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.

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.

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.

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).

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 control of desmosome gene expression and adhesion is compromised in AEC syndrome.

Ankyloblepharon, ectodermal defects, cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder caused by mutations in the p63 gene, essential for embryonic development of stratified epithelia. The most severe cutaneous manifestation of this disorder is the long-lasting skin fragility associated with severe skin erosions after birth. Using a knock-in mouse model for AEC syndrome, we found that skin fragility was associated with microscopic blistering between the basal and suprabasal compartments of the epidermis and reduced desmosomal contacts. Expression of desmosomal cadherins and desmoplakin was strongly reduced in AEC mutant keratinocytes and in newborn epidermis. A similar impairment in desmosome gene expression was observed in human keratinocytes isolated from AEC patients, in p63-depleted keratinocytes and in p63 null embryonic skin, indicating that p63 mutations causative of AEC syndrome have a dominant-negative effect on the wild-type p63 protein. Among the desmosomal components, desmocollin 3, desmoplakin and desmoglein 1 were the most significantly reduced by mutant p63 both at the RNA and protein levels. Chromatin immunoprecipitation experiments and transactivation assays revealed that p63 controls these genes at the transcriptional level. Consistent with reduced desmosome function, AEC mutant and p63-deficient keratinocytes had an impaired ability to withstand mechanical stress, which was alleviated by epidermal growth factor receptor inhibitors known to stabilize desmosomes. Our study reveals that p63 is a crucial regulator of a subset of desmosomal genes and that this function is impaired in AEC syndrome. Reduced mechanical strength resulting from p63 mutations can be alleviated pharmacologically by increasing desmosome adhesion with possible therapeutic implications.

Exome sequence identifies RIPK4 as the Bartsocas-Papas syndrome locus.

Pterygium syndromes are complex congenital disorders that encompass several distinct clinical conditions characterized by multiple skin webs affecting the flexural surfaces often accompanied by craniofacial anomalies. In severe forms, such as in the autosomal-recessive Bartsocas-Papas syndrome, early lethality is common, complicating the identification of causative mutations. Using exome sequencing in a consanguineous family, we identified the homozygous mutation c.1127C>A in exon 7 of RIPK4 that resulted in the introduction of the nonsense mutation p.Ser376X into the encoded ankyrin repeat-containing kinase, a protein that is essential for keratinocyte differentiation. Subsequently, we identified a second mutation in exon 2 of RIPK4 (c.242T>A) that resulted in the missense variant p.Ile81Asn in the kinase domain of the protein. We have further demonstrated that RIPK4 is a direct transcriptional target of the protein p63, a master regulator of stratified epithelial development, which acts as a nodal point in the cascade of molecular events that prevent pterygium syndromes.

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.

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.

Human skin-derived keratinocytes and fibroblasts co-cultured on 3D poly ε-caprolactone scaffold support in vitro HSC differentiation into T-lineage committed cells.

In humans, the thymus is the primary lymphoid organ able to support the development of T cells through its three-dimensional (3D) organization of the thymic stromal cells. Since a remarkable number of similarities are shared between the thymic epithelial cells (TECs) and skin-derived keratinocytes and fibroblasts, in this study we used human keratinocytes seeded with fibroblasts on the 3D poly ε-caprolactone scaffold to evaluate their ability to replace TECs in supporting T-cell differentiation from human haematopoietic stem cells (HSCs). We observed that in the multicellular biocomposite, early thymocytes expressing CD7(+)CD1a(+), peculiar markers of an initial T-cell commitment, were de novo generated. Molecular studies of genes selectively expressed during T-cell development revealed that TAL1 was down-regulated and Spi-B was up-regulated in the cell suspension, consistently with a T-cell lineage commitment. Moreover, PTCRA and RAG2 expression was detected, indicative of a recombinant activity, required for the generation of a T-cell receptor repertoire. Our results indicate that in the multicellular biocomposite, containing skin-derived elements in the absence of thymic stroma, HSCs do start differentiating toward a T-cell lineage commitment. In conclusion, the construct described in this study exerts some properties of a lymphoid organoid, suitable for future clinical applications in cell-based therapies.

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.

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.

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.

Direct targets of Klf5 transcription factor contribute to the maintenance of mouse embryonic stem cell undifferentiated state.

BACKGROUND: A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed. RESULTS: By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes. CONCLUSIONS: Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype. See: http://www.biomedcental.com/1741-7007/8/125.

p63 Suppresses non-epidermal lineage markers in a bone morphogenetic protein-dependent manner via repression of Smad7.

p63, a p53 family member, plays an essential role in epidermal development by regulating its transcriptional program. Here we report a previously uncovered role of p63 in controlling bone morphogenetic protein (BMP) signaling, which is required for maintaining low expression levels of several non-epidermal genes. p63 represses transcription of the inhibitory Smad7 and activates Bmp7, thereby sustaining BMP signaling. In the absence of p63, compromised BMP signaling leads to inappropriate non-epidermal gene expression in postnatal mouse keratinocytes and in embryonic epidermis. Reactivation of BMP signaling by Smad7 knockdown and/or, to a lesser extent, by BMP treatment suppresses expression of non-epidermal genes in the absence of p63. Canonical BMP/Smad signaling is essential for control of non-epidermal genes as use of a specific inhibitor, or simultaneous knockdown of Smad1 and Smad5 counteract suppression of non-epidermal genes. Our data indicate that p63 prevents ectopic expression of non-epidermal genes by a mechanism involving Smad7 repression and, to a lesser extent, Bmp7 induction, with consequent enhancement of BMP/Smad signaling.

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.

An autoregulatory loop directs the tissue-specific expression of p63 through a long-range evolutionarily conserved enhancer.

p63, a p53 family member, is essential for the development of various stratified epithelia and is one of the earliest markers of many ectodermal structures, including the epidermis, oral mucosa, apical ectodermal ridge, and mammary gland. Genetic regulatory mechanisms controlling p63 spatial expression during development have not yet been defined. Using a genomic approach, we identified an evolutionarily conserved cis-regulatory element, located 160 kb downstream of the first p63 exon, which functions as a keratinocyte-specific enhancer and is sufficient to recapitulate expression of the endogenous gene during mouse embryogenesis. Dissection of the p63 enhancer activity revealed a positive autoregulatory loop in which the p63 proteins directly bind to and are essential regulators of the enhancer. Accordingly, transactivating p63 isoforms induce endogenous p63 expression in cells that do not normally express this gene, whereas dominant negative isoforms suppress p63 expression in keratinocytes. In addition the transcription factor AP-2 also binds to the enhancer and cooperates with p63 to induce its activity. These results demonstrate that a long-range autoregulatory loop is involved in the regulation of p63 expression during embryonic development and in adult cells.

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.

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.