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Human papillomaviruses (HPVs) are agents of the most common sexually transmitted diseases. Both in men and in women, HPVs cause a variety of clinical symptoms ranging from warts to cancer. It has been shown that infection with oncogenic HPV is the major cause of development of cervical cancer in women and less frequently of penile cancers in men. HPV infects epithelial cells and can bind other cell types. Its presence in semen is widely known, but the effects on fertility and reproductive function are still controversial. Partners of women with high-grade squamous intraepithelial lesions were enrolled. From each subject seminal parameters were evaluated. HPV was detected and genotyped both in total semen and in swim up-fraction. In order to verify the cause-effect relationship between HPV presence in semen and sperm quality, we developed a new approach (differential cell lysis, DNA extraction and nested PCR) to evaluate virus localization in the different semen components. Our data indicate a correlation between HPV infection of exfoliated cells and reduction of sperm cell motility. Moreover, we analyzed the physical state of HPV18 DNA in sperm cells by qPCR. Our results suggest a possible integration event during which a disruption of the HPV genome occurs, causing the deletion of E1 and E2 genes. The data support the proposed role of HPV in decreased male and couple fertility and prompt new possible consequences of the viral infection in semen.

In order to understand the mechanisms of responses to changes in the physical and chemical environment, as well as the mechanisms of developmental pathways, we investigated by RT-qPCR assays and light microscopy observations the impact of antibiotics and cadmium in P. lividus sea urchin embryos. In particular we inspected development and biomarkers for free radical damage and apoptosis. During development embryos were exposed to an antibiotic mix (Ab mix, usually added to sea urchin cultures) or to sulfamethoxazole/trimethoprim mix (TMP/SMX, usually added to aquacultures) and/or levels of 10-5, 10-4, 10-3 M CdCl2. Even though treatment with TMP/SMX apparently did not affect development, it stimulated a remarkable molecular response to oxidative stress. Moreover, when embryos were exposed to TMP/SMX and CdCl2, even development was seriously impaired and the antioxidant defense was blocked. On the other hand, Ab mix and Cd co-treatment made the cadmium effect on development worse, but the antioxidant molecular defense was unchanged, rising the possibility that another stress pathway is involved. This study leads to the conclusion that co-exposure to antibiotics and cadmium induces synergistic effects on sea urchin embryos. Thus, in cadmium contaminated areas, antibiotic discharge can be an important environmental factor which might play an important role in survival of P. lividus populations.

Metallothioneins (MTs) constitute a heterogeneous superfamily of cysteine rich proteins, which coordinate divalent (Zn2+, Cd2+) or monovalent (Cu+) metal ions. Several functions have been proposed for these peptides, ranging from toxic metal protection to physiological metal homeostasis, free radical scavenging, oxidative stress protection, antiapoptotic defense, control of the redox status of the cell and also a role during development. Regarding the MT system in vertebrates’ nearest kin, little information is available at present. Recently MTs were also characterized in cephalochordates. Hence in order to shed some light on MT origin and functional differentiation through evolution, we studied MT genes in sea urchin P. lividus. Here we report the characterization of five different sea urchin MT genes (PlMT4 through PlMT8) and their regulation pattern during development. By Southern blot hybridization using MT4 through MT8 cDNA fragments as probes (Ragusa et al, 2013), we determined the number of MT genes in the genome, showing the presence of at least two different PlMT8 genes. Using primers based on P. lividus (v2.0) genome sequences, we amplified by PCR, cloned and sequenced the MT genes. PlMT gene structures are different from both vertebrates and cephalochordates. The genes are composed of 4 exons separated by 3 introns, the last intron is into the 3'UTR. In PlMT7 gene there are two predicted polyadenylation signals and in fact two species of mRNA transcripts exist. By RT-qPCR we showed that MT4 to MT6 are not expressed during development, while MT7 mRNA level rises throughout embryonic development and MT8 rises until gastrula stage and decreases thereafter. Analyzing MT promoters in silico, a considerable number of transcription factor binding elements can be identified, comprising putative metal response elements (MRE), antioxidant response elements (ARE), but their copy number and positions are different between constitutive (MT7-8) and induced (MT4/6) genes.

Metallothioneins are metal binding proteins that play a pivotal role in metal homeostasis and detoxification. Since their initial discovery, they have been extensively studied in a variety of organisms ranging from microbes to plants and animals. Organisms often possess multiple genes encoding metallothionein homologs with distinct properties, such as varying affinities for different metals, and in many cases different functions. Despite the plethora of available studies, very little information is known about sea urchin P. lividus MT (1, 2). We previously identified five Pl-MT embryonic cDNAs and we studied their induction after cadmium treatment (3). Now we studied their expression during embryo development by RT-qPCR. MT4 to MT6 are not expressed during normal development (very low levels of MT5 at pluteus stage), while MT7 mRNA level strikingly increases throughout embryonic development and MT8 rises until gastrula stage and decreases thereafter. Preliminary in situ hybridization experiment results indicate that both MT7 and MT8 are expressed in the archenteron at pluteus stage, but MT7 is expressed also in the oral ectoderm. We also isolated and sequenced the corresponding MT genes: all the Pl-MT genes have a similar structure (4 exons separated by 3 introns, the last intron into the 3'UTR) having introns in the same positions as S. purpuratus genes, but they are different from other deuterostomes. MT promoters were analyzed in silico: putative metal response elements (MRE), antioxidant response elements (ARE) were identified, but their copy number and positions are different between constitutive (MT7-8) and induced (MT4-5-6) genes. 1 Russo, R, et al. (2003) Cell Stress Chaperones, 8, 232-41 2 Russo, R, et al. (2013) Gene, 519, 305-10 3 Ragusa, MA, et al. (2013) Mol Biol Rep, 40, 2157-67

Echinoderms play a key role in the maintenance of the integrity of the ecosystem where they live. They are constantly exposed to pollutants, particularly in their early planktonic life stages. Sulfamethoxazole with trimethoprim (TMP/SMX) is a fixed antibiotic combination whose concentration is significantly increasing in the coastal waters due to human medicine and also intensive husbandry and aquaculture activities. Previously, we studied the defense strategies activated by P. lividus embryos in response to sublethal doses of CdCl2. Although toxic effects of cadmium on embryo development are not morphologically detectable before 24 hours of exposure, we found upregulation in mRNAs related to intracellular signaling component pathways, redox enzymes, and metal detoxification (including 5 metallothioneins) in response to cadmium. In this study, we investigated the putative morphological and molecular malfunctions in embryos exposed to different doses of TMP/SMX and cadmium, with the aim of simulating embryonic vulnerability that could occurred in polluted seawaters. The morphological analysis showed that embryos grown in the presence of TMP/SMX at the concentration used in aquaculture develop normally and gastrulate. Instead the embryos grown with a combination of TMP/SMX and low concentrations of cadmium show inhibition of development and arrest at the morula/blastula transition. RT-qPCR analysis was used to assess the extent of gene expression. Target mRNA were selected from the previously isolated mRNAs or from an in silico EST database search of sequences coding for apoptosis and stress markers. RT-qPCR results show that TMP/SMX treatment causes a remarkable increase in mRNAs coding for enzymes of Nitric Oxide pathway, detox enzymes and antiproliferative proteins. Co-treatment with TMP/SMX and cadmium blocks the response impairing development. Combined effect of TMP/SMX and cadmium could suppress the physiological protective response and activate the apoptotic program.

Cadmium (Cd) is a toxic heavy metal contaminating coastal environments. It has been suggested that the mechanisms of acute Cd toxicity involve the depletion of glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of ROS. Cd-increased ROS in turn produces lipid peroxidation, and results in DNA damage. However, little is known about direct evidence and mechanism for Cd-generated radicals until recently. In order to study the early defense strategies activated by P. lividus 30 hours embryos, in response to exposition to sub-lethal doses of Cd (100μM), we analyzed the induced transcriptome comparing it to that of control embryos by Suppression Subtractive Hybridization (SSH) technique. By this technique, we isolated five metallothionein (MT) cDNA and we analyzed their levels by RT-qPCR at different Cd concentrations. Two MT (MT7 and MT8) isogenes appeared to be constitutively expressed and upregulated upon high Cd concentration treatment (100μM). Three isogenes (MT4/MT6) are not transcribed in control embryos and they are specifically activated in response to low doses of Cd (0.1-1 μM). Moreover, in Cd upregulated group, there were: 10 genes related to intracellular signaling pathway components and transcription factors, 9 to oxidative, reductive and conjugative biotransformation, 7 to RNA maturation and protein synthesis, 6 to metal detoxification (including 5 MTs), 5 conserved hypothetical proteins with unknown functions and 16 unidentified sequences. In Cd down-regulated group, there were: 4 genes related to transcription regulation, 2 to oxidative phosphorilation, 1 to protein synthesis, 3 other hypothetical proteins and 4 unidentified sequences. We analyzed cDNA levels for 20 genes by RT-qPCR. Preliminary results show significant upregulation for apolipoprotein D, molybdopterin oxidoreductase, splicing coactivator subunit and ER lumen protein retaining receptor, TGFβ family receptor and TGFβ-activated kinase1

Expression of PlTa2 gene during sea urchin P. lividus development, is spatially confined to the neural territory and temporally activated from the blastula stage. To evaluate a possible involvement of chromatin modifications in regulation of PlTa2 gene expression we first searched for DNaseI hypersentive sites. We found four sites localized in the introns of the gene, when we used chromatin extracted from embryos at gastrula stage but not from morula stage. This result suggests a possible functional role of the introns in the activation of the expression of PlTa2 gene. Moreover, we used specific antibodies for RNA polymerase II and for different modified form of lysine 9, lysine 27 and lysine 4 of the H3 histone in quantitative Chromatin Immuno Precipitation experiments to emphasize the different state of chromatin during embryo development. Our analysis shows high H3K9 acetylation and H3K4 trimethylation degree in nucleosomes located at a2 promoter region in P. lividus embryos at gastrula stage, when the PlTa2 expression level is high. This observation agrees with conventional positive role assumed by these post-translational modifications in chromatin remodeling leading to increase promoter accessibility. Furthermore ChIP analysis shows also high H3K27 dimethylation degree during all development stage but ReChIP analysis shows no co-occupancy of this modification with RNA polymerase II in promoter region in embryos at gastrula stage. This observation is consistent with the hypothesis of a general repressive role of this modification in the non-neural territory of the embryo.

Heavy metals are common marine pollutants that emanate from such sources as industrial and sewage treatment discharges and anti-fouling paints. Cadmium (Cd2+) serves no essential function in biological organisms and it is a highly toxic and carcinogenic metal. In Paracentrotus lividus sea urchin embryos cadmium causes development arrest or severe malformations. At lower doses, cadmium activates different responses (HSPs synthesis, autophagic or apoptotic processes) that can allow embryo survival. This study was conducted to elucidate the gene transcription activation/upregulation or repression/downregulation inducted in sea urchin embryos grown under cadmium stress. Comparison between transcriptomes of 30 hours embryos treated or untreated with 100 mM cadmium chloride (CdCl2) was conducted by RDA technique. Results showed increased transcription levels, in treated embryos, of metallothionein genes, and genes coding for detoxification enzymes. Bioinformatic analysis of metallothionein sequences showed at least five different transcripts (MT-4 toMT -8) and only one of them was already new (MT-8 probably corresponds to Pl-MT). Quantitative real-time RT-PCR experiments confirmed RDA results and showed the specific activation of MT-4, MT-5 and MT-6 genes and the upregulation (or mRNA stabilization) of MT-7 and MT-8 genes. To investigate the threshold for gene activation, we are going to check the variations of transcript levels in embryos exposed to different doses of CdCl2.

P38 conveys a variety of signals, including conventional growth, migratory and death signals, as well as responding to environmental and mechanical stimuli. These signals induce phosphorylation of p38, which triggers both its translocation to the nucleus and the activation of its catalytic function. In Paracentrotus lividus we have shown that for a correct cilia re-generation the p38 activation is essential; we also showed, by time course Western blotting, two activation peaks during development (morula and early gastrula stages) with a precise localization of activated p38 in micromere and micromere derived cells (Nichel treatment higlighted this feature), and in the oral apical region. These results suggest that, like in Lytechinus variegatus, p38 activation/inactivation is involved in O-A axis specification, in gastrulation and in skeletogenesis. We found, in fact, that inhibiting (with SB203580) the p38 activation during cleavage (up to the morula stage) anomalies were observed in gastrulation (exogastrulae); later inhibition treatment (morula/blastula transition) impaired skeleton formation. Finally we was also able to demonstrate that in P. lividus are present four p38 isoforms differentially involved in different stress responses. All these features driven us to analyze if p38 activation is involved in high density population response in the “Ustica Island” MPA.

The sea urchin Arbacia lixula is a crucial species in driving the algal assemblages dynamic of the infralittoral zone. A. lixula is considered a thermophilic species and its increase in abundance in western Mediterranean waters, probably is due to an increase in seawater temperature. The aim of this study was to examine the reproductive success of A. lixula by larval development. The study was conducted within two Sicilian MPA (Capo Gallo-Isola delle Femmine and Ustica), characterized by photophilic algae, the same exposure and seawater temperature. In each area, twenty random specimens was sampled from May to September 2010, and mature gametes were collected from six males and six females to prepare embryos reared in experimental conditions. The larvae were monitored for 48h after the fertilization to assess the different stages and times of development. Our results showed a different larval development within two MPA, in time and quality of embryos. The samples from Ustica had a slower developmental rate and a higher mortality than samples from Capo Gallo. We hypothesize that in Ustica, A. lixula allocates almost resources into body components (Aristotele’s lantern) affecting reproductive success, than in Capo Gallo, probably due to an intra-specific competition relative to others factors here not investigated.

L’endotossina è una tossina microbica, parte integrante della membrana esterna della parete di batteri Gram-negativi che viene rilasciata completamente con la lisi del batterio. È costituita da sub-unita’ molecolari che hanno dimensioni comprese tra 10.000 e 20.000 Daltons, mentre le aggregazioni hanno dimensioni di circa 100.000 Daltons. Un esempio tipico di endotossina è rappresentato dal lipopolisaccaride (LPS) che è presente nella membrana esterna dei batteri Gram-negativi. Le endotossine sono le principali responsabili delle conseguenze cliniche delle infezioni da batteri gram-negativi. Infatti, l’endotossina è ritenuta responsabile della patogenesi della sepsi, dello shock settico e della conseguente malattia multiorgano (MOF) (1). A causa della sua natura particolarmente aggressiva e multifattoriale, la sepsi conduce rapidamente a morte e costituisce la principale causa di decesso nelle terapie intensive non coronariche di tutto il mondo. Da qui la necessità, per la cura della sepsi, di rimuovere e/o disattivare le endotossine dal corpo del paziente prima che la malattia degeneri. Infatti, in relazione alla presenza di endotossina nel sangue si innesca una complessa attivazione immunologica che coinvolge vari sistemi biologici (immunitario e reticoloendoteliale) e una serie di mediatori, liberati principalmente dall’attivazione di macrofagi, monociti e altre cellule. Inoltre, le endotossine sono contaminanti frequenti del DNA plasmidico estratto dai batteri e di tutti quei prodotti che sono estratti e/o sono venuti a contatto con essi. Le endotossine devono essere rimosse da questi prodotti per evitare reazioni infiammatorie nel corso delle applicazioni in vivo, quali ad esempio la terapia genica. Dal punto di vista terapeutico, negli ultimi anni hanno assunto un ruolo rilevante una classe di proteine e/o peptidi (e loro derivati) che, come prodotti dell’immunità innata, sono in grado di legare e neutralizzare componenti della parete batterica tra cui l’LPS. I peptidi che sono in grado di neutralizzare questo tipo di processo vengono oggi definiti con un termine ampio quale quello di Host Defense Peptide (HDP). Quest’ultima classe di proteine sono di particolare interesse in quanto oltre all’attività antimicrobica diretta, sono in grado di potenziare la risposta immunitaria dell’organismo, promuovendo in vitro varie risposte di difesa (ad es. migrazione di leucociti, maturazione delle cellule dendritiche) e contribuendo così al potenziamento della risposta immunitaria adattativa. Queste proteine sono generalmente caratterizzate dall’avere un peso molecolare compreso tra 2 ed 80 kDa e in maniera preferenziale, contenenti amino-acidi caricati positivamente (da qui il termine di proteine cationiche) (2). Le proteine denominate non specific Lipid Transfer proteins (ns-LTPs) sono piccole molecole proteiche particolarmente stabili di approssimativamente 10 KDa solitamente presenti in tutti gli organismi vegetali sino ad oggi studiati (3). Tali proteine sono accomunate dalla capacità di promuovere, in vitro, il trasferimento di molecole lipidiche attraverso membrane anche se, studi recenti, hanno dimostrato che esse sembrano svolgere una funzione protettiva in quanto capaci di agire come peptidi ad attività antimicrobica. In alcune specie vegetali è stata dimostrata una loro capacità allergenica come nel caso delle Rosaceae Prunoideae (pesca, albicocca, prugna), Pomoideae (mela) e le Urticacee quali la Parietaria (4). La tecnologia del DNA ricombinante ha permesso l’isolamento di vari allergeni della famiglia delle proteine ns- LTPs, tra questi quelli della Parietaria denominati Parj1 e Parj2. In particolare, sono state ad oggi isolate due isoforme dell’allergene Parj1 denominate (secondo la nomenclatura internazionale) Parj1.01 e Par1.02 (5, 6). Tali isoforme differiscono essenzialmente per la presenza di una regione di 37 aminoacidi presente esclusivamente nell’isoforma Parj1.01 (7). Caratterizzazione di un peptide in grado di legare LPS batterico Una analisi in silico condotta mediante l’algoritmo messo a disposizione dall’Antimicrobial Peptide Database ha messo in evidenza che la porzione carbossiterminale del Parj1.01 presenta caratteristiche biochimiche peculiari. Tale regione presenta una carica netta totale positiva (+5) e una elevata percentuale di residui di prolina suggerendo che tale peptide possa avere la capacità di agire come peptide ad attività antimicrobica. I dati riportati in Tabella 1 descrivono la diversa capacità delle due isoforme ricombinanti dell’allergene maggiore Parj1 (Parj1.01 e Parj1.02) di legare LPS di origine batterica. I dati mostrati evidenziano come l’isoforma Parj1.01 presenti una spiccta capacità di legare LPS. Tabella 1. Saggi di determinazione della quantità di endotossina endogena Quantità di proteina analizzata Positività al LAL test rParj1.01 rParj1.02 1 μg