Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes

The insufficient ability of specialized cells such as neurons, cardiac myocytes, and epidermal cells to regenerate after tissue damage poses a great challenge to treat devastating injuries and ailments. Recent studies demonstrated that a diverse array of cell types can be directly derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or somatic cells by combinations of specific factors. The use of iPSCs and direct somatic cell fate conversion, or transdifferentiation, holds great promise for regenerative medicine as these techniques may circumvent obstacles related to immunological rejection and ethical considerations. However, producing iPSC-derived keratinocytes requires a lengthy two-step process of initially generating iPSCs and subsequently differentiating into skin cells, thereby elevating the risk of cellular damage accumulation and tumor formation. In this study, we describe the reprogramming of mouse embryonic fibroblasts into functional keratinocytes via the transient expression of pluripotency factors coupled with directed differentiation. The isolation of an iPSC intermediate is dispensable when using this method. Cells derived with this approach, termed induced keratinocytes (iKCs), morphologically resemble primary keratinocytes. Furthermore they express keratinocyte-specific markers, downregulate mesenchymal markers as well as the pluripotency factors Oct4, Sox2, and Klf4, and they show important functional characteristics of primary keratinocytes. iKCs can be further differentiated by high calcium administration in vitro and are capable of regenerating a fully stratified epidermis in vivo. Efficient conversion of somatic cells into keratinocytes could have important implications for studying genetic skin diseases and designing regenerative therapies to ameliorate devastating skin conditions.COST (European Cooperation in Science and Technology) (EU-COST Action BM1302 “Joining Forces in Corneal Regeneration Research”)University of Cypru

Histone acetyltransferase NAA40 modulates acetyl-CoA levels and lipid synthesis

Work in the A.K. laboratory was co-funded by the European Regional Development Fund and the Republic of Cyprus through the Research & Innovation Foundation (Projects: EXCELLENCE/0918/0081, EXCELLENCE/0918/0105 and EXCELLENCE/1216/0215) and was also supported by a Marie Skłodowska-Curie individual fellowship grant (no. 890750) to E.C. JLG’s laboratory is supported by the Wellcome Trust (Equipment grant 093,148/Z/10/Z)), the Medical Research Council (G0801841 & UD99999906), and UK Dementia Research Institute. The K.S. laboratory is co-funded by the European Regional Development Fund and the Republic of Cyprus through the Research & Innovation Foundation (Projects: OPPORTUNITY/0916/ERC-StG/003,INFRASTRUCTURES/1216/0034POST-DOC/0916/0111, INTERNATIONAL/OTHER/0118/0018).Peer reviewe

Histone acetyltransferase NAA40 modulates acetyl-CoA levels and lipid synthesis.

BACKGROUND: Epigenetic regulation relies on the activity of enzymes that use sentinel metabolites as cofactors to modify DNA or histone proteins. Thus, fluctuations in cellular metabolite levels have been reported to affect chromatin modifications. However, whether epigenetic modifiers also affect the levels of these metabolites and thereby impinge on downstream metabolic pathways remains largely unknown. Here, we tested this notion by investigating the function of N-alpha-acetyltransferase 40 (NAA40), the enzyme responsible for N-terminal acetylation of histones H2A and H4, which has been previously implicated with metabolic-associated conditions such as age-dependent hepatic steatosis and calorie-restriction-mediated longevity. RESULTS: Using metabolomic and lipidomic approaches, we found that depletion of NAA40 in murine hepatocytes leads to significant increase in intracellular acetyl-CoA levels, which associates with enhanced lipid synthesis demonstrated by upregulation in de novo lipogenesis genes as well as increased levels of diglycerides and triglycerides. Consistently, the increase in these lipid species coincide with the accumulation of cytoplasmic lipid droplets and impaired insulin signalling indicated by decreased glucose uptake. However, the effect of NAA40 on lipid droplet formation is independent of insulin. In addition, the induction in lipid synthesis is replicated in vivo in the Drosophila melanogaster larval fat body. Finally, supporting our results, we find a strong association of NAA40 expression with insulin sensitivity in obese patients. CONCLUSIONS: Overall, our findings demonstrate that NAA40 affects the levels of cellular acetyl-CoA, thereby impacting lipid synthesis and insulin signalling. This study reveals a novel path through which histone-modifying enzymes influence cellular metabolism with potential implications in metabolic disorders

Changing Stem Cell Dynamics during Papillomavirus Infection: Potential Roles for Cellular Plasticity in the Viral Lifecycle and Disease

Stem cells and cellular plasticity are likely important components of tissue response to infection. There is emerging evidence that stem cells harbor receptors for common pathogen motifs and that they are receptive to local inflammatory signals in ways suggesting that they are critical responders that determine the balance between health and disease. In the field of papillomaviruses stem cells have been speculated to play roles during the viral life cycle, particularly during maintenance, and virus-promoted carcinogenesis but little has been conclusively determined. I summarize here evidence that gives clues to the potential role of stem cells and cellular plasticity in the lifecycle papillomavirus and linked carcinogenesis. I also discuss outstanding questions which need to be resolved

Protocol for in vivo lineage tracing of the mouse-papillomavirus-type 1-infected cells in mice

Summary: Here, we present a protocol to create an in vivo lineage-tracing mouse model for mouse-papillomavirus-type 1 (MmuPV1)-infected cells. We describe the steps to generate and deliver the MmuPV1 lox-Cre-lox plasmid for the infection of mice, followed by skin tissue extraction and processing. We then detail how to use flow cytometry to trace, quantify, and analyze MmuPV1-harboring cells and their progeny. This model is suitable to investigate the early effects of papillomavirus on the target cells.For complete details on the use and execution of this protocol, please refer to Yilmaz et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Special Issue “New Frontiers in Small DNA Virus Research”

Scientific progress in understanding, preventing, treating, and managing viral infections and associated diseases exemplifies the extent to which research on small DNA tumor viruses has impacted human health [...

Terc is dispensable for most of the short-term HPV16 oncogene-mediated phenotypes in mice.

High-risk human papillomaviruses (HPVs) have been shown in vitro to impinge on telomere homeostasis in a number of ways. However, the in vivo interaction of viruses with the telomere homeostasis apparatus has not been previously explored. Since E6 and E7 are the main viral oncogenes and key for viral replication, we have explored here the short-term phenotypes of the genes in the context of defective telomere homeostasis. We examined the short-term phenotypes of E6 and E7 in a context where the Terc component of the telomerase holoenzyme was knocked out. We determined that Terc was dispensable for most oncogene-mediated phenotypes. Surprisingly, E7-mediated reduction of label retaining cells was found to be in part dependent on the presence of Terc. Under the conditions examined here, there appears to be no compelling evidence Terc is required for most short-term viral oncogene mediated phenotypes. Further studies will elucidate its role in longer-term phenotypes

Whole transcriptome sequence data of 5-FU sensitive and 5-FU resistant tumors generated in a mouse model of de novo carcinogenesis

We have performed whole transcriptome sequencing of 5-FU resistant and 5-FU sensitive tumors generated in a mouse model of de novo carcinogenesis that closely recapitulates tumor initiation, progression and maintenance in vivo. Tumors were generated using the DMBA/TPA model of chemically induced carcinogenesis [1], tumor-bearing mice were subsequently treated with 5-FU, and tumor growth as well as response to treatment was monitored by measuring tumor volume twice a week. Based on these measurements, we selected two 5-FU resistant and two 5-FU sensitive tumors and performed whole transcriptome sequencing and in order to identify differentially expressed transcripts between the two sets. Data obtained is deposited and available through NCBI SRA (reference number SRP155180 – https://www.ncbi.nlm.nih.gov/sra/?term=SRP155180)