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A sensitive, fast and robust liquid chromatography--tandem mass spectrometry (LC–MS-MS) method was developed and validated for the determination of usaramine (URM) and usaramine N-oxide (UNO) in rat plasma. The separation was conducted on an ACQUITY UPLC BEH C18 Column (50 × 2.1 mm, 1.7 μm) and gradient eluted with mobile phase A (0.1% formic acid with 5 mM ammonium acetate in water) and B (0.1% formic acid in acetonitrile/methanol, 9/1, v/v). The method was linear over the range of 1–2,000 ng/mL for both analytes. The validated method was applied to investigate the pharmacokinetic behaviors and sex differences of URM and its N-oxide metabolite in rats. After intravenous administration of URM at 1 mg/kg, the AUC0-t values for URM and UNO were 363 ± 65 and 172 ± 32 ng/mL*h in male rats, while 744 ± 122 and 30.7 ± 7.4 ng/mL*h in females, respectively. The clearance of URM was significantly higher in male rats than in females (2.77 ± 0.50 vs 1.35 ± 0.19 L/h/kg, P

Ascorbic acid is often used to enhance iron absorption in nutritional interventions, but it produces pro-oxidant effects in the presence of iron. This study aimed to evaluate ascorbate's role in iron toxicity on intestinal resistance against foodborne pathogens during iron supplementation/fortification. In polarized Caco-2 cell monolayers, compared to the iron-alone treatment, the iron-ascorbate co-treatment caused more than 2-fold increase in adhesion, invasion and translocation of Salmonella enterica serovar Typhimurium. According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase release and transepithelial electrical resistance, the iron-ascorbate co-treatment resulted in reduced cell viability and increased impairment of cell membrane and paracellular permeability compared to the iron-alone treatment. Butylated hydroxytoluene protected cells against these prooxidant toxicities of ascorbate. Ascorbate completely restored iron-induced intracellular oxidant burst and depletion of cytosolic antioxidant reserve, according to dichlorodihydrofluorescein fluorescence and intracellular reduced glutathione levels. In Salmonella-infected C57BL/6 mice, iron-ascorbate co-supplementation resulted in greater loss of body weight and appetite, lower survival rate, shorter colon length, heavier intestinal microvilli damage, and more intestinal pathogen colonization and translocation than the iron-alone supplementation. Overall, ascorbate would exacerbate iron toxicity on intestinal resistance against Salmonella infection through pro-oxidant impairment of intestinal epithelial barrier from extracellular side and/or by facilitating intestinal pathogen colonization.

In this study, we successfully synthesized a thin-slice layer of graphitic carbon nitride (g-C3N4) with abundant irregular holes by a facile two-step method using Pluronic P123 as a template (CN-P1...

Monocrotaline (MCT) is a pyrrolizidine alkaloid that can induce hepatic sinusoidal damage, pulmonary hypertension, renal toxicity, and heart disease. Monocrotaline N-oxide (MNO), the primary metabolite of MCT, is less toxic; however, it can convert back to MCT to exhibit its toxicity. This study developed and validated a rapid and sensitive LC-MS/MS method for the simultaneous determination of MCT and monocrotaline N-oxide in rat plasma. The method has a linearity over the concentration range of 1-2000 ng/mL with correlation coefficients (r) >0.997 for each analyte. The results of selectivity, matrix effect, accuracy and precision, and recovery were all within the acceptance criteria. The validated method has been successfully applied to study pharmacokinetic behaviors and bioavailability of MCT in rats. MCT was rapidly absorbed (Tmax : 0.400 ± 0.149 h) after oral administration, and the absolute bioavailability of MCT was 78.2%.

Sulfur-containing metabolites are related to several physiologic disorders and metabolic diseases. In this study, a simultaneous identification and quantification strategy in one batch for determination of sulfhydryl-containing metabolites was developed using stable isotope labeling combined with liquid chromatography-tandem mass spectrometry (SIL-LC-MS). In the proposed method, a pair of isotope labeling reagents, D0/D5-N-ethylmaleimide (D0/D5-NEM), was used to derivatize sulfhydryl-containing metabolites in blood and plasma of normal- and high-fat-diet (NFD and HFD) hamsters for reduced (-SH) and total (-SH, -S-S-, S-glutathionylated proteins) analysis. Quality control (QC) samples and test samples were prepared for LC-MS analysis. First, both QC samples and stable isotope labeled internal standards were used to monitor the status of the instrument and ensure the reliability of the analysis. Subsequently, an inhouse database containing 45 sulfhydryl-containing metabolites was established by MS1 based on QC samples. Then, qualitatively differential sulfhydryl-containing metabolites were found by MS2 between the NFD and HFD hamsters of the test samples, including 3 in reduced and 8 in total analysis of blood samples, and 2 in reduced and 2 in total analysis of plasma samples. Next, in quantitative analysis, satisfied linearities for 6 sulfhydryl-containing metabolites were obtained with the correlation coefficient (R2) > 0.99 and absolute quantification was carried out. The results showed that glutathione and cysteine have different concentrations in blood and plasma of hamsters. Finally, the correlation of sulfhydryl-containing metabolites with blood lipid and oxidative stress levels was determined, which provided insight into the hyperlipidemia-related oxidative stress. Taken together, the developed method of simultaneous identification with the inhouse database and MS2 and quantification with standards in one batch provides a promising strategy for the analysis of sulfhydryl-containing metabolites in biological samples, which may promote the in-depth investigation on sulfhydryl-containing metabolites and related diseases.

Developing efficient and robust materials for emerging electrodegradation of organic pollutants has attracted broad interests. In this study, a novel controlled pyrolysis approach was employed to fabricate a quasi-MOF derivative-based electrode by pyrolyzing MIL-101(Fe) anchored on a polyaniline-modified carbon fiber paper at 400 °C. The construction of the accessible Fe-O sites, and the in situ generation of Fe3O4 nanoparticles with graphene-like carbon layers coated, would enhance the electro-Fenton activity of the electrode, which was used as the cathode. The results showed that 100 % of 50 mg L−1 p-nitrophenol and 52 % total organic carbon were removed in 120 min under a current density of 5 mA cm-2, suggesting that the prepared electrode had a more efficient mineralization current efficiency and less energy consumption compared with electrodes before pyrolysis. Notably, the stability of the electrode was greatly improved, maintaining its outstanding performance even after ten runs. The plausible reaction mechanism and degradation pathway were also proposed. This new pyrolysis strategy is expected to serve as a paradigm for designing efficient electrode in electro-Fenton remediation field.

In this work, the bimetallic iron oxide self-supported electrode was prepared by a simple solvothermal as well as thermal method. CoFe2O4 magnetic nanoparticles were grown in situ on the CFP surface and characterized to reveal the morphology, composition, and electrochemical properties of the electrode. Compared to CFP and CFP@Co-Fe, CFP@CoFe2O4 equipped more efficient mineralization current efficiency and lower energy consumption due to the improved electrocatalytic capacity of CoFe2O4 properly grown on the conductive substrate surface. Further studies showed that the manufactured electrode maintained a high level of stability after continuous operation. According to the free radical trapping experiment, EPR, and liquid mass spectrometry analysis, the rational reaction mechanism of p-nitrophenol was finally proposed, in which ·OH and SO4·- were considered as the main active oxidants. This work demonstrated the great potential of establishing an electro-Fenton system based on CoFe2O4 immobilized self-supporting cathode for environmental remediation.

Iron fortification has been associated with a modest increase in diarrhea risk among children. Herein, we investigate the correction for this unwanted side effect with probiotic/prebiotic supplementation in weaning mice. Iron fortification with 250 ppm and 500 ppm ferrous sulfate for 30 days significantly increased the species richness of the mouse gut microbiota compared to controls. The 500 ppm-FeSO4 diet caused a significantly decreased abundance of potentially beneficial Lactobacillus. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), mice on the 500 ppm-FeSO4 diet showed earlier appearance of poisoning symptoms, higher rates of weight and appetite loss, and lower survival rates, all of which were effectively reversed by supplementation with a probiotic (Lactobacillus acidophilus) or a prebiotic (inulin) for 7 days before infection. Iron fortification with 500 ppm ferrous sulfate also increased fecal shedding and spleen and liver load of viable S. Typhimurium, suggesting its promoting effect on pathogen colonization and translocation, and this negative effect was found to be well corrected by supplementation with Lactobacillus acidophilus or inulin. Light and transmission electron microscopic observation on the ileal villus structure revealed the histopathological impairment of the intestine by iron fortification with both 250 ppm and 500 ppm ferrous sulfate, and the intestinal lesions were markedly alleviated by supplementation with Lactobacillus acidophilus or inulin. These results provide experimental evidence for the increased diarrhea risk upon iron fortification with high pathogen load, and demonstrate that probiotic or prebiotic supplementation can be used to eliminate the potential harm of iron fortification on gut health.

Using nanomaterials to achieve functional enzyme mimics (nanozymes) is attractive for both applied and fundamental research. Laccases are multicopper oxidases highly important for biotechnology and environmental remediation. In this work, we report an exceptionally simple yet functional laccase mimic based on guanosine monophosphate (GMP) coordinated copper. It forms an amorphous metal–organic framework (MOF) material. The ratio of copper and GMP is 3:4 as determined by isothermal titration calorimetry. It has excellent laccase-like activity and converts a diverse range of phenol containing substrates such as hydroquinone, naphthol, catechol and epinephrine. Comparative work shows that the activity is originated from guanosine coordination instead of phosphate binding in GMP. Cu2+ is required and cannot be substituted by other metal ions. At the same mass concentration, the Cu/GMP nanozyme has a higher Vmax and similar Km compared to the protein laccase. To achieve the same catalytic efficiency, the cost ...

In the intestine, several phenols and aromatic acids are generated by microbiota and are highly related to the formation of uremic toxins. Herein, we developed a new derivatization reagent, 2-bromo-1-[4-(dimethylamino)phenyl] ethyl ketone (BDAPE), that reacted simultaneously with phenols and aromatic acids. Following a reaction within 2 h at 60 °C in the presence of 200 mM potassium carbonate (K2CO3), the obtained BDAPE derivatives were separated on a reversed-phase C18 column and quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in positive electrospray ionization mode. This method allowed a lower limit of quantification (LLOQ) of 0.090 μΜ for 3-indolepropionic acid (3IPA), indole-3-acetic acid (3IAA), p-cresol (PC), benzoic acid (BA), and phenol (PN); 0.30 μΜ for phenylacetic acid (PAA); 0.45 μΜ for 4-hydroxyphenylacetic acid (4HPAA); and 0.60 μΜ for 3-phenylpropionic acid (PPA). Methodological validation further demonstrated acceptable accuracy (%RE

Rationale Short-chain fatty acids (SCFAs) are associated with intestinal microbiota and diseases in humans. SCFAs have a low response in mass spectrometry, and in order to increase sensitivity, reduce sample consumption, shorten analysis time, and simplify sample preparation steps, a derivatization method was developed. Methods We converted seven SCFAs into amide derivatives with 4-aminomethylquinoline. The reaction occurred for 20 min at room temperature. The analytes were separated on a reversed-phase C18 column and quantitated in the positive ion electrospray ionization mode using multiple reaction monitoring. Acetic acid-d4 was used as the stable-isotope-labeled surrogate analyte for acetic acid in the working solutions, while the other stable-isotope-labeled standards were used as internal standards (ISs). Results Method validation showed that the intra-day and inter-day precision of quantitation for the seven SCFAs over the whole concentration range was ≤3.8% (n = 6). The quantitation accuracy ranged from 85.5% to 104.3% (n = 6). Most important, the collected feces were vortexed immediately with ethanol. Conclusions This study provides a new derivatization method for a precise, accurate, and rapid quantitation of SCFAs in human feces using ultra-performance liquid chromatography/tandem mass spectrometry. This method successfully determined the concentration of SCFAs in human feces and could assist in the exploration of intestinal microbiota and diseases.

In this study, we report a novel magnetic biomimetic nanozyme (Fe3O4@Cu/GMP (guanosine 5′-monophosphate)) with high laccase-like activity, which could oxidize toxic o-phenylenediamine (OPD) and remove phenolic compounds. The magnetic laccase-like nanozyme was readily obtained via complexed Cu2+ and GMP that grew on the surface of magnetic Fe3O4 nanoparticles. The prepared Fe3O4@Cu/GMP catalyst could be magnetically recycled for at least five cycles while still retaining above 70% activity. As a laccase mimic, Fe3O4@Cu/GMP had more activity and robust stability than natural laccase for the oxidization of OPD. Fe3O4@Cu/GMP retained about 90% residual activity at 90°C and showed little change at pH 3–9, and the nanozyme kept its excellent activity after long-term storage. Meanwhile, Fe3O4@Cu/GMP had better activity for removing phenolic compounds, and the removal of naphthol was more than 95%. Consequently, the proposed Fe3O4@Cu/GMP nanozyme shows potential for use as a robust catalyst for applications in environmental remediation.

Graphitic carbon nitride (g-C3N4) possess great potential applications in photocatalytic wastewater purification and hydrogen production. However, its activity is moderate because of the sluggish electron transfer and insufficient light absorption. Here, a series of hydroxyl carbon quantum dots modified with fiber-like carbon nitride composites (OH-CQDs/CNF) were synthesized by a solvothermal approach to enhance the photocatalytic activity. Compared to g-C3N4, the prepared OH-CQDs/CNF exhibited significantly improved visible light absorption (UV-VIS), low recombination electron-hole pairs (PL), and dramatically enhanced conductivity (EIS). Therefore, the optimized OH-CQDs/CNF-4 composite (derived from 4 mL OH-CQDs) displayed excellent photodegradation activity, and its rate constant for tetracycline degradation was 11.3 times higher than that in the presence of g-C3N4, even higher than some noble metal or metal oxides modified g-C3N4. In addition, OH-CQDs/CNF-4 rendered around 4 times H2 evolution rate than CNF, mainly because the OH-CQDs were highly dispersed on the CNF surface and formed an intact interface through hydrogen bond interactions. Quenching experiments and ESR results indicated that ·O2− and h+ were the main free radicals generated during TC degradation. This study provides guidance for the design of hydrogen bonding-based metal-free photocatalysts for wastewater purification and hydrogen evolution.

BACKGROUND: Transplantation with bone marrow-derived mesenchymal stem cells (BMSCs) improves the survival of neurons and axonal outgrowth after stroke remains undetermined. Here, we investigated whether PI3K/AKT signaling pathway is involved in these therapeutic effects of BMSCs. METHODOLOGY/PRINCIPAL FINDINGS: (1) BMSCs and cortical neurons were derived from Sprague-Dawley rats. The injured neurons were induced by Oxygen-Glucose Deprivation (OGD), and then were respectively co-cultured for 48 hours with BMSCs at different densities (5×10(3), 5×10(5)/ml) in transwell co-culture system. The average length of axon and expression of GAP-43 were examined to assess the effect of BMSCs on axonal outgrowth after the damage of neurons induced by OGD. (2) The injured neurons were cultured with a conditioned medium (CM) of BMSCs cultured for 24 hours in neurobasal medium. During the process, we further identified whether PI3K/AKT signaling pathway is involved through the adjunction of LY294002 (a specific phosphatidylinositide-3-kinase (PI3K) inhibitor). Two hours later, the expression of pAKT (phosphorylated AKT) and AKT were analyzed by Western blotting. The length of axons, the expression of GAP-43 and the survival of neurons were measured at 48 hours. RESULTS: Both BMSCs and CM from BMSCs inreased the axonal length and GAP-43 expression in OGD-injured cortical neurons. There was no difference between the effects of BMSCs of 5×10(5)/ml and of 5×10(3)/ml on axonal outgrowth. Expression of pAKT enhanced significantly at 2 hours and the neuron survival increased at 48 hours after the injured neurons cultured with the CM, respectively. These effects of CM were prevented by inhibitor LY294002. CONCLUSIONS/SIGNIFICANCE: BMSCs promote axonal outgrowth and the survival of neurons against the damage from OGD in vitro by the paracrine effects through PI3K/AKT signaling pathway.