Study of the Antimicrobial Activity of the Human Peptide SQQ30 against Pathogenic Bacteria.

Given the continuous increase in antibiotic resistance, research has been driven towards the isolation of new antimicrobial molecules. Short, charged, and very hydrophobic antimicrobial peptides have a direct action against biological membranes, which are less prone to developing resistance. Using a bioinformatic tool, we chose the SQQ30 peptide, isolated from the human SOGA1 protein. The antimicrobial activity of this peptide against various Gram-negative and Gram-positive bacterial strains and against a fungal strain was studied. A mechanism of action directed against biological membranes was outlined. When administered in combination with the antibiotic ciprofloxacin and with the TRS21 (buforin II), another antimicrobial peptide, SQQ30 can be used with a lower MIC, showing additivity and synergism, respectively. Particularly interesting is the ability of SQQ30 to bind LPS in Gram-negative strains, preventing the eukaryotic cell from releasing inflammatory mediators. Our study indicates SQQ30 as a novel and promising antimicrobial agent.

NAD+ Metabolism-Related Gene Profile Can Be a Relevant Source of Squamous Cell Carcinoma Biomarkers.

Poor survival rates of squamous cell carcinomas (SCCs) are associated with high recurrence, metastasis, and late diagnosis, due in part to a limited number of reliable biomarkers. Thus, the identification of signatures improving the diagnosis of different SCC types is mandatory. Considering the relevant role of NAD+ metabolism in SCC chemoprevention and therapy, the study aimed at identifying new biomarkers based on NAD+ metabolism-related gene (NMRG) expression. Gene expression of 18 NMRGs and clinical-pathological information for patients with head and neck SCC (HNSCC), lung SCC (LuSCC), and cervix SCC (CeSCC) from The Cancer Genome Atlas (TCGA) were analyzed by several bioinformatic tools. We identified a 16-NMRG profile discriminating 3 SCCs from 3 non-correlated tumors. We found several genes for HNSCC, LuSCC, and CeSCC with high diagnostic power. Notably, three NMRGs were SCC-type specific biomarkers. Furthermore, specific signatures displayed high diagnostic power for several clinical-pathological characteristics. Analyzing tumor-infiltrating immune cell profiles and PD-1/PD-L1 levels, we found that NMRG expression was associated with suppressive immune microenvironment mainly in HNSCC. Finally, the evaluation of patient survival identified specific genes for HNSCC, LuSCC, and CeSCC with potential prognostic power. Therefore, our analyses indicate NAD+ metabolism as an important source of SCC biomarkers and potential therapeutic targets.

Thymus richardii subsp. nitidus (Guss.) Jalas Essential Oil: An Ally against Oral Pathogens and Mouth Health.

The genus Thymus L., belonging to the Lamiaceae family, contains about 220 species with a distribution that mainly extends in Europe, northwest Africa, Ethiopia, Asia, and southern Greenland. Due to their excellent biological properties, fresh and/or dried leaves and aerial parts of several Thymus ssp. have been utilized in the traditional medicine of many countries. To evaluate not only the chemical aspects but also the biological properties, the essential oils (EOs), obtained from the pre-flowering and flowering aerial parts of Thymus richardii subsp. nitidus (Guss.) Jalas, endemic to Marettimo Island (Sicily, Italy), were investigated. The chemical composition of the EOs, obtained by classical hydrodistillation and GC-MS and GC-FID analyses, showed the occurrence of similar amounts of monoterpene hydrocarbons, oxygenated monoterpenes, and sesquiterpene hydrocarbons. The main constituents of the pre-flowering oil were ß-bisabolene (28.54%), p-cymene (24.45%), and thymol methyl ether (15.90%). The EO obtained from the flowering aerial parts showed as principal metabolites ß-bisabolene (17.91%), thymol (16.26%), and limonene (15.59%). The EO of the flowering aerial parts, and its main pure constituents, ß-bisabolene, thymol, limonene, p-cymene, and thymol methyl ether were investigated for their antimicrobial activity against oral pathogens and for their antibiofilm and antioxidant properties.

Disease-related p63 DBD mutations impair DNA binding by distinct mechanisms and varying degree.

The transcription factor p63 shares a high sequence identity with the tumour suppressor p53 which manifests itself in high structural similarity and preference for DNA sequences. Mutations in the DNA binding domain (DBD) of p53 have been studied in great detail, enabling a general mechanism-based classification. In this study we provide a detailed investigation of all currently known mutations in the p63 DBD, which are associated with developmental syndromes, by measuring their impact on transcriptional activity, DNA binding affinity, zinc binding capacity and thermodynamic stability. Some of the mutations we have further characterized with respect to their ability to convert human dermal fibroblasts into induced keratinocytes. Here we propose a classification of the p63 DBD mutations based on the four different mechanisms of DNA binding impairment which we identified: direct DNA contact, zinc finger region, H2 region, and dimer interface mutations. The data also demonstrate that, in contrast to p53 cancer mutations, no p63 mutation induces global unfolding and subsequent aggregation of the domain. The dimer interface mutations that affect the DNA binding affinity by disturbing the interaction between the individual DBDs retain partial DNA binding capacity which correlates with a milder patient phenotype.

Loss of p53 activates thyroid hormone via type 2 deiodinase and enhances DNA damage.

The Thyroid Hormone (TH) activating enzyme, type 2 Deiodinase (D2), is functionally required to elevate the TH concentration during cancer progression to advanced stages. However, the mechanisms regulating D2 expression in cancer still remain poorly understood. Here, we show that the cell stress sensor and tumor suppressor p53 silences D2 expression, thereby lowering the intracellular THs availability. Conversely, even partial loss of p53 elevates D2/TH resulting in stimulation and increased fitness of tumor cells by boosting a significant transcriptional program leading to modulation of genes involved in DNA damage and repair and redox signaling. In vivo genetic deletion of D2 significantly reduces cancer progression and suggests that targeting THs may represent a general tool reducing invasiveness in p53-mutated neoplasms.

YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation.

The Yes-associated protein (YAP), one of the major effectors of the Hippo pathway together with its related protein WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ), mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ) regulate a large number of target genes, acting as coactivators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis, we identified two molecules that could have a role in the altered genome-wide methylation profile: the long noncoding RNA ephemeron, whose rapid upregulation is crucial for the transition of ESCs into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency.

Author Correction: Thyroid hormone induces progression and invasiveness of squamous cell carcinomas by promoting a ZEB-1/E-cadherin switch.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Thyroid hormone induces progression and invasiveness of squamous cell carcinomas by promoting a ZEB-1/E-cadherin switch.

Epithelial tumor progression often involves epithelial-mesenchymal transition (EMT). We report that increased intracellular levels of thyroid hormone (TH) promote the EMT and malignant evolution of squamous cell carcinoma (SCC) cells. TH induces the EMT by transcriptionally up-regulating ZEB-1, mesenchymal genes and metalloproteases and suppresses E-cadherin expression. Accordingly, in human SCC, elevated D2 (the T3-producing enzyme) correlates with tumor grade and is associated with an increased risk of postsurgical relapse and shorter disease-free survival. These data provide the first in vivo demonstration that TH and its activating enzyme, D2, play an effective role not only in the EMT but also in the entire neoplastic cascade starting from tumor formation up to metastatic transformation, and supports the concept that TH is an EMT promoter. Our studies indicate that tumor progression relies on precise T3 availability, suggesting that pharmacological inactivation of D2 and TH signaling may suppress the metastatic proclivity of SCC.

Positive selection in Europeans and East-Asians at the ABCA12 gene.

Natural selection acts on genetic variants by increasing the frequency of alleles responsible for a cellular function that is favorable in a certain environment. In a previous genome-wide scan for positive selection in contemporary humans, we identified a signal of positive selection in European and Asians at the genetic variant rs10180970. The variant is located in the second intron of the ABCA12 gene, which is implicated in the lipid barrier formation and down-regulated by UVB radiation. We studied the signal of selection in the genomic region surrounding rs10180970 in a larger dataset that includes DNA sequences from ancient samples. We also investigated the functional consequences of gene expression of the alleles of rs10180970 and another genetic variant in its proximity in healthy volunteers exposed to similar UV radiation. We confirmed the selection signal and refine its location that extends over 35 kb and includes the first intron, the first two exons and the transcription starting site of ABCA12. We found no obvious effect of rs10180970 alleles on ABCA12 gene expression. We reconstructed the trajectory of the T allele over the last 80,000 years to discover that it was specific to H. sapiens and present in non-Africans 45,000 years ago.

Isolation and Enrichment of Newborn and Adult Skin Stem Cells of the Interfollicular Epidermis.

The interfollicular epidermis regenerates from a heterogeneous population of basal cells undergoing either self-renewal or terminal differentiation, thereby balancing cell loss in tissue turnover or in wound repair. In this chapter, we describe a reliable and simple method for isolating interfollicular epithelial stem cells from the skin of newborn mice or from tail and ear skin of adult mice using fluorescence-activated cell sorting (FACS). We also provide a detailed protocol for culturing interfollicular epidermal stem cells and to assess their proliferative potential and self-renewing ability. These techniques are useful for directly evaluating epidermal stem cell function in normal mice under different conditions or in genetically modified mouse models.

Functional and Mechanistic Insights into the Pathogenesis of P63-Associated Disorders.

The p53 family member p63 is a master regulator of gene expression in stratified epithelia, such as the epidermis. One of the main functions of p63 is to sustain mechanical resistance, positively regulating several epidermal genes involved in cell-matrix adhesion and cell-cell adhesion (Ferone et al., 2015).

Protein aggregation of the p63 transcription factor underlies severe skin fragility in AEC syndrome.

The p63 gene encodes a master regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the C-terminal domain of the p63 gene can cause ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility and severe, long-lasting skin erosions. Despite deep knowledge of p63 functions, little is known about mechanisms underlying disease pathology and possible treatments. Here, we show that multiple AEC-associated p63 mutations, but not those causative of other diseases, lead to thermodynamic protein destabilization, misfolding, and aggregation, similar to the known p53 gain-of-function mutants found in cancer. AEC mutant proteins exhibit impaired DNA binding and transcriptional activity, leading to dominant negative effects due to coaggregation with wild-type p63 and p73. Importantly, p63 aggregation occurs also in a conditional knock-in mouse model for the disorder, in which the misfolded p63 mutant protein leads to severe epidermal defects. Variants of p63 that abolish aggregation of the mutant proteins are able to rescue p63's transcriptional function in reporter assays as well as in a human fibroblast-to-keratinocyte conversion assay. Our studies reveal that AEC syndrome is a protein aggregation disorder and opens avenues for therapeutic intervention.

Research Techniques Made Simple: Identification and Characterization of Long Noncoding RNA in Dermatological Research.

Long noncoding RNAs (lncRNAs) are a functionally heterogeneous and abundant class of RNAs acting in all cellular compartments that can form complexes with DNA, RNA, and proteins. Recent advances in high-throughput sequencing and techniques leading to the identification of DNA-RNA, RNA-RNA, and RNA-protein complexes have allowed the functional characterization of a small set of lncRNAs. However, characterization of the full repertoire of lncRNAs playing essential roles in a number of normal and dysfunctional cellular processes remains an important goal for future studies. Here we describe the most commonly used techniques to identify lncRNAs, and to characterize their biological functions. In addition, we provide examples of these techniques applied to cutaneous research in healthy skin, that is, epidermal differentiation, and in diseases such as cutaneous squamous cell carcinomas and psoriasis. As with protein-coding RNA transcripts, lncRNAs are differentially regulated in disease, and can serve as novel biomarkers for the diagnosis and prognosis of skin diseases.

p63 in Squamous Cell Carcinoma of the Skin: More Than a Stem Cell/Progenitor Marker.

The p63 gene is often overexpressed in squamous cell carcinomas; however, how its overexpression contributes to tumor formation and expansion is still incompletely understood. Devos et al. report the development of a versatile mouse model demonstrating that p63 facilitates squamous cell carcinoma formation in skin and providing an excellent tool to dissect the relevance of its downstream signaling pathways in tumorigenesis.

HUR protects NONO from degradation by mir320, which is induced by p53 upon UV irradiation.

UV radiations challenge genomic stability and are a recognized cancer risk factor. We previously found that the RNA-binding protein NONO regulates the intra-S phase checkpoint and its silencing impaired HeLa and melanoma cell response to UV-induced DNA damage. Here we investigated the mechanisms underlying NONO regulation upon UVC treatment. We found that UVC rays induce the expression of mir320a, which can indeed target NONO. However, despite mir320a induction, NONO mRNA and protein expression are not affected by UVC. We found through RNA immunoprecipitation that UVC rays induce the ubiquitous RNA-binding protein HUR to bind NONO 5'UTR in a site overlapping mir320a binding site. Both HUR silencing and its pharmacological inhibition induced NONO downregulation following UVC exposure, whereas concomitant mir320a silencing restored NONO stability. UVC-mediated mir320a upregulation is triggered by p53 binding to its promoter, which lies within a region marked by H3K4me3 and H3K27ac signals upon UVC treatment. Silencing mir320a sensitizes cells to DNA damage. Overall our findings reveal a new mechanism whereby HUR protects NONO from mir320-mediated degradation upon UVC exposure and identify a new component within the complex network of players underlying the DNA damage response adding mir320a to the list of p53-regulated targets upon genotoxic stress.

Research Techniques Made Simple: Skin Carcinogenesis Models: Xenotransplantation Techniques.

Xenotransplantation is a widely used technique to test the tumorigenic potential of human cells in vivo using immunodeficient mice. Here we describe basic technologies and recent advances in xenotransplantation applied to study squamous cell carcinomas (SCCs) of the skin. SCC cells isolated from tumors can either be cultured to generate a cell line or injected directly into mice. Several immunodeficient mouse models are available for selection based on the experimental design and the type of tumorigenicity assay. Subcutaneous injection is the most widely used technique for xenotransplantation because it involves a simple procedure allowing the use of a large number of cells, although it may not mimic the original tumor environment. SCC cell injections at the epidermal-to-dermal junction or grafting of organotypic cultures containing human stroma have also been used to more closely resemble the tumor environment. Mixing of SCC cells with cancer-associated fibroblasts can allow the study of their interaction and reciprocal influence, which can be followed in real time by intradermal ear injection using conventional fluorescent microscopy. In this article, we will review recent advances in xenotransplantation technologies applied to study behavior of SCC cells and their interaction with the tumor environment in vivo.

A composite enhancer regulates p63 gene expression in epidermal morphogenesis and in keratinocyte differentiation by multiple mechanisms.

p63 is a crucial regulator of epidermal development, but its transcriptional control has remained elusive. Here, we report the identification of a long-range enhancer (p63LRE) that is composed of two evolutionary conserved modules (C38 and C40), acting in concert to control tissue- and layer-specific expression of the p63 gene. Both modules are in an open and active chromatin state in human and mouse keratinocytes and in embryonic epidermis, and are strongly bound by p63. p63LRE activity is dependent on p63 expression in embryonic skin, and also in the commitment of human induced pluripotent stem cells toward an epithelial cell fate. A search for other transcription factors involved in p63LRE regulation revealed that the CAAT enhancer binding proteins Cebpa and Cebpb and the POU domain-containing protein Pou3f1 repress p63 expression during keratinocyte differentiation by binding the p63LRE enhancer. Collectively, our data indicate that p63LRE is composed of additive and partly redundant enhancer modules that act to direct robust p63 expression selectively in the basal layer of the epidermis.

p63-dependent and independent mechanisms of nectin-1 and nectin-4 regulation in the epidermis.

Nectins are immunoglobulin-like cell adhesion molecules mainly localized in adherens junctions. The transcription factor p63 is a master regulator of gene expression in stratified epithelia and controls several molecular processes. As mutations in the Pvrl1 and Pvrl4 genes encoding for nectins cause genetic disorders with phenotypes similar to p63-related syndromes, we investigated whether these proteins might be under p63 transcriptional control. Here, we show that in p63-null skin, Pvrl1 gene expression is strongly reduced, whereas Pvrl4 expression is unaffected. In human and mouse primary keratinocytes p63 depletion leads to a specific downregulation of the Pvrl1 gene. Consistent with a direct regulation, chromatin immunoprecipitation experiments (ChIP) indicate that p63 binds to two conserved intronic Pvrl1 enhancer regions. Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder, caused by mutations in p63 gene, mainly characterized by skin fragility. To test whether nectins may be affected in AEC syndrome, their expression was measured in keratinocytes obtained from patients with AEC or from a conditional mouse model for AEC syndrome. Pvrl1 expression was reduced in AEC keratinocytes, consistent with impaired p63 function. Surprisingly, Pvrl4 expression was similarly affected, in parallel with decreased expression of the transcription factor Irf6. Consistent with the well-characterized role of Irf6 in keratinocyte differentiation and its strong downregulation in AEC syndrome, Irf6 depletion caused reduced expression of Pvrl4 in wild-type keratinocytes. Taken together, our results indicate that Pvrl1 is a bona fide target gene of the transcription factor p63, whereas Pvrl4 regulation is linked to epidermal differentiation and is under Irf6 control.

The desmosomal protein desmoglein 1 aids recovery of epidermal differentiation after acute UV light exposure.

Epidermal structure is damaged by exposure to UV light, but the molecular mechanisms governing structural repair are largely unknown. UVB (290-320 nm wavelengths) exposure before induction of differentiation reduced expression of differentiation-associated proteins, including desmoglein 1 (Dsg1), desmocollin 1 (Dsc1), and keratins 1 and 10 (K1/K10), in a dose-dependent manner in normal human epidermal keratinocytes (NHEKs). The UVB-induced reduction in both Dsg1 transcript and protein was associated with reduced binding of the p63 transcription factor to previously unreported enhancer regulatory regions of the Dsg1 gene. As Dsg1 promotes epidermal differentiation in addition to participating in cell-cell adhesion, the role of Dsg1 in aiding differentiation after UVB damage was tested. Compared with controls, depleting Dsg1 via short hairpin RNA resulted in further reduction of Dsc1 and K1/K10 expression in monolayer NHEK cultures and in abnormal epidermal architecture in organotypic skin models recovering from UVB exposure. Ectopic expression of Dsg1 in keratinocyte monolayers rescued the UVB-induced differentiation defect. Treatment of UVB-exposed monolayer or organotypic cultures with trichostatin A, a histone deacetylase inhibitor, partially restored differentiation marker expression, suggesting a potential therapeutic strategy for reversing UV-induced impairment of epidermal differentiation after acute sun exposure.

Crosstalk among p53 family members in cutaneous carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer with a frequency increasing worldwide. The risk of developing cSCC has been strongly associated with chronic sun exposure, especially in light skin people. The aim of this viewpoint is to discuss the contribution of the tumor suppressor p53 and its homologues p63 and p73 in the formation and progression of cSCC. Mutations in the p53 gene are early and frequent events in skin carcinogenesis mainly as a consequence of UV light exposure, often followed by loss of function of the second allele. Although rarely mutated in cancer, p63 and p73 play key roles in human cancers, with their truncated isoforms lacking the N-terminal transactivating domain (∆N) being often upregulated as compared to normal tissues. ∆Np63 is abundantly expressed in cSCC, and it is likely to favour tumor initiation and progression. The function of p73 in cSCC is more enigmatic and awaits further studies. Interestingly, an intimate interplay exists between both p53 and p63, and the Notch signalling pathway, often inactivated in cSCC. Here, we summarize our current knowledge about the biological activities of p53 family members in cSCC and propose that integration of their signalling with Notch is key to cSCC formation and progression.