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.

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.

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.

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.

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).

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.

Immunophenotyping and transcriptional profiling of in vitro cultured human adipose tissue derived stem cells.

Adipose-derived stem cells (ASCs) have become an important research model in regenerative medicine. However, there are controversies regarding the impact of prolonged cell culture on the ASCs phenotype and their differentiation potential. Hence, we studied 10 clinical ASCs replicates from plastic and oncological surgery patients, in six-passage FBS supplemented cultures. We quantified basic mesenchymal cell surface marker transcripts and the encoded proteins after each passage. In parallel, we investigated the differentiation potential of ASCs into chondrocytes, osteocytes and adipocytes. We further determined the effects of FBS supplementation and subsequent deprivation on the whole transcriptome by comprehensive mRNA and miRNA sequencing. Our results show that ASCs maintain differentiation potential and consistent profile of key mesenchymal markers, with apparent expression of distinct isoforms, in long-term cultures. No significant differences were observed between plastic and oncological surgery cohorts. ASCs in FBS supplemented primary cultures are almost committed to mesenchymal lineages as they express key epithelial-mesenchymal transition genes including early mesenchymal markers. Furthermore, combined mRNA/miRNA expression profiling strongly supports a modulatory role for the miR-30 family in the commitment process to mesenchymal lineages. Finally, we propose improvements to existing qPCR based assays that address alternative isoform expression of mesenchymal markers.

Transcriptional profile of in vitro expanded human epidermal progenitor cells for the treatment of non-healing wounds.

BACKGROUND: Epidermal progenitor cells (EPCs) have been under extensive investigation due to their increasing potential of application in medicine and biotechnology. Cultured human EPCs are used in the treatment of chronic wounds and have recently became a target for gene therapy and toxicological studies. One of the challenges in EPCs culture is to provide a high number of undifferentiated, progenitor cells displaying high viability and significant biological activity. OBJECTIVES: The goal of this study was to characterize the in vitro cultured progenitor cells and to assess whether the cells with the progenitor phenotype are able to enhance wound healing. Additionally, we aimed to establish the complete procedure of the culture, analysis and clinical application of epidermal progenitor cells. METHODS: In this study we present a method of cell isolation and culture followed by a technique of transplantation of the cultured cells onto the wound bed. The applied isolation technique involves two enzymatic steps (dispase, trypsin) and it is characterized by a high yield of cells. The obtained cells were cultured in vitro up to the second passage in serum-free and xeno-free keratinocytes-dedicated medium. Key stem cell markers were determined with means of flow cytometry and quantitative real-time PCR. RESULTS: The in vitro expanded cells displayed high proliferative activity without features of neither apoptosis nor necrosis. The flow cytometry and transcriptomic analyses showed enhanced expression of stem cell markers (i.e. proteins: ΔNp63, CD29, CD49f and BNC1, CDKN1A transcripts) in the expanded cells. In the presented compassionate use study, cultured autologous cells from an oncological patient were suspended in fibrin sealant and transplanted directly to a non-healing wound, resulting in wound closure within 2 months. CONCLUSION: The cells cultured in serum-free media display epidermal stem cells features and a potential to stimulate wound healing. This promising procedure of isolation, culture and application warrants further clinical trials in the treatment of chronic wounds.

Transcriptomic Effects of Estrogen Starvation and Induction in the MCF7 Cells. The Meta-analysis of Microarray Results.

Estrogen is one of the most important signaling molecules which targets a number of genes. Estrogen levels regulate cell proliferation and a plethora of metabolic processes, which may interfere with a range of medical conditions and drug metabolism. The MCF7 breast cancer cell line, expressing the estrogen receptor α, is a well-studied model of cellular answer to estrogen. The aim of this study was to characterize transcriptomic responses to estrogen in a broad time range. We performed a meta-analysis of microarray data on gene expression in the MCF7 cells under estrogen exposure and deprivation. As the result we distinguished three major phases of transcriptomic response to stimulation with 17ß- estradiol: the early (1-2 h), with the activation of the MAPK signaling pathway; the intermediate (3-12 h), with enhanced expression of genes participating in cell surface receptor linked signal transduction and cellular homeostasis; and the late one (24-48 h), with the induction of genes involved in mitotic cell division. Two main phases under estrogen starvation were indicated as the early (1-3 days), with elevated expression of genes associated with cell projection and repression of those responsible for cell cycle regulation, and the late (15-180 days), with increased expression of genes of cell adhesion proteins. The meta-analysis displayed how different gene sets are either induced or repressed following either estrogen exposure or deprivation, and how the gene expression changes are orchestrated by estrogen in time dependent manner, indicating that proper understanding of estrogen impact on transcriptional gene activity requires an extensive time perspective.

Conserved motifs of MutL proteins.

The MutL protein is best known for its function in DNA mismatch repair (MMR). However, there is evidence to suggest that MutL is not only the linker connecting the functions of MutS and MutH in MMR, but that it also participates in other repair systems, such as Very Short Patch (VSP), Base Excision (BER) and Nucleotide Excision Repair (NER). This study set out to identify the most highly conserved amino acid sequence motifs in MutL proteins. We analyzed 208 MutL amino acid sequences of 199 representative prokaryotic species belonging to 28 classes of bacteria and archaea. The analysis revealed 16 conserved motifs situated in the ATPase and endonuclease domains, as well as within the disordered loop, and in the MutL regions interacting with the ß clamp of DNA polymerase III. The conserved sequence motifs thus determined constitute a structural definition of MutL and they may be used in site-directed mutagenesis studies. We found conserved residues within the potential regions where binding with MutS occurs. However, the existing data does not provide clues as to the possible sites of MutL interactions with the proteins involved in other DNA repair systems such as NER, BER and VSP. We determined the 57 most highly conserved amino acid residues, including 43 which were identical in all the sequences analyzed. The greater part of the most predominantly conserved amino acid residues identified in MutL are identical to the corresponding residues reported as mutational hot-spots in one of its human homologues, MLH1, but not in the other, PMS2. This is the first study to present the conserved sequence motifs of MutL widespread in bacteria and archaea and the classification of MutLs into five groups distinguished on the basis of differences in the C-terminal region. Our analysis is of use in better understanding MutL functions.

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.

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.

MutS as a tool for mutation detection.

MutS, a DNA mismatch-binding protein, seems to be a promising tool for mutation detection. We present three MutS based approaches to the detection of point mutations: DNA retardation, protection of mismatched DNA against exonuclease digestion, and chimeric MutS proteins. DNA retardation in polyacrylamide gels stained with SYBR-Gold allows mutation detection using 1-3 microg of Thermus thermophilus his6-MutS protein and 50-200 ng of a PCR product. The method enables the search for a broad range of mutations: from single up to several nucleotide, as mutations over three nucleotides could be detected in electrophoresis without MutS, due to the mobility shift caused by large insertion/deletion loops in heteroduplex DNA. The binding of DNA mismatches by MutS protects the complexed DNA against exonuclease digestion. The direct addition of the fluorescent dye, SYBR-Gold, allows mutation detection in a single-tube assay. The limited efficiency of T4 DNA polymerase as an exonuclease hampers the application of the method in practice. The assay required 300-400 ng of PCR products in the range of 200-700 bp and 1-3 microg of MutS. MutS binding to mismatched DNA immobilised on a solid phase can be observed thanks to the activity of a reporter domain linked to MutS. We obtained chimeric bifunctional proteins consisting of T. thermophilus MutS and reporter domains, like beta-galactosidase or GFP. Very low detection limits for beta-galactosidase could theoretically enable mutation detection not only by the examination of PCR products, but even of genomic DNA.

Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples.

MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 microg of Thermus thermophilus his6-MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations.

Construction and purification of his6-Thermus thermophilus MutS protein.

The mutS gene from the thermophilic bacterium Thermus thermophilus was PCR amplified, cloned, and expressed in Escherichia coli. The recombinant MutS protein containing an oligohistidine domain at the N-terminus was purified in a single step by Ni(2+) affinity chromatography to apparent homogeneity. The mismatch recognition properties of the his(6)-tagged MutS protein were confirmed by DNA protection against exonuclease digestion and retardation assays. The results of analytical gel filtration indicate that the predominant form of T. thermophilus MutS at micromolar concentrations is a tetramer.