tumorigenesis, deacetylase sirtuin 1 (Sirt1) was shown to interact with USP31 by co-immunoprecipitation and blocking the function of Sirt1 by knockdown or the inhibitor nicotinamide could increase the acetylation of USP31. When Lys1264 of USP31 mutated, Sirt1 could not remove its acetylation and alter the expression level of p65. Finally, inhibition or knockdown of Sirt1 suppressed USP31 activity in HeLa cell line, leading to cisplatin-induced apoptosis resistance. Therefore, acetylation at Lys1264 suppresses USP31 activity and plays a protective role in cancer cell growth. Our study contributes to understanding the mechanism of USP31 activity regulation and its role in tumorigenesis.

Dopamine and Melamine Binding to Gold Nanoparticles Dominates Their Aptamer-Based Label-Free Colorimetric Sensing.

Target-directed aptamer adsorption by gold nanoparticles (AuNPs) has been widely used to develop label-free colorimetric biosensors. However, the potential interactions between target molecules and AuNPs have not been considered, which may lead to misinterpretation of analytical results. In this work, the detection of dopamine, melamine, and K+ was studied as model systems to address this problem. First, dopamine and two control molecules all induced the aggregation of citrate-capped AuNPs with apparent Kd's of 5.8 µM dopamine, 51.6 µM norepinephrine, and 142 µM tyramine. Isothermal titration calorimetry measured the aptamer Kd to be 1.9 µM dopamine and 16.8 µM norepinephrine, whereas tyramine cannot bind. Surface enhanced Raman spectroscopy confirmed direct adsorption of dopamine, and the adsorbed dopamine inhibited the adsorption of DNA. Using a typical salt-induced colorimetric detection protocol, a similar color response was observed regardless of the sequence of DNA, indicating the observed color change reflected the adsorption of dopamine by the AuNPs instead of the binding of dopamine by the aptamer. For this label-free sensor to work, the interaction between the target molecule and AuNPs should be very weak, while dopamine represents an example of strong interactions. For the other two systems, the melamine detection did not reflect aptamer binding either but the K+ detection did, suggesting melamine also strongly interacted with AuNPs, whereas K+ had very weak interactions with AuNPs. Since each target molecule is different, such target/AuNP interactions need to be studied case-by-case to ensure the sensing mechanism.

Tunable dielectric polarization and breakdown behavior for high energy storage capability in P(VDF-TrFE-CFE)/PVDF polymer blended composite films.

Polymer dielectrics with high dielectric performances and superior discharge energy capability are highly desirable for advanced electrostatic capacitor applications. However, the paradoxical relationship between dielectric polarization and electric breakdown behavior generally hinder their further enhancement in energy storage performances. Herein, polymer blended composite films with high energy storage capability were successfully fabricated by blending together poly(vinylidene fluoride) (PVDF) polymer and poly(vinylidene fluoride-trifluoroethylene-chlorofluoroethylene) (P(VDF-TrFE-CFE)) terpolymer. The P(VDF-TrFE-CFE) terpolymer has a high dielectric constant to provide a large electric displacement under an applied electric field far below its breakdown field, which is anticipated to modulate the dielectric polarization behavior of PVDF polymer when blended in different proportions. Consequently, the polymer blended composite film consisting of 20 wt% (P(VDF-TrFE-CFE)) terpolymer exhibits a high discharge energy density of 13.63 J cm-3 at an enhanced breakdown strength of 480 MV m-1. This obtained high discharge energy density is 84% higher than the pure PVDF film and 582% higher than a commercialized biaxially oriented polypropylene (BOPP). Large interfacial polarization and strong interaction of polymer chains between the PVDF polymer and P(VDF-TrFE-CFE) terpolymer may contribute to the tunable dielectric constant and electric breakdown strength, thus promoting the energy storage capability. This work establishes a facile, but effective approach to achieve the high energy storage capability of PVDF polymer-based flexible composite films for capacitive energy storage applications.

Production of a specific monoclonal antibody for 1-naphthol based on novel hapten strategy and development of an easy-to-use ELISA in urine samples.

1-naphthol (1-NAP) is the main metabolite of pesticide carbaryl and naphthalene, and is also a genotoxic and carcinogenic intermediate in the synthesis of organic compound, dyes, pigment and pharmaceutical industry. In this work, two novel haptens were designed and synthesized for developing a competitive indirect enzyme-linked immunosorbent assay (ciELISA) method for 1-NAP in urine samples. The assay showed a limit of detection of 2.21 ng/mL and working range from 4.02 ng/mL to 31.25 ng/mL for 1-NAP in optimized working buffer. The matrix effect of samples was eliminated via 15-fold dilution of optimized working buffer. Good average recoveries (102.4%-123.4%) with a coefficient of variation from 11.7% to 14.7% was obtained for spiked urine samples. Subsequent instrument verification test showed good correlation between the results of ciELISA and high-performance liquid chromatography. The developed ciELISA is a high-throughput tool to monitor 1-NAP in urine, which can provide technical support for the establishment of biological exposure level for the exposure to carbaryl, naphthalene and other related pollutants.

Detection of chloramphenicol with an aptamer-based colorimetric assay: critical evaluation of specific and unspecific binding of analyte molecules.

A chloramphenicol (CAP)-binding aptamer of 80 nucleotides (nt) was reported in 2011. In 2014, it was truncated to 40 nt and has since been used by most researchers, although a careful binding study is still lacking. In this work, binding assays using isothermal titration calorimetry and various DNA-staining dyes were performed. By comparing the truncated aptamer with three control sequences, no specific binding of CAP was observed in each case. The secondary structures of the original and truncated aptamers were analyzed, and it was shown that the likelihood of the truncated aptamer to retain the same binding mechanism as the original sequence is low. We further examined gold nanoparticle (AuNP)-based label-free colorimetric assays. By quantifying the extinction ratio at 620 nm over that at 520 nm, a similar color response was observed regardless of the sequence of DNA, suggesting the color change mainly reflected other events such as the adsorption of CAP by the AuNPs, instead of aptamer binding to CAP. Salt-induced aggregation experiments suggested direct adsorption of CAP on AuNPs. CAP only weakly inhibited DNA adsorption by AuNPs but did not displace pre-adsorbed DNA. Therefore, CAP adsorption by AuNPs needs to be considered when designing related sensors, for example, by using non-aptamer sequences as controls. This work calls for careful confirmation of aptamer binding and control experiments for designing aptamer and AuNP-based biosensors.

Selection of Aptamers Specific for DEHP Based on ssDNA Library Immobilized SELEX and Development of Electrochemical Impedance Spectroscopy Aptasensor.

A selection of aptamers specific for di(2-ethylhexyl) phthalate (DEHP) and development of electrochemical impedance spectroscopy (EIS) aptasensor are described in this paper. The aptamers were selected from an immobilized ssDNA library using the systematic evolution of ligands by exponential enrichment (SELEX). The enrichment was monitored using real-time quantitative PCR (Q-PCR), and the aptamers were identified by high-throughput sequencing (HTS), gold nanoparticles (AuNPs) colorimetric assay, and localized surface plasmon resonance (LSPR). The EIS aptasensor was developed to detect DEHP in water samples. After eight rounds of enrichment, HTS, AuNPs colorimetric assay, and LSPR analysis indicated that four aptamers had higher binding activity, and aptamer 31 had the highest affinity (Kd = 2.26 ± 0.06 nM). The EIS aptasensor had a limit of detection (LOD) of 0.103 pg/mL with no cross-reactivity to DEHP analogs and a mean recovery of 76.07% to 141.32% for detection of DEHP in water samples. This aptamer is novel with the highest affinity and sensitivity.

Highly Stable, New, Organic-Inorganic Perovskite (CH3 NH3 )2 PdBr4 : Synthesis, Structure, and Physical Properties.

Lead halide perovskites have attracted striking attention recently, due to their appealing properties. However, toxicity and stability are two main factors restricting their application. In this work, a less toxic and highly stable Pd-based hybrid perovskite was experimentally synthesized, after exploring different experimental conditions. This new hybrid organic-inorganic perovskite (CH3 NH3 )2 PdBr4 was found to be an orthorhombic crystal (Cmce, Z=4) with lattice parameters a=8.00, b=7.99, c=18.89 Å. The Cmce symmetry and lattice parameters were confirmed using Pawley refinement and the atoms positions were confirmed based on DFT calculation. This perovskite compound was determined to be a p-type semiconductor, with a resistivity of 102.9 kΩ cm, a carrier concentration of 3.4 ×1012 cm-3 , and a mobility of 23.4 cm2 (V s)-1 . Interestingly, XRD and UV/Vis measurements indicated that the phase of this new perovskite was maintained with an optical gap of 1.91 eV after leaving in air with a high humidity of 60 % for 4 days, and unchanged for months in N2 atmosphere; much more stable than most existing organic-inorganic perovskites. The synthesis and various characterizations of this work further the understanding of this (CH3 NH3 )2 PdBr4 organic-inorganic hybrid perovskite material.

Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor.

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5'-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33⁻97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

Aptamers for nanobodies: A nontoxic alternative to toxic ochratoxin A in immunoassays.

To measure toxins using immunoassays, hazardous toxin standards need to be added for quantification. To solve this problem, we propose to use aptamers as competitors to replace toxin standards. In this work, aptamers specific for ochratoxin A (OTA) nanobodies were selected using a DNA library containing a 36 nucleotide random region. The obtained sequences were highly aligned and the best competitor was identified to be a sequence named apt2-OT based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). The Kd of apt2-OT was measured to be 2.86 µM using local surface plasmon resonance spectroscopy. The optimal apt2-OT was identified to substitute the OTA standard with a concentration needed for 50% inhibition of binding (IC50) of 3.26 µM based on a nontoxic direct competitive ELISA. The equivalence relationship between the aptamer and OTA was established in a flour sample, and a recovery experiment was performed. The detection limit for this method was 0.23 ng/mL, with a linear range from 0.25 to 10.50 ng/mL. The recovery rate was 97.5%-115.5%. This study provides a low-cost, rapid and environmentally friendly alternative to the development of immunoassays for toxins.

Probiotics Alleviate Microcystin-LR-Induced Developmental Toxicity in Zebrafish Larvae.

Microcystin-LR (MCLR) poses a significant threat to aquatic ecosystems and public health. This study investigated the protective effects of the probiotic Lactobacillus rhamnosus against MCLR-induced developmental toxicity in zebrafish larvae. Zebrafish larvae were exposed to various concentrations of MCLR (0, 0.9, 1.8, and 3.6 mg/L) with or without L. rhamnosus from 72 to 168 h post-fertilization (hpf). Probiotic supplementation significantly improved survival, hatching, and growth rates and reduced malformation rates in MCLR-exposed larvae. L. rhamnosus alleviated MCLR-induced oxidative stress by reducing reactive oxygen species (ROS) levels and enhancing glutathione (GSH) content and catalase (CAT) activity. Probiotics also mitigated MCLR-induced lipid metabolism disorders by regulating key metabolites (triglycerides, cholesterol, bile acids, and free fatty acids) and gene expression (ppara, pparb, srebp1, and nr1h4). Moreover, 16S rRNA sequencing revealed that L. rhamnosus modulated the gut microbiome structure and diversity in MCLR-exposed larvae, promoting beneficial genera like Shewanella and Enterobacter and inhibiting potential pathogens like Vibrio. Significant correlations were found between gut microbiota composition and host antioxidant and lipid metabolism parameters. These findings suggest that L. rhamnosus exerts protective effects against MCLR toxicity in zebrafish larvae by alleviating oxidative stress, regulating lipid metabolism, and modulating the gut microbiome, providing insights into probiotic-based strategies for mitigating MCLR toxicity in aquatic organisms.

Ferulic acid alleviates high fat diet-induced cognitive impairment by inhibiting oxidative stress and apoptosis.

Cognitive impairment has become a major public health problem. Growing evidence suggests that high-fat diet (HFD) can cause cognitive dysfunction and increase the risk of dementia. However, effective treatment for cognitive impairment is not available. Ferulic acid (FA) is a single phenolic compound with anti-inflammatory and antioxidant properties. Nevertheless, its role in regulating learning and memory in HFD-fed mice and the underlying mechanism remains unclear. In this study, we aimed to identify the neuroprotective mechanisms of FA in HFD induced cognitive impairment. We found that FA improved the survival rate of HT22 cells treated with palmitic acid (PA), inhibited cell apoptosis, and reduced oxidative stress via the IRS1/PI3K/AKT/GSK3ß signaling pathway; Furthermore, FA treatment for 24 weeks improved the learning and memory of HFD-fed mice and decreased hyperlipidemia. Moreover, the expression of Nrf2 and Gpx4 proteins were decreased in HFD-fed mice. After FA treatment, the decline of these proteins was reversed. Our study showed that the neuroprotective effect of FA on cognitive impairment was related to the inhibition of oxidative stress and apoptosis and regulation of glucose and lipid metabolism. These findings suggested that FA can be developed as a potential agent for the treatment of HFD-induced cognitive impairment.

Treatment of posterior interosseous nerve entrapment syndrome with ultrasound-guided hydrodissection: A case report.

BACKGROUND: Posterior interosseous nerve (PIN) entrapment syndrome is one of the causes of weakness and pain of the arm muscles, which is prone to missed diagnosis and misdiagnosis in clinic practice. This paper reports a case of PIN entrapment syndrome, with PIN injury indicated by electrophysiology. Musculoskeletal ultrasound was applied to identify that the entrapment point was located at the inlet of the Frohse arch and the outlet of the supinator muscle. Treatment with ultrasound-guided nerve hydrodissection was performed on the entrapment point, which significantly improved the symptoms. Ultrasound-guided nerve hydrodissection is an effective therapeutic method for PIN entrapment syndrome. CASE SUMMARY: A male patient, 35 years old, worked as an automobile mechanic. He felt slightly weak extension activity of his right fingers 2 years ago but sought no treatment. Later, the symptoms gradually became aggravated and led to finger drop, particularly severe in the right middle finger, accompanied by supination weakness of the right forearm. Neural electrophysiological examination showed that the patient had partial PIN injury of the right radius. Musculoskeletal ultrasound examination indicated PIN entrapment at the inlet of the Frohse arch and the outlet of the supinator muscle. Therefore, PIN entrapment syndrome was diagnosed. After treatment with ultrasound-guided nerve hydrodissection around the entrapment point, the dorsiflexion weakness of the right hand was significantly improved compared with before treatment. CONCLUSION: Ultrasound-guided hydrodissection is efficacious for PIN entrapment syndrome, with high clinical value and great application prospects.

Selection of a Novel DNA Aptamer Specific for 5-Hydroxymethylfurfural Using Capture-SELEX.

A capture systematic evolution of ligands by exponential enrichment (Capture-SELEX) was described to discover novel aptamers specific for 5-hydroxymethylfurfural (5-HMF), and a biosensor based on molecular beacon was constructed to detect 5-HMF. The ssDNA library was immobilized to streptavidin (SA) resin to select the specific aptamer. The selection progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing (HTS). Candidate and mutant aptamers were selected and identified by Isothermal Titration Calorimetry (ITC). The FAM-aptamer and BHQ1-cDNA were designed as the quenching biosensor to detect 5-HMF in milk matrix. After the 18th round selection, the Ct value decreased from 9.09 to 8.79, indicating that the library was enriched. The HTS results indicated that the total sequence numbers for 9th, 13th, 16th, and 18th were 417054, 407987, 307666, and 259867, but the number of sequences for the top 300 sequences was gradually increased from 9th to 18th, and the ClustalX2 analysis showed that there were four families with high homology rate. ITC results indicated that the Kd values of H1 and its mutants H1-8, H1-12, H1-14, and H1-21 were 2.5 µM, 1.8 µM, 1.2 µM, 6.5 µM, and 4.7 µM. The linear range of the quenching biosensor was from 0 µM to 75 µM, and it had a similar linear range in the 0.1% milk matrix. This is the first report to select a novel aptamer specific for 5-HMF and develop quenching biosensor for the rapid detection of 5-HMF in milk matrix.

Molecular Mechanisms of Tebuconazole Affecting the Social Behavior and Reproduction of Zebrafish.

The aim of this study was to explore the underlying mechanism of adverse effects caused by tebuconazole (TEB) on the reproduction of aquatic organisms In the present study, in order to explore the effects of TEB on reproduction, four-month-old zebrafish were exposed to TEB (0, DMSO, 0.4 mg/L, 0.8 mg/L, and 1.6 mg/L) for 21 days. After exposure, the accumulations of TEB in gonads were observed and the cumulative egg production was evidently decreased. The decline of fertilization rate in F1 embryos was also observed. Then the changes in sperm motility and histomorphology of gonads were discovered, evaluating that TEB had adverse effects on gonadal development. Additionally, we also found the alternations of social behavior, 17ß-estradiol (E2) level, and testosterone (T) level. Furthermore, the expression levels of genes involved in the hypothalamic-pituitary-gonadal (HPG) axis and social behavior were remarkably altered. Taken together, it could be concluded that TEB affected the egg production and fertilization rate by interfering with gonadal development, sex hormone secretion, and social behavior, which were eventually attributed to the disruption of the expressions of genes associated with the HPG axis and social behavior. This study provides a new perspective to understanding the mechanism of TEB-induced reproductive toxicity.

Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli.

Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)(1). Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ∼27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC(50) value of 1.46±0.01 ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.

Comparison of the Effects of 5-Hydroxymethylfurfural in Milk Powder Matrix and Standard Water on Oxidative Stress System of Zebrafish.

5-Hydroxymethylfurfural (5-HMF) and furfural (FF) are products of the maillard reaction (MR) in milk powder and their safety is controversial. The concentration changes of 5-HMF and FF after a period of cold storage were determined by high-performance liquid chromatography (HPLC). Then, we compared the toxicity effects of 5-HMF (2, 20, or 200 µM) in milk powder matrix and standard water on the oxidative stress system of zebrafish embryos. The results showed that the concentration of 5-HMF was stable, and the concentration of FF degraded over time. 5-HMF-exposed zebrafish embryos had a LC50 value of 961 µM for 120 h. High-concentration of 5-HMF exposure resulted in developmental toxicity and induced oxidative stress. 5-HMF exposure resulted in low expression of gstr gene at 200 µM in both matrices. Moreover, sod, cat, gstr, and gpxla genes were differentially highly expressed in other groups or showed no significant difference. Residual levels in all groups were well below the exposed dose, with a maximum value of only 0.4‱. These results provided a theoretical basis for understanding the effects of 5-HMF exposure in milk powder matrix on the oxidative stress system and suggested that the presence of 5-HMF in our daily consumption of milk powder does not produce significant toxic effects and need not be overstressed.