Resveratrol has neuroprotective effects and plays an anti-inflammatory role through Dectin-1/p38 pathway in Aspergillus fumigatus keratitis.

PURPOSE: To determine the antifungal, anti-inflammatory and neuroprotective effects of resveratrol (RES) in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: Cytotoxicity assay and Draize eye assay were performed to assess the toxicity of RES. The antifungal effect of RES was assessed by minimal inhibitory concentration, scanning or transmission electron microscopy, propidium iodide uptake assay, and Calcofluor white staining. Phosphorylation of p38 MAPK, mRNA and protein levels of Dectin-1 and related inflammatory factors were measured by qRT-PCR, ELISA and Western blot in vitro and in vivo. Clinical score, HE staining, plate count, and myeloperoxidase test were used to observe the progress of fungal keratitis. IF staining, qRT-PCR, and the Von Frey test were selected to assess the neuroprotective effects of RES. RESULTS: RES suppressed A. fumigatus hyphae growth and altered hyphae morphology in vitro. RES decreased the expression of Dectin-1, IL-1ß and TNF-α, as well as p38 MAPK phosphorylation expression, and also decreased clinical scores, reduced inflammatory cell infiltration and neutrophil activity, and decreased fungal load. RES also protected corneal basal nerve fibers, down-regulated mechanosensitivity thresholds, and increased the mRNA levels of CGRP and TRPV-1.. CONCLUSION: These evidences revealed that RES could exert antifungal effects on A. fumigatus and ameliorate FK through suppressing the Dectin-1/p38 MAPK pathway to down-regulate IL-1ß, IL-6, etc. expression and play protective effect on corneal nerves.

Rosmarinic acid alleviates fungal keratitis caused by Aspergillus fumigatus by inducing macrophage autophagy.

Fungal keratitis (FK) is an infectious keratopathy can cause serious damage to vision. Its severity is related to the virulence of fungus and response of inflammatory. Rosmarinic acid (RA) extracted from Rosmarinus officinalis exhibits antioxidant, anti-inflammatory and anti-viral properties. The aim of this study was to investigate the effect of RA on macrophage autophagy and its therapeutic effect on FK. In this study, we demonstrated that RA reduced expression of proinflammatory cytokine, lessened the recruitment of inflammatory cells in FK. The relative contents of autophagy markers, such as LC3 and Beclin-1, were significantly up-regulated in RAW 264.7 cells and FK. In addition, RA restored mitochondrial membrane potential (MMP) of macrophage to normal level. RA not only reduced the production of intracellular reactive oxygen species (ROS) but also mitochondria ROS (mtROS) in macrophage. At the same time, RA induced macrophage to M2 phenotype and down-regulated the mRNA expression of IL-6, IL-1ß, TNF-α. All the above effects could be offset by the autophagy inhibitor 3-Methyladenine (3-MA). Besides, RA promote phagocytosis of RAW 264.7 cells and inhibits spore germination, biofilm formation and conidial adherence, suggesting a potential therapeutic role for RA in FK.

Telomere heterogeneity linked to metabolism and pluripotency state revealed by simultaneous analysis of telomere length and RNA-seq in the same human embryonic stem cell.

BACKGROUND: Telomere length heterogeneity has been detected in various cell types, including stem cells and cancer cells. Cell heterogeneity in pluripotent stem cells, such as embryonic stem cells (ESCs), is of particular interest; however, the implication and mechanisms underlying the heterogeneity remain to be understood. Single-cell analysis technology has recently been developed and effectively employed to investigate cell heterogeneity. Yet, methods that can simultaneously measure telomere length and analyze the global transcriptome in the same cell have not been available until now. RESULTS: We have established a robust method that can simultaneously measure telomere length coupled with RNA-sequencing analysis (scT&R-seq) in the same human ESC (hESC). Using this method, we show that telomere length varies with pluripotency state. Compared to those with long telomere, hESCs with short telomeres exhibit the lowest expressions of TERF1/TRF1, and ZFP42/REX1, PRDM14 and NANOG markers for pluripotency, suggesting that these hESCs are prone to exit from the pluripotent state. Interestingly, hESCs ubiquitously express NOP10 and DKC1, stabilizing components of telomerase complexes. Moreover, new candidate genes, such as MELK, MSH6, and UBQLN1, are highly expressed in the cluster of cells with long telomeres and higher expression of known pluripotency markers. Notably, short telomere hESCs exhibit higher oxidative phosphorylation primed for lineage differentiation, whereas long telomere hESCs show elevated glycolysis, another key feature for pluripotency. CONCLUSIONS: Telomere length is a marker of the metabolic activity and pluripotency state of individual hESCs. Single cell analysis of telomeres and RNA-sequencing can be exploited to further understand the molecular mechanisms of telomere heterogeneity.

The role of group III, IV elements in Nb4AC3 MAX phases (A = Al, Si, Ga, Ge) and the unusual anisotropic behavior of the electronic and optical properties.

Niobium based Nb4AlC3, Nb4SiC3, Nb4GeC3 and Nb4GaC3 were investigated by means of density functional theory. Together with the known Nb4AlC3, the role of group III, IV elements in various properties of Nb4AC3 (A = Al, Si, Ga, Ge) was systematically investigated, and particularly the bulk moduli, shear moduli, and Young's moduli helped us to approach the ductility. All the studied compounds were found to be mechanically stable, and they also exhibit the metallic nature that results from the Nb-4d states being dominant at the Fermi level. The typical 4d-2p hybridization leads to strong Nb-C covalent bonding and a relatively weaker 4d-3p (4p) hybridization between Nb and A is identified. The latter does perturb the performance of materials. By varying A elements in Nb4AC3, the position and the width of the p states as well as hybridizations are altered, which determine the covalency and the ionicity of the chemical bonds. A high density of states at the Fermi level and the nesting effects in the Fermi surface are identified in Nb4SiC3 and linked to its unusual anisotropic behavior. Furthermore, Nb4GeC3 is predicted to be a very promising candidate solar heating barrier material. Overall, the present work gives insights into the role of A elements in the electronic structure and the physical properties of Nb4AC3 compounds. The tendencies and rules established here will help in the designing of functional ceramic materials with desirable properties.

Structural and functional assessment after intravitreal injection of ranibizumab in diabetic macular edema.

PURPOSE: To evaluate structure and function improvement in central retina by optical coherence tomography (OCT) and multifocal electroretinography (mf-ERG) in diabetic macular edema (DME) patients after intravitreal injection of ranibizumab (IVR) treatment. METHODS: Twenty-seven eyes in 27 patients with DME received three consecutive monthly injections of IVR (0.05 ml, 10 mg/ml) and as needed thereafter. The clinical parameters of best-corrected visual acuity (BCVA), central foveal thickness (CFT) and mf-ERG were monitored for 6 months before and after IVR. The findings at baseline, 1, 3 and 6 months were analyzed. Correlation and regression analyses were performed on BCVA, CFT, mf-ERG amplitude and implicit time of the N1 and P1 waves. RESULTS: IVR significantly improved visual acuity from the beginning of the treatment (P < 0.05). There were significant decreases in the CFT compared with the baseline after IVR (P < 0.05). The mean amplitude of P1 and N1 in the central ring at all examinations increased significantly compared with the baseline (P < 0.05). The mean P1 and N1 implicit times in the central ring were shortened, but not significantly (P > 0.05). There were significant correlations of BCVA with CFT, P1 and N1 amplitudes in the central retina (P < 0.05). CONCLUSION: In addition to the improvement in BCVA and the reduction in CFT, IVR improved macular retinal function, as assessed by mf-ERG, in diabetic eyes. The combination of OCT and mf-ERG for macular evaluation may better assess DME.

Effect of the Coal Fly Ash Blending Ratio on Biomass Slagging Structure Modification and Alkali Metal Migration.

The analysis of ash deposition samples by electron microscopy (EM), scanning electron microscopy (SEM)/energy dispersive scanning (EDS), and X-ray diffraction (XRD) was carried out at three sampling points of a one-dimensional furnace (sampling temperatures were TL5 = 960 °C, TL6 = 855 °C, and TL7 = 820 °C, respectively). The improvement of ash deposition characteristics of biomass combustion with the different addition ratios of coal fly ash (CFA) (Mr = 1:1, 1.5:1 or 2:1) and the heterogeneous removal of gas phase alkali metal heterogeneous were studied. The results showed that under the condition of Mr = 1:1, the ash sample presented a three-dimensional porous loose structure like "coral reef" with the rice husk ash as the skeleton and the combustion products of CFA as the bonding point. The spherical and filamentous crystals mainly composed of SiO2, Al2O3, and silicates were distributed in the slag, and the surfaces of these crystals were accompanied by the eutectic composite salt of K-Al-Si with a high melting point, reflecting the heterogeneous curing effect of CFA on the gas phase migration of the alkali metal (K). With the decreasing addition ratio of CFA (Mr = 1.5:1 or 2:1), the spherical and filamentous crystals were increased in the slag. MgO, Fe2O3, and Al2O3 may be involved in the formation of high-temperature refractory Mg-Fe-aluminate, Ca- and Mg-silicate, which were the precursors of the rigid slag. The alkali metals mostly presented in the form of K2O and KCl in the gas-solid conversion under the temperature effect, serving as binders for bridging, agglomeration, and slagging of the above precursors. In addition, the kalium enrichment was accompanied by Al and S in the CFA, indicating that the Al- and S-active sites have obvious adsorption to kalium.

Integrated Computing Accelerates Design and Performance Control of New Maraging Steels.

This paper mainly used database technology, machine learning, thermodynamic calculation, experimental verification, etc., on integrated computational materials engineering. The interaction between different alloying elements and the strengthening effect of precipitated phases were investigated mainly for martensitic ageing steels. Modelling and parameter optimization were performed by machine learning, and the highest prediction accuracy was 98.58%. We investigated the influence of composition fluctuation on performance and correlation tests to analyze the influence of elements from multiple perspectives. Furthermore, we screened out the three-component composition process parameters with composition and performance with high contrast. Thermodynamic calculations studied the effect of alloying element content on the nano-precipitation phase, Laves phase, and austenite in the material. The heat treatment process parameters of the new steel grade were also developed based on the phase diagram. A new type of martensitic ageing steel was prepared by selected vacuum arc melting. The sample with the highest overall mechanical properties had a yield strength of 1887 MPa, a tensile strength of 1907 MPa, and a hardness of 58 HRC. The sample with the highest plasticity had an elongation of 7.8%. The machine learning process for the accelerated design of new ultra-high tensile steels was found to be generalizable and reliable.

Exploring the conformational dynamics and thermodynamics of EGFR S768I and G719X + S768I mutations in non-small cell lung cancer: An in silico approaches.

Non-small cell lung cancer (NSCLC) is often driven by mutations in the epidermal growth factor receptor (EGFR) gene. However, rare mutations such as G719X and S768I lack standard anti-EGFR targeted therapies. Understanding the structural differences between wild-type EGFR and these rare mutants is crucial for developing EGFR-targeted drugs. We performed a systematic analysis using molecular dynamics simulations, essential dynamics (ED), molecular mechanics Poisson-Boltzmann surface area, and free energy calculation methods to compare the kinetic properties, molecular motion, and free energy distribution between wild-type EGFR and the rare mutants' structures G719X-EGFR, S768I-EGFR, and G719X + S768I-EGFR. Our results showed that S768I-EGFR and G719X + S768I-EGFR have higher global and local conformational flexibility and lower thermal and global structural stability than WT-EGFR. ED analysis revealed different molecular motion patterns between S768I-EGFR, G719X + S768I-EGFR, and WT-EGFR. The A-loop and αC-helix, crucial structural elements related to the active state, showed a tendency toward active state development, providing a molecular mechanism explanation for NSCLC caused by EGFR S768I and EGFR G719C + S768I mutations. The present study may be helpful in the development of new EGFR-targeted drugs based on the structure of rare mutations. Our findings may aid in developing new targeted treatments for patients with EGFR S768I and EGFR G719X + S768I mutations.

[The correlation between BRAF mutations, RET/PTC rearrangements and platelet-derived growth factor B expression in papillary thyroid carcinomas].

OBJECTIVE: To investigate the prevalence of BRAF T1799A mutation and RET/PTC rearrangement in Qingdao and detect the expression of platelet-derived growth factor B (PDGF-B) in order to investigate the correlation between gene mutation and PDGF-B. METHODS: Fresh tissue from 48 papillary thyroid carcinomas (PTC) patients was examined for BRAF mutation RET rearrangements (RET/PTC1 and RET/PTC3) by PCR, followed by direct-sequence analysis. The expression of PDGF was analyzed by immunohistochemistry. RESULTS: Among the 48 patients, 14 (29.2%) were micro PTC; 18 (37.5%) had BRAF T1799A mutations and 23(47.9%) had RET/PTC rearrangement. There were 17 (35.4%) cases of RET/PTC1 and 6 (12.5%) of RET/PTC3, with no multiple rearrangements. Both BRAF T1799A mutation and RET/PTC rearrangement were present in 6 (12.5%) cases of non-micro PTC. The level of PDGF-B expression in BRAF T1799A positive was higher than that in the negative, and the level of PDGF-B expression in RET/PTC3 was higher than that in RET/PTC1 (P < 0.05). The more advanced neoplasm stage was, the stranger PDGF-B expression was. CONCLUSIONS: The incidence of BRAF T1799A mutation and RET/PTC rearrangement is higher in Qingdao. BRAF T1799A mutation and RET/PTC3 rearrangement in patients suggests a poorer prognosis than the negative one. The BRAF T1799A mutation and RET/PTC3 rearrangement may strengthen the expression of PDGF-B. Both variations suggest a poor prognosis.

Effect of plasma-derived extracellular vesicles on angiogenesis and the ensuing proliferative diabetic retinopathy through a miR-30b-dependent mechanism.

BACKGROUND/PURPOSE: Proliferative diabetic retinopathy (PDR) is a major diabetic microvascular complication, characterized by pathological angiogenesis. This study sets out to investigate the potential molecular mechanism in the angiogenesis during PDR. METHODS: The expression of microRNA-30b (miR-30b) was quantified in a streptozotocin (STZ)-induced mouse model of PDR. The binding affinity between SIRT1 and miR-30b was then identified and validated. After transduction with In-miR-30b or combined with sh-SIRT1, high-glucose (HG)-induced retinal microvascular endothelial cells (RMECs) were co-cultured with extracellular vesicles (EVs) derived from the plasma of PDR mice (plasma-EVs). The proliferation and angiogenesis of RMECs were then detected in vitro. RESULTS: miR-30b expression was upregulated in the retinal tissue of PDR mice. SIRT1 was a target gene of miR-30b and under the negative regulation by miR-30b in RMECs. In contrast, inhibition of miR-30b resulted in elevated SIRT1 expression, thus alleviating the angiogenesis of RMECs. miR-30b was enriched in the plasma-EVs and could be delivered to RMECs, in which miR-30b exerted pro-angiogenic effects. Furthermore, inhibition of miR-30b arrested the progression of PDR in mice by promoting the expression of SIRT1. CONCLUSION: Collectively, the present study pinpointed the involvement of miR-30b delivered by plasma-EVs in PDR angiogenesis, thus laying the basis for the development of novel therapeutic targets for the treatment of PDR.

Exploring Hydrogen Incorporation into the Nb4AlC3 MAX Phases: Ab Initio Calculations.

The Nb4AlC3 MAX phase can be regarded as a TMC structure with stacking faults, which has great potential as a novel solid hydrogen storage material. Herein, we used ab initio calculations for understanding the hydrogen incorporation into Nb4AlC3 MAX phases, including equilibrium structural characteristics, energy changes, electronic structures, bonding characteristics, and diffusion paths. According to the calculated results, H has thermal stability in the interstice of the Nb-Al layer, and the most probable insertion site is an octahedron (3-site) composed of three Nb atoms and three Al atoms. When C vacancies are introduced, the Nb-C layer has a specific storage capacity for H. In addition, Al vacancies can also be used as possible sites for H incorporation. Moreover, the introduction of vacancies significantly increase the hydrogen storage capacity of the MAX phase. According to the electronic structure and bonding characteristics, the excellent hydrogen storage ability of the Nb4AlC3 structure may be due to the formation of ionic bonds between H and Nb/Al. It is worth noting that the H-Al bond in the 1-site is a covalent bond and an ionic bond key mixture. The linear synchronous transit optimization study shows that only H diffusion in Al vacancies is not feasible. In conclusion, the Nb-Al layer in Nb4AlC3 can provide favorable conditions for the continuous insertion and subsequent extraction of H, while the vacancy structure is more suitable for H storage. Our work provides solid theoretical results for understanding the hydrogen incorporation into Nb4AlC3 MAX phases that can be helpful for the design of advanced hydrogen storage materials.

Unique characteristics of G719X and S768I compound double mutations of epidermal growth factor receptor (EGFR) gene in lung cancer of coal-producing areas of East Yunnan in Southwestern China.

BACKGROUND: The principal objective of this project was to investigate the Epidermal Growth Factor Receptor (EGFR) gene mutation characteristics of lung cancer patients, which can provide a molecular basis for explaining the clinicopathological features, epidemiology and use of targeted therapy in lung cancer patients in the coal-producing areas of East Yunnan. METHODOLOGY: We collected 864 pathologically confirmed lung cancer patients' specimens in First People's Hospital of Qujing City of Yunnan Province from September 2016 to September 2021. We thereafter employed Next Generation Sequencing (NGS) technology to detect all exons present in the EGFR gene. RESULTS: The overall mutation frequency of the EGFR gene was 47.22%. The frequency of EGFR gene mutations in the tissue, plasma, and cytology samples were found to be 53.40%, 23.33%, and 62.50%, respectively. Univariate analysis indicated that the coal-producing areas and Fuyuan county origin were significantly associated with relatively low EGFR gene mutation frequency. Female, non-smoking history, adenocarcinoma, non-brain metastasis, and tissue specimens were found to be related to high EGFR gene mutation frequency. Multivariate logistic regression analysis suggested the lung cancer patients in the central area of Qujing City, stage Ia, non-coal-producing areas, non-Fuyuan origin, and non-Xuanwei origin were more likely to develop EGFR gene mutations. The most common mutations were L858R point mutation (33.09%) and exon 19 deletion (19-del) (21.32%). Interestingly, the mutation frequency of G719X (p = 0.001) and G719X + S768I (p = 0.000) in the coal-producing areas were noted to be more significant than those in non-coal-producing regions. CONCLUSION: This findings of this study might be important in establishing the correlation between routine using NGS for EGFR gene mutation diagnosis and clinical practice in the lung cancer patients.

Silenced SNHG1 Inhibited Epithelial-Mesenchymal Transition and Inflammatory Response of ARPE-19 Cells Induced by High Glucose.

PURPOSE: The lncRNA small nucleolar RNA host gene 1 (SNHG1) is a cerebral infarction-associated gene, its biological role and mechanism in diabetic retinopathy remain to be illuminated. The present study was designed to investigate the role of SNHG1 in high glucose induced human retinal pigment epithelial cells (ARPE-19). METHODS: ARPE-19 cells were cultured and exposed to 60 mM high glucose for 48h, and 5.5mM glucose-exposed ARPE-19 cells were used as the control. The levels of the epithelial-mesenchymal transition (EMT) markers E-cadherin, ZO-1, vimentin and α-SMA were measured, and the Cell inflammatory response was evaluated by detecting IL-6 and IL-1ß levels. Then, cell migration, proliferation and apoptosis were detected. The expression of the lncRNA SNHG1 in ARPE-19 cells was detected by quantitative real-time PCR. SNHG1 was knocked down by small interfering RNA (siRNA) transfection. The effects of SNHG1 inhibition on inflammation, EMT, migration, proliferation and apoptosis were observed. RESULTS: The results showed that the expression of SNHG1 was significantly increased in ARPE-19 cells exposed to high glucose. Silencing SNHG1 reduced the expression of vimentin, α-SMA, and the expression of inflammatory chemokines IL-6 and IL-1ß, inhibited migration and proliferation, elevated the expression of E-cadherin and ZO-1, and promoted apoptosis in ARPE-19 cells. CONCLUSION: The lncRNA SNHG1 is involved in hyperglycemia-induced EMT and the inflammatory response of ARPE-19 cells and provides a new understanding of the pathogenesis of DR.

High-Temperature Oxidation Behavior of AlTiNiCuCox High-Entropy Alloys.

In this study, the high-temperature oxidation behavior of a series of AlTiNiCuCox high-entropy alloys (HEAs) was explored. The AlTiNiCuCox (x = 0.5, 0.75, 1.0, 1.25, 1.5) series HEAs were prepared using a vacuum induction melting furnace, in which three kinds of AlTiNiCuCox (x = 0.5, 1.0, 1.5) alloys with different Co contents were oxidized at 800 °C for 100 h, and their oxidation kinetic curves were determined. The microstructure, morphology, structure, and phase composition of the oxide film surface and cross-sectional layers of AlTiNiCuCox series HEAs were analyzed using scanning electron microscopy (SEM), energy-dispersive spectrometry (EDS), and X-ray diffraction (XRD). The influence of Co content on the high-temperature oxidation resistance of the HEAs was discussed, and the oxidation mechanism was summarized. The results indicate that, at 800 °C, the AlTiNiCuCox (x = 0.5, 1.0, 1.5) series HEAs had dense oxide films and certain high-temperature oxidation resistance. With increasing Co content, the high-temperature oxidation resistance of the alloys also increased. With increasing time at high temperature, there was a significant increase in the contents of oxide species and Ti on the oxide film surface. In the process of high-temperature oxidation of AlTiNiCuCox series HEAs, the interfacial reaction, in which metal elements and oxygen in the alloy form ions through direct contact reaction, initially dominated, then the diffusion process gradually became the dominant oxidation factor as ions diffused and were transported in the oxide film.

Role of CD133 in human embryonic stem cell proliferation and teratoma formation.

BACKGROUND: Pluripotent stem cells (PSCs), including human embryonic stem cells (hESCs), hold great potential for regenerative medicine and cell therapy. One of the major hurdles hindering the clinical development of PSC-based therapy is the potential risk of tumorigenesis. CD133 (Prominin 1, PROM1) is a transmembrane protein whose mRNA and glycosylated forms are highly expressed in many human cancer cell types. CD133 also serves as a cancer stem cell (CSC) marker associated with cancer progression and patient outcome. Interestingly, CD133 is highly expressed in hESCs as well as in human preimplantation embryos, but its function in hESCs has remained largely unknown. METHODS: CD133 knockout hESC WA26 cell line was generated with CRISPR/Cas9. CD133 knockout and wide type hESC lines were subjected to pluripotency, proliferation, telomere biology, and teratoma tests; the related global changes and underlying mechanisms were further systemically analyzed by RNA-seq. RESULTS: CD133 deficiency did not affect hESC pluripotency or in vivo differentiation into three germ layers but significantly decreased cell proliferation. RNA-seq revealed that CD133 deficiency dysregulated the p53, PI3K-Akt, AMPK, and Wnt signaling pathways. Alterations in these pathways have been implicated in tumor proliferation and apoptotic escape. CONCLUSIONS: Our data imply that CD133 could be an additional target and used as a selective marker to sort and eliminate undifferentiated cells in reducing potential teratoma formation risk of hESCs in regenerative medicine.

Corrigendum to "Identification of In Vivo Metabolites of Dictamnine in Mice Using HPLC-LTQ-Orbitrap Mass Spectrometry".

[This corrects the article DOI: 10.1155/2018/3567647.].

Telomere Maintenance-Associated PML Is a Potential Specific Therapeutic Target of Human Colorectal Cancer.

Telomere length maintenance is essential for cell proliferation, which is particularly prominent in cancer. We validate that the primary colorectal tumors exhibit heterogeneous telomere lengths but mostly (90%) short telomeres relative to normal tissues. Intriguingly, relatively short telomeres are associated with tumor malignancy as indicated by poorly differentiated state, and these tumors contain more cancer stem-like cells (CSLCs) identified by several commonly used markers CD44, EPHB2 or LGR5. Moreover, promyelocytic leukemia (PML) and ALT-associated PML nuclear bodies (APBs) are frequently found in tumors with short telomeres and high proliferation. In contrast, distant normal tissues rarely or only minimally express PML. Inhibition of PML and APBs by an ATR inhibitor decreases proliferation of CSLCs and organoids, suggesting a potential therapeutic target to progressive colorectal tumors. Together, telomere maintenance underling tumor progression is connected with CSLCs.

Identification of In Vivo Metabolites of Dictamnine in Mice Using HPLC-LTQ-Orbitrap Mass Spectrometry.

Dictamnine (4-methoxyfuro[2,3-b]quinolone, DIC), a common furoquinoline alkaloid in the family of Rutaceae, showed diverse biological activities. To investigate the in vivo metabolic pathways of DIC, metabolism of DIC in mice was studied using a high-performance liquid chromatography coupled to electrospray ionization of hybrid linear trap quadrupole orbitrap (HPLC-LTQ-Orbitrap) mass spectrometer. Nine metabolites were identified in the DIC-treated mouse urine, plasma, and fecal samples, of which two were identified as new metabolites. The major metabolic pathways of DIC in animal and human liver microsomes were confirmed in the present study, including o-demethylation, monohydroxylation, N-oxidation, and 2,3-olefinic epoxidation pathways. For the first time, a mono-acetylcysteine conjugate of DIC (M9) was detected from DIC-treated mouse urine and plasma samples, and 4-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (M10) and 2-(2,8-dihydroxy-4-methoxyquinolin-3-yl)acetaldehyde (M11) were identified as new metabolites of DIC; furthermore, using an in vitro human fecal incubation model, furo[2,3-b]quinolin-4-ol (M1) was verified to be a microbial demethylated metabolite of DIC. Collectively, the present study provided new information on the in vivo metabolic fate of DIC.

Detection and Structural Characterization of Nucleophiles Trapped Reactive Metabolites of Limonin Using Liquid Chromatography-Mass Spectrometry.

Limonin (LIM), a furan-containing limonoid, is one of the most abundant components of Dictamnus dasycarpus Turcz. Recent studies demonstrated that LIM has great potential for inhibiting the activity of drug-metabolizing enzymes. However, the mechanisms of LIM-induced enzyme inactivation processes remain unexplored. The main objective of this study was to identify the reactive metabolites of LIM using liquid chromatography-mass spectrometry. Three nucleophiles, glutathione (GSH), N-acetyl cysteine (NAC), and N-acetyl lysine (NAL), were used to trap the reactive metabolites of LIM in in vitro and in vivo models. Two different types of mass spectrometry, a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometry and a LTQ velos Pro ion trap mass spectrometry, were employed to acquire structural information of nucleophile adducts of LIM. In total, six nucleophile adducts of LIM (M1-M6) with their isomers were identified; among them, M1 was a GSH and NAL conjugate of LIM, M2-M4 were glutathione adducts of LIM, M5 was a NAC and NAL conjugate of LIM, and M6 was a NAC adduct of LIM. Additionally, CYP3A4 was found to be the key enzyme responsible for the bioactivation of limonin. This metabolism study largely facilitates the understanding of mechanisms of limonin-induced enzyme inactivation processes.