Advanced techniques to investigate the internalization mechanism of TiO2 NPs in the roots grown in a biosolid-amended agricultural soil

Plants play an important role in introducing the engineered nanoparticles (ENPs) into the food chain. The pathway of ENPs uptake from soil, their distribution in the edible plant parts, and their impact in the food production are important issues to be investigated. In the present study, Pisum sativum plants were grown at microcosm scale under medium-term TiO2 NPs exposure, to possibly mime environmental conditions in an agricultural soil amended with biosolids from a wastewater treatment plant in Pisa, Italy. TiO2 NPs were applied as pure rutile, pure anatase and a mixture of both crystalline phases in the biosolid amended-soil. Micro-XRF and μ-XANES from ID21 beamline were used for Ti elemental mapping and crystalline phase identification to indicate a relative distribution/localization of TiO2 crystalline phases within a given cross-section of roots, as well as the possible speciation and preferential crystalline phase uptake in the roots. Titanium in roots showed a main localization in the rizoderma, independently of the crystalline phase. Fewer Ti spots were found localized in the cortex or in vessel, however the roots grown in presence of a mixture of both phases showed a main presence of anatase, suggesting a preferential adsorption and translocation of this crystalline form through the roots. Our data indicated also a reduced translocation of Ti to the aerial part of the plant, confirming the chemical analysis of shoots and roots separately, which showed that Ti concentration was about 40 times lower in the upper part than in the below ground tissues. The TiO2 NPs were characterized on the basis of their size and shape by TEM analysis. Moreover, observations on cell ultrastructure of control and of anatase, rutile and mixture of both crystalline phases treated roots were performed. The root cells of plant grown in the presence of all NPs treatments shared the same alterations of ultrastructure: mitochondria with swollen cristae, nuclei with condensed chromatin, and part of the cytoplasm degraded, probably in consequence of an autophagic process. As detected by μ-XRF and μ-XANES, electron dense prismatic or round profiled particles of about 30-40 nm were observed mainly in form of aggregates in the intercellular spaces or crossing the wall of the cells next to rizoderma and in the cortex cells. Furthermore, the anatase treated cells were mostly damaged in respect to control and rutile treated roots, and more frequently internalized NPs were observed in these samples

De-inked paper sludge and mature compost as high-valuecomponents of soilless substrate to support tree growth

The recycled paper industry produces tons of waste whose disposal is a cost for industry and the environment. This research examines the suitability of de-inked paper sludge (DPS), after pelletization, as a sustainable alternative component to a peat-based growing media, creating ideal root conditions for tree development (e.g. high water storage, low compaction). DPS, tested on Lepidium sativum L. germination, did not show toxicity effects. Three species, Quercus ilex L., Lagerstroemia indica L. and Prunus serrulata “Kanzan”, were planted in 40 cm Ø pots filled with a control (peat, pumice and zeolite) and the experimental substrate (compost, DPS pellets, pumice and zeolite). After two years in the nursery, the trees were planted in situ. The physical and chemical properties of substrates were analyzed. Plant morphological and physiological parameters were monitored: trunk diameter, leaf dry matter, leaf nitrogen, chlorophyll, and photosynthetic efficiency. The new substrate showed higher Corg (+135%), total N (+73%) and easily available water (+19%), compared to the control substrate used in the nursery. In this new substrate, the trees showed similar radial growth values to the control in the nursery and after transplanting in situ improved their photosynthetic performance in terms of quantum yield of photosystem II (+36%, and +29% in P. serrulata and L. indica, respectively) and electron transport rate (+39%, +25%, and +32% in P. serrulata, Q. ilex and L. indica, respectively). Pelletization represents an attractive amendment for growing media, which enhances the plant’s physiological health status. This study proposes alternative recovery methods for paper industry waste with low environmental impact. As the process is developed locally, it should also contribute to reducing energy-related CO2 emissions from transport. Pelletization represents an attractive novelty in the use of DPS as amendment for growing media, which enhances the plant’s physiological health status. This study proposes alternative recovery methods for paper industry waste with low environmental impact. As the process is developed locally, it should also contribute to reducing energy-related CO2 emissions from transport

Identification of stably expressed reference small non-coding RNAs for microRNA quantification in high-grade serous ovarian carcinoma tissues

MicroRNAs (miRNAs) belong to a family of small non‐coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high‐grade serous ovarian carcinoma (HGS‐OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS‐OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR‐92a‐3p, let‐7a‐5p, SNORD61, SNORD72, SNORD68, miR‐103a‐3p, miR‐423‐3p, miR‐191‐5p, miR‐16‐5p) were analysed on a total of 75 HGS‐OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS‐OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR‐191‐5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS‐OvCa and normal controls, including the first time both the normal tissues supposed to be HGS‐OvCa progenitors. In addition, we recommend miR‐191‐5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS‐OvCa tissues

Evaluation of a novel human IgG1 anti-claudin3 antibody that specifically recognizes its aberrantly localized antigen in ovarian cancer cells and that is suitable for selective drug delivery

Membrane protein claudin3 has been recently suggested as a marker for biologically aggressive tumors and a possible target for the therapeutic delivery of active anti-cancer compounds. Claudin3-binding molecules such as the Clostridium perfringens enterotoxin (CPE), CPE-related molecules, and murine and chimeric antibodies have shown promising antitumor efficacy in preclinical oncological settings. We first engineered a fully human anti-claudin3 IgG1 antibody (IgGH6) by fusing the human IgG1 Fc-domain to the anti-claudin3 scFvH6 previously isolated from a pre-immune phage display library. The construct was expressed in mammalian cells and specifically targeted claudin3 endogenously expressed on the surface of different human ovarian cancer cell lines. No detectable cross-reactivity with other homologous claudins was observed. The epitope recognized by IgGH6 is located within the minor extracellular domain of claudin3 and becomes accessible only in tumor cells characterized by incomplete junction formation. Confocal microscopy experiments demonstrated that IgGH6 was actively internalized in tumor cells after binding to native claudin3 and co-localized, likely within intracellular vesicles, with the C-CPE peptide. Preliminary results indicate that IgGH6 accumulated in vivo in free claudin3 ovarian carcinoma xenografts. For its selective uptake in tumor cells and its human nature, IgGH6 represents a valuable candidate for antibody-drug conjugate therapeutic applications in ovarian cancer patients

Mammaglobin B is an independent prognostic marker in epithelial ovarian cancer and its expression is associated with reduced risk of disease recurrence

<p>Abstract</p> <p>Background</p> <p>Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients.</p> <p>Methods</p> <p>MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome.</p> <p>Results</p> <p>MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08–0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17–0.99, p = 0.048) had an independent prognostic value for disease-free survival.</p> <p>Conclusion</p> <p>This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.</p

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells

IntroductionDespite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity.MethodsIn this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs).ResultsActivated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature.ConclusionThese data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches

Cytomegalovirus-specific T cells restricted for shared and donor human leukocyte antigens differentially impact on cytomegalovirus reactivation risk after allogeneic hematopoietic stem cell transplantation

After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P<0.01) and multivariate (P<0.05) analyses. Dextramer quantification correlated with functional assays measuring interferon-γ production, and allowed earlier identification of high-risk patients. In mismatched transplants, the comparative analysis of lymphocytes restricted by shared, donor- and host-specific HLA revealed the dominant role of thymic-independent CMV-specific reconstitution. Shared and donor-restricted CMV-specific T cells reconstituted with similar kinetics in recipients of CMV-seropositive donors, while donor-restricted T-cell reconstitution from CMV-seronegative grafts was impaired, indicating that in primary immunological responses the emergence of viral-specific T cells is largely sustained by antigen encounter on host infected cells rather than by cross-priming/presentation by non-infected donor-derived antigen-presenting cells. Multiparametric flow cytometry and high-dimensional analysis showed that shared-restricted CMV-specific lymphocytes display a more differentiated phenotype and increased persistence than donor-restricted counterparts. In this study, monitoring CMV-specific cells by Dextramer assay after allogeneic HSCT shed light on mechanisms of immune reconstitution and enabled risk stratification of patients, which could improve the clinical management of post-transplant CMV reactivations

Preparação e caracterização de um material elastomerico, eletroativo e eletrocromico

Orientador : Marco-Aurelio De PaoliTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuimicaDoutorad

La canapa nella riqualificazione di suoli contenenti metalli pesanti a Villadossola: vantaggi e prospettive

Not availableLe piante rivestono un ruolo fondamentale nella produzione di ossigeno e nel sequestro di anidride carbonica, sono parte integrante dell\u27ecosistema e mettono in relazione l\u27atmosfera e tutto ci? che ? disperso in essa con il suolo e l\u27acqua. La canapa (Cannabis sativa L.), una specie erbacea di coltivazione annuale appartenente alla famiglia delle Cannabaceae, suscita particolare interesse per le sue numerose funzioni, la pi? nota consiste nella produzione di materie prime alimentari per uso umano e zootecnico (foglie, fiori, frutti, semi, germogli, fibra), nella produzione di fibre tessili e bioplastiche, pannelli isolanti, biodiesel e/o bioetanolo, legna, pellet etc. (Bouloc et al., 2013). Inoltre, l\u27utilizzo delle piante per la riqualificazione di ambienti degradati e/o inquinati (phytoremediation) suscita grande attenzione per il basso impatto ambientale e costi di intervento relativamente ridotti. La phytoremediation, termine anglosassone ormai utilizzato anche nella lingua italiana, consiste nell\u27utilizzo di piante con particolari capacit? di contenere, rimuovere o degradare i contaminanti presenti nei suoli, nei sedimenti e nelle acque. Le tecnologie che utilizzano piante, chiamate anche "fitotecnologie", possono essere impiegate per rimuovere o contenere inquinanti inorganici (metalli pesanti: piombo (Pb), nichel (Ni), zinco (Zn) etc., elementi radioattivi: cesio (Cs), uranio (U) etc.); per rimuovere o degradare inquinanti organici (idrocarburi policiclici aromatici (IPA), composti clorurati, etc.); possono essere utilizzate per riqualificare substrati solidi (suoli e sedimenti) e substrati liquidi (acque superficiali e falde sotterranee

Phosphate-assisted phytoextraction in as-contaminated soil

In this paper, the effect of phosphate on As phytoextraction was examined. The effect of phosphate on As dissolution, the As uptake of plants, the internal plant translocation, and phytotoxic effect were investigated. Lupine plants were grown on As-contaminated soil collected from an industrial site containing 670 mg/kg As and were treated with biammonium phosphate (BAP). Two different BAP application procedures were tested: single-dose and multiple split additions. BAP was found to be effective in increasing the water-soluble As concentration in the test soil. As the concentration of water-soluble As increased, the Lupine plants responded accordingly with an increased As uptake. The As content in the shoots and the translocation factor were the highest when BAP was added in multiple split additions. On the contrary, a single application caused the highest As content in the roots and consequently the lowest translocation factor. In addition, it was established that the single-application method significantly reduced the plant biomass by twofold, this reduction being an evident phytotoxic symptom. Measurements of the combined biomass production and As content values revealed that the highest As phytoextraction is obtained with BAP applied in multiple doses which is about 14-fold higher than in the control plants, whereas a single-dose BAP application increased the phytoextraction rate only 1.6 times. These results demonstrate that significant improvements in the current phosphate-assisted phytoextraction of As could be achieved