Effects of praziquantel isomers on the proliferation and activation of the LX-2 human hepatic stellate cell line / 中国血吸虫病防治杂志

Objective To compare the effects of levo-praziquantel (L-PZQ) and dextro-praziquantel (D-PZQ) on the proliferation and activation of the human hepatic stellate cell line LX-2 in vitro. Methods LX-2 cells were stimulated with transforming growth factor-β (TGF-β). LX-2 cell proliferation was measured using the CCK-8 assay after 24 h stimulation with 0 to 50 μg/mL concentrations of praziquantel, and the gene and protein expression of type Ⅰ collagen (collagen Ⅰ), type Ⅲ collagen (collagen Ⅲ) and α-smooth muscle actin (α-SMA) was quantified in LX-2 cells using quantitative real-time PCR (qPCR) and Western blotting assays 24 h and 48 h following stimulation with 15 μg/mL praziquantel to detect LX-2 cell activation. Results There were significant differences in the survival rate of LX-2 cells between L-PZQ and D-PZQ treatments at all concentrations (F = 6.119 and 79.180, both P values < 0.05). Either L-PZQ or D-PZQ at a concentration of < 30 μg/mL showed no remarkableeffectsonthe LX-2 cell proliferation (both P values > 0.05), and L-PZQ at a concentration of > 50 μg/mL and D-PZQ at a concentration of > 40 μg/mL inhibited the LX-2 cell proliferation (both P values < 0.05), while D-PZQ at concentrations of 40 μg/mL and 50 μg/mL showed greater inhibition on LX-2 cell proliferation than L-PZQ (t = 3.419 and 8.776, both P values < 0.05). There were significant differences in the collagen Ⅰ, collagen Ⅲ and α-SMA expression in LX-2 cells at both transcriptional (F = 21.55, 79.99 and 46.70, all P values < 0.05) and translational levels (F = 20.12, 30.29 and 32.93, all P values < 0.05) among the blank control group, TGF-β stimulation group, L-PZQ treatment group and D-PZQ treatment group. L-PZQ treatment resulted in remarkable inhibition on collagen Ⅲ and α-SMA gene expression in LX-2 cells (both P values < 0.05); however, the treatment showed no remarkable inhibition collagen Ⅰ gene expression or collagen Ⅰ, collagen Ⅲ or α-SMA protein expression in LX-2 cells (all P values > 0.05). In addition, D-PZQ treatment resulted in significant inhibition on collagen Ⅰ, collagen Ⅲ and α-SMA expression in LX-2 cells at both translational and transcriptional levels (all P values < 0.05), and D-PZQ showed higher inhibition on collagen Ⅰ, collagen Ⅲ and α-SMA gene expression in LX-2 cells than L-PZQ (all P values < 0.05). Conclusions Both L-PZQ and D-PZQ inhibit the proliferation and activation of LX-2 cells, and D-PZQ shows a higher inhibitory activity than L-PZQ.

Molluscicidal activity of the secondary metabolites from Streptomyces nigrogriseolus XD 2-7 against Oncomelania hupensis and its preliminary mechanisms of molluscicidal actions / 中国血吸虫病防治杂志

OBJECTIVE@#To evaluate the storage stability of metabolites from actinomycetes Streptomyces nigrogriseolus XD 2-7 and the mollcuscicidal activity against Oncomelania hupensis in the laboratory, and to preliminarily explore the mechanisms of the molluscicidal activity.@*METHODS@#The fermentation supernatant of S. nigrogriseolus XD 2-7 was prepared and stored at -20, 4 °C and 28 °C without light for 10 d; then, the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The fermentation supernatant was boiled in a 100 °C water bath for 30 min and recovered to room temperature, and then the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The pH values of the fermentation supernatant were adjusted to 4.0, 6.0 and 9.0 with concentrated hydrochloric acid and sodium hydroxide, and the fermentation supernatant was stilled at room temperature for 12 h, with its pH adjusted to 7.0; then, the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The fermentation product of S. nigrogriseolus XD 2-7was isolated and purified four times with macroporous resin, silica gel and octadecylsilane bonded silica gel. The final products were prepared into solutions at concentrations of 10.00, 5.00, 2.50, 1.25 mg/L and 0.63 mg/L, and the molluscicidal effect of the final productswas tested against O. hupensis following immersion for 72 h, while dechlorination water served as blank controls, and 0.10 mg/L niclosamide served as positive control. The adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels were measured in in O. hupensis soft tissues using high performance liquid chromatography (HPLC) following exposure to the final purified fermentation products of S. nigrogriseolus XD 2-7.@*RESULTS@#After the fermentation supernatant of S. nigrogriseolus XD 2-7 was placed at -20, 4 °C and 28 °C without light for 10 d, immersion in the stock solution and solutions at 10- and 50-fold dilutions for 72 h resulted in a 100% (30/30) O. hupensis mortality. Following boiling at 100 °C for 30 min, immersion in the stock solution and solutions at 10- and 50-fold dilutions for 72 h resulted in a 100.00% (30/30) O. hupensis mortality. Following storage at pH values of 4.0 and 6.0 for 12 h, immersion in the fermentation supernatant of S. nigrogriseolus XD 2-7 for 72 h resulted in a 100.00% (30/30) O. hupensis mortality, and following storage at a pH value of 9.0 for 12 h, immersion in the fermentation supernatant of S. nigrogriseolus XD 2-7 for 72 h resulted in a 33.33% (10/30) O. hupensis mortality (χ2 = 30.000, P < 0.05). The minimum concentration of the final purified fermentation products of S. nigrogriseolus XD 2-7 was 1.25 mg/L for achieving a 100% (30/30) O. hupensis mortality. The ATP level was significantly lower in O. hupensis soft tissues exposed to 0.10 mg/L and 1.00 mg/L of the final purified fermentation products of S. nigrogriseolus XD 2-7 than in controls (F = 7.274, P < 0.05), while no significant difference was detected in the ADP level between the treatment group and controls (F = 2.485, P > 0.05).@*CONCLUSIONS@#The active mollcuscicidal ingredients of the S. nigrogriseolus XD 2-7 metabolites are maintained stably at -20, 4 °C and 28 °C for 10 d, and are heat and acid resistant but not alkali resistant. The metabolites from S. nigrogriseolus XD 2-7 may cause energy metabolism disorders in O. hupensis, leading to O. hupensis death.

Activity of aromatic pyrrole-based compounds against of Schistosoma japonicum cercariae and acute toxicity to fish / 中国血吸虫病防治杂志

Objective To test the activity of aromatic pyrrole-based compounds against cercariae of Schistosoma japonicum and test their acute toxicity to fish. Methods A series of aromatic pyrrole-based compounds were synthesized using 4-benzyl-5-(trifluoromethyl)-1H-pyrrole-3-nitrile as the lead compound. The synthesized compounds were prepared into solutions at concentrations of 10.00, 1.00, 0.10, 0.01 mg/L, and the activity of these solutions against S. japonicum cercariae was tested in 30 min, while 0.10 mg/L and 0.01 mg/L niclosamide solutions served as a positive control and dechlorinated water with 1% dimethyl sulfoxide (DMSO) was used as a negative control, with 10 to 30 cercariae of S. japonicum in each group. In addition, the compounds were prepared into solutions at concentrations of 0.50, 0.25, 0.12, 0.06, 0.03 mg/L, and their toxicity to zebrafish was tested in 72 h, while 0.15 mg/L and 0.30 mg/L niclosamide solutions served as a positive control and dechlorinated water with 1% DMSO was used as a negative control, with 10 zebrafishes in each group. Results A total of 7 aromatic pyrrole-based compounds were successfully synthesized. Treatment with compounds 102, 104 and 106 at a concentration of 0.01 mg/L for 30 min killed all S. japonicum cercariae, and compounds 105 and 107 showed no activity against cercariae. No death of cercariae was found in the blank control group, while treatment with 0.10 mg/L niclosamide for 10 min caused a 100% mortality rate of S. japonicum cercariae and 0.01 mg/L niclosamide failed to kill S. japonicum cercariae. No zebrafish death was found 72 h post-treatment with compounds 101, 104 and 105 at a concentration of 0.03 mg/L, and exposure to compounds 102, 103 and 106 at a concentration of 0.03 mg/L for 12 h resulted in a 100% mortality rate of zebrafish. No zebrafish death occurred 72 h post-treatment with 0.50 mg/L Compound 104, and no zebrafish death was found in the blank control group, while treatment with 0.30 mg/L niclosamide for 24 h resulted in a 100% mortality rate of zebrafish. Conclusions Compound 104 achieves a 100% mortality rate against S. japonicum cercariae at a concentration of 0.01 mg/L for 30 min, and causes no death of zebrafish at a concentration of 0.50 mg/L for 72 h, which may serve as a cercaricide candidate.

Studies on resistance of Schistosoma to praziquantel XVIII Sensitivity to praziquantel in filial generations of praziquantel-resistant and -sensitive Schistosoma japonicum mixed infections / 中国血吸虫病防治杂志

Objective To investigate the sensitivity of adult worms of filial generations from praziquantel-resistant and -sensitive Schistosoma japonicum mixed infections to praziquantel. Methods Mice were infected with the cercariae of an experimentally generated praziquantel-resistant S. japonicum isolate [median effective dose (ED50) = 277.4 mg/kg] and a laboratory-maintained praziquantel-sensitive S. japonicum isolate (ED50 = 99.6 mg/kg) at a mixture ratio of 1:1 and 2:1, which was maintained in the laboratory via the mouse-snail cycle for 8 generations. Then, mice were infected with the cercariae of the 8th filial-generation parasite, and grouped 35 days post-infection. Mice in the 5 treatment groups were given praziquantel treatment by gavage at a single oral dose of 37.5, 75, 150, 300 mg/kg and 600 mg/kg, while animals in the control group was administered orally with 2.5% cremophor EL. All mice were sacrificed 14 days post-treatment and adult worms were collected by perfusion of the portal vein. The worm burden reductions and praziquantel ED50 values were calculated. The praziquantel-resistant S. japonicum isolate generated from experimental induction with 12 rounds of praziquantel treatment with sub-curative doses was maintained in the laboratory via the mouse-snail cycle, and mice were infected with the cercariae of the 8th filial-generation parasite. The praziquantel ED50 value against the 8th filial-generation adults was measured. Results After mice were infected with the mixture of cercariae of PZQ-resistant and -sensitive S. japonicum isolates at a ratio of 1:1, the praziquantel ED50 was 135.2 mg/kg against the adults of the 8th filial-generation parasite. After mice were infected with the mixture of cercariae of PZQ-resistant and -sensitive S. japonicum isolates at a ratio of 2:1, the praziquantel ED50 was 129.2 mg/kg against the adults of the 8th filial-generation parasite. In addition, the praziquantel ED50 was 208.4 mg/kg against the adults of the 8th filial-generation S. japonicum without the selection pressure of praziquantel. Conclusions Compared with the experimentally induced praziquantel-resistant S. japonicum isolate, the adult worms of the filial-generation S. japonicum show a reduced sensitivity to praziquantel in the same host following infection with the mixture of cercariae of praziquantel-resistant and -sensitive S. japonicum isolates. The adult worms of the filial generation of the praziquantel-resistant S. japonicum isolate without the selection pressure of praziquantel may still maintain the resistance to praziquantel.

Study on the factors affecting the degradation of niclosamide in the soil / 中国血吸虫病防治杂志

ObjectiveTo investigate the factors affecting the degradation of niclosamide in the soil, so as to provide the evidence for the assessment of the environmental safety in the field snail control with niclosamide. MethodsA high performance liquid chromatography was established for the determination of niclosamide in the field. Then, the degradation of niclosamide was investigated in soils with different moistures (10%, 30%, 50%, 70% and 90%), temperatures [(15 ± 1), (25 ± 1), (35 ± 1) °C], initial concentrations (1, 5, 10 mg/kg) and in sterilized and non-sterilized soils. In addition, the degradation of niclosamide was fitted with the first-order kinetics equation, and the degradation half-life was calculated. Results The niclosamide residues gradually decreased over time in soils with different moistures, and a higher rate of degradation was seen in soils with a higher moisture. The degradation half-life of niclosamide reduced from 4.258 d in the soil with a 10% moisture to 2.412 d in the soil with a 90% moisture. The niclosamide residues gradually decreased over time in soils with different temperatures, and a higher rate of degradation was seen in soils with a higher temperature. The degradation half-life of niclosamide reduced from 4.398 d in the soil with a temperature of (15 ± 1) °C to 2.828 d in the soil with a temperature of (35 ± 1) °C. The degradation half-lives of niclosamide were 3.212, 3.333 d and 3.448 d in soils containing niclosamide at initial concentrations of 1, 5 mg/kg and 10 mg/kg, and > 30 d and 3.273 d in sterilized and non-sterilized soils. Multiple linear regression analysis revealed that soil microorganisms (P = 0.010), moisture (P = 0.000) and temperature (P = 0.002) affected the half-life of niclosamide degradation. Conclusions The degradation of niclosamide in soils fits the first-order kinetics equation, and presence of microorganisms, a high temperature and high moisture may accelerate the degradation of niclosamide in the soil.

Relation of ASXL2 Gene Mutation with Clinical Characteristics, Prognosis and C-KIT Gene Mutation in AML Patients with AML1- ETO Fusion Gene / 中国实验血液学杂志

OBJECTIVE@#To analyze relation of ASXL2 gene mutation with the clinical characteristics, prognosis and C-KIT gene mutation in acute myeloid leukemia (AML) patients with AML1-ETO fusion gene.@*METHODS@#The clinical data of 63 primary AML patients with AML1-ETO fusion gene were collected and retrospectively analyzed. The mutation of ASXL2 gene was directly sequenced by PCR. The clinical characteristics, C-KIT mutation rate and prognosis were compared between the patients with ASXL2 gene mutation (group A) and non-mutation (group B).@*RESULTS@#Among 63 patients, 8 (12.70%) cases of ASXL2 mutation gene was detected. Hemoglobin level in peripheral blood of patients in group A was significantly lower than that in group B (P＜0.01). There was no significant difference in sex, ages proportion of bone marrow blasts, lymph node enlargement, peripheral blood leukocytes count and platelets between the two groups (P＞0.05). The infiltration of central nervous system, liver and spleen was not found in both groups. The expression of CD33 in group A was significantly lower than that in group B (P＜0.05), but the results of other immunophenotype analysis were not significantly different between the two groups (P＞0.05). The remission rate and median survival time were not significantly different between two groups (P＞0.05). The detection rate of C-KIT gene mutation were not significantly different between group A and group B (P＞0.05).@*CONCLUSION@#Among AML patients with AML1-ETO fusion gene, ASXL2 gene mutation accounts for a certain ratio, and the peripheral blood hemoglobin concentration and CD33 expression in these patients are often low. At the same time, ASXL2 gene mutation may not be closely related with C-KIT gene mutation.

Study on biological characteristics: reproduction and viability of Biomphalaria glabrata as an intermediate host of Schistosoma mansoni / 中国血吸虫病防治杂志

Objective To compare the difference of fertility of Biomphalaria glabrata snails between self-fertilization and cross-fertilization and to observe the circadian rhythm of laying eggs, the effect of light on laying eggs and the tolerance of the snail to water and food deficiency, so as to provide the evidence for control and elimination of B. glabrata snails in the field. Methods Under laboratory conditions, a single B. glabrata egg for self-fertilization was separated and hatched individually, and young snails were raised in different plastic boxes individually. The eggs for cross-fertilization were hatched and the young snails were fed in the same plastic box. The ability of spawn, the development of the eggs, and the number of snails growing from young to adult snails were compared between the self-fertilization and cross-fertilization. The snails were in the water under four environments, all day illumination, all day without illumination, daytime lighting and night without illumination, and daytime without illumination but night lighting. The eggs were collected and counted daily. The circadian rhythm of spawn and the effect of illumination on spawn were observed. The adult snails were divided into 6 groups and exposed to the environments with relative humidity of 0, 65%, 87% and 100%, respectively. The survival rates of the adult snails exposed to the different environments after different time were observed. The adult snails were placed at 25 °C in the oven to remove water content from the soft body of snails. When the dehydration rates of the soft bodies achieved 10%, 20%, 30%, 40%, 50%, 52%, 55%, 57%, 60%, and 70% respectively, the survival rates of the adult snails exposed to the oven were observed. Results In the 25 °C water, the average laying egg number for 15 days per snail was (8.77 ± 16.92) eggs/snail in the self-fertilization snail. The average laying egg number for 15 days per snail was (149.71 ± 142.28) eggs/snail in the cross-fertilization snails. There was a significant difference between the self-fertilization snail and cross-fertilization snail (t = 0.999 999, P < 0.01). The hatching rate and reproductive maturation rate of the self-fertilization snails and cross-fertilization snails were 50.1% and 78.9%, and 19.3% and 3.8%, respectively, There was a significant difference (the hatching rate: χ2 = 18.18, P < 0.01, the reproductive maturation rate: χ2 = 11.83, P < 0.01) . In the natural environment of daytime with illumination and nighttime with darkness, the amount of laying 20 eggs of B. glabrata snail was (944.07 ± 392.53) eggs/day during a whole day, among them the amount of laying eggs during daytime account for 10.1% and the amount of laying eggs during nighttime account for 89.9%, and the laying egg was given priority to with the night. The above results suggested that the dark environment was conducive to B. glabrata snails to lay eggs. The above results suggested that light can promote the increase of spawning of B. glabrata. When B. glabrata was exposed to the environments with the relative humidity of 0, 65%, 87% and 100% at 25 °C, respectively, and the longest survival times of snails were 7, 70, 150 d and 100 d, respectively. In the 25 °C water, the snails could survive for 50 days without food. The adult snails were placed at 25 °C in the oven to remove water content from the soft body of snails. When the dehydration rates of the soft bodies achieved 10%, 20%, 30%, 40%, 50%, 52%, 55%, 57%, 60%, and 70% respectively, the survival rates of the adult snails exposed to the oven were 100%, 100%, 100%, 100%, 70%, 30%, 0, 0, 0 and 0, respectively. Conclusions B. glabrata can achieve the reproductive process by cross-fertilization or self-fertilization. There is a significant difference in reproductive ability between the cross-fertilization snail and self-fertilization snail, cross-fertilization is stronger than self-fertilization, but the rate of reproduction in the self-fertilization is higher than that in the cross-fertilization. It is indicated that B. glabrata that survive after the dry season plays an important role in the maintenance of local snail populations and transmission of schistosomiasis mansoni.

Study on biological characteristics: reproduction and viability of Biomphalaria glabrata as an intermediate host of Schistosoma mansoni / 中国血吸虫病防治杂志

Objective To compare the difference of fertility of Biomphalaria glabrata snails between self-fertilization and cross-fertilization and to observe the circadian rhythm of laying eggs, the effect of light on laying eggs and the tolerance of the snail to water and food deficiency, so as to provide the evidence for control and elimination of B. glabrata snails in the field. Methods Under laboratory conditions, a single B. glabrata egg for self-fertilization was separated and hatched individually, and young snails were raised in different plastic boxes individually. The eggs for cross-fertilization were hatched and the young snails were fed in the same plastic box. The ability of spawn, the development of the eggs, and the number of snails growing from young to adult snails were compared between the self-fertilization and cross-fertilization. The snails were in the water under four environments, all day illumination, all day without illumination, daytime lighting and night without illumination, and daytime without illumination but night lighting. The eggs were collected and counted daily. The circadian rhythm of spawn and the effect of illumination on spawn were observed. The adult snails were divided into 6 groups and exposed to the environments with relative humidity of 0, 65%, 87% and 100%, respectively. The survival rates of the adult snails exposed to the different environments after different time were observed. The adult snails were placed at 25 °C in the oven to remove water content from the soft body of snails. When the dehydration rates of the soft bodies achieved 10%, 20%, 30%, 40%, 50%, 52%, 55%, 57%, 60%, and 70% respectively, the survival rates of the adult snails exposed to the oven were observed. Results In the 25 °C water, the average laying egg number for 15 days per snail was (8.77 ± 16.92) eggs/snail in the self-fertilization snail. The average laying egg number for 15 days per snail was (149.71 ± 142.28) eggs/snail in the cross-fertilization snails. There was a significant difference between the self-fertilization snail and cross-fertilization snail (t = 0.999 999, P < 0.01). The hatching rate and reproductive maturation rate of the self-fertilization snails and cross-fertilization snails were 50.1% and 78.9%, and 19.3% and 3.8%, respectively, There was a significant difference (the hatching rate: χ2 = 18.18, P < 0.01, the reproductive maturation rate: χ2 = 11.83, P < 0.01) . In the natural environment of daytime with illumination and nighttime with darkness, the amount of laying 20 eggs of B. glabrata snail was (944.07 ± 392.53) eggs/day during a whole day, among them the amount of laying eggs during daytime account for 10.1% and the amount of laying eggs during nighttime account for 89.9%, and the laying egg was given priority to with the night. The above results suggested that the dark environment was conducive to B. glabrata snails to lay eggs. The above results suggested that light can promote the increase of spawning of B. glabrata. When B. glabrata was exposed to the environments with the relative humidity of 0, 65%, 87% and 100% at 25 °C, respectively, and the longest survival times of snails were 7, 70, 150 d and 100 d, respectively. In the 25 °C water, the snails could survive for 50 days without food. The adult snails were placed at 25 °C in the oven to remove water content from the soft body of snails. When the dehydration rates of the soft bodies achieved 10%, 20%, 30%, 40%, 50%, 52%, 55%, 57%, 60%, and 70% respectively, the survival rates of the adult snails exposed to the oven were 100%, 100%, 100%, 100%, 70%, 30%, 0, 0, 0 and 0, respectively. Conclusions B. glabrata can achieve the reproductive process by cross-fertilization or self-fertilization. There is a significant difference in reproductive ability between the cross-fertilization snail and self-fertilization snail, cross-fertilization is stronger than self-fertilization, but the rate of reproduction in the self-fertilization is higher than that in the cross-fertilization. It is indicated that B. glabrata that survive after the dry season plays an important role in the maintenance of local snail populations and transmission of schistosomiasis mansoni.

Simultaneous Determination of Twelve Kinds of Residual Solvents in A New Drug CBT108 By Headspace Gas Chromatography / 分析化学

A simple and sensitive method for simultaneous determination of 12 kinds of residual solvents in a new drug CBT108 was established and validated by headspace gas chromatographic technology. The rationality,accuracy and feasibility of the analytical method were verified. Under the optimized conditions, simultaneous separation and determination of 12 kinds of residual solvents, including methanol, ethanol, ether, acetone, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran, heptane, toluene and carbon tetrachloride was carried out by using a DB624 capillary column(30 m×0.53 mm×3.0 μm) for separation, a flame ionization detector for detection and internal standard method for quantitation. Good linearity was obtained for 12 solvents with the correlation coefficients(R2) of more than 0.997. The limits of quantitation and detection were defined at S/N=3 and S/N=10,respectively. LOQ and LOD for 12 solvents were given as 0.024 μg/mL and 0.0072 μg/mL for methanol,0.1 μg/mL and 0.012 μg/mL for ethanol, 0.01 μg/mL and 0.005 μg/mL for ether, 0.1 μg/mL and 0.008 μg/mL for acetone, 1.025 μg/mL and 0.0615 μg/mL for acetonitrile, 0. 09 μg/mL and 0. 06 μg/mL for dichloromethane, 0. 09 μg/mL and 0.06 μg/mL for n-hexane, 0. 25 μg/mL and 0. 008 μg/mL for ethyl acetate, 0. 108 μg/mL and 0.014 μg/mL for tetrahydrofuran,0.16 μg/mL and 0.0004 μg/mL for carbon tetrachloride,0.0075 μg/mL and 0.005 μg/mL for heptane, and 0.0445 μg/mL and 0.0014 μg/mL for toluene. The adding standards recoveries for 12 residual solvents at three spiked levels were in the range of 90.96%-108.67%,with relative standard deviations of 0.1%-5.7%. This simple,high accuracy and good repeatability method is feasible for rapidly determination of 12 residual solvents in drug candidate CBT108. Meanwhile, this simple method provides a consulted value for detection of residual solvents in other medicines.

Chiral Probe for Recognition of Landiolol Hydrochloride and Its Stereoisomer by Mass Spectrometry / 分析化学

In this work,two chiral chloride probes were used to differentiate landiolol hydrochloride by mass spectrometry. Two chiral chloride probe reagents, N-(p-Tosyl)-L-phenylalaninyl chloride (TSPC) and (-)-Camphanic acid chloride, were chosen to react with landiolol hydrochloride and its stereoisomers to form covalent bonding derivatives, which enlarged the difference of stereo structure between landiolol and its stereoisomers. Result of tandem mass spectrometry showed that fragment from derivative products prefers to losing water to form fragment ions m/z 793 and m/z 672. The relative abundance of ions m/z 793 and m/z 672 was quite different in each isomer. The fragment ions m/z 603 from (-)-Camphanic acid chloride derivative products showed distinction relative abundance because of the different stability of each stereoisomers, which gave rise to the enlarged difference of stereo structure between landiolol and its stereoisomers. By comparing the different relative abundance ratio of analyte and each stereoisomer in MS/MS spectra, we could realize recognization landiolol hydrochloride and its stereoisomers. Accurate masses of precursor and fragment ions were confirmed on an IT-TOF mass spectrometer. This method by using ion-trap mass spectrometry could rapidly and simply differentiate landiolol hydrochloride and its stereoisomers. This work could also contribute to differentiation and discrimination of landiolol hydrochloride and its stereoisomers.

Studies on colonization risk and potential geographical distribution of Biom-phalaria glabrata as an intermediate host of Schistosoma mansoni in Main-land China / 中国血吸虫病防治杂志

Objective To predict the colonization risk and potential geographical distribution of Biomphalaria glabrata in the Mainland China based on the past period temperature data.Methods The survival extreme high temperatures and low tem-peratures of B.glabrata eggs,young and adult B.glabrata snails and the average effective accumulated temperature of genera-tion development were determined in laboratory conditions.The temperature data in January and July from 1955 to 2010 were collected from the national meteorological monitoring sites in the southern part of China,including Chongqing,Zhejiang,Yun-nan,Sichuan,Jiangxi,Hunan,Hainan,Guizhou,Guangdong,Guangxi and Fujian provinces(11 provinces).A database of ambient temperature related to B.glabrata was established based on the Geographic Information System(GIS).The colonization risk and potential geographical distribution of B.glabrata in the southern part of China were analyzed and predicted by ArcGIS 10.1 software.Results The half lethal low temperatures of B.glabrata eggs,young and adult B.glabrata snails were 6.80,6.34℃ and 6.60℃ respectively;the half lethal high temperatures of B.glabrata eggs,young and adult B.glabrata snails were 35.99,33.59℃ and 32.20℃,respectively.The developmental threshold temperature was 7.16℃;the average effective accumu-lated temperature of generation development was(1 970.07 ± 455.10)days-degree.The GIS overlay analysis of the half lethal low and high temperatures of B.glabrata showed that the local temperature conditions in all Hainan and part regions in Yunnan,Guangxi,Guangdong and Fujian were conformed to the survival temperature of B.glabrata snails.The regions,where the aver-age effective accumulated temperature was more than the average effective accumulated temperature of generation development of B.glabrata,were Guangdong and Hainan,and part regions of other 9 provinces.The overlay analysis of GIS maps of the sur-vival extreme high temperatures and low temperatures of B.glabrata with the GIS map of the average effective accumulated tem-perature of generation development in 2010 showed that the whole region of Hainan and part regions of Guangdong,Guangxi,Yunnan and Fujian were potential geographical distribution regions of colonization risk of B.glabrata.The overlay analysis of GIS maps of the survival extreme high temperatures and low temperatures of B.glabrata with the GIS map of the average effective accumulated temperature of generation development from 1955 to 2010 showed that the potential geographical distribution re-gions of B.glabrata was expanding from the whole region of Hainan and part regions of Guangdong in 1955 to the whole region of Hainan and part regions of Guangdong,Guangxi,Yunnan and Fujian in 2010.Conclusions If B.glabrata snails were intro-duced into the Mainland China,the potential geographical distribution regions would be the whole region of Hainan and part re-gions of Guangdong,Guangxi and Yunnan.The changes of risk range and risk intensity present the trends of expanding and in-creasing from the south to the north gradually.

Prevalence,antimicrobial resistance patterns and virulence gene analysis of Salmonella spp.originated from ducks in Chengdu area,China / 中国人兽共患病学报

We investigated the contamination,antimicrobial resistance and the virulence genes carriage of Salmonella spp. in duck slaughter chain.Suspected strains were isolated from slaughterhouse samples according to GB 4789.4-2010 and identi-fied by duplex PCR,and then the positive strains were used for serotype analysis.Subsequently,positive strains were tested against 10 different antimicrobial agents using Kirby-Bauer disk diffusion method,the results were determined on the basis of CLSI standard.Finally,9 virulence genes were detected among positive strains by PCR.The results showed that 9 9 Salmonella isolates were recovered from 343 samples and total isolation rate was 28.86%.The prevalence of Salmonella before slaughte-ring,at depilation stage,at evisceration stage,in duck meat and after slaughtering was 45.71%,22.68%,24.72%, 38.33% and 25.81%,respectively.Seven serotypes were de-tected and most of them were S.Indiana,S.Newlands,S. Anatum.The Salmonella isolates were most frequently re-sistant to nalidixic acid(91.92%),the resistance rates of tet-racycline (43.43%),ampicillin (42.42%),trimethoprim-sulfamethoxazole (34.34%),ciprofloxacin (29.29%),ceftri-axone (27.27%),gentamycin (24.24%),and kanamycin (22.22%)were at a medium level.The resistance rates of amoxicillin-clavulanic acid (9.09%)and minocycline (6.06%)were relatively low.The multi-drug resistance rate of Salmonella isolates,which was 47.47%,showed a high especially in S.Indi-ana,S.Typhi and S.Typhimurium.It was notable that the harboring rates of virulence gene spvR(94.95%),avrA (93.94%),ssaQ(90.91%),mgtC(87.88%),sopB(83.84%),bcfC(80.81%),siiD(77.78%)among Salmonella isolates were at high level,in contrast to the lower carriage rates of spvB(29.29%),spvC (11.11%).In summary,the results indica-ted that the duck slaughter chain was easily contaminated by Salmonella spp.with different serotypes,different antibiotic re-sistant patterns and high virulence genes harboring rate.Relevant slaughterhouse and departments should strengthen supervi-sion in sanitation and manage the use of antimicrobial agents,to guarantee food safety and public health．

Intestinal Microbiota Metabolism and Atherosclerosis / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>This review aimed to summarize the relationship between intestinal microbiota metabolism and cardiovascular disease (CVD) and to propose a novel CVD therapeutic target.</p><p><b>DATA SOURCES</b>This study was based on data obtained from PubMed and EMBASE up to June 30, 2015. Articles were selected using the following search terms: "Intestinal microbiota", "trimethylamine N-oxide (TMAO)", "trimethylamine (TMA)", "cardiovascular", and "atherosclerosis".</p><p><b>STUDY SELECTION</b>Studies were eligible if they present information on intestinal microbiota metabolism and atherosclerosis. Studies on TMA-containing nutrients were also included.</p><p><b>RESULTS</b>A new CVD risk factor, TMAO, was recently identified. It has been observed that several TMA-containing compounds may be catabolized by specific intestinal microbiota, resulting in TMA release. TMA is subsequently converted to TMAO in the liver. Several preliminary studies have linked TMAO to CVD, particularly atherosclerosis; however, the details of this relationship remain unclear.</p><p><b>CONCLUSIONS</b>Intestinal microbiota metabolism is associated with atherosclerosis and may represent a promising therapeutic target with respect to CVD management.</p>

Nanometer detection of self-assembly morphology of sodium hyaluronate injection on mica / 南方医科大学学报

<p><b>OBJECTIVE</b>To detect the self-assembly morphology of sodium hyaluronate injection on mica using atomic force microscopy(AFM).</p><p><b>METHODS</b>Atomic force microscopy with nanometer resolution was used to observe the self-assembly morphology of different concentrations of sodium hyaluronate injection on mica at room temperature.</p><p><b>RESULTS</b>The self-assembly morphology of 0.001, 0.01, and 0.1 mg/ml sodium hyaluronate injection on mica featured piebald, reticular and dendritic structures, respectively. At 1 and 5 mg/ml, sodium hyaluronate injection displayed bacilliform and spherical structures on mica, respectively; the diameter and height of the particles of 5 mg/ml sodium hyaluronate was 197.97±78.48 nm and 30.79±18.67 nm, significantly greater than those of 0.1 mg/ml sodium hyaluronate injection (49.52±11.93 nm and 5.37±1.59 nm, respectively, P<0.05).</p><p><b>CONCLUSION</b>The self-assembly morphology of sodium hyaluronate injection on mica varies with its concentration. The piebald and reticular structure may facilitate the function of sodium hyaluronate, and the dendritic feature resembles the representative model of diffusion-limited aggregation (DLA).</p>

Curcumin induces apoptosis through the mitochondria-mediated apoptotic pathway in HT-29 cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2, Bax, Bad, Bcl-xL, caspase-3, poly ADP-ribose polymerase (PARP), and survivin of HT-29 cells.</p><p><b>METHODS</b>HT-29 cells were treated with curcumin (0 approximately 80 micromol/L) for 24 h. The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax, Bcl-2, Bcl-xL, Bad, caspase-3, PARP, and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Curcumin significantly induced the growth inhibition and apoptosis of HT-29 cells. A decrease in expressions of Bcl-2, Bcl-xL and survivin was observed after exposure to 10 approximately 80 micromol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cytochrome c, the activation of caspase-3, and the cleavage of PARP in a dose-dependent manner.</p><p><b>CONCLUSION</b>These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitochondria-mediated pathway.</p>

Fatty acid profiles of muscle from large yellow croaker (Pseudosciaena crocea R.) of different age / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We investigated the fatty acid profiles of muscle from large yellow croaker (Pseudosciaena crocea R.) of different age. One- and two-year-old fish were cultured in floating net cages and sampled randomly for analysis. Moisture, protein, lipid and ash contents were determined by methods of Association of Analytical Chemist (AOAC) International. Fatty acid profile was determined by gas chromatography. Crude protein, fat, moisture and ash contents showed no significant differences between the two age groups. The contents of total polyunsaturated fatty acids and docosahexaenoic acid (DHA) were significantly higher and eicosapentaenoic acid (EPA) content was significantly lower in the two-year-old large yellow croaker than in the one-year-old (P<0.05). No significant differences were observed in the contents of total saturated fatty acids and monounsaturated fatty acids, or the ratio of n-3/n-6 fatty acids among the large yellow croakers of the two age groups. We conclude that large yellow croakers are good food sources of EPA and DHA.

Effects of fish protein hydrolysate on growth performance and humoral immune response in large yellow croaker (Pseudosciaena crocea R.) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We investigated the effects of fish protein hydrolysate (FPH) on growth performance and humoral immune response of the large yellow croaker (Pseudosciaena crocea R.). One thousand and two hundred large yellow croakers [initial average weight: (162.75+/-23.85) g] were divided into four groups and reared in floating sea cages (3 m x 3 m x 3 m). The animals were fed with 4 diets: basal diet only (control) or diets supplemented with 5%, 10% and 15% (w/w) FPH. The results show that dietary FPH levels significantly influenced the growth and immunity of the large yellow croaker. Compared with the control group, total weight gain (TWG) in all treatment groups, relative weight gain (RWG) and specific growth rate (SGR) in fish fed with diets supplemented with 10% and 15% FPH were significantly increased (P<0.05). Similar results were observed in immune parameters [lysozyme activity, serum complements, immunoglobulin M (IgM)]. Lysozyme activity, complement C4 and IgM were also significantly increased (P<0.05) in fish fed with diets supplemented with 10% and 15% FPH, while complement C3 level was significantly increased (P<0.05) in all treatment groups. In general, with the supplementation of FPH, particularly at dose of 10%, the growth performance and immunity of the large yellow croaker can be improved effectively.

Influence of dietary conjugated linoleic acid on growth, fatty acid composition and hepatic lipogenesis in large yellow croaker (Pseudosciaena crocea R.) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We examined the effects of conjugated linoleic acid (CLA) on growth, fatty acid composition and enzyme activity of fatty acid oxidation in the liver of large yellow croaker. We divided 1600 fish (average initial weight 150 g) into 4 groups and reared them in 8 cages. Four dietary treatments were formulated to contain 0%, 1%, 2% and 4% (w/w) CLA, respectively. The fish were fed for 10 weeks ad libitum twice daily. We found that the dietary CLA had no effect on growth, biometric parameters and whole body proximate (P>0.05), but showed some significant effects on the fatty acid composition in both muscle and the liver. The activities of lipogenic enzymes were slightly depressed in fish fed with increasing levels of CLA when compared with control (P>0.05). Dietary CLA supplementation had no effects on liver lipid content, but significantly increased the contents of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) (P<0.05) and decreased monounsaturated fatty acid (MUFA) content in both muscle and the liver. Dietary CLA inclusion resulted in significant increases of the biologically active cis-9, trans-11 and trans-10, cis-12 isomers in both tissues (P<0.05). The total accumulation of CLA was higher in the liver (3.83%, w/w) than in muscle (3.77%, w/w) when fed with 4% (w/w) CLA. This study demonstrates that large yellow croakers are capable of absorbing and depositing CLA and long-chain n-3 PUFA in the liver and muscle, showing that this species fed with CLA could be an important human food source for these healthful fatty acids.

Effects of alpha-tocopheryl acetate supplementation in preslaughter diet on antioxidant enzyme activities and fillet quality of commercial-size Sparus macrocephalus / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

This study examined the effects of dietary alpha-tocopheryl acetate supplementation on antioxidant enzyme activities and fillet quality in commercial-size Sparus macrocephalus. Three hundred fish [main initial weight (350+/-12) g] were divided into three groups (E250, E500 and E1000) and reared in 9 cages. The fish were fed for 8 weeks with three diets containing different levels of dietary alpha-tocopheryl acetate (289, 553, 1 069 mg/kg). Over the experimental period, fish were fed to satiation and reached a final mean weight of (465+/-28) g without significant body weight difference and proximate composition difference. Fillet alpha-tocopherol was significantly (P<0.05) different between groups, reaching levels of 14.2, 22.1, 30.9 microg/mg fillet for groups E250, E500 and E1000, respectively. Total serum superoxide dismutase (SOD) activity increased significantly (P<0.05) in fish fed the diets high in alpha-tocopheryl acetate, but serum glutathione peroxidase (GPX) activity was unaffected. In storage on ice, fillets of fish fed the diets high in alpha-tocopheryl acetate exhibited significantly lower (P<0.05) levels of oxidation. These results suggested that increased dietary alpha-tocopheryl acetate could increase its flesh deposition, increase the activity of SOD and prevent lipid peroxidation of Sparus macrocephalus fillets in retail storage on ice.

Influence of fasting on muscle composition and antioxidant defenses of market-size Sparus macrocephalus / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The study was conducted to investigate fasting effects on flesh composition and antioxidant defenses of market-size Sparus macrocephalus. Two hundred fish (main initial weight 580 g) were divided into two groups (control and fasted) and reared in 6 cages. After two weeks of adaptation, group I fasted for 28 d; group II was fed normally as a control. In 3, 7, 14, 21 and 28 d, 6 fish per group were sampled for proximate flesh composition, liver antioxidant enzyme activities and malondialdehyde flesh content analyses. In fasted fish, the reduction of lipid content in muscle occurred after day 3, and, compared to controls, the content of protein decreased from day 14, the activities of liver antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) increased from day 3, and flesh malondialdehyde levels increased from day 21. Flesh fat reduction shows that fasting may be used as a technique to reduce flesh lipid content in Sparus macrocephalus. However, considering flesh protein loss and the subsequent oxidative stress, the fasting technique should be used with precautions.