[An algorithm for three-dimensional plumonary parenchymal segmentation by integrating surfacelet transform with pulse coupled neural network].

In order to overcome the difficulty in lung parenchymal segmentation due to the factors such as lung disease and bronchial interference, a segmentation algorithm for three-dimensional lung parenchymal is presented based on the integration of surfacelet transform and pulse coupled neural network (PCNN). First, the three-dimensional computed tomography of lungs is decomposed into surfacelet transform domain to obtain multi-scale and multi-directional sub-band information. The edge features are then enhanced by filtering sub-band coefficients using local modified Laplacian operator. Second, surfacelet inverse transform is implemented and the reconstructed image is fed back to the input of PCNN. Finally, iteration process of the PCNN is carried out to obtain final segmentation result. The proposed algorithm is validated on the samples of public dataset. The experimental results demonstrate that the proposed algorithm has superior performance over that of the three-dimensional surfacelet transform edge detection algorithm, the three-dimensional region growing algorithm, and the three-dimensional U-NET algorithm. It can effectively suppress the interference coming from lung lesions and bronchial, and obtain a complete structure of lung parenchyma.

Characterization of gelsevirine metabolites in rat liver S9 by accurate mass measurements using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

RATIONALE: Gelsemium elegans Benth. belongs to the family Loganiaceae and is widely distributed in northern America, east Asia, and southeast Asia. It has attracted wide attention for its diverse biological effects and complex architectures. Gelsevirine is one of the major components in G. elegans. Compared with other alkaloids from G. elegans, gelsevirine exhibits equally potent anxiolytic effects but with less toxicity. However, the metabolism of gelsevirine has not been clearly elucidated. METHODS: The metabolism of gelsevirine was investigated using liver S9 fractions derived from rat liver homogenates by centrifugation at 9000 g. A rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOF-MS) method was applied to characterize the gelsevirine metabolites. RESULTS: We discovered a total number of four metabolites of gelsevirine. The metabolic pathways of gelsevirine consisted of hydrogenation, N-demethylenation and oxidation in rat liver S9. CONCLUSIONS: This is the first study on the metabolism of gelsevirine. We proposed possible metabolic pathways of gelsevirine. These findings may warrant future studies of the in vivo metabolism of gelsemine in animals.

Deoxidation rates play a critical role in DNA damage mediated by important synthetic drugs, quinoxaline 1,4-dioxides.

Quinoxaline 1,4-dioxides (QdNOs) are synthetic agents with a wide range of biological activities. However, the mechanism of DNA damage mediated by QdNOs is far from clear. Five classical QdNOs, quinocetone (QCT), mequindox (MEQ), carbadox (CBX), olaquindox (OLA), and cyadox (CYA), were used to investigate the genotoxicity of QdNOs. The deoxidation rate of QdNOs was presumed to play a role in their genotoxicity. Deoxidation rates of QdNOs in both rat and pig liver microsomes were investigated using LC/MS-IT/TOF, and their relative quantification was achieved with HPLC. To reveal the relationships between the deoxidation rate and genotoxicity, cell damage, oxidative stress, and DNA damage were detected. Under low oxygen conditions, the rank order of the desoxy and bidesoxy rates in rat and pig liver microsomes was QCT < CBX < MEQ < OLA < CYA and QCT < MEQ < CBX < OLA < CYA, respectively. Only desoxy-quinoxalines were detected under aerobic conditions. The concentrations of deoxidized metabolites under low oxygen conditions were at least 6 times higher than those under aerobic conditions. In rats, porcine primary hepatocytes, and HepG2 cells, oxidative stress indices and DNA damage showed inverse relationships with the deoxidation rate, indicating that the deoxidation rate of QdNOs, especially bidesoxy rates, might play a critical role in mediating their ability to promote DNA damage. These results indicated that faster deoxidation of QdNOs results in lower DNA-damage-induced toxicity. Our results shed new light on the prevention of DNA damage mediated by QdNOs and help to understand the relationships among the chemical structures, metabolism, and DNA damage of QdNOs.

Identification of sanguinarine metabolites in pig liver preparations by accurate mass measurements using electrospray ionization hybrid ion trap/time-of-flight mass spectrometry.

RATIONALE: Sanguinarine (SA) is currently used in veterinary medicine for animal husbandry as a natural component of feed additive Sangrovit. To date, SA metabolism in food-producing animals has not yet been reported. Therefore, the purpose of the present study was to investigate the metabolism of SA in pig liver microsomes and cytosol. METHODS: The SA incubations mixtures of microsomes and cytosol were processed by trichloroacetic acid (TCA) and acetonitrile. Then, the samples were analyzed using a sensitive and reliable method based on liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC-IT/TOFMS). The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and product ions generated from precursor ions with those of the parent drug. RESULTS: Seven metabolites were identified in pig liver preparations. Dihydrosanguinarine (DHSA, m/z 334) was the main metabolite formed in liver microsomes and the only one in cytosol. One oxidative metabolite and two O-demethylenated metabolites of SA (m/z 320) were found in the TCA-treated microsomal samples. However, SA pseudobase and two additional O-demethylenated metabolites of DHSA (m/z 322) were found only in the acetonitrile-treated microsomal samples. CONCLUSIONS: It was demonstrated that different metabolites of SA were identified depending on the acidic or neural extraction conditions. A metabolic pathway of SA in pig was tentatively proposed based on these characterized metabolites and early reports.

Ultrasound promotes germination of aging Pinus tabuliformis seeds is associated with altered lipid metabolism.

Seed germination is an essential biological process for establishing new organisms of higher plants, it is especially significant for those aged seeds stored in gene banks for years. In this study, we investigated ultrasound treatment induced germination for aged Pinus tabuliformis seeds, which has been used in large scale aircraft sowing based afforestation in North China over 30 years' ago without knowing possible mechanisms. We showed certain strength of ultrasound could increase the germination rate of aged seeds for about 3 times compare with control. Interestingly, although our transcriptome and lipidome analysis showed the differences between control and ultrasound treatments can be observed 1 day after germination by partial least squares discriminant analysis (PLSDA) analysis, majority (75 % or 69 %) of the significantly altered genes or lipids were commonly shared between them. Further analysis for the commonly altered lipids between both treatments showed ultrasound provoked the variations of lipids during germination process. Our investigation also revealed large amount of ultrasound-related genes and lipids that might be involved in germination promotion process. We hypothesis ultrasound provokes seed lipidome which further increases seed germination of Pinus tabuliformis. Our study provides new insides into the role of ultrasound induced lipidome change in seed germination. Moreover, we provide a new method to improve germination of aged seeds which might benefit preservation of seeds in gene banks.

Production of high-affinity monoclonal antibody and development of immunoassay for 3-methyl-quinoxaline-2-carboxylic acid detection in swine muscle and liver.

Production of high-affinity and specific antibodies to small molecules with molecular weight (MW) lower than 200 Da is challenging. Here, we designed a novel hapten, named hapten H6, for the detection of 3-methyl-quinoxaline-2-carboxylic acid (MQCA, MW of 189 Da), a residual marker of olaquindox, one of important veterinary antibiotics. The hapten H6 maintained all structural features of MQCA, especially in mulliken atomic charge distribution. Then, a monoclonal antibody (mAb) named 8C9 was obtained with an IC50 value of 0.2 µg/L, yielding a 15.5- to 88.5-fold improvement compared to previously prepared specific antibodies against MQCA. In addition, mAb 8C9 exhibited ignorable cross-reactivity with other structural analogs. Finally, a highly sensitive and specific indirect competitive ELISA based on mAb 8C9 was developed for the detection of MQCA in swine muscle and liver samples with limit of detection values of 0.04 µg/kg and 0.09 µg/kg, respectively.

An integrated method for degradation products detection and characterization using hybrid ion trap/time-of-flight mass spectrometry and data processing techniques: Application to study of the degradation products of danofloxacin under stressed conditions.

A new strategy using hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography and post-acquisition data mining techniques was developed and applied to the detection and characterization of degradation products of danofloxacin. The degradation products formed under different forced conditions were separated using an ODS-C18 column with gradient elution. Accurate full-scan MS data were acquired in the first run and processed with the combination of extracted ion chromatograms and LC-UV chromatograms. These processes were able to find accurate molecular masses of possible degradation products. Then, the accurate MS/MS data acquired through data-dependent analysis mode in another run facilitated the structural elucidations of degradation products. As a result, a total of 11 degradation products of danofloxacin were detected and characterized using the developed method. Overall, this analytical strategy enables the acquisition of accurate-mass LC/MS data, search of a variety of degradation products through the post-acquisition processes, and effective structural characterization based on elemental compositions of degradation product molecules and their product ions. The ability to measure degradation products via tandem mass spectrometry coupled with accurate mass measurement, all in only two experimental runs, is one of the most attractive features of this methodology. The results demonstrate that use of the LC/MS-IT-TOF approach appears to be rapid, efficient and reliable in structural characterization of drug degradation products.

The metabolism of gelsevirine in human, pig, goat and rat liver microsomes.

Gelsemium is a small genus of flowering plants from the family Loganiaceae comprising five species, three of which, Gelsemium sempervirens (L.) J. St.-Hil., G. elegans Benth and G. rankinii Small, are particularly popular. Compared with other alkaloids from G. elegans, gelsemine, gelsevirine and koumine exhibit equally potent anxiolytic effects and low toxicity. Although the pharmacological activities and metabolism of koumine and gelsemine have been reported in previous studies, the species differences of gelsevirine metabolism have not been well studied. In this study, the metabolism of gelsevirine was investigated by using liver microsomes of humans, pigs, goats and rats by means of HPLC-QqTOF/MS. The results indicated that the metabolism of gelsevirine in liver microsomes had qualitative and quantitative species differences. Based on the results, the possible metabolic pathways of gelsevirine in liver microsomes were proposed. Investigation of the metabolism of gelsevirine will provide a basis for further studies of the in vivo metabolism of this drug.

mRNA expression profiles of P450 3A enzymes in the liver and small intestine of the domestic pig.

The cytochrome P450 (P450) 3A family is considered to be the most important and abundantly expressed P450 subfamily in mammals. The mRNA expression levels of four P450 3A enzymes in porcine liver and small intestine were investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of P450 3A mRNAs (P450 3A pool) was higher in the liver than that in the small intestine. In the small intestine, the P450 3A mRNAs were gradually decreased from the duodenum to the ileum. P450 3A29 and P450 3A22 were predominantly expressed both in liver and small intestine tissues with larger ratios in the P450 3A pool than the other P450 3A enzymes. These results demonstrate that P450 3A29 and P450 3A22 probably serve as the major P450 3A contributors for both the hepatic and intestinal P450 3A pool. This work provides a deeper comprehension of the contribution of P450 3A enzymes to xenobiotic metabolism in pigs.

The metabolism of olaquindox in rats, chickens and pigs.

Olaquindox is a growth-promoting feed additive for food-producing animals. Its toxicities were reported to be closely related to the metabolism. To provide the interpretation of toxicities in animals, this study explored the metabolism of olaquindox in rats, chickens and pigs of different genders by qualitative metabolite profiling. Animals were fed olaquindox in an oral dose, and then their urine, plasma, feces, liver, kidney and muscle were collected. Liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry was used for structural investigation and identification of metabolites. The structures of metabolites were elucidated based on the accurate MS² spectra and comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite. A total of 18, 18 and 16 metabolites of rats, chickens and pigs were identified, respectively. Among the identified metabolites, 8 known metabolites were confirmed as an early study had stated, and 15 metabolites were found for the first time in vivo. The major metabolic pathways of olaquindox were proposed to be N-O reduction and oxidation of hydroxyl to carboxylic acid followed by N-O reduction. The qualitative species difference on the metabolite profiles of olaquindox among the three species was observed. However, metabolite profiles of olaquindox appeared to be qualitatively similar between female and male for the same species. The proposed metabolic pathways of olaquindox in animals will provide comprehensive data to clarify the metabolism of olaquindox among different species, and will give scientific explanation for toxicities and residues of olaquindox.

Development of a liquid chromatography-tandem mass spectrometry with pressurized liquid extraction for determination of glucocorticoid residues in edible tissues.

A multi-residues method using pressurized liquid extraction (PLE) and liquid chromatography combined with mass spectrometry (LC-MS/MS) has been developed for determination of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, fludrocortisone) in muscle of swine, cattle, and sheep. Parameters affecting PLE extraction including extraction solvent, extraction temperature, extraction pressure and extraction cycles were optimized. The optimized method employed 11 ml extraction cells, hexane-ethyl acetate (50:50, v/v) as extraction solvent, 1500 psi of extraction pressure and 50°C of extraction temperature. The samples were detected by LC-ESI-MS/MS in negative mode with selected reaction monitoring (SRM) mode. The recovery of glucocorticoids spiked at levels of 0.5-6 µg kg(-1) ranged from 70.1% to 103.1%; the between-day relative standard deviations were no more than 9.6%. The limits of quantification were 0.5-2 µg kg(-1) in muscle. The results demonstrated that the method is simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken.

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu(2)SO(4). The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min(-1), and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 µg g(-1) ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0-2.0 µg g(-1). This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.

Development of a liquid chromatography-tandem mass spectrometry with pressurized liquid extraction method for the determination of benzimidazole residues in edible tissues.

A confirmatory and quantitative method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with a pressure liquid extraction (PLE) was developed for the determination of 11 benzimidazole and 10 metabolites of albendazole, fenbendazole and mebendazole in the muscles and livers of swine, cattle, sheep and chicken. For sample preparation, we used an automated technique of PLE method. The optimum extraction conditions were obtained using an 11 ml Accelerated Solvent Extraction (ASE) cells, acetonitrile/hexane as the extraction solvent. HPLC analysis was performed on a C18 column with gradient elution using acetonitrile and 5 mmol l(-1) formic ammonium as mobile phase. The analytes were detected in the positive ion multiple reaction monitoring (MRM) mode by the LC-ESI-MS/MS analysis. The recoveries of benzimidazole (BZDs) spiked at the levels of 0.5 µg kg(-1) ranged from 70.1% to 92.7%; the between-day relative standard deviations were no more than 10%. The limits of quantification were 0.02-0.5 µg kg(-1). The optimized method was successfully applied to monitor real samples containing BZDs, demonstrating the method to be simple, fast, robust and suitable for identification and quantification of BZDs residues in animal products.

Metabolites and JAK/STAT pathway were involved in the liver and spleen damage in male Wistar rats fed with mequindox.

Mequindox (MEQ) is a novel synthetic quinoxaline 1,4-dioxides antibacterial agent and growth promoter in animal husbandry. This study was to investigate whether reactive oxygen species (ROS), the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway, suppressors of cytokine signaling (SOCS) and inflammatory cytokines were involved in toxicities of MEQ. Our data demonstrated that high dose of MEQ (275 mg/kg) apparently led to tissue impairment combined with imbalance of redox in liver. In liver and spleen samples, hydroxylation metabolites and desoxymequindox were detected, directly confirming the potential link of N→O group reduction metabolism with its organ toxicity. Moreover, up-regulation of JAK/STAT, SOCS family, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) were also observed in the high-dose group. Meanwhile, significant changes of oxidative stress indices in liver were observed in the high-dose group. As for NADPH subunit, the mRNA levels of many subunits were significantly up-regulated at low doses but down-regulated in a dose-dependent manner in liver and spleen, suggesting an involvement of NADPH in MEQ metabolism and ROS generation. In conclusion, we reported the dose-dependent long-term toxicity as well as the discussion of the potential mechanism and pathways of MEQ, which raised further awareness of its toxicity following with the dose change.

ROS mediated cytotoxicity of porcine adrenocortical cells induced by QdNOs derivatives in vitro.

Quinoxaline 1,4-dioxides (QdNOs) derivatives, the potent synthetic antibacterial group used in food-producing animals, are assumed to have pro-oxidant properties. However, how oxidative stress mediated their adrenal toxicity is far from clear. The aim of this study was to assess the ability of three QdNOs, i.e. olaquindox (OLA), mequindox (MEQ), and cyadox (CYA), to produce reactive oxygen species (ROS) and oxidative cell damage in porcine adrenocortical cells. Multiple approaches such as cell activity assay, biochemical detectation, flow cytometry and fluorescent were used to study the integrated role of ROS homeostasis, mitochondrial redox metabolism and cell apoptosis as well as chemical stability of these drugs. The results showed that OLA and MEQ treatment evoked a significant dose and time-dependent cell damage in adrenocortical cells, well CYA displayed much less toxicity. As for the intracellular ROS production, OLA irritated a persistent and utmost release of ROS while MEQ made a similar but weaker reaction. CYA, however, had a short and unstable release of intracellular ROS. On the other hand, quinoxalinine-2-carboxylie acid (QCA), one of the metabolites of OLA and MEQ, did not cause any significant production of ROS and showed relatively lower toxicity than its parents. Moreover, an imbalance in the redox metabolism and mitochondrial membrane damage has been implicated in adrenal toxicity of QdNOs. ROS scavengers partially reversed QdNOs-induced mitochondrial damage, indicating that mitochondria may be a major target and critical for ROS-mediated cell death. In a word, these results suggested that ROS is a key mediator of QdNOs-induced cell death via mitochondria-dependent pathway in adrenocortical cells. The results provide a mechanism approach in understanding the characterize of adrenal damage caused by QdNOs in vitro, which would in turn, help in designing the appropriate therapeutic strategies of these kind of feed additives.