ASPP2 binds to hepatitis C virus NS5A protein via an SH3 domain/PxxP motif-mediated interaction and potentiates infection.

Hepatitis C virus (HCV) infects millions of people worldwide and is a leading cause of liver disease. Despite recent advances in antiviral therapies, viral resistance can limit drug efficacy and understanding the mechanisms that confer viral escape is important. We employ an unbiased interactome analysis to discover host binding partners of the HCV non-structural protein 5A (NS5A), a key player in viral replication and assembly. We identify ASPP2, apoptosis-stimulating protein of p53, as a new host co-factor that binds NS5A via its SH3 domain. Importantly, silencing ASPP2 reduces viral replication and spread. Our study uncovers a previously unknown role for ASPP2 to potentiate HCV RNA replication.

Long non-coding RNA uc.291 controls epithelial differentiation by interfering with the ACTL6A/BAF complex.

The mechanisms that regulate the switch between epidermal progenitor state and differentiation are not fully understood. Recent findings indicate that the chromatin remodelling BAF complex (Brg1-associated factor complex or SWI/SNF complex) and the transcription factor p63 mutually recruit one another to open chromatin during epidermal differentiation. Here, we identify a long non-coding transcript that includes an ultraconserved element, uc.291, which physically interacts with ACTL6A and modulates chromatin remodelling to allow differentiation. Loss of uc.291 expression, both in primary keratinocytes and in three-dimensional skin equivalents, inhibits differentiation as indicated by epidermal differentiation complex genes down-regulation. ChIP experiments reveal that upon uc.291 depletion, ACTL6A is bound to the differentiation gene promoters and inhibits BAF complex targeting to induce terminal differentiation genes. In the presence of uc.291, the ACTL6A inhibitory effect is released, allowing chromatin changes to promote the expression of differentiation genes. Thus, uc.291 interacts with ACTL6A to modulate chromatin remodelling activity, allowing the transcription of late differentiation genes.

Transglutaminase 3 Reduces the Severity of Psoriasis in Imiquimod-Treated Mouse Skin.

Four transglutaminase (TG) isoforms have been detected in epidermal keratinocytes: TG1, TG2, TG3, and TG5. Except for TG1 and TG3, their contribution to keratinocyte development and structure remains undefined. In this paper, we focused on the roles of TG2 and TG3 in imiquimod-induced psoriasis in mouse skin. We evaluated the severity of psoriasis markers in the skin of imiquimod-treated TG3 null and TG2 null mice. Our results showed that compromised TG3KO mouse skin was more responsive than WT or TG2KO mouse skin to the action of the pro-inflammatory drug imiquimod.

p63 Is a Promising Marker in the Diagnosis of Unusual Skin Cancer.

Skin cancer is the most common type of cancer worldwide. Ozone depletion and climate changes might cause a further increase in the incidence rate in the future. Although the early detection of skin cancer enables it to be treated successfully, some tumours can evolve and become more aggressive, especially in the case of melanoma. Therefore, good diagnostic and prognostic markers are needed to ensure correct detection and treatment. Transcription factor p63, a member of the p53 family of proteins, plays an essential role in the development of stratified epithelia such as skin. In this paper, we conduct a comprehensive review of p63 expression in different types of skin cancer and discuss its possible use in the diagnosis and prognosis of cutaneous tumours.

Kruppel-like factor 4 regulates keratinocyte senescence.

Keratinocyte replicative senescence has an important role in time-related changes of epidermis. Previous studies demonstrated that miRNAs play key roles in inhibiting proliferation and in the acquisition of the keratinocyte senescent phenotype as well as in individual ageing. Kruppel-like factor 4 is a transcription factor with dual functions in keratinocytes, being a stemness factor and a pro-differentiation factor. Interestingly, in skin squamous cell carcinomas KLF4 expression is strongly down-regulated or absent. While KLF4 involvement in senescence and ageing has not been investigated yet. Here, we show that Klf4 protein decreases during keratinocyte replicative senescence and during physiological skin aging, while its mRNA level does not change. We demonstrated that the senescence-associated miR-34a regulates post-transcriptionally Klf4 expression. KLF4 silencing is sufficient to induce a senescent phenotype in primary keratinocytes and ectopic miR-34a over-expression phenocopies this result. Our findings identify a novel regulatory loop between miR-34a and KLF4 during keratinocytes replicative senescence. This regulatory loop, beside aging, may play a role in age-related pathologies.

Metabolic pathways regulated by p63.

The transcription factor p63 belongs to the p53-family and is a master regulator of proliferative potential, lineage specification, and differentiation in epithelia during development and tissue homeostasis. In cancer, p63 contribution is isoform-specific, with both oncogenic and tumour suppressive roles attributed, for ΔNp63 and TAp63, respectively. Recently, p53 and TAp73, in line with other tumour suppressor genes, have emerged as important regulators of energy metabolism and metabolic reprogramming in cancer. To date, p63 contributions in controlling energy metabolism have been partially investigated; given the extensive interaction of the p53 family members, these studies have potential implications in tumour cells for metabolic reprogramming. Here, we review the role of p63 isoforms, TAp63 and ΔNp63, in controlling cell metabolism, focusing on their specific metabolic target genes and their physiological/functional context of action.

A comprehensive molecular characterization of a claudin-low luminal B breast tumor.

Breast cancer is the most common cause of death from cancer in women. Here, we present the case of a 43-year-old woman, who received a diagnosis of claudin-low luminal B breast cancer. The lesion revealed to be a poorly differentiated high-grade infiltrating ductal carcinoma, which was strongly estrogen receptor (ER)/progesterone receptor (PR) positive and human epidermal growth factor receptor (HER2) negative. Her tumor underwent in-depth chromosomal, mutational and gene expression analyses. We found a pathogenic protein truncating mutation in the TP53 gene, which is predicted to disrupt its transcriptional activity. The patient also harbors germline mutations in some mismatch repair (MMR) genes, and her tumor displays the presence of immune infiltrates, high tumor mutational burden (TMB) status and the apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) associated signatures, which, overall, are predictive for the use of immunotherapy. Here, we propose promising prognostic indicators as well as potential therapeutic strategies based on the molecular characterization of the tumor.

Thioredoxin-interacting protein (TXNIP) is a substrate of the NEDD4-like E3 ubiquitin-protein ligase WWP1 in cellular redox state regulation of acute myeloid leukemia cells.

The HECT-type E3 ubiquitin WWP1 (also known as NEDD4-like E3 ubiquitin-protein ligase WWP1) acts as an oncogenic factor in acute myeloid leukemia (AML) cells. WWP1 overexpression in AML confers a proliferative advantage to leukemic blasts (abnormal immature white blood cells) and counteracts apoptotic cell death and differentiation. In an effort to elucidate the molecular basis of WWP1 oncogenic activities, we identified WWP1 as a previously unknown negative regulator of thioredoxin-interacting protein (TXNIP)-mediated reactive oxygen species (ROS) production in AML cells. TXNIP inhibits the disulfide reductase enzymatic activity of thioredoxin (Trx), impairing its antioxidant function and, ultimately, leading to the disruption of cellular redox homeostasis. In addition, TXNIP restricts cell growth and survival by blocking glucose uptake and metabolism. Here, we found that WWP1 directly interacts with TXNIP, thus promoting its ubiquitin-dependent proteasomal proteolysis. As a result, accumulation of TXNIP in response to WWP1 inactivation in AML blasts reduces Trx activity and increases ROS production, hence inducing cellular oxidative stress. Increased ROS generation in WWP1-depleted cells culminates in DNA strand breaks and subsequent apoptosis. Coherently with TXNIP stabilization following WWP1 inactivation, we also observed an impairment of both glucose up-take and consumption. Hence, a contribution to the increased cell death observed in WWP1-depleted cells also possibly arises from the attenuation of glucose up-take and glycolytic flux resulting from TXNIP accumulation. Future studies are needed to establish whether TXNIP-dependent deregulation of redox homeostasis in WWP1-overexpressing blasts may affect the response of leukemic cells to chemotherapeutic drugs.

Take a breath: oxygen sensing of epidermal differentiation.

Skin serves as a barrier to protect our body from injury, pathogens and trans-epidermal water loss. It is the only tissue directly exposed to oxygen besides lungs. Air exposure is an essential step of in vitro generation skin graft. However, the role of oxygen in this process remains hitherto unclear. Teshima et al. unveiled the impact of the hypoxia-inducible factor (HIF) pathway on epidermal differentiation in three-dimensional skin models. The authors of this work describe how air-lifting of organotypic epidermal cultures impairs HIFs activity, leading to a proper terminal differentiation of keratinocytes and stratification.

Gene expression in organoids: an expanding horizon.

Recent development of human three-dimensional organoid cultures has opened new doors and opportunities ranging from modelling human development in vitro to personalised cancer therapies. These new in vitro systems are opening new horizons to the classic understanding of human development and disease. However, the complexity and heterogeneity of these models requires cutting-edge techniques to capture and trace global changes in gene expression to enable identification of key players and uncover the underlying molecular mechanisms. Rapid development of sequencing approaches made possible global transcriptome analyses and epigenetic profiling. Despite challenges in organoid culture and handling, these techniques are now being adapted to embrace organoids derived from a wide range of human tissues. Here, we review current state-of-the-art multi-omics technologies, such as single-cell transcriptomics and chromatin accessibility assays, employed to study organoids as a model for development and a platform for precision medicine.

A novel neural network model of Earth's topside ionosphere.

The Earth's ionosphere affects the propagation of signals from the Global Navigation Satellite Systems (GNSS). Due to the non-uniform coverage of available observations and complicated dynamics of the region, developing accurate models of the ionosphere has been a long-standing challenge. Here, we present a Neural network-based model of Electron density in the Topside ionosphere (NET), which is constructed using 19 years of GNSS radio occultation data. The NET model is tested against in situ measurements from several missions and shows excellent agreement with the observations, outperforming the state-of-the-art International Reference Ionosphere (IRI) model by up to an order of magnitude, especially at 100-200 km above the F2-layer peak. This study provides a paradigm shift in ionospheric research, by demonstrating that ionospheric densities can be reconstructed with very high fidelity. The NET model depicts the effects of numerous physical processes governing the topside dynamics and can have wide applications in ionospheric research.

p63 orchestrates serine and one carbon metabolism enzymes expression in head and neck cancer.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is characterized by high proliferation and limited differentiation. The altered expression of the p53 family members, and specifically of p63, represents a pivotal event in the pathogenesis of HNSCC. Physiologically, p63 affects metabolism through the direct transactivation of the enzyme hexokinase 2, and subsequently controls the proliferation of epithelial cells; nonetheless, its role in cancer metabolism is still largely unclear. The high energetic demand of cancer and the consequent needs of a metabolic reshape, also involve the serine and glycine catabolic and anabolic pathways, including the one carbon metabolism (OCM), to produce energetic compounds (purines) and to maintain cellular homeostasis (glutathione and S-adenosylmethionine). RESULTS: The involvement in serine/glycine starvation by other p53 family members has been reported, including HNSCC. Here, we show that in HNSCC p63 controls the expression of the enzymes regulating the serine biosynthesis and one carbon metabolism. p63 binds the promoter region of genes involved in the serine biosynthesis as well as in the one carbon metabolism. p63 silencing in a HNSCC cell line affects the mRNA and protein levels of these selected enzymes. Moreover, the higher expression of TP63 and its target enzymes, negatively impacts on the overall survival of HNSCC patients. CONCLUSION: These data indicate a direct role of p63 in the metabolic regulation of HNSCC with significant clinical effects.

A primary luminal/HER2 negative breast cancer patient with mismatch repair deficiency.

Here, we present the case of a 47-year-old woman diagnosed with luminal B breast cancer subtype and provide an in-depth analysis of her gene mutations, chromosomal alterations, mRNA and protein expression changes. We found a point mutation in the FGFR2 gene, which is potentially hyper-activating the receptor function, along with over-expression of its ligand FGF20 due to genomic amplification. The patient also harbors somatic and germline mutations in some mismatch repair (MMR) genes, with a strong MMR mutational signature. The patient displays high microsatellite instability (MSI) and tumor mutational burden (TMB) status and increased levels of CTLA-4 and PD-1 expression. Altogether, these data strongly implicate that aberrant FGFR signaling, and defective MMR system might be involved in the development of this breast tumor. In addition, high MSI and TMB in the context of CTLA-4 and PD-L1 positivity, suggest the potential benefit of immune checkpoint inhibitors. Accurate characterization of molecular subtypes, based on gene mutational and expression profiling analyses, will be certainly helpful for individualized treatment and targeted therapy of breast cancer patients, especially for those subtypes with adverse outcome.

Epigenetic priming of an epithelial enhancer by p63 and CTCF controls expression of a skin-restricted gene XP33.

The transcription factor p63 is a renowned master regulator of gene expression of stratified epithelia. While multiple proteins have been identified as p63 bona fide targets, little is known about non-coding RNAs (ncRNAs) whose transcription is controlled by p63. Here, we describe a skin-specific non-coding RNA XP33 as a novel target of p63. XP33 levels are increased during keratinocyte differentiation in vitro, while its depletion results in decreased expression of late cornified gene LCE2D. By using publicly available multi-omics data, we show that CTCF and p63 establish an epithelial enhancer to prime XP33 transcription in a tissue-restricted manner. XP33 promoter and enhancer form a chromatin loop exclusively in keratinocytes but not in other cell types. Moreover, the XP33 enhancer is occupied by differentiation-specific factors that control XP33 transcription. Altogether, we identify a tissue-specific non-coding RNA whose expression is epigenetically regulated by p63 and CTCF.

A BRCA2 germline mutation and high expression of immune checkpoints in a TNBC patient.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of mammary carcinoma. Here, we describe a case of an 81-year-old female diagnosed with ductal triple negative breast cancer with a germline pathogenic variant in BReast CAncer gene2 (BRCA2). Genetic testing also revealed the presence of four somatic mutations in the ephrin type-A receptor 3 (EphA3), TP53, BRCA1-associated protein (BAP1), and MYB genes. The BRCA2, TP53, and BAP1 gene mutations are highly predictive of a defective homologous recombination repair system and subsequent chromosomal instability in this patient. Coherently, the patient displayed a strong homologous recombination deficiency signature and high tumor mutational burden status, which are generally associated with increased probability of immune neoantigens formation and presentation, and with tumor immunogenicity. Analysis of immune checkpoint revealed high expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed death 1 (PD1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA 4), suggesting that the patient might likely benefit from immunotherapies. Altogether, these findings support an unveiled link between BRCA2 inactivation, HR deficiency and increased expression of immune checkpoints in TNBC. This clinical case highlights the importance of screening TNBC patients for genetic mutations and TMB biomarkers in order to predict the potential efficacy of immunotherapy.

Hippo Pathway Activation in Aged Mesenchymal Stem Cells Contributes to the Dysregulation of Hepatic Inflammation in Aged Mice.

Aging is always accompanied by chronic diseases which probably attribute to long-term chronic inflammation in the aging body. Whereas, the mechanism of chronic inflammation in aging body is still obscure. Mesenchymal stem cells (MSCs) are capable of local chemotaxis to sites of inflammation and play a powerful role in immune regulation. Whether degeneration of MSCs in the aging body is associated with unbalanced inflammation is still not clear. In this study, immunosuppressive properties of aged MSCs are found to be repressed. The impaired immunosuppressive function of aged MSCs is associated with lower expression of the Hippo effector Yes-associated protein 1 (YAP1) and its target gene signal transducer and activator of transcription 1 (STAT1). YAP1 regulates the transcription of STAT1 through binding with its promoter. In conclusion, a novel YAP1/STAT1 axis maintaining immunosuppressive function of MSCs is revealed and impairment of this signal pathway in aged MSCs probably resulted in higher inflammation in aged mice liver.

Combined model of topside ionosphere and plasmasphere derived from radio-occultation and Van Allen Probes data.

In the last years, electron density profile functions characterized by a linear dependence on the scale height showed good results when approximating the topside ionosphere. The performance above 800 km, however, is not yet well investigated. This study investigates the capability of the semi-Epstein functions to represent electron density profiles from the peak height up to 20,000 km. Electron density observations recorded by the Van Allen Probes were used to resolve the scale height dependence in the plasmasphere. It was found that the linear dependence of the scale height in the topside ionosphere cannot be directly used to extrapolate profiles above 800 km. We find that the dependence of scale heights on altitude is quadratic in the plasmasphere. A statistical model of the scale heights is therefore proposed. After combining the topside ionosphere and plasmasphere by a unified model, we have obtained good estimations not only in the profile shapes, but also in the Total Electron Content magnitude and distributions when compared to actual measurements from 2013, 2014, 2016 and 2017. Our investigation shows that Van Allen Probes can be merged to radio-occultation data to properly represent the upper ionosphere and plasmasphere by means of a semi-Epstein function.

17ß-Oestradiol Protects from Hepatitis C Virus Infection through Induction of Type I Interferon.

BACKGROUND AND AIMS: Sex hormones are widely recognised to act as protective factors against several viral infections. Specifically, females infected by the hepatitis C virus display higher clearance rates and reduced disease progression than those found in males. Through modulation of particle release and spread, 17ß-oestradiol controls HCV's life cycle. We investigated the mechanism(s) behind oestrogen's antiviral effect. METHODS: We used cell culture-derived hepatitis C virus in in vitro assays to evaluate the effect of 17ß-oestradiol on the innate immune response. Host immune responses were evaluated by enumerating gene transcripts via RT-qPCR in cells exposed to oestrogen in the presence or absence of viral infection. Antiviral effects were determined by focus-forming unit assay or HCV RNA quantification. RESULTS: Stimulation of 17ß-oestradiol triggers a pre-activated antiviral state in hepatocytes, which can be maintained for several hours after the hormone is removed. This induction results in the elevation of several innate immune genes, such as interferon alpha and beta, tumour necrosis factor, toll-like receptor 3 and interferon regulatory factor 5. We demonstrated that this pre-activation of immune response signalling is not affected by a viral presence, and the antiviral state can be ablated using an interferon-alpha/beta receptor alpha inhibitor. Finally, we proved that the oestrogen-induced stimulation is essential to generate an antiviral microenvironment mediated by activation of type I interferons. CONCLUSION: Resulting in viral control and suppression, 17ß-oestradiol induces an interferon-mediated antiviral state in hepatocytes. Oestrogen-stimulated cells modulate the immune response through secretion of type I interferon, which can be countered by blocking interferon-alpha/beta receptor alpha signalling.

Macrophage Activation and M2 Polarization in Wound Bed of Diabetic Patients Treated by Dermal/Epidermal Substitute Nevelia.

Clinical evidences have shown good results using dermal/epidermal substitutes (DESs) to treat diabetic foot ulcers. Recent studies suggest that, in addition to their scaffold action, DESs may favor wound healing by influencing wound bed inflammatory cells. This study aims to investigate whether DES may influence the inflammatory infiltrate and macrophages polarization toward a reparative phenotype. Fifteen diabetic patients with chronic foot ulcers have been randomly enrolled: 5 treated only by standard of care, served as control group (CG), and 10 treated with DES composed of type 1 bovin collagen (Nevelia, SYMATESE) considered as test group (TG). A biopsy was taken at baseline (T0) and after 30 days (T1). From bioptic paraffin specimen histological, immunohistochemical, and immunofluorescence analysis was performed. Immunohistochemistry reactions evaluated the number of M1 macrophage (CD38+) and M2 macrophage (CD163+). TG patients displayed general macrophage activation and their greater polarization toward M2 subpopulation 30 days after DES implant, compared with CG. From T0 to T1 there was a significant decrease of CD38+ (230 ± 42 and 135 ± 48 mm2, respectively; P < .001) and significant increase of CD163+ (102 ± 21 positive cells/mm2 and 366 ± 42 positive cells/mm2, respectively; P < .001). Confocal microscopy confirmed an increase of M2 cells as expressed by the reduced CD68+/CD163+ ratio. After 6 months of observation 6 patients (60%) of the TG completely healed, while only 1 patient (20%) healed in the CG (P < .01). The tested DES makes possible to treat diabetic foot ulcers inducing tissue reparative processes through macrophage activation and M2 reparative polarization.

Mutant Ras and inflammation-driven skin tumorigenesis is suppressed via a JNK-iASPP-AP1 axis.

Concurrent mutation of a RAS oncogene and the tumor suppressor p53 is common in tumorigenesis, and inflammation can promote RAS-driven tumorigenesis without the need to mutate p53. Here, we show, using a well-established mutant RAS and an inflammation-driven mouse skin tumor model, that loss of the p53 inhibitor iASPP facilitates tumorigenesis. Specifically, iASPP regulates expression of a subset of p63 and AP1 targets, including genes involved in skin differentiation and inflammation, suggesting that loss of iASPP in keratinocytes supports a tumor-promoting inflammatory microenvironment. Mechanistically, JNK-mediated phosphorylation regulates iASPP function and inhibits iASPP binding with AP1 components, such as JUND, via PXXP/SH3 domain-mediated interaction. Our results uncover a JNK-iASPP-AP1 regulatory axis that is crucial for tissue homeostasis. We show that iASPP is a tumor suppressor and an AP1 coregulator.