Optimization of Stachydrine Hydrochloride in Leonurus Extraction Method by HPLC-ELSD

The study was aimed to extract stachydrine hydrochloride in leonurus by HPLC-ELSD,and establish hydrochloride stachydrine determination methods for providing theoretical basis of leonurus extraction process optimization.The chromatographic conditions:venusil HILIC hydrophilic column;Mobile phase was acetonitrile -0.2% acetic acid solution (volume ratio 80:20);The flow rate was 1.0 mL/min;And the column temperature was 30 ℃;The detector was ELSD and drift tube temperature was 80 ℃ with the atomization chamber temperature 50 ℃ and carrier gas volume flow 1.0000 mL/min.The results showed that there was a good linear relationship when the stachydrine hydrochloride content was 0.49 to 4.91 μg (R²=0.9993);There was the highest rate of active ingredients extraction of when the extraction time was 1 h,material to water ratio was 20:1 and extracted for 2 times.Combined with production practice,the final optimization scheme was that the extraction time was 1.5 h,material to water ratio was 20:1 and extracted for 2 times.In summary,the determination of stachydrine hydrochloride in leonurus should use HPLC-ELSD method which had high specificity,good stability,response and reproducibility and easy to handle.This method could provide the theoretical basis for optimizing the leonurus extraction process.     

Nutritional Contribution of Biofloc within the Diet of Growout and Broodstock of Litopenaeus vannamei,Determined by Stable Isotopes and Fatty Acids

The relative contributions of feed sources were determined through the isotopic signal (δ¹³C and δ¹⁵N) and fatty acid profile of feed items, shrimp muscle, and eggs of Litopenaeus vannamei reared in a biofloc system. In the growout phase, the isotope analysis showed the biofloc particle size class ≥250 μm contributed 55–100%; size ≥50 < 250 μm contributed 0–22%; and artificial feed contributed 0–45%. Principal component analysis applied to fatty acid profiles showed that biofloc ≥250 μm and artificial feed were the most important items in shrimp growout. For the egg production, isotope analysis suggested that the most important feed sources according to their relative contributions were polychaetes (0–100%), followed by artemia biomass (0–86%) and semi‐moist feed (0–66%), with lower contributions from squid, mussel, and the muscle of L. vannamei broodstock that had been cultured in biofloc. In terms of fatty acids, the most important items were artemia, polychaetes, and semi‐moist feeds. This work clarified the importance of feed sources for shrimp during culture in biofloc systems and during reproduction. Analysis of stable isotopes and fatty acids can be successfully used to trace the assimilation of nutrients during the nutrition of shrimp.     

Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes

This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.     

Concurrent immune-mediated haemolytic anaemia and severe thrombocytopenia in 21 dogs

The medical records of 21 dogs with concurrent immune-mediated haemolytic anaemia (imha) and severe thrombocytopenia (defined as an automated platelet count of less than 50x10(9)/l, confirmed by the examination of a blood smear) were reviewed. Their mean (sd) age was 5.8 (2.5) years. When compared with the 24,759 dogs in the hospital population for the same period Airedale terriers and dobermanns appeared to be over-represented with odds ratios of 22.5 (95 per cent confidence interval [ci] 5.2 to 97.9) and 7.6 (95 per cent ci 1.8 to 32.7) respectively. The median duration of the dogs' clinical signs was seven days, with a range from one to 17 days. Eleven of the dogs had a history of a tendency to bleed, and 15 had evidence of bleeding when examined. Twenty of the 21 dogs had been treated with glucocorticoids, nine with vincristine, and seven with azathioprine. Their median stay in hospital was four days, with a range from one to 17 days. The median period for which they survived after admission to hospital was five days, with a range from one to 558 days, and 16 of the 21 dogs had died or been euthanased within 30 days of their admission.     

袋鼠疱疹病毒1型实时荧光PCR方法的建立

为实现袋鼠临床样本中疱疹病毒1型(Macropodid herpesvirus 1,MaHV-1)的出入境快速检测,基于MaHV-1的gB基因序列设计特异引物和TaqMan探针,建立了一种MaHV-1荧光PCR检测方法。试验结果显示,该方法只对MaHV-1 gB基因呈现特异性扩增,与禽传染性喉气管炎病毒、伪狂犬病毒和牛传染性鼻气管炎病毒不发生交叉反应,对阳性标准质粒对照(pCR-MaHV-1-gB)的最低检测限为8个拷贝数/反应。该方法的组内和组间试验的Ct值变异系数介于0.17%～0.96%之间,具有良好的重现性。试验结果表明,本研究建立的实时荧光PCR方法可用于袋鼠MaHV-1的病原学检测。     

Seroepidemiology and genotyping of hepatitis E virus in Singapore reveal rise in number of cases and similarity of human strains to those detected in pig livers

Hepatitis E virus (HEV) causes 20 million infections worldwide yearly, of which only about 3.3 million are symptomatic. In developed Asian countries, HEV strains detected in human sera and in food sources were genetically similar, suggesting that indigenous HEV infections may be largely food‐borne. To assess the burden of hepatitis E in Singapore, we performed a seroepidemiologic study of the infection. Additionally, we carried out HEV genotyping on archived, residual HEV IgM‐positive serum samples collected between 2014 and 2016 (n = 449), and on pig liver samples (n = 36) purchased from wet markets and supermarkets. Our study shows a rise in hepatitis E incidence (IgM) from 1.7 to 4.1 cases per 100,000 resident population from 2012 to 2016 and an increase in hepatitis E IgG positivity rate among residents from 14% in 2007 to 35% in 2016. Other findings also suggest the epidemiology of hepatitis E in Singapore has shifted, from it being mainly a disease imported from the Indian subcontinent, to one that is now increasingly prevalent in our resident population. Genotypes obtained from 143 human samples identified the majority to be genotype 3 (n = 121), 21 to be genotype 1 and one to be genotype 4. Further phylogenetic analyses suggest genotype 3a to be the cause of indigenous infections in residents, which showed genetic similarity to the genotype 3a strains detected in pig livers. This link between the strains in the majority of human samples and those in pig livers consumed by the public suggests a possible food‐borne route of HEV infection in Singapore.     

Biofilm production by pathogens associated with canine otitis externa,and the antibiofilm activity of ionophores and antimicrobial adjuvants

Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N‐acetylcysteine (NAC), Tris‐EDTA and disodium EDTA) were investigated spectrophotometrically (OD_570nm) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S. pseudintermedius clinical isolates were weak biofilm producers. 19/20 P. aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000–10,000 µg/ml and from 20,000–80,000 µg/ml against S. pseudintermedius and P. aeruginosa, respectively. Tris‐EDTA eradicated P. aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800 µg/ml. The MBEC was up to 16‐fold and eightfold higher than the MIC/MBC of NAC and Tris‐EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470 µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris‐EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm‐associated OE in dogs.     

Mechanism of AKT1 negatively regulating breast cancer cell migration

AIM: To investigate the molecular mechanism and downstream signaling pathway by which AKT1 inhibition regulates breast cancer cell migration. METHODS: RNA interference was used to knockdown the expression of AKT1. Western blot assay was performed to examine the expression of AKT1 total protein, β-catenin total protein and β-catenin nuclear protein. Immunofluorescence was used to examine the cellular localization of β-catenin. Transwell assay was used to investigate whether β-catenin nuclear accumulation as an alternative pathway was responsible for breast cancer metastasis induced by AKT1 inhibition. RESULTS: The total protein expression of AKT1 was decreased in MCF-7 and MDA-MB-231 cells treated with AKT1 siRNA. A significant increase in the protein expression of β-catenin was observed in MCF-7 cells and MDA-MB-231 cells treated with AKT1 siRNA. Immunofluorescence staining showed that MCF-7 cells and MDA-MB-231 cells displayed strong β-catenin staining in the cytoplasm and nucleus after knockdown of AKT1 expression. The ability of tumor cell migration increased dramatically after treated with AKT1 specific siRNA in the breast cancer MCF-7 cells and MDA-MB-231 cells in Transwell assay. XAV-939 reversed breast cancer cell migration induced by knockdown of AKT1 expression. CONCLUSION: β-catenin nuclear accumulation contributes to AKT1 inhibition-mediated breast cancer cell migration.     

Perinatal hypoxia induces subsequent retinal degeneration in the offspring of ovoviviparous fish, Xiphophorous maculates

OBJECTIVE: This experiment evaluated the perinatal hypoxic effect on the retina of offspring of the ovoviviparous fish. ANIMAL STUDIED: The ovoviviparous fish Xiphophorous maculates was used for the experiment. PROCEDURE: The mothers were kept in a hypoxic environment of 3.5% oxygen for 6 h, starting 30 h before hatching. Subsequently, the retinae of the offspring were fixed, sectioned at 6 microm and evaluated microscopically from the age of 1 to 35 days. RESULTS: Degeneration of the outer nuclear layer of the retina was noted on the 3rd day and severe retinal degeneration was observed on the 35th day. Immunocytochemistry confirmed apoptosis by TUNEL reaction. There was no difference in neovascularization, as revealed by vascular endothelial growth factor, between controls (group 1) and hypoxic fish (group 2). CONCLUSIONS: Perinatal hypoxia could have long-lasting effects on the central nervous system in some species.     

Effects of Landfill Gas on Growth and Nitrogen Fixation of Two Leguminous Trees (Acacia Confusa,Leucaena Leucocephala)

A study was made on the effects of landfill gas on ARA (acetylene reducing activity) of nodules of two woody legumes (Acacia confusa and Leucaena leucocephala) widespread on landfill sites in Hong Kong. The effects of the three main components of landfill gas, O₂, CO₂ and CH4, were first measured separately over a 1-hr period. Maximum ARA was found at 20% O₂ (close to atmospheric partial pressure) and ARA decreased as the O₂ decreased in the range of 16–1%. Acacia confusa nodular ARA was significantly inhibited at 30–50% CO₂, but not Leucaena leucocephala nodular ARA. CH₄ had no significant effect on ARA of either species. As the landfill gas concentrations in the landfill topsoil were mostly > 10% O₂ and < 10% CO₂, root nodules should fix N₂ effectively over these ranges of gases. A four-week test was conducted to assess the long-term influence of landfill gas on seedlings of the two legumes. Landfill gas and elevated CO₂ both suppressed their growth and their nodular ARA. Even under the influence of the gases, however, seedlings with nodules formed a higher biomass than seedlings lacking nodules. The growth of the two legumes under actual landfill conditions was investigated by transplanting non-inoculated and pre-inoculated seedlings to two landfill sites in Hong Kong: Junk Bay and Shuen Wan Landfill. After six months, most of the non-inoculated seedlings became infected: Acacia confusa 63 and 70%, Leucaena leucocephala 17 and 89%, respectively, at the test sites. The results indicate that there were free rhizobia at these landfill sites to infect the legumes and they had formed effective nodules to fix N₂ under landfill conditions.