Ameliorative Effect of Faidherbia albida Against 2.4-Dinitrophenylhydrazine Induced Hyperbilirubinemia in Wistar Albino Rats.

Background: Faidherbia albida, popularly known as gawo in Hausa, is traditionally used to treat jaundice in Zuru emirate of Kebbi State. Herein, the ameliorative effect of F. albida against 2.4-dinitrophenylhydrazine-induced hyperbilirubinemia in Wistar albino rats was investigated. Methods: Thirty healthy rats were administered 75 mg of 2.4-dinitrophenylhydrazine to induce hyperbilirubinemia. Thereafter, groups 1-3 received 500, 750, and 1000 mg/kg body weight of the methanol stem-bark extract, and 15 mg/kg of phenobarbitone (standard drug) was administered to group 4. Groups 5 and 6 served as the untreated and normal controls, respectively. The phytochemical composition was evaluated using standard methods, and acute oral toxicity was evaluated using standard OECD 2008 guidelines. Results: Phytochemical analysis revealed the presence of alkaloids, phenols, and a substantial amount of tannins. A significant (P<0.05) reduction of direct bilirubin, total bilirubin, and total protein levels for all the doses of the extract and standard drug compared to untreated groups was observed. Similarly, there were significant reductions in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels of the group treated with the standard drug and all extract-treated groups compared to elevated levels observed in untreated controls. However, a significant (P<0.05) increase in serum albumin (ALB) levels, red blood cells, hemoglobin, and pack cell volume was observed in all extract-treated compared to the untreated control in contrast to a significant decrease in MCH levels in treated groups compared to the untreated group. Conclusion: F. albida ameliorated the hyperbilirubinemia induced by 2.4-dinitrophenylhydrazine in Wistar albino rats, thus providing some support for its use in traditional medicine to treat jaundice.

Cryoprotective effect of soluble soybean polysaccharides and enzymatic hydrolysates on the myofibrillar protein of Nemipterus virgatus surimi.

Cryoprotective effect and potential mechanism of soluble soybean polysaccharides (SSPS) and enzymatic hydrolysates on surimi was investigated. After hydrolysis, the molecular weight of SSPS significantly decreased, and the hydrolysates prepared by endo-polygalacturonase (EPG-SSPS) was the lowest (154 kDa). Infrared spectrum analysis revealed that enzymatic hydrolysis didn't alter the functional groups of SSPS, but it did augment the exposure to hydroxyl groups. Surimi containing 5 % EPG-SSPS had the lowest freezable water after 20 days of frozen storage. Furthermore, the 5 % EPG-SSPS group manifested the highest metrics in total sulfhydryl (8.0 × 10-5 mol/g), active sulfhydryl content (6.7 × 10-5 mol/g), Ca2+-ATPase activity, and exhibited the lowest level in carbonyl content, surface hydrophobicity (153 µg). Notably, the 5 % EPG-SSPS maintained the stability of protein structure. Conclusively, SSPS enzymatic hydrolysate using endo-polygalacturonase imparted superior cryoprotective effect on the myofibrillar protein of surimi, and the mechanism might be a decrease in molecular weight and exposure of hydroxyl groups.

Rational design of Bi2Sn2O7/Bi5O7I S-scheme heterojunction for visible photocatalytic oxidation of emerging pollutants.

The construction of an S-scheme heterostructure is considered as a promising strategy for enhancing photocatalytic performance. Herein, a three-dimensional Bi5O7I (BOI) microsphere decorated with Bi2Sn2O7 (BSO) nanoparticles was prepared for the first time via a simple ultrasonic-assisted electrostatic self-assembly strategy and used for the degradation of 2,4-dinitrophenylhydrazine. 3 wt% Bi2Sn2O7/Bi5O7I has the highest degradation activity (93.7 %), with an apparent rate constant of 0.0848 min-1, which is 2.55 times that of the original Bi5O7I (0.0333 min-1). Moreover, the optimal binary heterojunction photocatalyst has good reusability and universal applicability. The results of cyclic voltammetry tests clarify that the optimal photocatalyst can provide more surface reactive sites. The results of radical trapping experiments and electron spin resonance indicate that holes (h+) and superoxide radicals are the main active radicals in the degradation process of 2,4-dinitrophenylhydrazine. Photoelectrochemical and photoluminescence confirm that 3 wt% Bi2Sn2O7/Bi5O7I composites exhibit the highest separation rate of photogenerated carriers. Finally, based on the results of experimental studies and theoretical calculations, the S-scheme charge transfer path on Bi2Sn2O7/Bi5O7I composite is determined. This work provides a new perspective on how to design high-performance S-scheme bismuth oxyhalide-based heterojunction photocatalysts for solar energy conversion.

Carbon-coated copper nanocrystals with enhanced peroxidase-like activity for sensitive colorimetric determination of 2,4-dinitrophenylhydrazine.

Carbon-coated copper nanocrystals (CuNCs) with peroxidase-like activity were hydrothermally prepared by using copper acetate, citric acid (CA) and histidine (His) as the precursors. Various shaped CuNCs, including urchin-like, slab-like and spherical appearance were facilely prepared by addition of different amount of NaNO2 in the precursor solutions. When 3,3',5,5'-tetramethylbenzidine (TMB) was used as the substrate, the CuNCs with urchin-like appearance have greatest peroxidase-like activity and their Michaelis-Menten constant (Km) and the maximum rate constant (νmax) are respectively 8.8 and 1.2 times higher than that obtained from horseradish peroxidase (HRP). The production of reactive oxygen species (ROS) was confirmed by radical quenching and electron spin resonance (ESR) tests. Subsequent studies have found that the CuNCs catalyzed color reaction of TMB can be selectively quenched by the environmental pollutant 2,4-dinitrophenylhydrazine (2,4-DNPH). Thus a new colorimetric method for the determination of 2,4-DNPH with a linear range of 0.60-20 µM was developed and a limit of detection (LOD) as low as 0.166 µM was achieved. The results obtained not only reveal the tunability of the peroxidase-like activity of Cu-based nanomaterials, but also provide a new method for the sensitive determination of environmental contaminate.

Detection and quantification of UV-transparent keto-androgens by dinitrophenylhydrazine derivatization for discontinuous kinetic assays.

Kinetic assays with recombinant enzymes are critical to determine the steady state kinetic parameters for androgen conversion to understand their role in androgen biosynthesis and metabolism. Detection and quantification of 5α-reduced androgens remain difficult to assay because they are UV-transparent compounds. Therefore, radioactive isotopic versions of these compounds are often required to conduct steady-state kinetics. Here we developed a derivatization protocol with dinitrophenylhydrazine (DNPH) to form hydrazones on the ketones of androgens enabling them to be detected by UV-reverse phase high performance liquid chromatography (RP-HPLC). We determined the kinetic parameters for the conversion of 5α-androstane-3,17-dione (5AD) to 5α-dihydrotestosterone (DHT), 11-keto-5α-androstane-3,17-dione (11K-5AD) to 11-keto-5α-dihydrotestosterone (11K-DHT), and 11ß-hydroxy-5α-androstane-3,17-dione (11ß-OH-5AD) to 11ß-hydroxy-5α-dihydrotestosterone (11ß-OH-DHT) catalyzed by recombinant aldo-keto reductase 1C3 (AKR1C3) as measured by product formation post DNPH derivatization.

Applicability of 2,4-dinitrophenylhydrazine (DNPH) method of protein oxidative damage measurement in the seminal plasma of canine ( Canis lupus familiaris) and stallion ( Equus caballus).

Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content of carbonyl groups in the SP was measured on the basis of the reactions with DNPH. The following reagent variants were used to dissolve protein precipitates: Variant 1 (V1) - 6M Guanidine solution and Variant 2 (V2) - 0.1M NaOH solution. It has been shown that to obtain reliable results for the measurement of protein carbonylated groups in the dog and horse SP, both 6M Guanidine and 0.1M NaOH may be used. A correlation was also found between the number of carbonyl groups and the total protein content in the canine (V1: r = -0.724; V2: r = -0.847) and stallion (V1: r = -0.336; V2: r = -0.334) SP. Additionally, the study showed a higher content (p≤0.05) of protein carbonyl groups in the stallion SP in the non-breeding season compared to the breeding season. The method based on the reaction with DNPH, due to its simplicity and cost effectiveness, appears to be suitable for large-scale application in the determination of the SP proteins oxidative damage in dog and horse semen.

Oxime blot: A novel method for reliable and sensitive detection of carbonylated proteins in diverse biological systems.

Oxidative stress and oxidative protein damage occur in various biological processes and diseases. The carbonyl group on amino acid side chains is the most widely used protein oxidation biomarker. Carbonyl groups are commonly detected indirectly through their reaction with 2,4-dinitrophenylhydrazine (DNPH) and subsequent labeling with an anti-DNP antibody. However, the DNPH immunoblotting method lacks protocol standardization, exhibits technical bias, and has low reliability. To overcome these shortcomings, we have developed a new blotting method in which the carbonyl group reacts with the biotin-aminooxy probe to form a chemically stable oxime bond. The reaction speed and the extent of the carbonyl group derivatization are increased by adding a p-phenylenediamine (pPDA) catalyst under neutral pH conditions. These improvements are crucial since they ensure that the carbonyl derivatization reaction reaches a plateau within hours and increases the sensitivity and robustness of protein carbonyl detection. Furthermore, derivatization under pH-neutral conditions facilitates a good SDS-PAGE protein migration pattern, avoids protein loss by acidic precipitation, and is directly compatible with protein immunoprecipitation. This work describes the new Oxime blot method and demonstrates its use in detecting protein carbonylation in complex matrices from diverse biological samples.

Selective Colorimetric Detection of Novichok Agents with Hydrazone Chemosensors.

Novichok is a recently identified class of neurotoxic organophosphorus compounds that have subsequently been banned by the Organization for Prohibition of Chemical Weapons (OPCW) as chemical warfare agents. Selective, rapid detection of Novichok remains a challenge. Several colorimetric paper-based detection devices have recently been developed for the rapid and selective detection of sarin, VX, and sulfur mustard. In this article, we present a similar colorimetric device designed specifically for the detection of Novichok, based on hydrazone derivatives from 2,4-dinitrophenylhydrazine (2,4-DNPH) impregnated on a glass fiber substrate. Results show that these compounds rapidly and selectively reveal the presence of Novichok agents to the naked eye. The low cost, ease of use, portability, and high selectivity to Novichok of this device complete the detection range of colorimetric paper-based sensors for chemical warfare agents.

Effects of tetrasodium pyrophosphate coupled with soy protein isolate on the emulsion gel properties of oxidative myofibrillar protein.

The effects of protein oxidation on the emulsion gel properties of myofibrillar protein (MP) in the presence of tetrasodium pyrophosphate (TSPP) and soybean protein isolate (SPI) were investigated from the perspective of interfacial protein interactions. The results showed that the emulsifying activity and emulsion stability of MP increased by 35.2 %-181.6 % with elevated H2O2 concentrations (1-20 mM), while the gel strength and water holding capacity of MP emulsions first increased to a maximum at 5 mM H2O2 and then decreased. TSPP and SPI further reinforced the effects caused by oxidation. The emulsifying properties of MP and its emulsion gel properties were closely related to surface hydrophobicity/hydrogen bonds/hydrophobic interactions and disulfide bonds among interfacial proteins, respectively. However, these correlations became difficult to define when TSPP and SPI were introduced. The study provides a theoretical basis for the strategy development to reduce protein oxidation damage on meat product quality.

Reductive amination of aldehyde 2,4-dinitrophenylhydrazones using cyanoborohydride for determination of selected carbonyl compounds in Port wines, table wines, and wine spirits.

Carbonyl compounds are mainly analysed by HPLC with ultraviolet detection after reaction with DNPH, yielding carbonyl-hydrazones. The presence of geometrical isomers whose relative abundance is dependent on the reaction condition introduces analytical errors and increases the complexity of the chromatograms. In this work, a simple, fast, and robust method for determining pyruvic acid, acetaldehyde, and α-ketobutyric acid in Port wines, dry table wines, and wine spirits by reductive amination of aldehyde 2,4-dinitrophenylhydrazones using cyanoborohydride followed by HPLC-DAD was developed, optimised and validated. The developed method was simple and showed good performance in several parameters such as linearity (0.025-200 mg/L), detection limits (0.0043-0.0097 mg/L), quantification (0.014-0.032 mg/L), precision (1.1-4.2 %) and accuracy (99.02 %). The method was applied to five different wine matrices, White, Tawny, and Ruby Port wines, white and red table wines, and wine spirits. The quantification of these carbonyl compounds simultaneously was achieved in Port wines for the first time.

Overall analyses of the reactions catalyzed by acetohydroxyacid synthase/acetolactate synthase using a precolumn derivatization-HPLC method.

A precolumn derivatization-HPLC method using 2,4-dinitrophenylhydrazine and 4-nitro-o-phenylenediamine as respective labeling reagents for comprehensive analyses of the reactions catalyzed by acetohydroxyacid synthase (AHAS)/acetolactate synthase (ALS) is developed and evaluated in this research. Comparison with the classic Bauerle' UV assay which can analyze the enzymes only through measurement of acetoin production, the HPLC method shows advantages because it can analyze the enzymes not only via determination of consumption of the substrate pyruvate, but also via measurement of formation of the products including acetoin, 2,3-butanedione, and acetaldehyde in the enzymatic reactions. Thus the results deduced from the HPLC method can reflect the trait of each enzyme in a more precise manner. As far as we know, this is the first time that the reactions mediated by AHAS/ALS using pyruvate as a single substrate are globally analyzed and the features of the enzymes are properly discussed.

2,4-dinitrophenylhydrazine capturing combined with mass defect filtering strategy to identify aliphatic aldehydes in biological samples.

Aliphatic aldehydes are toxic substances that correlate with the onset of many diseases. However, up to date, the methods to identify aliphatic aldehydes in biological samples are less selectivity and/or robustness. In this study, a strategy based on 2,4-dinitrophenylhydrazine (DNPH) capturing combined with mass defect filtering (MDF) was established and validated to identify aliphatic aldehydes in two biological samples (serum of immunosuppressed rats and oxidative damaged cells). Firstly, the mass spectrometric characteristic ions (m/z 163.01, 163.02 and 191.04) and fragmentation pathways of aldehyde-DNPHs were acquired through analyzing the standard references. Then, biological samples were derivatized by DNPH, a routine reagent, and subsequently assessed on an ultra-performance liquid chromatography coupled time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). Thirdly, the raw chromatogram was processed by MDF method to obtain interference-free chromatogram. Fourthly, the aldehyde-DNPHs were characterized through investigating the mass spectrometric information of each peak referred to the identified characteristic ions and fragmentation pathways. Finally, 6 and 8 aliphatic aldehydes were exclusively identified in serum of immunosuppressed rats and supernatant of oxidative damaged cells. Among which, propanal and butanal were positively correlate with immunosuppression, while formalin was more relevant to oxidative stress. The results demonstrated that the established strategy could robustly characterize the aliphatic aldehydes in biological samples, which would be helpful to evaluate the physical conditions of subjects.

Analysis of Content of 2-Oxoacids in Rat Brain Extracts Using High-Performance Liquid Chromatography.

2-Oxoacids are involved in a number of important metabolic processes and can be used as biomarkers in some human diseases. A new optimized method for quantification of 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids using high-performance liquid chromatography was developed based on available techniques for quantification of 2-oxoacids in mammalian brain. The use of the 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids was shown to be more advantageous in comparison with the previously used phenylhydrazine derivatives, due to a high chemical stability of the former. Here, we determined the concentrations of pyruvate, glyoxylate, 2-oxoglutarate, 2-oxomalonate, and 4-methylthio-2-oxobutyrate in the methanol/acetic acid extracts of the rat brain using the developed method, as well discussed the procedures for the sample preparation in analysis of mammalian brain extracts. The validation parameters of the method demonstrated that the quantification limits for each of the analyzed of 2-oxoacids was 2 nmol/mg tissue. The developed method facilitates identification of subtle changes in the tissue and cellular content of 2-oxoacids as (patho)physiological biomarkers of metabolism in mammalian tissues.

A sensitive simultaneous detection approach for the determination of 30 atmospheric carbonyls by 2,4-dinitrophenylhydrazine derivatization with HPLC-MS technique and its preliminary application.

Atmospheric carbonyls are important precursors of PM2.5 and ground-level ozone, and some carbonyls are toxic and harmful; thus, it is crucial to obtain accurate information on the ambient levels of carbonyls. However, the detection of carbonyls is difficult due to their relatively higher reactivities and chemical instabilities; therefore, accurate determination of atmospheric carbonyls is important. In this study, an analytical method for atmospheric carbonyls with high concentration or reactivity was developed, the precursor ion of each carbonyl compound was selected, and the declustering potential (DP) and entrance potential (EP) for each precursor ion were optimized. A 2,4-dinitrophenylhydrazine cartridge derivatization-high performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (DNPH-HPLC/APCI-MS) method for the determination of 30 carbonyls was established. The results showed that the linear range of 24 carbonyls was 1.2-600 ng/mL, while other 6 carbonyls was 1.2-300 ng/mL, and the detection limits of 30 carbonyls ranged from 0.092 to 0.947 ng/mL (0.005-0.049 µg/m3 with an ambient air sampling volume of 96 L). The intra-day and inter-day repeatability ranges were 0.55-4.20% and 1.40-12.48%, respectively. A preliminary application of the method was carried out in the urban area of Beijing in spring and summer of 2021, and it was found that the mean total mass concentration of 30 carbonyls was 35.894 µg/m3. This study provided additional concentration information for 14 atmospheric carbonyls, including mono-, di-, oxygen-containing and heterocyclic carbonyls, which accounted for 38% and 35% of the total mass concentration and OH radical reactivities of 30 carbonyls, respectively. This is the first investigation of simultaneous quantitative analysis of multiple atmospheric carbonyls based on commercial standard derivatives. The optimized method could provide more comprehensive information for atmospheric carbonyls and further support research concerning the role of chemical reaction processes and health effects than traditional measuring techniques.

Novel allyl-hydrazones including 2,4-dinitrophenyl and 1,2,3-triazole moieties as optical sensor for ammonia and chromium ions in water.

It is critical to take safety action if carcinogenic heavy metals and ammonia can be detected quickly, cheaply, and selectively in an environmental sample. As a result, compound 4a [4-(1-(2-(2,4-Dinitrophenyl)hydrazineylidene)-3-(naphthalen-2-yl)allyl)-5-methyl-1-phenyl-1 H-1,2,3-triazole] and compound 4b [4-(1-(2-(2,4-Dinitrophenyl)hydrazineylidene)-3-(naphthalen-2-yl)allyl)-1-(4-fluorophenyl)-5-methyl-1 H-1,2,3-triazole] were prepared. The aldol condensation process of 4-acetyl-1,2,3-triazoles 1a,b (Ar = C6H4; 4-FC6H4) with 2-naphthaldehyde yields 1-acetyl-1,2,3-triazoles 1a,b (Ar = C6H4; 4-FC6H4) (5-methyl-1-aryl-1 H-1,2,3-triazol-4-yl) -3-(naphthalen-2-yl)prop-2-en-1-ones 3a,b with a yield of around 95%. The target compounds 4a,b are obtained in around 88% yield by condensation of 3a,b with (2,4-dinitrophenyl)hydrazine in a refluxing acidic medium. Compounds 4a,b exhibited possible colorimetric detection for chromium ion in the range of 0-14 ppm and ammonia in the range of 0-20 ppm. As a result, this research suggests that strong electron-withdraw groups in related probes can improve anion detection ability, while the conjugation effect should also be considered while building structures.

Comparative study of the antioxidant activity of the essential oils of five plants against the H2O2 induced stress in Saccharomyces cerevisiae.

The purpose of this work was to investigate the protective effect of five essential oils (EOs); Rosmarinus officinalis, Thymus vulgaris, Origanum compactum Benth., Eucalyptus globulus Labill. and Ocimum basilicum L.; against oxidative stress induced by hydrogen peroxide in Saccharomyces cerevisiae. The chemical composition of the EOs was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The in vitro antioxidant activity was evaluated and the protective effect of EOs was investigated. Yeast cells were pretreated with different concentrations of EOs (6.25-25 µg/ml) for an hour then incubated with H2O2 (2 mM) for an additional hour. Cell viability, antioxidants (Catalase, Superoxide dismutase and Glutathione reductase) and metabolic (Succinate dehydrogenase) enzymes, as well as the level of lipid peroxidation (LPO) and protein carbonyl content (PCO) were evaluated. The chemical composition of EOs has shown the difference qualitatively and quantitatively. Indeed, O. compactum mainly contained Carvacrol, O. basilicum was mainly composed of Linalool, T. vulgaris was rich in thymol, R. officinalis had high α-Pinene amount and for E. globulus, eucalyptol was the major compound. The EOs of basil, oregano and thyme were found to possess the highest amount of total phenolic compounds. Moreover, they have shown the best protective effect on yeast cells against oxidative stress induced by H2O2. In addition, in a dose dependent manner of EOs in yeast medium, treated cells had lower levels of LPO, lower antioxidant and metabolic enzymes activity than cells exposed to H2O2 only. The cell viability was also improved. It seems that the studied EOs are efficient natural antioxidants, which can be exploited to protect against damages and serious diseases related to oxidative stress.

Diversity of Carbonyl Compounds in Biogas and Natural Gas Revealed Using High-Resolution Mass Spectrometry and Nontarget Analysis.

Airborne carbonyl compounds such as formaldehyde, acrolein, and methyl ethyl ketone have long been chemicals-of-concern in the environment due to their reactivity and their potential for negative health effects. Standard methods for determining carbonyls in air, which focus on a set of 15 or fewer compounds, involve derivatization to form nonvolatile hydrazones, which can readily be analyzed via liquid chromatography (LC) with ultraviolet detectors. Here, we apply a new LC-high-resolution mass spectrometry (HRMS) method to natural gas and a variety of upgraded biofuels to better assess their total carbonyl profile using the inherent selectivity of the standard sampling methodology and the selectivity and sensitivity of HRMS. The standard method accounted for only 64% of the total carbonyl content in natural gas and between 26 and 45% of the total carbonyl content in biogas sources, with the balance detected by the new LC/HRMS method. An additional 540 compounds with molecular formulas consistent with carbonyl compounds were detected compared to only 14 target compounds using the standard method. These results demonstrate that the established method dramatically under-reports both the total carbonyl load and the diversity of carbonyl species in natural gas and biogas samples.

Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins.

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [14C]O2 release from labeled [14C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.

High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase.

A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485 nm. Site-directed saturation mutagenesis libraries of D-threonine aldolase from Alcaligenes xylosoxidans CGMCC 1.4257 (AxDTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants AxDTAD321C and AxDTAN101G were identified. Substrate specificity of AxDTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore, AxDTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral ß-hydroxy-α-amino acids.

The flavoring and not the nicotine content is a decisive factor for the effects of refill liquids of electronic cigarette on the redox status of endothelial cells.

Electronic cigarettes are constantly gaining ground as they are considered less harmful than conventional cigarettes, and there is also the perception that they may serve as a potential smoking cessation tool. Although the acute effects of electronic cigarette use have been extensively studied, the long-term potential adverse effects on human health remain largely unknown. It has been well-established that oxidative stress is involved in the development of various pathological conditions. So far, most studies on e-cigarettes concern the effects on the respiratory system while fewer have focused on the vascular system. In the present study, we attempted to reveal the effects of electronic cigarette refill liquids on the redox state of human endothelial cells (EA.hy926 cell line). For this purpose, the cytotoxic effect of three e-liquids with different flavors (tobacco, vanilla, apple/mint) and nicotine concentrations (0, 6, 12, 18 mg/ml) were initially examined for their impact on cell viability of EA.hy926 cells. Then, five redox biomarkers [reduced form of glutathione (GSH), reactive oxygen species (ROS), total antioxidant capacity (TAC), thiobarbituric acid reactive substances (TBARS) and protein carbonyls (CARBS)] were measured. The results showed a disturbance in the redox balance in favor of free radicals in tobacco flavored e-liquids while vanilla flavored e-liquids exhibited a more complex profile depending on the nicotine content. The most interesting finding of the present study concerns the apple/mint flavored e-liquids that seemed to activate the cellular antioxidant defense and, thus, to protect the cells from the adverse effects of free radicals. Conclusively, it appears that the flavorings and not the nicotine content play a key role in the oxidative stress-induced toxicity of the e-liquids.