Free-amino acid metabolic profiling of visceral adipose tissue from obese subjects.

Interest in adipose tissue pathophysiology and biochemistry have expanded considerably in the past two decades due to the ever increasing and alarming rates of global obesity and its critical outcome defined as metabolic syndrome (MS). This obesity-linked systemic dysfunction generates high risk factors of developing perilous diseases like type 2 diabetes, cardiovascular disease or cancer. Amino acids could play a crucial role in the pathophysiology of the MS onset. Focus of this study was to fully characterize amino acids metabolome modulations in visceral adipose tissues (VAT) from three adult cohorts: (i) obese patients (BMI 43-48) with metabolic syndrome (PO), (ii) obese subjects metabolically well (O), and (iii) non obese individuals (H). 128 metabolites identified as 20 protein amino acids, 85 related compounds and 13 dipeptides were measured by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) and gas chromatography-/mass spectrometry GC/MS, in visceral fat samples from a total of 53 patients. Our analysis indicates a probable enhanced BCAA (leucine, isoleucine, valine) degradation in both VAT from O and PO subjects, while levels of their oxidation products are increased. Also PO and O VAT samples were characterized by: elevated levels of kynurenine, a catabolic product of tryptophan and precursor of diabetogenic substances, a significant increase of cysteine sulfinic acid levels, a decrease of 1-methylhistidine, and an up regulating trend of 3-methylhistidine levels. We hope this profiling can aid in novel clinical strategies development against the progression from obesity to metabolic syndrome.

Molecular Mechanisms and Function of the p53 Protein Family Member - p73.

Over 20 years after identification of p53 and its crucial function in cancer progression, two members of the same protein family were identified, namely p63 and p73. Since then, a body of information has been accumulated on each of these genes and their interrelations. Biological role of p73 has been elucidated thanks to four distinct knockout mice models: (i) with deletion of the entire TP73 gene, (ii) with deletion of exons encoding the full length TAp73 isoforms, (iii) with deletions of exons encoding the shorter DNp73 isoform, and (iv) with deletion of exons encoding C-terminal of the alpha isoform. This work, as well as expression studies in cancer and overwhelming body of molecular studies, allowed establishing major role of TP73 both in cancer and in neuro-development, as well as ciliogenesis, and metabolism. Here, we recapitulate the major milestones of this endeavor.

Role of the TAp63 Isoform in Recurrent Nasal Polyps.

The pathogenic molecular mechanisms underlying the insurgence of nasal polyps has not been completely defined. In some patients, these lesions can have a recurrence after surgery removal, and the difference between recurrent and not recurrent patients is still unclear. To molecularly characterize and distinguish between these two classes, a cohort of patients affected by nasal polyposis was analysed. In all patients we analysed the p63 isoform expression using fresh tissues taken after surgery. Moreover, confocal immunofluorescence analysis of fixed sections was performed. The results show high ΔNp63 expression in samples from the nasal polyps of patients compared to the normal epithelia. Analysis of the expression level of the TAp63 isoform shows differential expression between the patients with recurrence compared to those not recurring. The data, considered as the ΔN/TAp63 ratio, really discriminate the two groups. In fact, even though ΔNp63 is expressed in non-recurrent patients, the resulting ratio ΔN/TAp63 is significantly lower in these patients. This clearly indicates that the status of TAp63 expression, represented by the ΔN/TAp63 ratio, could be considered a prognostic marker of low recurrence probability. In these samples we also investigated the expression of OTX2 transcription factor, known to be a selective activator of TAp63, detecting a significant correlation. Database analysis of HNSCC patients showed increased survival for the patients presenting OTX2 amplification and/or overexpression. These results, together with the fact that TAp63 can be selectively upregulated by HDAC inhibitors, open the possibility to consider local treatment of recurrent nasal polyps with these molecules.

Arterial ageing: from endothelial dysfunction to vascular calcification.

Complex structural and functional changes occur in the arterial system with advancing age. The aged artery is characterized by changes in microRNA expression patterns, autophagy, smooth muscle cell migration and proliferation, and arterial calcification with progressively increased mechanical vessel rigidity and stiffness. With age the vascular smooth muscle cells modify their phenotype from contractile to 'synthetic' determining the development of intimal thickening as early as the second decade of life as an adaptive response to forces acting on the arterial wall. The increased permeability observed in intimal thickening could represent the substrate on which low-level atherosclerotic stimuli can promote the development of advanced atherosclerotic lesions. In elderly patients the atherosclerotic plaques tend to be larger with increased vascular stenosis. In these plaques there is a progressive accumulation of both lipids and collagen and a decrease of inflammation. Similarly the plaques from elderly patients show more calcification as compared with those from younger patients. The coronary artery calcium score is a well-established marker of adverse cardiovascular outcomes. The presence of diffuse calcification in a severely stenotic segment probably induces changes in mechanical properties and shear stress of the arterial wall favouring the rupture of a vulnerable lesion in a less stenotic adjacent segment. Oxidative stress and inflammation appear to be the two primary pathological mechanisms of ageing-related endothelial dysfunction even in the absence of clinical disease. Arterial ageing is no longer considered an inexorable process. Only a better understanding of the link between ageing and vascular dysfunction can lead to significant advances in both preventative and therapeutic treatments with the aim that in the future vascular ageing may be halted or even reversed.

Carmine Melino and the Institute of Hygiene.

Throughout his 94 years of life, Carmine Melino brilliantly pursued different professional paths, his life being a constant stimulus for students, colleagues, friends and the family. Following the early formative years of study, here, we briefly list his scientific achievements in Occupational Medicine and Hygiene as well as his broad literary interests. Carmine was an inspiration to his generation not only because of his professional achievements, but also for his warm personality, exemplary hard-playing life and unbounded enthusiasm. A polymath, post-enlightenment ethos flowed to all his friends and colleagues, creating an ambience where intellectual excellence was highly appreciated and avidly pursued.

Cutaneous mosaicism, in KRT1 pI479T patient, caused by the somatic loss of the wild-type allele, leads to the increase in local severity of the disease.

BACKGROUND: Epidermolytic ichthyosis (BCIE, OMIM 113800), is an autosomal dominant disorder of the skin caused by mutations in keratin genes KRT1 and KRT10. We present two sporadic patients showing a mild diffuse ichthyosis with palmoplantar keratoderma. Interestingly, one of them shows a significant hyperkeratosis of palms and soles similar to those present in the Meleda disease (OMIM 248300). OBJECTIVE: In this paper we would clarify the genetic difference between the two patients, giving rise to the different phenotype. METHODS: Clinical evaluation, followed by histological and molecular analysis has been established for these patients. RESULTS: We demonstrated the presence of a genetic cutaneous mosaicism. Both patients carry the KRT1 pI479T substitution, but in the palmoplantar areas of one of them, only the mutated allele is expressed (hemizygous). This leads to highlight a new type of cutaneous mosaic, the palmoplantar mosaicism.

AMP-activated protein kinase: structure, function, and role in pathological processes.

Recently, AMP-activated protein kinase (AMPK) has emerged as a key regulator of energy balance at cellular and whole-body levels. Due to the involvement in multiple signaling pathways, AMPK efficiently controls ATP-consuming/ATP-generating processes to maintain energy homeostasis under stress conditions. Loss of the kinase activity or attenuation of its expression leads to a variety of metabolic disorders and increases cancer risk. In this review, we discuss recent findings on the structure of AMPK, its activation mechanisms, as well as the consequences of its targets in regulation of metabolism. Particular attention is given to low-molecular-weight compounds that activate or inhibit AMPK; the perspective of therapeutic use of such modulators in treatment of several common diseases is discussed.

[TUMOR SUPPRESSOR p63 REGULATES EXPRESSION OF UBIQUITIN LIGASE Pirh2].

Transcription factor p63 is a member of the p53 protein family. Due to the high degree of structural similarity p53, p63, and p73 are known to have overlapping functions relating to cell cycle regulation, apoptosis and tumor transformation. Furthermore, p63 plays crucial role in epidermal tissue development and differentiation. Pirh2 (product of RCHY1 gene) is an E3 ubiquitin ligase modifying all three members of the p53 family resulting in their subsequent proteasomal degradation. Our results demonstrate that p63, similar to p53, is able to regulate expression levels of Pirh2. Importantly, Pirh2 expression is activated only by transcriptionally active isoform of p63--TAp63, but not the N-terminally truncated ΔNp63.

Apoptin induces apoptosis by changing the equilibrium between the stability of TAp73 and ΔNp73 isoforms through ubiquitin ligase PIR2.

Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ∆Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ∆Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.

MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation.

Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-ß-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3'UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

Involvement of transcribed lncRNA uc.291 and SWI/SNF complex in cutaneous squamous cell carcinoma.

While non-melanoma skin cancers (NMSCs) are the most common tumours in humans, only the sub-type cutaneous squamous cell carcinoma (cSCC), might become metastatic with high lethality. We have recently identified a regulatory pathway involving the lncRNA transcript uc.291 in controlling the expression of epidermal differentiation complex genes via the interaction with ACTL6A, a component of the chromatin remodelling complex SWI/SNF. Since transcribed ultra-conserved regions (T-UCRs) are expressed in normal tissues and are deregulated in tumorigenesis, here we hypothesize a potential role for dysregulation of this axis in cSCC, accounting for the de-differentiation process observed in aggressive poorly differentiated cutaneous carcinomas. We therefore analysed their expression patterns in human tumour biopsies at mRNA and protein levels. The results suggest that by altering chromatin accessibility of the epidermal differentiation complex genes, down-regulation of uc.291 and BRG1 expression contribute to the de-differentiation process seen in keratinocyte malignancy. This provides future direction for the identification of clinical biomarkers in cutaneous SCC. Analysis of publicly available data sets indicates that the above may also be a general feature for SCCs of different origins.

Two new p73 splice variants, gamma and delta, with different transcriptional activity.

p73 has been recently identified as a new structural and functional homologue of the transcription factor p53. It is expressed in either a full-length form, alpha, or a shorter beta mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of two new p73 splicing variants, gamma (splicing out exon 11) and delta (splicing out exons 11, 12, and 13). Both gamma and delta p73 variants are expressed in human peripheral blood lymphocytes, primary keratinocytes, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs in both primary cells of different lineage and established cell lines even within the same type of tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73gamma nor p73delta interact with p53, whereas p73gamma showed strong interactions with all p73 isoforms, and p73delta binds efficiently p73alpha and p73gamma but only weakly p73beta. At the functional level, p73gamma is significantly less efficient in activating transcription of the p21(Waf1/Cip1) promoter than p53 or p73beta, whereas the effect of p73delta is intermediate and comparable to that of p73alpha. The ability of the different p73 variants to affect cell growth in p53 null osteosarcoma SAOS-2 cells correlates with their transcriptional activity on the p21(Waf1/Cip1) promoter: p73beta is the most efficient in inhibiting colony formation, whereas p73gamma is almost ineffective. Our results suggest that p73 isoforms may be differentially regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and growth suppression activities by affecting heterodimer formation.

[Vincenzo Cuomo, health officer and general practitioner on the Isle of Capri]. / Vincenzo cuomo medico condotto ed ufficiale sanitario a Capri.

We provide a broad review of the scientific output of Vincenzo Cuomo, general practitioner and health officer on the Isle of Capri. As well as his work as a doctor he was a close observer of the environment and in particular of the island's splendid climate and its benefits not only for the local population in general but also for visitors from northern Europe's colder climes. These mainly included tuberculosis sufferers in search of the beneficial effects of the Mediterranean climate, as well as those suffering from asthma and chronic bronchitis. Vincenzo Cuomo understood the beneficial effects of the island's climate and campaigned successfully to have the island formally recognized as a major health resort. To add scientific rigour to his observations, he installed a fully operational meteorological station at the top of his villa, supplying data as part of Italy's network of meteorological and climatological stations over some 50 years. Given his contribution to science and medicine, we believe it appropriate to consider Vincenzo Cuomo a precursor of modern clinical climatology.

miR-203 represses 'stemness' by repressing DeltaNp63.

The epidermis, the outer layer of the skin composed of keratinocytes, is a stratified epithelium that functions as a barrier to protect the organism from dehydration and external insults. The epidermis develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where epithelial cells with high clonogenic and proliferative capacity reside. Deletion of p63 in mice results in a dramatic loss of all keratinocytes and loss of stratified epithelia, probably due to a premature proliferative rundown of the stem and transient amplifying cells. Here we report that microRNA (miR)-203 is induced in vitro in primary keratinocytes in parallel with differentiation. We found that miR-203 specifically targets human and mouse p63 3'-UTRs and not SOCS-3, despite bioinformatics alignment between miR-203 and SOCS-3 3'-UTR. We also show that miR-203 overexpression in proliferating keratinocytes is not sufficient to induce full epidermal differentiation in vitro. In addition, we demonstrate that miR-203 is downregulated during the epithelial commitment of embryonic stem cells, and that overexpression of miR-203 in rapidly proliferating human primary keratinocytes significantly reduces their clonogenic capacity. The results suggest that miR-203, by regulating the DeltaNp63 expression level, is a key molecule controlling the p63-dependent proliferative potential of epithelial precursor cells both during keratinocyte differentiation and in epithelial development. In addition, we have shown that miR-203 can regulate DeltaNp63 levels upon genotoxic damage in head and neck squamous cell carcinoma cells, thus controlling cell survival.

Brn-3a/POU4F1 interacts with and differentially affects p73-mediated transcription.

The Brn-3a/POU4F1 POU transcription factor is critical for the survival and differentiation of specific sensory neurons during development or upon injury; by regulating expression of target genes, either directly or indirectly upon interaction with other proteins. In this study, we demonstrated the physical interaction of Brn-3a with different p73 isoforms and showed co-localization in sensory neurons arising from the neural crest. The biological effects of p73/ Brn-3a interaction depend on the particular p73 isoform, because co-expression of Brn-3a with TAp73 enhanced cell cycle arrest, whereas Brn-3a and DeltaNp73 cooperated to increase protection from apoptosis. Brn-3a antagonized TAp73 transactivation of pro-apoptotic Bax, but co-operated to increase transcription of the cell cycle regulator p21 CIP1/Waf1. The region 425-494 amino acids within the TAp73 C terminus were critical for Brn-3a to repress Bax transactivation, but not for cooperation on the p21 CIP1/Waf1 promoter. Our results suggest that co-factors binding to the p73 C terminus facilitate maximal activation on the Bax but not p21 CIP1/Waf1 promoter and that Brn-3a modulates this interaction. Thus, the physical interaction of Brn-3a with specific p73 isoforms will be critical for determining cell fate during neuronal development or in injured neurons expressing both factors.

Itch: a HECT-type E3 ligase regulating immunity, skin and cancer.

The HECT-type E3 ubiquitin ligase (E3) Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease, including lung and stomach inflammation and hyperplasia of lymphoid and hematopoietic cells. The involvement of Itch in multiple signaling pathways and pathological conditions is presently an area of extensive scientific interest. This review aims to bring together a growing body of work exploring Itch-regulated biological processes, and to highlight recent discoveries on the regulatory mechanisms modulating its catalytic activity and substrate recognition capability. Our contribution is also an endeavor to correlate Itch substrate specificity with the pathological defects manifested by the mutant Itchy mice.

Transglutaminase-2 differently regulates cartilage destruction and osteophyte formation in a surgical model of osteoarthritis.

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodeling. Transglutaminases catalyze a calcium-dependent transamidation reaction that produces covalent cross-linking of available substrate glutamine residues and modifies the extracellular matrix. Increased transglutaminases-mediated activity is reported in osteoarthritis, but the relative contribution of transglutaminases-2 (TG2) is uncertain. We describe TG2 expression in human femoral osteoarthritis and in wild-type and homozygous TG2 knockout mice after surgically-induced knee joint instability. Increased TG2 levels were observed in human and wild-type murine osteoarthritic cartilage compared to the respective controls. Histomorphometrical but not X-ray investigation documented in osteoarthritic TG2 knockout mice reduced cartilage destruction and an increased osteophyte formation compared to wild-type mice. These differences were associated with increased TGFbeta-1 expression. In addition to confirming its important role in osteoarthritis development, our results demonstrated that TG2 expression differently influences cartilage destruction and bone remodeling, suggesting new targeted TG2-related therapeutic strategies.

FXIIIA and TGF-beta over-expression produces normal musculo-skeletal phenotype in TG2-/- mice.

Transglutaminase (TGs) enzymes and proteins crosslinking have for long time been implicated in the formation of hard tissue development, matrix maturation and mineralization. Among the TGs family members, in the context of connective tissue formation, TG2 and Factor XIII are expressed in cartilage by hypertrophic chondrocytes. Here, we analyse the morphological consequences of TG2 deficiency, during the development of skeletal elements. When TG2 is absent, there are not gross abnormalities in the development of the skeletal system, probably from compensatory mechanisms resulting in increased expression of FXIIIA and TGF-beta 1. In vivo other TGs may be involved in promoting chondrocytes and osteoblast differentiation and matrix mineralisation.

The E3 ubiquitin ligase WWP1 sustains the growth of acute myeloid leukaemia.

The E3 ubiquitin ligase (E3) WWP1 is an oncogenic factor implicated in the maintenance of different types of epithelial cancers. The role of WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in haematological neoplasms remains unknown. Acute myeloid leukaemia (AML) is characterized by the expansion of malignant myeloid cells blocked at different stages of differentiation. Here we report that the expression of WWP1 is significantly augmented in a large cohort of primary AML patients and in AML cell lines, compared with haematopoietic cells from healthy donors. We show that WWP1 inactivation severely impairs the growth of primary AML blasts and cell lines in vitro. In vivo, we observed a reduced leukaemogenic potential of WWP1-depleted AML cells upon transplantation into immunocompromised mice. Mechanistically, WWP1 inactivation induces the accumulation of its protein substrate p27Kip1, which ultimately contributes to G0/G1 cell cycle arrest of AML blasts. In addition, WWP1 depletion triggers the autophagy signalling and reduces survival of leukaemic cells. Collectively, our findings provide molecular insights into the anti-cancer potential of WWP1 inhibition, suggesting that this E3 is a promising biomarker and druggable target in AML.