Liver-enriched transcription factors are critical for the expression of hepatocyte marker genes in mES-derived hepatocyte-lineage cells

<p>Abstract</p> <p>Background</p> <p>Induction of stem cell differentiation toward functional hepatocytes is hampered by lack of knowledge of the hepatocyte differentiation processes. The overall objective of this project is to characterize key stages in the hepatocyte differentiation process.</p> <p>Results</p> <p>We established a mouse embryonic stem (mES) cell culture system which exhibited changes in gene expression profiles similar to those observed in the development of endodermal and hepatocyte-lineage cells previously described in the normal mouse embryo. Transgenic mES cells were established that permitted isolation of enriched hepatocyte-lineage populations. This approach has isolated mES-derived hepatocyte-lineage cells that express several markers of mature hepatocytes including albumin, glucose-6-phosphatase, tyrosine aminotransferase, cytochrome P450-3a, phosphoenolpyruvate carboxykinase and tryptophan 2,3-dioxygenase. In addition, our results show that the up-regulation of the expression levels of hepatocyte nuclear factor-3α, -4α, -6, and CCAAT-enhancer binding protein-β might be critical for passage into late-stage differentiation towards functional hepatocytes. These data present important steps for definition of regulatory phenomena that direct specific cell fate determination.</p> <p>Conclusion</p> <p>The mES cell culture system generated in this study provides a model for studying transition between stages of the hepatocyte development and has significant potential value for studying the molecular basis of hepatocyte differentiation <it>in vitro</it>.</p

Engineering Chinese Hamster Ovary cells for enhanced protein secretion

Please click Additional Files below to see the full abstract

Developing methodologies to optimise production of recombinant antibodies at specific phases of Chinese Hamster Ovary (CHO) cell culture

Please click Additional Files below to see the full abstract

Effects of diets containing vegetable oil on expression of genes involved in highly unsaturated fatty acid biosynthesis in liver of Atlantic salmon (Salmo salar)

Fish are an important dietary source of the long-chain C20 and C22 highly unsaturated fatty acids (HUFA), arachidonate (20:4n-6), eicosapentaenoate (20:5n-3) and docosahexaenoate (22:6n-3), that are crucial to the health of higher vertebrates and that can be beneficial in human diets. Δ5 and Δ6 fatty acid desaturases, and fatty acid elongases are critical enzymes in the biosynthetic pathways of HUFA from shorter chain C18 polyunsaturated fatty acids (PUFA) such as linoleic (18:2n-6) and α-linolenic (18:3n-3) acids. Recently, full-length cDNAs for fatty acid desaturase and elongase enzymes have been cloned from Atlantic salmon. Functional characterisation of the desaturase revealed n-3 Δ5 desaturase activity, whereas the elongase had broad substrate specificity for PUFA with a range of chain lengths from C18 to C22. The study described here was primarily focused on the nutritional regulation of genes involved in the HUFA biosynthetic pathway in Atlantic salmon. A feeding trial was performed whereby salmon smolts in seawater pens were fed for 40 weeks on five different diets. The diets consisted of a control diet containing fish oil (FO) and four diets in which the FO was replaced in a graded manner by linseed oil (LO). Specifically, in terms of added oils, the five diets were 100% FO (FO), 100%LO (LO100) and FO/LO in ratios of 3:1 (LO25), 1:1 (LO50) and 1:3 (LO75). Fish were sampled at 20 and 40 weeks, and samples of liver were collected for lipid analyses and total RNA extraction. Hepatocytes were also prepared and the activity of the HUFA biosynthetic pathway determined. Expression of fatty acid desaturase and elongase genes was determined by quantitative real time PCR and the ratio of the copy number of the targeted gene against that of β-actin was calculated. The results showed that after 20 weeks of feeding, desaturase and elongase gene expression in liver was increased in a graded manner by increasing dietary LO. Expression of both genes was positively and negatively correlated with dietary 18:3n-3 and n-3 HUFA, respectively. By 40 weeks of feeding, expression of neither gene showed the same degree of correlation with dietary fatty acid composition. In contrast, activity of the HUFA biosynthetic pathway, which showed some association with diet at 20 weeks, was positively and significantly correlated with dietary LO after 40 weeks of feeding. Elongation activity reflected the overall activity of the HUFA biosynthetic pathway to a greater degree than Δ5 desaturation activity. The possible mechanisms underlying the observed results and the regulation of the HUFA biosynthetic pathway are discussed

Highly unsaturated fatty acid synthesis in vertebrates: new insights with the cloning and characterization of a delta6 desaturase of Atlantic salmon

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a ∆5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterized previously. Here we report cloning and functional characterisation of a ∆6 fatty acyl desaturase of Atlantic salmon, and describe its genomic structure, tissue expression and nutritional regulation. A salmon genomic library was screened with a salmon ∆5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106bp containing an ORF of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the haem-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly ∆6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the ∆6 desaturase gene comprised 13 exons in 7965 bp of genomic DNA. Quantitative real time PCR assay of gene expression in Atlantic salmon showed that both ∆6 and ∆5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver and brain, and less so in kidney, heart, gill, adipose tissue, muscle and spleen. Furthermore, expression of both ∆6 and ∆5 fatty acyl desaturase genes in intestine, liver, red muscle and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil

Use of a ‘molecular tug’ to overcome limitations in the production of ‘difficult to express’ recombinant proteins

In recent years there has been an increased drive towards the production of recombinant proteins in large amounts using rapid cell culture processes. Mammalian expression systems such as Chinese Hamster Ovary (CHO) cells have remained the preferred choice for large-scale recombinant protein production (Walsh, 2014). However, in mammalian cells certain recombinant targets can prove to be ‘difficult to express’ and require extensive upstream process optimisation which can have a negative impact on industrial processes. This study has investigated the molecular mechanisms that are responsible for poor recombinant protein production. Model proteins from the Tissue Inhibitors of Metalloproteinase (TIMP) family, TIMP-2, TIMP-3 and TIMP-4, were subject to detailed study to characterise the molecular mechanisms that limit production of recombinant proteins with high sequence homology (Hussain et al., 2017). TIMP-2, -3 and -4, share significant sequence/structural homology (Douglas et al., 1997, Garcia et al., 2012), but show differences when produced in a transient CHO expression system (Hussain et al., 2017) . A systematic screen of the protein expression pathway showed all three TIMPs were detectable at the mRNA and protein level within the cell but only TIMP-2 was secreted in significant amounts into the culture medium. Analysis of the intracellular protein suggested the post-translational processing of poorly expressed TIMPs was limiting. A protein engineering approach was employed to overcome challenges in the production of these ‘difficult to express’ TIMP proteins. This approach involved the attachment of a furin-cleavable pro-sequence from a secretory growth factor to recombinant targets. The pro-sequence was predicted to act as a ‘molecular tug’ to aid transit through the protein expression pathway and/or promote correct post-translational processing. Initially, the furin-cleavable pro-sequence was added to TIMP-3 (non-secreted), which resulted in secretion of TIMP-3, however incomplete processing of the pro-sequence was observed. The protein engineering approach was optimised further and applied in combination with cell engineering (furin overexpression) to TIMP-4 (poorly secreted), which was also successfully detected in significantly higher amounts in the culture medium (Hussain et al., 2017). Together, the described protein engineering approach presents a novel strategy to increase the production of ‘difficult-to-express’ recombinant targets. References: Douglas, D. A., Shi, Y. E. & Sang, Q. X. A. 1997. Computational sequence analysis of the tissue inhibitor of metalloproteinase family. Journal of Protein Chemistry, 16, 237-255. Garcia, M. P. S., Suarez-Penaranda, J. M., Gayoso-Diz, P., Barros-Angueira, F., Gandara-Rey, J. M. & Garcia-Garcia, A. 2012. Tissue inhibitor of metalloproteinases in oral squamous cell carcinomas - A therapeutic target? Cancer Letters, 323, 11-19. Hussain, H., Fisher, D. I., Abbott, W. M., Roth, R. G. & Dickson, A. J. 2017. Use of a protein engineering strategy to overcome limitations in the production of Difficult to Express recombinant proteins. Biotechnology and Bioengineering, 114, 2348-2359. Walsh, G. 2014. Biopharmaceutical benchmarks 2014. Nature Biotechnology, 32, 992-1000

Zebrafish cDNA encoding multifunctional fatty acid elongase involved in production of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids

Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids. The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina. A search of the Genbank database identified several EST sequences, including one obtained from zebrafish (Danio rerio), with high similarity to gLELO. The full-length transcript, ZfELO, encoding a polypeptide of 291 amino acid residues was isolated from zebrafish liver cDNA. The predicted amino acid sequence of the open reading frame (ORF) shared high similarity with the elongases of C. elegans and human. When expressed in Saccharomyces cerevisiae, the zebrafish ORF conferred the ability to lengthen the chain of a range of C18, C20 and C22 polyunsaturated fatty acids, indicating that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is not only feasible, but that one elongase enzyme can perform all three elongation steps required. The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously

Molecular cloning and functional characterization of fatty acyl desaturase and elongase cDNAs involved in the production of eicosapentaenoic and docosahexanoic acids from alpha-linolenic acid in Atlantic salmon (Salmo salar)

Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFA) and, with declining fisheries, farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term, and will constrain continuing growth of aquaculture activities. A greater understanding of how fish metabolise and biosynthesise HUFA may lead to effective use of more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimising the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterisation of two genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase cDNA, SalDes, was isolated that included an open reading frame (ORF) of 1362 bp specifying a protein of 454 amino acids. The protein sequence included all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing a haem-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast, Saccharomyces cerevisiae, showed SalDes is predominantly an omega-3 Δ5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from α-linolenic acid (18:3n-3). The desaturase showed only low levels of Δ6 activity towards C18 polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that includes an ORF of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo includes characteristic features of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast, SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C18 > C20 > C22. Thus, all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3 are displayed by this one polypeptide product

Graduates’ Attitudes to Research Skill Development in Undergraduate Media Education

This paper examines the attitudes of graduates employed in different segments of the media industry to the development of research skills during their studies. Qualitative interviews were conducted with ten graduates employed in different jobs approximately one year after graduation to understand how applicable they found generic and media-specific research skills to their employment. The study was conducted as part of a wider project evaluating the application of a systematic framework for research skills development, across whole degree programmes. The interviews demonstrate broad agreement regarding the value of research skills for media employment. However, there were divergent opinions about the need to articulate research skills explicitly and the value of media-specific skill for current employment situations. Interviewees also indicated varying levels of awareness regarding the relevance research skills have across different employment contexts. Therefore, it is important that media-educators understand how their students’ differing career destinations immediately after graduation influence formal and informal evaluations of the quality of their course. We conclude that this educational challenge is best addressed by implementing a consistent framework for research education that improves students’ metacognitive awareness of the transferability of this graduate attribute across multiple industries and career destinations

Elevated carbon dioxide and ozone alter productivity and ecosystem carbon content in northern temperate forests

Three young northern temperate forest communities in the north‐central United States were exposed to factorial combinations of elevated carbon dioxide ( CO 2 ) and tropospheric ozone (O 3 ) for 11 years. Here, we report results from an extensive sampling of plant biomass and soil conducted at the conclusion of the experiment that enabled us to estimate ecosystem carbon (C) content and cumulative net primary productivity ( NPP ). Elevated CO 2 enhanced ecosystem C content by 11%, whereas elevated O 3 decreased ecosystem C content by 9%. There was little variation in treatment effects on C content across communities and no meaningful interactions between CO 2 and O 3 . Treatment effects on ecosystem C content resulted primarily from changes in the near‐surface mineral soil and tree C, particularly differences in woody tissues. Excluding the mineral soil, cumulative NPP was a strong predictor of ecosystem C content ( r 2  = 0.96). Elevated CO 2 enhanced cumulative NPP by 39%, a consequence of a 28% increase in canopy nitrogen (N) content (g N m −2 ) and a 28% increase in N productivity ( NPP /canopy N). In contrast, elevated O 3 lowered NPP by 10% because of a 21% decrease in canopy N, but did not impact N productivity. Consequently, as the marginal impact of canopy N on NPP (∆ NPP /∆N) decreased through time with further canopy development, the O 3 effect on NPP dissipated. Within the mineral soil, there was less C in the top 0.1 m of soil under elevated O 3 and less soil C from 0.1 to 0.2 m in depth under elevated CO 2 . Overall, these results suggest that elevated CO 2 may create a sustained increase in NPP , whereas the long‐term effect of elevated O 3 on NPP will be smaller than expected. However, changes in soil C are not well‐understood and limit our ability to predict changes in ecosystem C content.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108065/1/gcb12564.pd