Ziwen Zhao

Technical note: Selection of suitable reference genes for studying gene expression in milk somatic cell of yak (Bos grunniens) during the lactation cycle

Quantitative real-time PCR is the most sensitive technique for gene expression analysis. Data normalization is essential to correct for potential errors incurred in all steps from RNA isolation to PCR amplification. The commonly accepted approach for normalization is the use of reference gene. Until now, no suitable reference genes have been available for data normalization of gene expression in milk somatic cells of lactating yaks across lactation. In the present study, we evaluated the transcriptional stability of 10 candidate reference genes in milk somatic cells of lactating yak, including ACTB, B2M, GAPDH, GTP, MRPL39, PPP1R11, RPS9, RPS15, UXT, and RN18S1. Four genes, RPS9, PPP1R11, UXT, and MRPL39, were identified as being the most stable genes in milk somatic cells of lactating yak. Using the combination of RPS9, PPP1R11, UXT, and MRPL39 as reference genes, we further assessed the relative expression of 4 genes of interest in milk somatic cells of yak across lactation, including ELF5, ABCG2, SREBF2, and DGAT1. Compared with expression in colostrum, the overall transcription levels of ELF5, ABCG2, and SREBF2 in milk were found to be significantly upregulated in early, peak, and late lactation, and significantly downregulated thereafter, before the dry period. A similar pattern was observed in the relative expression of DGAT1, but no significant difference was revealed in its expression in milk from late lactation compared with colostrum. Based on these results, we suggest that the geometric mean of RPS9, PPP1R11, UXT, and MRPL39 can be used for normalization of real-time PCR data in milk somatic cells of lactating yak, if similar experiments are performed.

Molecular Characterization of Prolactin cDNA and Its Expression Pattern in Skin Tissue of Liaoning Cashmere Goat

The Liaoning Cashmere goat is a local breed found in the Buyun Mountains of the Liaotung Peninsula. It has an estimated population of 1.5 million individuals and is well adapted to the local environment. The goats are white with two horns in both males and females. The main purpose of this breed is the production of cashmere, which is of great economic importance to the local people. The breed is characterized by a high cashmere yield, averaging 0.54 kg for male adults and 0.47 kg for female adults. The diameter of the Liaoning Cashmere fiber, however, is relatively large, compared with that of some other cashmere goat breeds in China (Jin et al. 2011), and the thinner fibers bring a higher price in the market (Li et al. 2005). Breeders are now attempting to lower the fiber diameter of the Liaoning Cashmere goat through modern breeding and genetics technology. Little is known regarding the molecular regulatory mechanism of cashmere growth in this breed. Therefore, it is necessary for us to understand the growth biology of cashmere of

Effect of nisin and perilla oil combination against Listeria monocytogenes and Staphylococcus aureus in milk

In the present study, in vitro interaction of nisin and perilla oil (PO) against 20 food-borne isolates of L. monocytogenes and S. aureus were assessed using a checkerboard microdilution method. Synergism was observed in tested strains with the fractional inhibitory concentration indexs (FICIs) ranges from 0.125–0.25 and 0.19–0.375, respectively. Scanning electron microscopy was carried out to investigate the effect of nisin and PO on the integrity of cell wall and membrane of L. monocytogenes and S. aureus. The results showed that nisin and PO were more effective in damaging cell wall and membrane in combination.

In vitro antimicrobial activity of honokiol against Staphylococcus aureus in biofilm mode.

Abstract Staphylococcus aureus (S. aureus) can attach to food, host tissues and the surfaces of medical implants and form a biofilm, which makes it difficult to eliminate. The purpose of this study was to evaluate the effect of honokiol on biofilm-grown S. aureus. In this report, honokiol showed effective antibacterial activity against S. aureus in biofilms. S. aureus isolates are capable of producing distinct types of biofilms mediated by polysaccharide intercellular adhesion (PIA) or extracellular DNA (eDNA). The biofilms’ susceptibility to honokiol was evaluated using confocal laser scanning microscopy (CLSM) analysis. The transcript levels of the biofilm-related genes, the expression of PIA, and the amount of eDNA of biofilm-grown S. aureus exposed to honokiol were also investigated. The results of this study show that honokiol can detach existing biofilms, kill bacteria in biofilms, and simultaneously inhibit the transcript levels of sarA, cidA and icaA, eDNA release, and the expression of PIA.

Effect of honokiol on exotoxin proteins listeriolysin O and p60 secreted by Listeria monocytogenes.

Listeria monocytogenes is considered one of the most important foodborne pathogens. The virulence-related proteins listeriolysin O (LLO) and p60 are critical factors involved in Listeria pathogenesis. In the present study, we investigated the effect of honokiol on LLO and p60 secreted from L. monocytogenes. A listeriolysin assay was used to investigate the haemolytic activities of L. monocytogenes exposed to honokiol, and the secretion of LLO and p60 was detected by immunoblot analysis. Additionally, the influence of honokiol on the transcription of LLO and p60 genes (hly and iap, respectively) was analysed by real-time reverse transcription PCR. TNF-α release assays were performed to elucidate the biological relevance of changes in LLO and p60 secretion induced by honokiol. According to the data, honokiol showed good anti-L. monocytogenes activity, with MICs of 8-16 μg ml(-1), and the secretion of LLO and p60 was decreased by honokiol. In addition, the transcription of hly and iap was inhibited by honokiol. Our results indicate that TNF-α production by RAW264.7 cells stimulated with L. monocytogenes supernatants was inhibited by honokiol. Based on these data, we propose that honokiol could be used as a promising natural compound against L. monocytogenes and its virulence factors.