Functionalized Peptide Fibrils as a Scaffold for Active Substances in Wound Healing.

Technological developments in the field of biologically active peptide applications in medicine have increased the need for new methods for peptide delivery. The disadvantage of peptides as drugs is their low biological stability. Recently, great attention has been paid to self-assembling peptides that can form fibrils. Such a formulation makes bioactive peptides more resistant to enzymatic degradation and druggable. Peptide fibrils can be carriers for peptides with interesting biological activities. These features open up prospects for using the peptide fibrils as long-acting drugs and are a valid alternative to conventional peptidic therapies. In our study, we designed new peptide scaffolds that are a hybrid of three interconnected amino acid sequences and are: pro-regenerative, cleavable by neutrophilic elastase, and fibril-forming. We intended to obtain peptides that are stable in the wound environment and that, when applied, would release a biologically active sequence. Our studies showed that the designed hybrid peptides show a high tendency toward regular fibril formation and are able to release the pro-regenerative sequence. Cytotoxicity studies showed that all the designed peptides were safe, did not cause cytotoxic effects and revealed a pro-regenerative potential in human fibroblast and keratinocyte cell lines. In vivo experiments in a dorsal skin injury model in mice indicated that two tested peptides moderately promote tissue repair in their free form. Our research proves that peptide fibrils can be a druggable form and a scaffold for active peptides.

PTD4 Peptide Increases Neural Viability in an In Vitro Model of Acute Ischemic Stroke.

Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH2 (R49KKRRQRRR57-amide) and its less basic analogue, PTD4 (Y47ARAAARQARA57-amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 µm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49-57)-NH2 adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49-57)-NH2) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide's ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.

Imunofan-RDKVYR Peptide-Stimulates Skin Cell Proliferation and Promotes Tissue Repair.

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today's science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name "Imunofan" (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p < 0.05) pro-proliferative activity (30-40% and 20-50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).

Transcriptomic responses to wounding: meta-analysis of gene expression microarray data.

BACKGROUND: A vast amount of microarray data on transcriptomic response to injury has been collected so far. We designed the analysis in order to identify the genes displaying significant changes in expression after wounding in different organisms and tissues. This meta-analysis is the first study to compare gene expression profiles in response to wounding in as different tissues as heart, liver, skin, bones, and spinal cord, and species, including rat, mouse and human. RESULTS: We collected available microarray transcriptomic profiles obtained from different tissue injury experiments and selected the genes showing a minimum twofold change in expression in response to wounding in prevailing number of experiments for each of five wound healing stages we distinguished: haemostasis & early inflammation, inflammation, early repair, late repair and remodelling. During the initial phases after wounding, haemostasis & early inflammation and inflammation, the transcriptomic responses showed little consistency between different tissues and experiments. For the later phases, wound repair and remodelling, we identified a number of genes displaying similar transcriptional responses in all examined tissues. As revealed by ontological analyses, activation of certain pathways was rather specific for selected phases of wound healing, such as e.g. responses to vitamin D pronounced during inflammation. Conversely, we observed induction of genes encoding inflammatory agents and extracellular matrix proteins in all wound healing phases. Further, we selected several genes differentially upregulated throughout different stages of wound response, including established factors of wound healing in addition to those previously unreported  in this context such as PTPRC and AQP4. CONCLUSIONS: We found that transcriptomic responses to wounding showed similar traits in a diverse selection of tissues including skin, muscles, internal organs and nervous system. Notably, we distinguished transcriptional induction of inflammatory genes not only in the initial response to wounding, but also later, during wound repair and tissue remodelling.

Neotenic phenomenon in gene expression in the skin of Foxn1- deficient (nude) mice - a projection for regenerative skin wound healing.

BACKGROUND: Mouse fetuses up to 16 day of embryonic development and nude (Foxn1- deficient) mice are examples of animals that undergo regenerative (scar-free) skin healing. The expression of transcription factor Foxn1 in the epidermis of mouse fetuses begins at embryonic day 16.5 which coincides with the transition point from scar-free to scar-forming skin wound healing. In the present study, we tested the hypothesis that Foxn1 expression in the skin is an essential condition to establish the adult skin phenotype and that Foxn1 inactivity in nude mice keeps skin in the immature stage resembling the phenomena of neoteny. RESULTS: Uninjured skin of adult C57BL/6J (B6) mice, mouse fetuses at days 14 (E14) and 18 (E18) of embryonic development and B6.Cg-Foxn1 nu (nude) mice were characterized for their gene expression profiles by RNA sequencing that was validated through qRT-PCR, Western Blot and immunohistochemistry. Differentially regulated genes indicated that nude mice were more similar to E14 (model of regenerative healing) and B6 were more similar to E18 (model of reparative healing). The up-regulated genes in nude and E14 mice were associated with tissue remodeling, cytoskeletal rearrangement, wound healing and immune response, whereas the down-regulated genes were associated with differentiation. E14 and nude mice exhibit prominent up-regulation of keratin (Krt23, -73, -82, -16, -17), involucrin (Ivl) and filaggrin (Flg2) genes. The transcription factors associated with the Hox genes known to specify cell fate during embryonic development and promote embryonic stem cells differentiation were down-regulated in both nude and E14. Among the genes enriched in the nude skin but not shared with E14 fetuses were members of the Wnt and matrix metalloproteinases (Mmps) families whereas Bmp and Notch related genes were down-regulated. CONCLUSIONS: In summary, Foxn1 appears to be a pivotal control element of the developmental program and skin maturation. Nude mice may be considered as a model of neoteny among mammals. The resemblance of gene expression profiles in the skin of both nude and E14 mice are direct or indirect consequences of the Foxn1 deficiency. Foxn1 appears to regulate the balance between cell proliferation and differentiation and its inactivity creates a pro-regenerative environment.

Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential.

BACKGROUND: The neonatal murine heart is able to regenerate after severe injury; this capacity however, quickly diminishes and it is lost within the first week of life. DNA methylation is an epigenetic mechanism which plays a crucial role in development and gene expression regulation. Under investigation here are the changes in DNA methylation and gene expression patterns which accompany the loss of regenerative potential. RESULTS: The MeDIP-chip (methylated DNA immunoprecipitation microarray) approach was used in order to compare global DNA methylation profiles in whole murine hearts at day 1, 7, 14 and 56 complemented with microarray transcriptome profiling. We found that the methylome transition from day 1 to day 7 is characterized by the excess of genomic regions which gain over those that lose DNA methylation. A number of these changes were retained until adulthood. The promoter genomic regions exhibiting increased DNA methylation at day 7 as compared to day 1 are significantly enriched in the genes critical for heart maturation and muscle development. Also, the promoter genomic regions showing an increase in DNA methylation at day 7 relative to day 1 are significantly enriched with a number of transcription factors binding motifs including those of Mfsd6l, Mef2c, Meis3, Tead4, and Runx1. CONCLUSIONS: The results indicate that the extensive alterations in DNA methylation patterns along the development of neonatal murine hearts are likely to contribute to the decline of regenerative capabilities observed shortly after birth. This conclusion is supported by the evidence that an increase in DNA methylation in the neonatal murine heart from day 1 to day 7 occurs in the promoter regions of genes playing important roles in cardiovascular system development.

A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction.

DNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference. We determined DNA methylation levels in several promoter regions in two setups implementing different references: mock-digested and treated with a restriction enzyme that has no recognition sites within examined amplicons. Fragmentation of reference templates allowed removing the overestimation effect, thereby improving measurement accuracy.

Transcriptome profiling reveals distinctive traits of retinol metabolism and neonatal parallels in the MRL/MpJ mouse.

BACKGROUND: The MRL/MpJ mouse is a laboratory inbred strain known for regenerative abilities which are manifested by scarless closure of ear pinna punch holes. Enhanced healing responses have been reported in other organs. A remarkable feature of the strain is that the adult MRL/MpJ mouse retains several embryonic biochemical characteristics, including increased expression of stem cell markers. RESULTS: We explored the transcriptome of the MRL/MpJ mouse in the heart, liver, spleen, bone marrow and ears. We used two reference strains, thus increasing the chances to discover the genes responsible for the exceptional properties of the regenerative strain. We revealed several distinctive characteristics of gene expression patterns in the MRL/MpJ mouse, including the repression of immune response genes, the up-regulation of those associated with retinol metabolism and PPAR signalling, as well as differences in expression of the genes engaged in wounding response. Another crucial finding is that the gene expression patterns in the adult MRL/MpJ mouse and murine neonates share a number of parallels, which are also related to immune and wounding response, PPAR pathway, and retinol metabolism. CONCLUSIONS: Our results indicate the significance of retinol signalling and neonatal transcriptomic relics as the distinguishing features of the MRL/MpJ mouse. The possibility that retinoids could act as key regulatory molecules in this regeneration model brings important implications for regenerative medicine.

Chemotherapy-Mediated Complications of Wound Healing: An Understudied Side Effect.

Significance: Chemotherapy is a primary method to treat cancer. While chemotherapeutic drugs are designed to target rapidly dividing cancer cells, they can also affect other cell types. In the case of dermal cells and macrophages involved in wound healing, cytotoxicity often leads to the development of chronic wounds. The situation becomes even more severe when chemotherapy is combined with surgical tumor excision. Recent Advances: Despite its significant impact on patients' recovery from surgery, the issue of delayed wound healing in individuals undergoing chemotherapy remains inadequately explored. Critical Issues: This review aims to analyze the harmful impact of chemotherapy on wound healing. The analysis showed that chemotherapy drugs could inhibit cellular metabolism, cell division, and angiogenesis and lead to nerve damage. They impede the migration of cells into the wound and reduce the production of extracellular matrix. At the molecular level, they interfere with replication, transcription, translation, and cell signaling. This work reviews skin problems that patients may experience during and after chemotherapy and demonstrates insights into the cellular and molecular mechanisms of these pathologies. Future Directions: In the future, the problem of impaired wound healing in patients treated with chemotherapy may be addressed by cell therapies like autologous keratinocyte transplantation, which has already proved effective in this case. Epigenetic intervention to mitigate the side effects of chemotherapy is also worth considering, but epigenetic consequences of chemotherapy on skin cells are largely unknown and should be investigated.

Between therapy effect and false-positive result in animal experimentation.

Despite the animal models' complexity, researchers tend to reduce the number of animals in experiments for expenses and ethical concerns. This tendency makes the risk of false-positive results, as statistical significance, the primary criterion to validate findings, often fails if testing small samples. This study aims to highlight such risks using an example from experimental regenerative therapy and propose a machine-learning solution to validate treatment effects. The example analysed was the pharmacological treatment of ear pinna punch wound healing in mice. Wound closure data analysed included eight groups treated with an epigenetic inhibitor, zebularine, and eight control groups receiving vehicle alone, of six mice each. We confirmed the zebularine healing effect for all 64 pairwise comparisons between treatment and control groups but also determined minor yet statistically significant differences between control groups in five of 28 possible comparisons. The occurrences of significant differences between the control groups, regardless of standardised experimental conditions, indicate a risk of statistically significant effects in the case a compound lacking the desired biological activity is tested. Since the criterion of statistical significance itself can be confusing, we demonstrate a machine-learning algorithm trained on datasets representing treatment and control experiments as a helpful tool for validating treatment outcomes. We tested two machine-learning approaches, Naïve Bayes and Support Vector Machine classifiers. In contrast to the Mann-Whitney U-test, indicating enhanced healing effects for some control groups receiving saline alone, both machine-learning algorithms faultlessly assigned all animal groups receiving saline to the controls.

Release systems based on self-assembling RADA16-I hydrogels with a signal sequence which improves wound healing processes.

Self-assembling peptides can be used for the regeneration of severely damaged skin. They can act as scaffolds for skin cells and as a reservoir of active compounds, to accelerate scarless wound healing. To overcome repeated administration of peptides which accelerate healing, we report development of three new peptide biomaterials based on the RADA16-I hydrogel functionalized with a sequence (AAPV) cleaved by human neutrophil elastase and short biologically active peptide motifs, namely GHK, KGHK and RDKVYR. The peptide hybrids were investigated for their structural aspects using circular dichroism, thioflavin T assay, transmission electron microscopy, and atomic force microscopy, as well as their rheological properties and stability in different fluids such as water or plasma, and their susceptibility to digestion by enzymes present in the wound environment. In addition, the morphology of the RADA-peptide hydrogels was examined with a unique technique called scanning electron cryomicroscopy. These experiments enabled us to verify if the designed peptides increased the bioactivity of the gel without disturbing its gelling processes. We demonstrate that the physicochemical properties of the designed hybrids were similar to those of the original RADA16-I. The materials behaved as expected, leaving the active motif free when treated with elastase. XTT and LDH tests on fibroblasts and keratinocytes were performed to assess the cytotoxicity of the RADA16-I hybrids, while the viability of cells treated with RADA16-I hybrids was evaluated in a model of human dermal fibroblasts. The hybrid peptides revealed no cytotoxicity; the cells grew and proliferated better than after treatment with RADA16-I alone. Improved wound healing following topical delivery of RADA-GHK and RADA-KGHK was demonstrated using a model of dorsal skin injury in mice and histological analyses. The presented results indicate further research is warranted into the engineered peptides as scaffolds for wound healing and tissue engineering.

Examination of epigenetic inhibitor zebularine in treatment of skin wounds in healthy and diabetic mice.

DNA methyltransferase inhibitor zebularine was proven to induce regeneration in the ear pinna in mice. We utilized a dorsal skin wound model to further evaluate this epigenetic inhibitor in wound healing. Full-thickness excisional wounds were made on the dorsum of 2 and 10-month-old healthy BALB/c and 3 and 8-month-old diabetic (db/db) mice, followed by topical or intraperitoneal zebularine delivery. Depending on the strain, age, dose, and delivery, the zebularine treatments either had no effect or accelerated or delayed wound closure. In principle, zebularine applied topically moderately promoted wound closure in the healthy but markedly delayed in the diabetic mice, which was in line with decreased viability of cultured keratinocytes from diabetic patients exposed to zebularine. The histological analysis revealed an improvement in the architecture of restored skin in zebularine-treated mice, manifested as a distinct layered pattern resembling panniculus carnosus. The finding corresponds with the zebularine-mediated activation of the Wnt5a gene, an essential regulator of Wnt signaling, the pathway involved in hair follicle development, the process which in turn is connected with regenerative skin healing. Although zebularine did not remarkably accelerate wound healing, zebularine and other epigenetic inhibitors deserve further testing as potential drugs to improve the quality of restored skin.

Regenerative Drug Discovery Using Ear Pinna Punch Wound Model in Mice.

The ear pinna is a complex tissue consisting of the dermis, cartilage, muscles, vessels, and nerves. Ear pinna healing is a model of regeneration in mammals. In some mammals, including rabbits, punch wounds in the ear pinna close spontaneously; in common-use laboratory mice, they remain for life. Agents inducing ear pinna healing are potential regenerative drugs. We tested the effects of selected bioactive agents on 2 mm ear pinna wound closure in BALB/c mice. Our previous research demonstrated that a DNA methyltransferase inhibitor, zebularine, remarkably induced ear pinna regeneration. Although experiments with two other demethylating agents, RG108 and hydralazine, were unsuccessful, a histone deacetylase inhibitor, valproic acid, was another epigenetic agent found to increase ear hole closure. In addition, we identified a pro-regenerative activity of 4-ketoretinoic acid, a retinoic acid metabolite. Attempts to counteract the regenerative effects of the demethylating agent zebularine, with folates as methyl donors, failed. Surprisingly, a high dose of methionine, another methyl donor, promoted ear hole closure. Moreover, we showed that the regenerated areas of ear pinna were supplied with nerve fibre networks and blood vessels. The ear punch model proved helpful in testing the pro-regenerative activities of small-molecule compounds and observations of peripheral nerve regeneration.

Conservation and diversity of MutS proteins.

The homologues of MutS, mismatch repair protein, exist in all prokaryotes, with the exception of Actinobacteria, Mollicutes and part of the Archaea. Multiple alignments of 316 MutS amino acid sequences from 169 species revealed conserved residues and sequence motifs distinguishing MutS homologues. All MutS homologues show high conservation within the ATPase domain. MutS1, the homologue responsible for DNA mismatch recognition, is common in Archaea and Bacteria. MutS1 is distinguished by the N-terminal mismatch binding domain containing the GXFXE motif shared by all MutS1 homologues and MSH6 homologues in eukaryotes. Less common than MutS1, MutS2, the suppressor of homologous recombination, is rendered distinctive by the C-terminal Smr endonuclease domain containing the conserved HGXG motif. MutS1 and MutS2 are of enormous significance in maintaining genome integrity. The functions of the other homologues: MutS2-like, MutS3, MutS4, and MutS5 have not yet been found. Each of these homologues exists in a narrower range of taxonomic groups than MutS1 or MutS2 and has neither the mismatch binding nor the Smr domain. The number of different MutS homologues in a single organism usually ranges from one to four; there are rarely five and six only occur exceptionally. The diversity of MutS types and structures begs the question as to how this diversity influenced the evolution of genomes.

Development of a Peptide Derived from Platelet-Derived Growth Factor (PDGF-BB) into a Potential Drug Candidate for the Treatment of Wounds.

Objective: This study evaluated the use of novel peptides derived from platelet-derived growth factor (PDGF-BB) as potential wound healing stimulants. One of the compounds (named PDGF2) was subjected for further research after cytotoxicity and proliferation assays on human skin cells. Further investigation included evaluation of: migration and chemotaxis of skin cells, immunological and allergic safety, the transcriptional analyses of adipose-derived stem cells (ASCs) and dermal fibroblasts stimulated with PDGF2, and the use of dorsal skin wound injury model to evaluate the effect of wound healing in mice. Approach: Colorimetric lactate dehydrogenase and tetrazolium assays were used to evaluate the cytotoxicity and the effect on proliferation. PDGF2 effect on migration and chemotaxis was also checked. Immunological safety and allergic potential were evaluated with a lymphocyte activation and basophil activation test. Transcriptional profiles of ASCs and primary fibroblasts were assessed after stimulation with PDGF2. Eight-week-old BALB/c female mice were used for dorsal skin wound injury model. Results: PDGF2 showed low cytotoxicity, pro-proliferative effects on human skin cells, high immunological safety, and accelerated wound healing in mouse model. Furthermore, transcriptomic analysis of ASCs and fibroblasts revealed the activation of processes involved in wound healing and indicated its safety. Innovation: A novel peptide derived from PDGF-BB was proved to be safe drug candidate in wound healing. We also present a multifaceted in vitro model for the initial screening of new compounds that may be potentially useful in wound healing stimulation. Conclusion: The results show that peptide derived from PDGF-BB is a promising drug candidate for wound treatment.

Data regarding a new, vector-enzymatic DNA fragment amplification-expression technology for the construction of artificial, concatemeric DNA, RNA and proteins, as well as biological effects of selected polypeptides obtained using this method.

Applications of bioactive peptides and polypeptides are emerging in areas such as drug development and drug delivery systems. These compounds are bioactive, biocompatible and represent a wide range of chemical properties, enabling further adjustments of obtained biomaterials. However, delivering large quantities of peptide derivatives is still challenging. Several methods have been developed for the production of concatemers - multiple copies of the desired protein segments. We have presented an efficient method for the production of peptides of desired length, expressed from concatemeric Open Reading Frame. The method employs specific amplification-expression DNA vectors. The main methodological approaches are described by Skowron et al., 2020 [1]. As an illustration of the demonstrated method's utility, an epitope from the S protein of Hepatitis B virus (HBV) was amplified. Additionally, peptides, showing potentially pro-regenerative properties, derived from the angiopoietin-related growth factor (AGF) were designed and amplified. Here we present a dataset including: (i) detailed protocols for the purification of HBV and AGF - derived polyepitopic protein concatemers, (ii) sequences of the designed primers, vectors and recombinant constructs, (iii) data on cytotoxicity, immunogenicity and stability of AGF-derived polypeptides.

A vector-enzymatic DNA fragment amplification-expression technology for construction of artificial, concatemeric DNA, RNA and proteins for novel biomaterials, biomedical and industrial applications.

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS SapI restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential.

Retained features of embryonic metabolism in the adult MRL mouse.

The MRL mouse is an inbred laboratory strain that was derived by selective breeding in 1960 from the rapidly growing LG/J (Large) strain. MRL mice grow to nearly twice the size of other commonly used mouse strains, display uncommonly robust healing and regeneration properties, and express later onset autoimmune traits similar to Systemic Lupus Erythematosis. The regeneration trait (heal) in the MRL mouse maps to 14-20 quantitative trait loci and the autoimmune traits map to 5-8 loci. In this paper we report the metabolic and biochemical features that characterize the adult MRL mouse and distinguish it from C57BL/6 control animals. We found that adult MRL mice have retained a number of features of embryonic metabolism that are normally lost during development in other strains. These include an emphasis on aerobic glycolytic energy metabolism, increased glutamate oxidation, and a reduced capacity for fatty acid oxidation. MRL tissues, including the heart, liver, and regenerating ear hole margins, showed considerable mitochondrial genetic and physiologic reserve, decreased mitochondrial transmembrane potential (DeltaPsi(m)), decreased reactive oxygen species (ROS), and decreased oxidative phosphorylation, yet increased mitochondrial DNA and protein content. The discovery of embryonic metabolic features led us to look for cells that express markers of embryonic stem cells. We found that the adult MRL mouse has retained populations of cells that express the stem cell markers Nanog, Islet-1, and Sox2. These are present in the heart at baseline and highly induced after myocardial injury. The retention of embryonic features of metabolism in adulthood is rare in mammals. The MRL mouse provides a unique experimental window into the relationship between metabolism, stem cell biology, and regeneration.

Transcriptional activity of epigenetic remodeling genes declines in keratinocytes after in vitro expansion.

PURPOSE: In vitro expansion is an invaluable method to obtain keratinocytes in amounts necessary for effective transplantation therapies. In vitro cell culturing provokes questions concerning potential epigenetic alterations occurring in expanded cells in the context of usefulness for transplantation and safety. The purpose of this study was to investigate as to whether keratinocyte expansion is associated with changes in the activity of genes responsible for the maintenance of epigenetic stability. MATERIALS AND METHODS: We focussed on the transcriptional activity of genes involved in different epigenetic mechanisms including DNA methylation and histone modifications. We used quantitative real-time PCR to determine transcript levels of 16 epigenetic remodelling markers in 14 patients in the epidermal cells directly after collection and after in vitro expansion. RESULTS: We observed a remarkable decline in the transcriptional activity of the epigenetic remodelling genes following in vitro expansion, while no further fall of expression with passaging. In whole skin, we found even higher expression levels of the epigenetic markers. CONCLUSIONS: Transmission to in vitro environment challenges cellular signalling and metabolism. The regulation of epigenetic remodelling maintains the balance between cellular plasticity and phenotype deviation. This preliminary research demonstrated reduced activity of genes responsible for epigenetic modifications of DNA and histones in in vitro expanded epidermal cells. This observation indicates that epigenome re-patterning in cultured epidermal cells is significantly less intensive than in the skin. Also, this observation may imply that after adaptation to in vitro conditions, the epigenome does not undergo extensive transformation during further cultivation.

Epigenetic inhibitor zebularine activates ear pinna wound closure in the mouse.

BACKGROUND: Most studies on regenerative medicine focus on cell-based therapies and transplantations. Small-molecule therapeutics, though proved effective in different medical conditions, have not been extensively investigated in regenerative research. It is known that healing potential decreases with development and developmental changes are driven by epigenetic mechanisms, which suggests epigenetic repression of regenerative capacity. METHODS: We applied zebularine, a nucleoside inhibitor of DNA methyltransferases, to stimulate the regenerative response in a model of ear pinna injury in mice. FINDINGS: We observed the regeneration of complex tissue that was manifested as improved ear hole repair in mice that received intraperitoneal injections of zebularine. Six weeks after injury, the mean hole area decreased by 83.2 ±â€¯9.4% in zebularine-treated and by 43.6 ±â€¯15.4% in control mice (p < 10-30). Combined delivery of zebularine and retinoic acid potentiated and accelerated this effect, resulting in complete ear hole closure within three weeks after injury. We found a decrease in DNA methylation and transcriptional activation of neurodevelopmental and pluripotency genes in the regenerating tissues. INTERPRETATION: This study is the first to demonstrate an effective induction of complex tissue regeneration in adult mammals using zebularine. We showed that the synergistic action of an epigenetic drug (zebularine) and a transcriptional activator (retinoic acid) could be effectively utilized to induce the regenerative response, thus delineating a novel pharmacological strategy for regeneration. The strategy was effective in the model of ear pinna regeneration in mice, but zebularine acts on different cell types, therefore, a similar approach can be tested in other tissues and organs.