A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

Zinc Fingers 10 and 11 of Miz-1 undergo conformational exchange to achieve specific DNA binding.

Miz-1 (ZBTB17) is a poly-zinc finger BTB/POZ transcription factor with 12 consecutive C2H2 zinc fingers (ZFs) that binds transcriptional start sites (TSSs) to regulate the expression of genes involved in cell development and proliferation. As of now, it is not known which of the 12 consecutive ZFs are responsible for the recognition of the 24 base pair consensus sequence found at these TSSs. Evidence suggests ZFs 7-12 plays this role. We provide validation for this and describe the structural and dynamical characterization of unprecedented conformational exchange in the linker between ZFs 10 and 11. This conformational exchange uncouples ZFs 7-10 from 11 and 12 and promotes a scanning-recognition mechanism through which the two segments cooperate to bind two sub-sites at both ends of the consensus. We further show that this can result in the coiling of TSSs as part of Miz-1's mechanism of transcriptional transactivation.

Effect of emulsifiers on linseed oil emulsion structure, lipolysis and oxidation during in vitro digestion.

Health benefits have been associated with the consumption of omega-3 polyunsaturated fatty acids (PUFA). Linseed oil is rich in long chain omega-3 PUFA, but can generate toxic compounds due to its high susceptibility to oxidation. The nature of the emulsifier can affect both lipolysis and oxidation during digestion since these phenomena occur at the oil-water interface. The objective of this study was to compare the effect of low-molecular weight surfactants (cetyltrimethylammonium bromide (CTAB), Citrem), protein (sodium caseinate, fish gelatin) and polysaccharides (gum arabic, modified starch) on the structure of linseed oil emulsions, lipolysis and formation of reactive oxidation species during in vitro digestion. The emulsion stabilized with Citrem underwent extensive coalescence in the gastric phase, which strongly decreased the extent of lipid digestion and reduced the formation of oxidation markers relative to other emulsions. Emulsions stabilized by proteins and modified starch showed aggregation with partial coalescence in the gastric phase, but protein-stabilized emulsions showed better resistance to oxidation. This study shows that emulsifier properties affect the susceptibility of the emulsion to aggregation and coalescence in the gastrointestinal environment, and strongly influence the extent of lipid digestion and the formation of reactive oxidation products. These findings point out the importance of the choice of the emulsifier to control the lipid digestibility and the protection of sensible lipids thus promoting optimal nutritional properties in omega-3-enriched foods.

Human Hepatocyte Nuclear Factor 4-α Encodes Isoforms with Distinct Transcriptional Functions.

HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.