Repeated Rounds of Gonadotropin Stimulation Induce Imbalance in the Antioxidant Machinery and Activation of Pro-Survival Proteins in Mouse Oviducts.

Controlled ovarian stimulation (COS) through gonadotropin administration has become a common procedure in assisted reproductive technologies. COS's drawback is the formation of an unbalanced hormonal and molecular environment that could alter several cellular mechanisms. On this basis, we detected the presence of mitochondrial DNA (mtDNA) fragmentation, antioxidant enzymes (catalase; superoxide dismutases 1 and 2, SOD-1 and -2; glutathione peroxidase 1, GPx1) and apoptotic (Bcl-2-associated X protein, Bax; cleaved caspases 3 and 7; phosphorylated (p)-heat shock protein 27, p-HSP27) and cell-cycle-related proteins (p-p38 mitogen-activated protein kinase, p-p38 MAPK; p-MAPK activated protein kinase 2, p-MAPKAPK2; p-stress-activated protein kinase/Jun amino-terminal kinase, p-SAPK/JNK; p-c-Jun) in the oviducts of unstimulated (Ctr) and repeatedly hyperstimulated (eight rounds, 8R) mice. While all the antioxidant enzymes were overexpressed after 8R of stimulation, mtDNA fragmentation decreased in the 8R group, denoting a present yet controlled imbalance in the antioxidant machinery. Apoptotic proteins were not overexpressed, except for a sharp increase in the inflammatory-related cleaved caspase 7, accompanied by a significant decrease in p-HSP27 content. On the other hand, the number of proteins involved in pro-survival mechanisms, such as p-p38 MAPK, p-SAPK/JNK and p-c-Jun, increased almost 50% in the 8R group. Altogether, the present results demonstrate that repeated stimulations cause the activation of the antioxidant machinery in mouse oviducts; however, this is not sufficient to induce apoptosis, and is efficiently counterbalanced by activation of pro-survival proteins.

Early genotoxic damage through micronucleus test in exfoliated buccal cells and occupational dust exposure in construction workers: a cross-sectional study in L'Aquila, Italy.

AIM: The city of L'Aquila (central Italy) was hit by a strong earthquake in 2009 that caused the collapse of several buildings, deaths and injured people. In the following years, a great number of building sites were activated, building workers resulted intensely exposed and represent a relevant target for research on environmental mutagenesis and epidemiological surveillance. Cells of buccal mucosa are considered an appropriate site for early detecting of cytogenetic damage, since it represents the first barrier in inhalation or ingestion and can metabolize carcinogenic agents into reactive chemicals. Our study is aimed 1) at comparing the early genotoxic damage as measured by the buccal mucosa micronucleus test in two subgroups of workers defined by different occupational exposure and 2) at evaluating possible confounding variables such as lifestyle factors. METHODS AND RESULTS: A cross-sectional study was conducted in L'Aquila, on 24 outdoor workers (OWs) highly exposed on the construction sites and 26 indoor workers (IWs), all subjected to the compulsory occupational surveillance system, in the period 2017-2018. Buccal cells samples were collected and, based on the Micronucleus test, the exfoliated cells were classified in respect of nuclear changes observed. Moreover, a self-report questionnaire composed of 84 items, was administered to the workers. RESULTS: Significant differences were observed between Exp+ (OWs) and Exp- (IWs) in the number of the analyzed cells (expressed as mean value out of 1000 cells): respectively 954.46 vs 990.06 normal cells, (p < 0.001); 19.79 vs 4.95 micronucleated cells, as marker of chromosomal damage (p < 0.001); 13.93 vs 8.96 binucleated cells, as marker of failed cytokinesis (p < 0.001); 2.09 vs 1.18 karyolytic cells, as marker of cell death and damaged DNA (p < 0.05). According with a multivariate regression analysis, in addition to the job exposure (OW vs IW, beta = 12.221, p < 0.001), the only variable independently associated with an increase in Micronuclei (MNs) is the smoking habit (OWs vs IWs, beta = 6.683, p < 0.001) which, even if not associated with dust exposure, worsens cell integrity. Moreover, this worsening effect is weaker in workers not exposed to the site dust (moderation effect). Within social demographic factors, the high educational level only apparently seems to affect MNs number: even if unbalanced in favor of IWs vs OWs, this variable resulted a confounder, since its effect disappears when the interaction between these two factors is considered, because it is a covariate of smoking habit as well as of the job condition. CONCLUSION: Despite some limitation, our findings clearly confirm the role of occupational exposure as a marker of cytogenetic damage associated with MNs number in construction workers. Moreover, smoking status appears as the only other investigated factor independently associated to the outcome. The statistical model, in addition, highlights possible moderation and confounding effects, such as interaction between smoking and occupational exposure and the unbalanced school education level in workers. Micronucleus test in exfoliated buccal cells would be considered a suitable method for studying the early genotoxic damage in the construction occupational setting as well as in evaluating the efficacy of preventive practices.

Evidence for a quadruplex structure in the polymorphic hs1.2 enhancer of the immunoglobulin heavy chain 3' regulatory regions and its conservation in mammals.

Regulatory regions in the genome can act through a variety of mechanisms that range from the occurrence of histone modifications to the presence of protein-binding loci for self-annealing sequences. The final result is often the induction of a conformational change of the DNA double helix, which alters the accessibility of a region to transcription factors and consequently gene expression. A ∼300 kb regulatory region on chromosome 14 at the 3' end (3'RR) of immunoglobulin (Ig) heavy-chain genes shows very peculiar features, conserved in mammals, including enhancers and transcription factor binding sites. In primates, the 3'RR is present in two copies, both having a central enhancer named hs1.2. We previously demonstrated the association between different hs1.2 alleles and Ig plasma levels in immunopathology. Here, we present the analysis of a putative G-quadruplex structure (tetraplex) consensus site embedded in a variable number tandem repeat (one to four copies) of hs1.2 that is a distinctive element among the enhancer alleles, and an investigation of its three-dimensional structure using bioinformatics and spectroscopic approaches. We suggest that both the role of the enhancer and the alternative effect of the hs1.2 alleles may be achieved through their peculiar three-dimensional-conformational rearrangement. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 768-778, 2016.

Transgenerational effects of NMs.

Nanomaterials are present in a number of commercially available products but there are uncertainties as to whether the unique properties that support their commercial use may also pose potential health risks. Information is missing concerning the influence of nanomaterials on the overall reproductive outcome and transgenerational effects in animals and plants. To obtain this information, long-term studies would be required using animal models phylogenetically close to humans and exposure conditions that reflect realistic scenarios with regard to dosages and admission. The nanoreprotoxicology literature published to date is largely descriptive in nature regarding the effects of nanoparticles. The mechanisms, which determine particle reproduction compatibility, are mostly elusive at the moment. Thus, it is recommended that future research explore the interactions between nanomaterials and transgenerational matter on a molecular level. It would, for instance, be of major importance to understand the behaviour of nanoparticles inside the cells but also their genotoxic and epigenetic effects. Recent studies have shown that intravenous and/or intra-abdominal administration of nanoparticles to mice results in their accumulation in the cells of many tissues, including the brain and the testis, suggesting that they easily pass through the blood-brain and blood-testis barriers. In parallel embryo development after exposure to nanoparticles should be comparatively investigated. The majority of studies on embryo toxicology have concentrated on piscine embryos, mostly derived from zebrafish. Plants for human food as an important component of the ecosystem need also to be taken into account when evaluating transgenerational effects of engineered nanomaterials in crops.

Cytotoxicity and genotoxicity of ceria nanoparticles on different cell lines in vitro.

Owing to their radical scavenging and UV-filtering properties, ceria nanoparticles (CeO(2)-NPs) are currently used for various applications, including as catalysts in diesel particulate filters. Because of their ability to filter UV light, CeO(2)-NPs have garnered significant interest in the medical field and, consequently, are poised for use in various applications. The aim of this work was to investigate the effects of short-term (24 h) and long-term (10 days) CeO(2)-NP exposure to A549, CaCo2 and HepG2 cell lines. Cytotoxicity assays tested CeO(2)-NPs over a concentration range of 0.5 µg/mL to 5000 µg/mL, whereas genotoxicity assays tested CeO(2)-NPs over a concentration range of 0.5 µg/mL to 5000 µg/mL. In vitro assays showed almost no short-term exposure toxicity on any of the tested cell lines. Conversely, long-term CeO(2)-NP exposure proved toxic for all tested cell lines. NP genotoxicity was detectable even at 24-h exposure. HepG2 was the most sensitive cell line overall; however, the A549 line was most sensitive to the lowest concentration tested. Moreover, the results confirmed the ceria nanoparticles' capacity to protect cells when they are exposed to well-known oxidants such as H(2)O(2). A Comet assay was performed in the presence of both H(2)O(2) and CeO(2)-NPs. When hydrogen peroxide was maintained at 25 µM, NPs at 0.5 µg/mL, 50 µg/mL, and 500 µg/mL protected the cells from oxidative damage. Thus, the NPs prevented H(2)O(2)-induced genotoxic damage.

Particle Debris Generated from Passenger Tires Induces Morphological and Gene Expression Alterations in the Macrophages Cell Line RAW 264.7.

Air pollution in the urban environment is a topical subject. Aero-suspended particles can cause respiratory diseases in humans, ranging from inflammation to asthma and cancer. One of the components that is most prevalent in particulate matter (PM) in urban areas is the set of tire microparticles (1-20 µm) and nanoparticles (<1 µm) that are formed due to the friction of wheels with asphalt and are increased in slow-moving areas that involve a lot of braking actions. In this work, we studied the effect that microparticles generated from passenger tires (PTWP, passenger tire wear particles) have in vitro on murine macrophages cells RAW 264.7 at two concentrations of 25 and 100 µg/mL, for 24 and 48 h. In addition to the chemical characterization of the material and morphological characterization of the treated cells by transmission electron microscopy, gene expression analysis with RT-PCR and active protein analysis with Western blotting were performed. Growth curves were obtained, and the genotoxic effect was evaluated with a comet assay. The results indicate that initially, an induction of the apoptotic process is observable, but this is subsequently reversed by Bcl2. No genotoxic damage is present, but mild cellular abnormalities were observed in the treated cells.

Estimation of DNA Degradation in Archaeological Human Remains.

The evaluation of the integrity and quantity of DNA extracted from archaeological human remains is a fundamental step before using the latest generation sequencing techniques in the study of evolutionary processes. Ancient DNA is highly fragmented and chemically modified; therefore, the present study aims to identify indices that can allow the identification of potentially amplifiable and sequenceable DNA samples, reducing failures and research costs. Ancient DNA was extracted from five human bone remains from the archaeological site of Amiternum L'Aquila, Italy dating back to the 9th-12th century and was compared with standard DNA fragmented by sonication. Given the different degradation kinetics of mitochondrial DNA compared to nuclear DNA, the mitochondrially encoded 12s RNA and 18s ribosomal RNA genes were taken into consideration; fragments of various sizes were amplified in qPCR and the size distribution was thoroughly investigated. DNA damage degree was evaluated by calculating damage frequency (λ) and the ratio between the amount of the different fragments and that of the smallest fragment (Q). The results demonstrate that both indices were found to be suitable for identifying, among the samples tested, those less damaged and suitable for post-extraction analysis; mitochondrial DNA is more damaged than nuclear, in fact, amplicons up to 152 bp and 253 bp, respectively are obtained.

Nanoporous silica gel can compete with the flower stigma in germinating and attracting pollen tubes.

To find nanoporous substrates with hydrodynamic properties useful for pollen hydration and germination, we used the glassy Silica gel and Vycor scales and pollen with different morphological and physiological traits, that of Crocus vernus, and that of Narcissus poeticus. For in vitro tests, the scales were spread on microscope slides, hand pollinated, and incubated. Pollen germination was evaluated with the stereomicroscope and the tube growth was explored with scanning electron microscopy (SEM). The in vivo tests were carried out by sprinkling the stigmas of the Crocus plants with Silica gel scales and immediately after having pollinated them by hand, the plants were incubated. Three hours later, the stigmas were removed and treated for observation with SEM. In vitro the pollen of both species germinated on Silica gel with percentages similar to those of the in vivo and in vitro controls, accumulating fibrillary material at the interface. The tubes grew perpendicular to the surface of the scales, trying to penetrate the scales to the point of flattening with the apex. On Crocus stigmas sprinkled with Silica gel scales, pollen developed tubes that grew to the scales rather than penetrating the papillae. The results underline the close interaction of pollen with nanoporous artificial material, so much so that its pollen tubes are attracted to the Silica scales more than to the stigma papillae that arises from a mechanism of natural selection.

The Impact of Metal Nanoparticles on Female Reproductive System: Risks and Opportunities.

Humans have always been exposed to tiny particles via dust storms, volcanic ash, and other natural processes, and our bodily systems are well adapted to protect us from these potentially harmful external agents. However, technological advancement has dramatically increased the production of nanometer-sized particles or nanoparticles (NPs), and many epidemiological studies have confirmed a correlation between NP exposure and the onset of cardiovascular diseases and various cancers. Among the adverse effects on human health, in recent years, potential hazards of nanomaterials on female reproductive organs have received increasing concern. Several animal and human studies have shown that NPs can translocate to the ovary, uterus, and placenta, thus negatively impacting female reproductive potential and fetal health. However, NPs are increasingly being used for therapeutic purposes as tools capable of modifying the natural history of degenerative diseases. Here we briefly summarize the toxic effects of few but widely diffused NPs on female fertility and also the use of nanotechnologies as a new molecular approach for either specific pathological conditions, such as ovarian cancer and infertility, or the cryopreservation of gametes and embryos.

A qPCR-duplex assay for sex determination in ancient DNA.

Molecular biology techniques are increasingly being used in sex identification of skeletal remains when traditional anthropometric analyzes are not successful in identifying sex of remains that are incomplete, fragmented and /or of immature individuals. In the present work, we investigated the possibility of determining sex by using the qPCR-duplex method for both ancient and modern DNA samples. This method involves the co-amplification of two genes in a single reaction system and the subsequent analysis of the fusion curves; the gene sequences used for the construction of suitable primers are those of steroid sulfatase (STS) and testis specific protein Y-linked 1 (TSPY) genes which turned out to be two sensitive markers as they have a detection limit of 60 pg and 20 pg respectively on modern DNA. The validity of the method was verified on modern DNA in which gender was identified in all the samples with 100% accuracy; thus, allowing for the same results as the classic method with amelogenin, but in a faster and more immediate way, as it allows for sex determination solely by analyzing the denaturation curves without having to perform an electrophoretic run. The proposed molecular technique proves to be sensitive and precise even on degraded DNA, in fact on 9 archaeological finds dating from the VII-XII century in which sex had been identified through anthropometric analysis, it confirmed the sex of 8 out of 9 finds correctly.

Genotoxicity Response of Fibroblast Cells and Human Epithelial Adenocarcinoma In Vitro Model Exposed to Bare and Ozone-Treated Silica Microparticles.

Indoor air pollutants (IAP), which can pose a serious risk to human health, include biological pollutants, nitric oxide (NO), nitrogen dioxide (NO2), volatile organic compounds (VOC), sulfur dioxide (SO2), carbon monoxide (CO), carbon dioxide (CO2), silica, metals, radon, and particulate matter (PM). The aim of our work is to conduct a multidisciplinary study of fine silica particles (<2.5 µm) in the presence or absence of ozone (O3), and evaluate their potential cytotoxicity using MTS, micronucleus, and the comet test in two cell lines. We analyzed A549 (human basal alveolar epithelial cell adenocarcinoma) and Hs27 (human normal fibroblasts) exposed to dynamic conditions by an IRC simulator under ozone flow (120 ppb) and in the presence of silica particles (40 µg/h). The viability of A549 and Hs27 cells at 48 and 72 h of exposure to silica or silica/ozone decreases, except at 72 h in Hs27 treated with silica/ozone. The micronucleus and comet tests showed a significant increase in the number of micronuclei and the % of DNA in the queue, compared to the control, in both lines in all treatments, even if in different cell times/types. We found that silica alone or with more O3 causes more pronounced genotoxic effects in A549 tumor cells than in normal Hs27 fibroblasts.

Fast and non-destructive neutron activation analysis for simultaneous determination of TiO2 and SiO2 in sunscreens with attention to regulatory and research issues.

A new instrumental neutron activation analysis (INAA) for the simultaneous determination of titanium (TiO2) and silica (SiO2) dioxide as UV-filters in sunscreens is described. Samples are encapsulated, neutron irradiated (30 s) and after a suitable decay (3 min), the induced 51Ti (T1/2 = 5.76 min) and 29Al (T1/2 = 6.56 min) radionuclides are measured for the emitted γ-ray fingerprint. Three applications were carried out: (i) screening study (analysis of commercial sunscreens in combination with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS); (ii) research study (development of innovative UV-filters such as titanium dioxide or bismuth titanate loaded inorganic mesoporous silica nanoparticles, MSN); (iii) validation study (intercalibration of a spectrochemical method - inductively coupled plasma optical emission spectrometry, ICP-OES). Collectively, the nuclear method appears a powerful tool adequate for quantifying TiO2 and SiO2 in the above studies. The limited accessibility at the nuclear reactor for neutron activation is probably one of the reasons why the excellent characteristics of the nuclear technique are not always fully known and exploited in the industrial and research chemical world.

Interplay of plastic pollution with algae and plants: hidden danger or a blessing?

In the era of plastic pollution, plants have been discarded as a system that is not affected by micro and nanoplastics, but contrary to beliefs that plants cannot absorb plastic particles, recent research proved otherwise. The presented review gives insight into known aspects of plants' interplay with plastics and how plants' ability to absorb plastic particles can be utilized to remove plastics from water and soil systems. Microplastics usually cannot be absorbed by plant root systems due to their size, but some reports indicate they might enter plant tissues through stomata. On the other hand, nanoparticles can enter plant root systems, and reports of their transport via xylem to upper plant parts have been recorded. Bioaccumulation of nanoplastics in upper plant parts is still not confirmed. The prospects of using biosystems for the remediation of soils contaminated with plastics are still unknown. However, algae could be used to degrade plastic particles in water systems through enzyme facilitated degradation processes. Considering the amount of plastic pollution, especially in the oceans, further research is necessary on the utilization of algae in plastic degradation. Special attention should be given to the research concerning utilization of algae with restricted algal growth, ensuring that a different problem is not induced, "sea blooming", during the degradation of plastics.

An Epigenetic Alphabet of Crop Adaptation to Climate Change.

Crop adaptation to climate change is in a part attributed to epigenetic mechanisms which are related to response to abiotic and biotic stresses. Although recent studies increased our knowledge on the nature of these mechanisms, epigenetics remains under-investigated and still poorly understood in many, especially non-model, plants, Epigenetic modifications are traditionally divided into two main groups, DNA methylation and histone modifications that lead to chromatin remodeling and the regulation of genome functioning. In this review, we outline the most recent and interesting findings on crop epigenetic responses to the environmental cues that are most relevant to climate change. In addition, we discuss a speculative point of view, in which we try to decipher the "epigenetic alphabet" that underlies crop adaptation mechanisms to climate change. The understanding of these mechanisms will pave the way to new strategies to design and implement the next generation of cultivars with a broad range of tolerance/resistance to stresses as well as balanced agronomic traits, with a limited loss of (epi)genetic variability.

Effects of single and multi walled carbon nanotubes on macrophages: cyto and genotoxicity and electron microscopy.

Production of nanotechnology-based materials is increasing worldwide: it is essential to evaluate their potential toxicity. Among these nanomaterials, carbon nanotubes (CNTs) have tremendous potential in many areas of research and applications. We have investigated the cyto- and genotoxic effects of single and multi-walled CNTs (SWCNTs, MWCNTs) and carbon black (CB) on the mouse macrophage cell line RAW 264.7. Specifically we have investigated inflammatory response, release of tumor necrosis factor-α (TNF-α), intracellular reactive oxygen species (ROS) production, cell death (both necrosis and apoptosis), chromosomal aberrations and cellular ultrastructural alteration caused by CB, MWCNTs and SWCNTs. Our data confirm that both CNTs and CB are cyto and geno-toxic to RAW 264.7 mouse macrophages. CNTs exposure induced ROS release, necrosis and chromosomal aberrations but did not cause an inflammatory response. In addition CNTs induce ultrastructural damage and apoptosis. CNTs penetrate the cell membrane and individual MWCNTs are seen associated with the nuclear envelope.

Atomic Force Microscope nanolithography on chromosomes to generate single-cell genetic probes.

BACKGROUND: Chromosomal dissection provides a direct advance for isolating DNA from cytogenetically recognizable region to generate genetic probes for fluorescence in situ hybridization, a technique that became very common in cyto and molecular genetics research and diagnostics. Several reports describing microdissection methods (glass needle or a laser beam) to obtain specific probes from metaphase chromosomes are available. Several limitations are imposed by the traditional methods of dissection as the need for a large number of chromosomes for the production of a probe. In addition, the conventional methods are not suitable for single chromosome analysis, because of the relatively big size of the microneedles. Consequently new dissection techniques are essential for advanced research on chromosomes at the nanoscale level. RESULTS: We report the use of Atomic Force Microscope (AFM) as a tool for nanomanipulation of single chromosomes to generate individual cell specific genetic probes. Besides new methods towards a better nanodissection, this work is focused on the combination of molecular and nanomanipulation techniques which enable both nanodissection and amplification of chromosomal and chromatidic DNA. Cross-sectional analysis of the dissected chromosomes reveals 20 nm and 40 nm deep cuts. Isolated single chromosomal regions can be directly amplified and labeled by the Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP-PCR) and subsequently hybridized to chromosomes and interphasic nuclei. CONCLUSIONS: Atomic force microscope can be easily used to visualize and to manipulate biological material with high resolution and accuracy. The fluorescence in situ hybridization (FISH) performed with the DOP-PCR products as test probes has been tested succesfully in avian microchromosomes and interphasic nuclei. Chromosome nanolithography, with a resolution beyond the resolution limit of light microscopy, could be useful to the construction of chromosome band libraries and to the molecular cytogenetic mapping related to the investigation of genetic diseases.

Genotoxicity and oxidative stress induction by polystyrene nanoparticles in the colorectal cancer cell line HCT116.

The potential risks of environmental nanoparticles (NPs), in particular Polystyrene Nanoparticles (PNPs), is an emerging problem; specifically, the interaction of PNPs with intestinal cells has not been characterized so far. The mechanism by which polystyrene particles are transferred to humans has not yet been clarified, whether directly through ingestion from contaminated food. We evaluated the interaction between PNPs and colorectal adenocarcinoma cells (HCT116). Cells were exposed to different concentrations of PNPs, metabolic activity and the consequent cytotoxic potential were assessed through viability test; we evaluated the PNP genotoxic potential through the Cytokinesis-Block Micronucleus cytome (CBMN cyt) assay. Finally, we detected Reactive Oxygen Species (ROS) production after NPs exposure and performed Western Blot analysis to analyze the enzymes (SOD1, SOD2, Catalase, Glutathione Peroxidase) involved in the cell detoxification process that comes into play during the cell-PNPs interaction. This work analyzes the cyto and genotoxicity of PNPs in the colorectal HCT116 cell line, in particular the potential damage from oxidative stress produced by PNPs inside the cells related to the consequent nuclear damage. Our results show moderate toxicity of PNPs both in terms of ROS production and DNA damage. Further studies will be needed on different cell lines to have a more complete picture of the impact of environmental pollution on human health in terms of PNPs cytotoxicity and genotoxicity.

Genotoxicity and Immunotoxicity of Titanium Dioxide-Embedded Mesoporous Silica Nanoparticles (TiO2@MSN) in Primary Peripheral Human Blood Mononuclear Cells (PBMC).

Background: TiO2 nanoparticles (TiO2 NPs) are the nanomaterial most produced as an ultraviolet (UV) filter. However, TiO2 is a semiconductor and, in nanoparticle size, is a strong photocatalyst, raising concerns about photomutagenesis. Mesoporous silica nanoparticles (MSN) were synthetized incorporating TiO2 NPs (TiO2@MSN) to develop a cosmetic UV filter. The aim of this study was to assess the toxicity of TiO2@MSN, compared with bare MSN and commercial TiO2 NPs, based on several biomarkers. Materials and Methods: Human peripheral blood mononuclear cells (PBMC) were exposed to TiO2@MSN, bare MSN (network) or commercial TiO2 NPs for comparison. Exposed PBMC were characterized for cell viability/apoptosis, reactive oxygen species (ROS), nuclear morphology, and cytokines secretion. Results: All the nanoparticles induced apoptosis, but only TiO2 NPs (alone or assembled into MSN) led to ROS and micronuclei. However, TiO2@MSN showed lower ROS and cytotoxicity with respect to the P25. Exposure to TiO2@MSN induced Th2-skewed and pro-fibrotic responses. Conclusions: Geno-cytotoxicity data indicate that TiO2@MSN are safer than P25 and MSN. Cytokine responses induced by TiO2@MSN are imputable to both the TiO2 NPs and MSN, and, therefore, considered of low immunotoxicological relevance. This analytical assessment might provide hints for NPs modification and deep purification to reduce the risk of health effects in the settings of their large-scale manufacturing and everyday usage by consumers.

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development.

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17-18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O2 consumption during B. bufo development.

Analysis of ancient mtDNA from the medieval archeological site of Amiternum (L'Aquila), central Italy.

Study of ancient DNA makes it possible to analyze genetic relationships between individuals and populations of past and present. In this paper we have analyzed remains of human bones, dating back to the 8th-10th century AD, from the burials found in the Cathedral of Santa Maria in Civitate, archaeological site of Amiternum, L'Aquila, Italy. As a genetic marker, the hypervariable region 1 of mitochondrial DNA (HVR1) was selected. To obtain reliable sequences from the hypervariable region 1 of mtDNA (HVR1) were performed: multiple extractions, template quantification and cloning of PCR products. The sequences obtained were compared with Anderson's sequence for the identification of polymorphisms (SNP) and haplogroups. The data obtained were analyzed with various software and phylogenetic methods. For the comparison between populations, ancient and modern sequences found in databases and literature have been used. This work provides preliminary information on the correlation between the population of Amiternum, the migrant populations transited and/or established in the territory of Amiternum such as Byzantines, Longobards (Lombards), which dominated the Italian peninsula between 568 and 774 AD, and the current populations of Italy. The study of haplogroups, the analysis of genetic variability and phylogenesis studies on the sequences considered show a genetic closeness between the individuals of Amiternum, the current population of central-northern Italy and the Germanic tribe of Longobards, however, also highlights genetic traits of Byzantines in some samples of Amiternum. Using the analysis of amelogenin gene fragments, we successfully determined the sex of the bone remains on all samples.