Three-dimensional perfused cell culture

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell–cell and cell–extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review.     

Evidence for Inbreeding and Genetic Differentiation among Geographic Populations of the Saprophytic Mushroom Trogia venenata from Southwestern China

During the past 40 years, more than 400 Sudden Unexplained Deaths (SUDs) have occurred in Yunnan, southwestern China. Epidemiological and toxicological analyses suggested that a newly discovered mushroom called Trogia venenata was the leading culprit for SUDs. At present, relatively little is known about the genetics and natural history of this mushroom. In this study, we analyzed the sequence variation at four DNA fragments among 232 fruiting bodies of T. venenata collected from seven locations. Our ITS sequence analyses confirmed that all the isolates belonged to the same species. The widespread presence of sequence heterozygosity within many strains at each of three protein-coding genes suggested that the fruiting bodies were diploid, dikaryotic or heterokaryotic. Within individual geographic populations, we found significant deviations of genotype frequencies from Hardy-Weinberg expectations, with the overall observed heterozygosity lower than that expected under random mating, consistent with prevalent inbreeding within local populations. The geographic populations were overall genetically differentiated. Interestingly, while a positive correlation was found between population genetic distance and geographic distance, there was little correlation between genetic distance and barium concentration difference for the geographic populations. Our results suggest frequent inbreeding, geographic structuring, and limited gene flow among geographic populations of T. venenata from southwestern China.     

RNA‐seq analysis reveals new candidate genes for drip loss in a Pietrain × Duroc × Landrace × Yorkshire population

Drip loss, one of the most important meat quality traits, is characterized by low heritability. To date, the genetic factors affecting the drip loss trait have not been clearly elucidated. The objective of this study was to identify critical candidate genes affecting drip loss. First, we generated a Pietrain × Duroc × Landrace × Yorkshire commercial pig population and obtained phenotypic values for the drip loss trait. Furthermore, we constructed two RNA libraries from pooled samples of longissimus dorsi muscles with the highest (H group) and lowest (L group) drip loss and identified the differentially expressed genes (DEGs) between these extreme phenotypes using RNA‐seq technology. In total, 25 883 genes were detected in the H and L group libraries, and none was specifically expressed in only one library. Comparative analysis of gene expression levels found that 150 genes were differentially expressed, of which 127 were upregulated and 23 were downregulated in the H group relative to the L group. In addition, 68 drip loss quantitative trait loci (QTL) overlapping with 63 DEGs were identified, and these QTL were distributed mainly on chromosomes 1, 2, 5 and 6. Interestingly, the triadin (TRDN) gene, which is involved in muscle contraction and fat deposition, and the myostatin (MSTN) gene, which has a role in muscle growth, were localized to more than two drip loss QTL, suggesting that both are critical candidate genes responsible for drip loss.     

A divergent lineage among Octopus minor (Sasaki, 1920) populations in the Northwest Pacific supported by DNA barcoding

Octopus minor (Sasaki, 1920) is an important economic fishery resource in China. In order to explore the stock information and the phylogeographic status of O. minor, mitochondrial DNA cytochrome c oxidase subunit I (COI, 565?bp) and 16S rRNA (493?bp) genes were amplified from 11 different sampling locations. Genetic diversity evaluated by haplotypic and nucleotidic diversity implied high diversity in Lianjiang, and relatively low diversity in Rongcheng, which suggests that effective measures to protect the O. minor resource in this area are urgently required. Private haplotypes and remarkable higher pairwise Φ_ST in Yilan are responsible for the deep genetic divergence between Yilan and the 10 other populations. Haplotypes networks and two clusters’ topological structure also support the distinct subgroups (lineages A and lineages B), which apparently possess smaller genetic variation than mean interspecies distance. Taiwan island and its strait may act as a natural barrier that restricts the gene flow from the mainland. Deep genetic divergence between mainland and Taiwanese east coasts suggests different genetic stock, indicating that different management strategies are required.     

Investigation of fullerenol-induced changes in poroelasticity of human hepatocellular carcinoma by AFM-based creep tests

In this study, atomic force microscopy (AFM) is used to investigate the alterations of the poroelastic properties of hepatocellular carcinoma (SMMC-7721) cells treated with fullerenol. The SMMC-7721 cells were subject to AFM-based creep tests, and a corresponding poroelastic indentation model was used to determine the poroelastic parameters by curve fitting. Comparative analyses indicated that the both permeability and diffusion of fullerenol-treated cells increased significantly while their elastic modulus decreased by a small amount. From the change in the trend of the determined parameter, we verified the corresponding alternations of cytoskeleton (mainly filaments actin), which was reported by the previous study using confocal imaging method. Our investigation on SMMC-7721 cell reveals that the poroelastic properties could provide a better understanding how the cancer cells are affected by fullerenol or potentially other drugs which could find possible applications in drug efficacy test, cancer diagnosis and secure therapies.     

Splenic gene expression profiling in White Leghorn layer inoculated with the Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (SE) is a foodborne pathogen that can threaten human health through contaminated poultry products. Live poultry, chicken eggs and meat are primary sources of human salmonellosis. To understand the genetic resistance of egg‐type chickens in response to SE inoculation, global gene expression in the spleen of 20‐week‐old White Leghorn was measured using the Agilent 4 × 44 K chicken microarray at 7 and 14 days following SE inoculation (dpi). Results showed that there were 1363 genes significantly differentially expressed between inoculated and non‐inoculated groups at 7 dpi (I7/N7), of which 682 were up‐regulated and 681 were down‐regulated genes. By contrast, 688 differentially expressed genes were observed at 14 dpi (I14/N14), of which 371 were up‐regulated genes and 317 were down‐regulated genes. There were 33 and 28 immune‐related genes significantly differentially expressed in the comparisons of I7/N7 and I14/N14 respectively. Functional annotation revealed that several Gene Ontology (GO) terms related to immunity were significantly enriched between the inoculated and non‐inoculated groups at 14 dpi but not at 7 dpi, despite a similar number of immune‐related genes identified between I7/N7 and I14/N14. The immune response to SE inoculation changes with different time points following SE inoculation. The complicated interaction between the immune system and metabolism contributes to the immune responses to SE inoculation of egg‐type chickens at 14 dpi at the onset of lay. GC, TNFSF8, CD86, CD274, BLB1 and BLB2 play important roles in response to SE inoculation. The results from this study will deepen the current understanding of the genetic response of the egg‐type chicken to SE inoculation at the onset of egg laying.