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Centenarians are the best model to study successful ageing. Unfortunately, they are rare and do not have an aged-matched control population to compare their exceptional characteristics with non-longevous people. Considering the complexity of molecular studies, the opportunity to analyse the centenarian phenotype with anthropometry could be an easy and no invasive interesting solution to identify peculiar measurable variables. In addition to the classic measurements, the bioelectrical impedance could be considered. This method permits to analyse the body composition, in terms of fat free mass and fat mass. Ageing is related to reduced fat free mass and increased body fat. The reduction of the first one is directly responsible for the onset of frailty and sarcopenia. These are typical conditions in elderly that leads to reduced physical strength and loss of immune competence. Thus, it is mandatory to identify specific values and range and perform multiple measurements in aged people and centenarians to address to weight variation modifiable causes, preventing premature death and avoiding diseases.

There is an urgent need to discover new antimalarials, due to the spread of chloroquine resistance and the limited number of available drugs. A number of quinones have been shown to be effective antimalarials; the observed effects are most likely related to the most prominent chemical feature of these kind of molecules, that is their ability to undergo redox cycling. Antiplasmodial activity of quinone structures of marine origin has been reported. Examples are xestoquinone and halenaquinone (Laurent et al., 2006, Bioorg. Med. Chem., 14:4477-82), their derivative orlaquinone (Longeon et al., 2010, Bioorg. Med. Chem., 18:6006-11), and ketoadociaquinones (Shmitz et al., 1988, J. Org. Chem., 53:3922-25; Cao et al., 2005, Bioorg. Med. Chem., 13:999-1003), isolated from Xestospongia sponges, and thiaplakortones (Davis et al., 2013, J. Org. Chem., 78: 9608-13), isolated from Plakortis lita. It has been also evidenced that the presence of a dioxothiazine moiety enhances the antiplasmodial activity (Figure 1). Within our search for bioactive compounds from Mediterranean ascidians, we have previously isolated a large group of thiazinoquinones (Menna et al., 2013, Eur. J. Org. Chem,, 16:3241-46; Aiello et al., 2005, Eur. J. Org. Chem., 23:5024-30; Aiello et al., 2005, J. Med. Chem., 48:3410-16; Aiello et al., 2003, Eur. J. Org. Chem., 5: 98-900). Some of these compounds have been used as lead structures for the synthesis of simplified analogues for pharmacological screening (Aiello et al., 2010, Bioorg. Med. Chem., 18: 719-27). Based on the structural analogy with the reported antimalarial quinone structures, the synthetic derivatives prepared using the natural thiazinoquinones aplidinones A and B as model structures (1-12, Figure 2) have been tested in vitro against D10 (chloroquine-sensitive) and W2 (chloroquine-resistant) strains of P. falciparum. The synthetic derivatives have shown a significant antiplasmodial activity and many structural requirements, critical for their activity, have been evidenced. Conformational (MD and MM) and electronic (tautomeric form, pka values, percentage of neutral/ionized forms at different pH values) analysis have been performed by computational means. The propensity for electron acceptance of the active compounds has been explored by LUMO orbital and anionic radical analysis. A correlation between electrochemistry (???direct??? and in the presence of Fe(III)-Heme) of the studied compounds and their antiplasmodial effects has been evidenced by using solid-state electrochemical assays (Dome??nech-Carbo?? et al., 2013, Anal. Chem., 85: 4014-21).

Chemical investigation of the Mediterranean ascidian A.conicum resulted in the isolation of two new meroterpenes, the conithiaquinones A (1) and B (2), in addition to two previously reported chromenols (3 and 4) and conicaquinones (5 and 6). The structures of conithiaquinones A and B were determined by interpretation of spectroscopic data, and regiochemical and stereochemical assignments were achieved with the aid of computational methods, including the recently developed DP4 NMR spectral prediction method. Both conithiaquinones A and B showed significant effects on the growth and viability of cells, with 1 showing interesting cytotoxicity against human breast cancer cells.

The sulphate group is a common feature of echinoderm metabolites, like sulphated saponins with triterpenoid or steroidal aglycones and has also been encountered in sponges’ metabolites, in the form of sulphated sterols and phenol sulphates. Only a few examples of sulfated alkanes/alkenes have been reported from marine sources and, recently, they have been shown to be commonly present in remarkably amounts in Mediterranean tunicates.1 Most of these compounds, often having quite simple structures, are cytotoxic or antimicrobial. More complex polysubstituted alkyl sulfates have also been isolated from ascidians; a remarkable example is turbinamide, a bioactive metabolite isolated few years ago in our laboratory from Sidnyum turbinatum.2 This molecule demonstrated a strong and selective cytotoxic effect against neuronal rather than immune system cells.3 An extensive investigation we have carried out on several species of Mediterranean ascidians evidenced that complex sulphated polyketides are quite widespread in these organisms. We have indeed isolated two new polysubstituted alkyl sulphates from two different Mediterranean ascidians (Sidnyum elegans and Ciona edwardsii) whose structures are referable to that of turbinammide. The new compounds also exhibited a strong cytotoxic activity against C6 glioma cells; their structural analogy with turbinamide allowed also evaluating the influence of slight structural modifications on the activity.

Ascidians belonging to the genus Aplidium of the Polyclinidae family are renowned for the variability of their metabolic content. A great chemiodiversity is devised from non-nitrogenous metabolites, mainly prenyl quinones or hydroquinones, to a vast array of nitrogenous metabolites. Within a research project directed to the study of the chemistry of marine ascidians from the Mediterranean sea we have investigated an Aplidium sp. collected in the Bay of Naples. It revealed a very unusual metabolic content, since it was completely lacking in meroterpenes, whilst the major component of its methanol extract was shown to be a group of unique alkyl sulfoxides. The structures of the novel metabolites have been elucidated through extensive spectroscopic analysis and their stereochemical analysis has been carried out in a three stages-approach, relying on the combination of NMR spectroscopy, CD studies, and computational methods

Killer Immunoglobulin like Receptors (KIR) are membrane proteins expressed on Natural Killer (NK) cells and on a small subset of CD8 lymphocytes. They influence the activation or inhibition of both cell types through interaction with their ligands, represented by Human Leukocyte Antigen (HLA) class I molecules. Several studies have shown that KIR/HLA interactions are involved in the pathogenesis and progression of different diseases as viral infections, autoimmune-disorders, or cancer, conditioning susceptibly to or protection against the outcome of the disease. HBV infection represents a major health problem with 2 billion people infected and 3 hundred and fifty million people with chronic diseases worldwide. The aim of this study is to assess whether KIR/HLA interaction is involved in immune response against hepatitis B infection. We are conducting a case-control study in Sicilian population, comparing subjects with chronic hepatitis B with subjects with previous HBV infection (HBc-Ab with or without HBeAb or HBsAb) as controls. A second control population was represented by HBcAb negative subjects. Peripheral blood samples were collected and genomic DNA was extracted from leukocytes and typed, using PCR-SSP, for KIR and HLA Preliminary data showed that HLA-A Bw4 was more frequent in cases than controls (1. cases, 23/35, 66%, vs. HBcAb positive controls, 8/35, 23%, OR 6.47, p=0.0003, and 2. cases, 23/35, 66%, vs. HBcAb negative controls, 6/60, 10%, OR 17.25, p=0.00001). No difference have been found in the frequencies HLA-C1, HLA-C2, HLA-Bw4I, and HLA-Bw4T; no difference have also been reported in the number of KIRs, both activating and inhibitory, and haplotype A and B between groups. Our data suggest that the HLA-A Bw4, likely through its interaction with the inhibitory KIR3DL1, is associated to the risk of developing chronic hepatitis B. Data on HBcAb negative subjects suggest also the possibility of selective genetic pressure.

Complex polysubstituted alkyl sulfates have been isolated from ascidians; a remarkable example is turbinamide, a bioactive metabolite isolated in our laboratory from Sidnyum turbinatum. This molecule demonstrated a strong and selective cytotoxic effect against neuronal rather than immune system cells. We have indeed isolated two new sulphated compounds (1 and 2) from another collection of S. turbinatum; as well as two new molecules (3 and 4) from two different Mediterranean ascidians (Sidnyum elegans and Ciona edwardsii). Their structural analogy with turbinamide allowed evaluating the influence of slight structural modifications on the activity.

The sulphate group is a common feature of echinoderm metabolites, like sulfated saponins with triterpenoid or steroidal aglycones and has also been encountered in sponges’ metabolites, in the form of sulphated sterols and phenol sulphates. Only a few examples of sulfated alkanes/alkenes have been reported from marine sources and, recently, they have been shown to be commonly present in remarkably amounts in Mediterranean tunicates. Most of these compounds, often having quite simple structures, are cytotoxic or antimicrobial. More complex polysubstituted alkyl sulfates have also been isolated from ascidians; a remarkable example is turbinamide, a bioactive metabolite isolated few years ago in our laboratory from Sidnyum turbinatum. This molecule demonstrated a strong and selective cytotoxic effect against neuronal rather than immune system cells. An extensive investigation we have carried out on several species of Mediterranean ascidians evidenced that complex sulphated polyketides are quite widespread in these organisms. We have indeed isolated two new compounds (1 and 2) very closely related to turbinammide from another collection of S. turbinatum; as well as two new molecules from two different Mediterranean ascidians (Sidnyum elegans and Ciona edwardsii) whose structures are referable to that of turbinammide as well.

Betaines and similar zwitterionic compounds are widely distributed in terrestrial plants and algae, as well as in marine invertebrates. In biological systems, these highly soluble dipolar molecules serve as organic osmolytes (osmoprotectants). Example of betaines isolated from marine invertebrates are homarine, nor- and aminozooanemonin, pyridinebetaines A and B. Most of these compounds are amphoteric low-molecular weight quaternary ammonium compounds deriving from proteinogenic or non- proteinogenic amino acids in which the nitrogen atom is fully methylated. Within a recent investigation of alkaloids from Axinella sponges, we have examined specimens of Axinella polypoides collected along Sardinian coasts and we have isolated three novel unique compounds, polyaxibetaines A-C (1-3), whose structure elucidations are the subjects of this paper.

Betaines and similar zwitterionic compounds are widely distributed in terrestrial plants and algae, as well as in marine invertebrates. In biological systems, these highly soluble dipolar molecules serve as organic osmolytes (osmoprotectants), namely substances synthesized or taken up from the environment by cells for protection against osmotic stress. Osmoprotectants can stabilize proteins and membranes when salt levels or temperatures are unfavourable; therefore, they could play important roles in the adaption of cells of marine invertebrates to various adverse environmental conditions. Examples of betaines isolated from marine invertebrates are homarine,1 trigonelline,2 taurines, baikiain betaine,3 β-stachydrine, 4 nor- and aminozooanemonin, 5 pyridinebetaines A and B.5 Most of these compounds are amphoteric low-molecular weight quaternary ammonium compounds deriving from proteinogenic or non- proteinogenic amino acids in which the nitrogen atom if fully methylated. Within a recent investigation of alkaloids from Axinella sponges, we have examined specimens of Axinella polypoides collected along Sardinian coasts and we have isolated three novel unique compounds, polyaxibetaines A-C (1-3), whose structure elucidations are the subject of this paper.Polyaxibetaines A-C extend the structural variety of the so far known betaines; in compounds 1 and 2, indeed, the nitrogen atom of the -amino group of phenylalanine and tyrosine, respectively, is comprised in a piperidine ring and further methylated, while compound 3 is a pyridine derivative sharing the nitrogen atom with the alfa-amino group of a tyrosine residue.

Tyrosine-derived halometabolites frequently occur in marine organisms and are known to play basic functions related to the survival of the living creatures producing them.1 Bromine is by far the halogen most frequently found in these metabolites; for example, bromotyrosine derivatives are constantly detected among the secondary metabolites of the Demospongiae; thus, they have been used for taxonomic purposes.2-4 Iodinated halometabolites, biosynthetically related to tyrosine, are much less widespread in the marine environment, since they have been isolated just in a few algae, microorganisms and marine invertebrates;5 however, recent research indicates this number will increase in the future. Many of the isolated and identified iodine-containing metabolites exhibited biological activities.6 A chemical investigation of secondary metabolites from the Mediterranean ascidia Ciona edwardsii has been performed and a new iodinated derivative, named iodocionin (1) has been isolated and chemically characterized. Structure of 1 has been elucidated through extensive spectroscopic analysis (1D- and 2D-NMR, and MS). Tests in vitro have been performed by Abteilung für Angewandte Molekularbiologie, Institut für Physiologische Chemie, in order to evaluate the capability of compound 1 to affect cells viability; these studies demonstrated that iodocionin exhibits acute toxicity against lymphoma cells with an IC50 of 7.75 μg/ml.

AGE6. Skin Ageing: Focus on the Role of Inflammatory Genetic Factors in Cutaneous Neoplasia C. M. Gambino1, F. Crapanzano1, G. Accardi1, A. Aiello1, C. Virruso1, G. Pistone1, M. R. Bongiorno1, D. Lio1, C. R. Balistreri1, G. Candore1 1University of Palermo, Palermo, Italy Background: Skin aging is a complex process that involves intrinsic and exogenous causes. Photo-oxidative damage caused by UV is the leading cause of extrinsic aging of the skin, known as photo-ageing. UV damages can be linked mostly to overproduction of ROS that induces a complex molecular cascade able to accelerate physiological aging, determining a typical dermal/epidermal inflammation with an increased risk of getting skin cancer. The skin response is strongly influenced by individual genetic background. Thus, polymorphisms in candidate inflammatory genes might play a role in photo-ageing and skin cancer. Methods: A total of 30 Sicilian subjects (12 females and 18 males; age range, 65–90 years) were enrolled. From histo-pathological data, 12 subjects showed skin cancer. Their blood samples have been used to obtain DNA samples and have been genotyped for TLR4 (+896 A/G; rs.4986790), TLR4 (+1196 C/T, rs.4986791), MMP2 (-1306 C/T, rs.243865) and MMP9 (-1562 C/T, rs.3918242) using a RFLP-PCR. Results: Allelic and genotypic frequencies of the SNPs analysed were evaluated by gene count. No significant differences in frequencies of these SNPs among cases and controls were observed. Conclusions: Our results are preliminary, so it is certainly necessary to increase the sample size of our study. In fact, the possible role of these SNPs in skin-ageing related neoplasia might open new perspectives for their analysis and prevention. However, it is imperative to underline the concept that functional effects of each SNP depend on the presence of one or different environmental causes (UV radiation, smoking, etc.).

Ascidians belonging to the Aplidium genus are characterized by a great chemiodiversity that includes nitrogenous and non-nitrogenous metabolites, this latter group being dominated by prenyl quinones/hydroquinones, linear or cyclic derivatives1. These compounds are generally known as meroterpenes. Recently, we analyzed a sample of Aplidium conicum collected in the Bay of Naples and, surprisingly, the pattern of its secondary metabolites resulted very different from that of other samples of A. conicum previously investigated. In fact, the ascidian is completely lacking in meroterpenoids, whilst the main components of its methanolic extract are a new class of molecules, unsaturated linear alkylsulfoxides, whose major constituents are Conisulfamines A, B, and C. The structures of the new compounds have been elucidated through an extensive application of MS, 1H, 13C and 2D homo-and heteronuclear-NMR techniques. The stereochemical analysis of Conisulfamine A has been carried out in a three stages–approach, relying on the combination of NMR spectroscopy and computational methods.

During our search for new biologically active and significant metabolites from Mediterranean ascidians, we have examined specimens of the tunicate Clavelina phlegraea Salfi 1929 collected in the Bay of Naples. This study led to the isolation of twelve new cytotoxic compounds, clavaminols A-N, whosw stwreostructures were established by analysis of spectroscopic data and chemical conversion. Clavaminol A was shown to be the more potent cytotoxic compound of this series inducing cell death through activation of the apoptotic machinery.

L'indagine chimica condotta ha portato all'isolamento e alla caratterizzazione strutturale di una serie di nuovi amminoalcoli. Tests in vitro hanno dimostrato che questi metaboliti sono attivi su linee cellulari di carcinoma gastrico (AGS), polmonare (A549), mammario (T-47D) e su cellule leucemiche (Jucart). Saggi condotti su ceppi di S. aureus, E coli e Candida albicans hanno evidenziato proprietà antibatteriche e antimicotiche

Il confronto tra il contenuto metabolico di un campione di A. conicum proveniente dal golfo di Napoli e i campioni precedentemente studiati provenienti da altre zone geografiche italiane.

L'articolo descrive l'isolamento di nuovi alcaloidi bromo-pirrolidi dalle spugne Axinella verrucosa e Axinella damicornis. Tali alcaloidi hanno dimostrato un'attività di tipo neuroprotettivo in quanto sono in grado di ridurre i livelli di calcio intraneuronali prevenendo il fenomeno dell'Eccitotossicità, ritenuto responsabile di alcune patologie del SNC.

L'analisi chimica di un tunicato del Mediterraneo ha portato al ritrovamento di due nuovi meroterpeni con uno scheletro carbonioso mai ritrovato precedentemente. I due composti hanno dimostrato attività pro-apoptotica.

Background: T-cells expressing the KIR2DS2 activating receptor were found to be prevalent in the coronary culprit atherosclerotic plaque in patients with acute coronary syndromes (ACS), suggesting a possible cytolytic activity against the endothelium of the plaque, leading to plaque rupture and in turn to superimposed thrombosis and sudden occlusion of the artery. To explore if a similar pathogenetic mechanism occurs in acute ischemic stroke, we analyzed the possible association between the genetic KIR and HLA repertoire and the susceptibility to ischemic stroke. Methods: Fourteen patients with ischemic stroke and ten controls with atherosclerotic disease without ischemic stroke matched for sex and age were genotyped for KIR and their HLA ligands, using PCR-SSP. Results: The frequency of the homozygous A haplotype (only KIR2DS4 as activating KIR) was 57% in patients and 80% controls (OR=0.33). The total number of activating and inhibitory KIR was not significantly different between cases and controls. The HLA-A was present in four cases and one control. The HLABw4T and HLABw4I alleles, as well as either the homozygous or the heterozygous form, were present in 12 cases and 6 controls, respectively. The putative interaction between HLABw4T/I allele and the KIR3DL1 inhibitory receptor was reported in 12 cases and 6 controls (OR=4). Conclusions: This descriptive preliminary analysis shows that subjects with ischemic stroke have a lower frequency of the A-haplotype, a higher frequency the HLABw4T or HLABw4I allele and a higher frequency of the HLABw4T/I-KIR3DL1 inhibitory interaction compared to controls, suggesting a stronger potential inhibition on NK activation.

Background: Dietary intervention and genetic alterations in gene encoding proteins involved in nutrient-sensing pathways can modulate lifespan, influencing longevity. It depends on under/over-expression of regulatory molecules that lead to different expression of homeostatic genes. Insulin/IGF-1 pathway was associated with longevity and lifespan modulation in model organisms. In humans, a key molecule in this pathway is FOXO3A that acts as a TF on homeostatic genes in response to decreased signaling increasing life span. Interestingly, other genes that increase lifespan interact with FOXO3A such as SIRT1, which modulates the oxidative stress response. Methods: We used meta-analytical and candidate-gene approaches to investigate the association of SNPs encoding proteins involved in Insulin/IGF-1 pathway with ageing and longevity. Results: Our study confirmed previous results related to the association between FOXO3A, IGF-1R, KLOTHO and longevity. No association was reported between IGF-1 and SIRT1 although all IGF-1 SNPs could affect its serum levels, known to modulate ageing and longevity. Moreover, we showed a new association between SHIP2 SNPs and longevity. Conclusions: Our results showed that specific SNPs of IGF-1R, FOXO3A, SHIP2 and KLOTHO influence ageing and longevity in different ethnic populations and that FOXO3A could be the most relevant gene in lifespan extension, particularly in males. Moreover, it could be speculated that a reduction of insulin signaling may decrease the activation of NF-kB slowing down inflammatory gene transcription. Thus, the intervention on ageing and longevity should be based both on nutrient-sensing and inflammatory pathways. The first simple step could be represented by the adherence to the Mediterranean diet with low glycaemic index and low animal proteins.

È noto che diverse specie di tunicati, appartenenti all’ordine Aplousobranchiata, famiglia Polyclinidae, genere Aplidium sono in grado di produrre metaboliti di tipo chinonico caratterizzati da interessanti attività biologiche. Abbiamo recentemente condotto uno studio su un campione di Aplidium conicum, proveniente dalle coste della Sardegna, che ha portato all’isolamento di diversi nuovi chinoni prenilati a conferma della presenza, nelle specie del genere Aplidium, di molecole a struttura chinonica. La delucidazione strutturale è basata sull’analisi dei dati spettroscopici, in special modo quelli ottenuti da esperimenti 2D-NMR; per l’assegnazione della regiochimica ci siamo avvalsi del confronto dei dati di chemical shift sperimentali con quelli stimati mediante calcoli quantomeccanici ab initio dei tensori di schermo NMR.

Lo studio chimico dell'ascidia A. conicum ha portato all'isolamento di due nuovi derivati terpene-chinonicicaratterizzati da un nucleo triciclico mai ritrovato precedentemente in natura originato dalla condensazione di un inusuale anello 1, 1-dioxo-1, 4-tiazinico con una porzione naftochinonica prenilata. La determinazione della struttura dei conicachinoni è stata realizzata mediante una approfondita indagine spettroscopica realizzata con l'ausilio di diverse tecniche NMR monodimensionali 1H e 13C NMR e bidimensionali ( COSY, HOHAHA, HSQC, HMBC). I conicachinoni A e B sono stati testati in vitro su due linee cellulari tumorali, le cellule della linea C6 (glioma di ratto) e della linea RBL-2H3 (leucemia basofila di ratto). Entrambi i composti hanno dimostrato un marcato e selettivo effetto citotossico nei confronti delle cellule gliomali di ratto.

L’indagine chimica condotta su un campione di Axinella verrucosa proveniente dal Mar mediterraneo ha portato all’isolamento, accanto alle già note mukanadine A-C5, manzacidine A e C, stevensina, spongiacidine A-B, 4,5-dibromo-palau’anina e la 2,3-dibromo styloguanidina, di alcuni nuovi alcaloidi mono-bromo-pirrolici. L’isolamento di questi nuovi mono bromo-pirrol-alcaloidi è stato realizzato mediante combinazione di diverse tecniche cromatografiche e la loro determinazione strutturale è stata eseguita mediante una approfondita indagine spettroscopica in particolare con l’ausilio di diverse tecniche NMR monodimensionali 1H e 13C NMR e bidimensionali (COSY, ROESY, HSQC, HMBC).

Un campione di Aplidium conicum proveniente da Capo Caccia in sardegna ha mostrato contenere oltre alla 6-bromo-ipaforina e alla plakaipaforina-A, un nuovo alcaloide, la conicamina, a struttura indolica. Saggi farmacologici hanno evidenziato una interessante attività antistaminica a concentrazioni micromolari.

L'analisi del tunicato Aplidium conicum ha portato all'isolamento di un nuovo ed inusuale meroterpene, la cui struttura è stata determinata attraverso la combinazione di metodi spettroscopici e computazionali

Descrizione dei risultati dell'analisi chimica di alcuni ascidiacei del Mediterraneo che ha portato all'isolamento di metaboliti secondari solfatati, piuttosto rari negli organismi marini, con attività antiproliferativa.

L'articolo descrive l'isolamento di esteri alchilsolforici da tunicati, la loro caratterizzazione stereostrutturale e l'attività farmacologica.

E' stato esaminato il Sidnyum turbinatum, una specie di ascidiaceo coloniale piuttosto raro presente nel mar Mediterraneo, il cui estratto butanolico si era dimostrato attivo in uno screening farmacologico preliminare di citotossicità sulle cellule WHEI 164. Il frazionamento “bioassay-guided” di tale estratto ha portato all’isolamento della turbinamide, una poliidrossidotriacontanammide solfatata. Tale metabolita, la cui struttura non ha precedenti in letteratura, presenta una notevole attività citotossica selettiva nei confronti di cellule tumorali derivanti da tessuti nervosi, In particolare, la turbinammide si è dimostrata particolarmente citotossica nei confronti delle cellule di glioma di ratto della linea C6 e nei confronti delle cellule di neuroblastoma umano della linea SH-SY5Y. D’altro canto, l’attività citotossica non sembra essere particolarmente esercitata nei confronti di altre linee cellulari come quelle immunitarie (macrofagi J774, IC50=220 g/ml). La determinazione della complessa struttura della turbinammide è stata realizzata mediante una approfondita indagine spettroscopica realizzata con l’ausilio di diverse tecniche NMR monodimensionali 1H e 13C NMR e bidimensionali (COSY, HOHAHA, HMQC, HMBA, HMQC-HOHAHA).