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The contemporary discovery of extremely versatile engineered nucleic acid-binding proteins has transformed a brave new world in the genome-editing scientific area. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated programmable nucleic acid-binding proteins have brought about a revolution in diagnostic platforms. The groundbreaking finding that bacteria and archaea that harbored prokaryotes have transmitted adaptive immunity through CRISPR and CRISPR-associated (Cas) proteins has driven revolutionary advances in molecular biology. Importantly, advances in gene editing focus how expanding visions in CRISPR-Cas biology are revolutionizing the area of molecular diagnostics for identifying DNA and RNA in emerging microbiological pathogens, for single nucleotide polymorphism (SNP) identifications, and for cell-free mutation. Recent advances, such as improvements in multiplexing and quantitative capabilities as well as instrument-free detection of nucleic acids, will potentially leverage the introduction of these novel technologies to detecting bacteria and viruses at the point of care (POC). In this review, we highlight the fundamental features of CRISPR/Cas-based molecular diagnostic technologies and summarize a vision of the next applications for identifying pathogens in POC settings.

The assessment of the actual impact of discharged wastewater on the whole ecosystem and, in turn, on human health requires the execution of bioassays. In effect, based on the chemical characterization alone, the synergistic/antagonistic effect of mixtures of pollutants is hardly estimable. The aim of this work was to evaluate the applicability of a battery of bioassays and to suggest a smart procedure for results representation. Two real wastewater treatment plants were submitted to analytical campaigns. Several baseline toxicity assays were conducted, together with tests for the determination of endocrine activity, genetic toxicity and carcinogenicity of wastewater. A “traffic light” model was adopted for an easy-to-understand visualization of the results. Although the legal prescriptions of chemical parameters are fully complied with, bioassays show that a certain biological activity still residues in the treated effluents. Moreover, influent and effluent responses are not always appreciably different. Some tests employing human cells were revealed to be only partially adequate for environmental applications. An interesting and helpful development of the present approach would consist in the estimation of biological equivalents of toxicity, as shown for the estrogenic compound 17-β-estradiol.

The human dental follicle (hDF) contains the developing tooth and is involved in regulating tooth maturation and eruption. To investigate the mesenchymal stromal cells of the dental follicle, 2 three-dimensional (3D) culture models were used, based on a dynamic bioreactor: the Rotary Cell Culture System (RCCS™) and the 3D culture of precursor cells isolated from follicular tissue (human dental follicle cells [hDFCs]). The hDFCs were obtained from impacted third molars of 20 patients. Two 3D culture models were tested. In the first model, intact hDF explants were cultured in 3D conditions, preserving the original tissue architecture; they were studied using histomorphological and molecular analyses. The second model involved the 3D culture of hDFCs, which were characterized to evaluate their multipotency in terms of differentiation capability. Of the biomarkers known to characterize hDFCs, hDF precursors were selected for our study. The immunophenotype and in situ immunocytochemistry were evaluated for markers CD44, CD90, CD146, CD105, CD31, CD34, and CD45 Ag. The results show that the conditions provided by the RCCS preserve the original organizational architecture of the cells. The 3D conditions of the model enhanced differentiation in response to adipogenic, osteogenic, and chondrogenic inductive growth media. The immunophenotype and the immunocytochemistry showed generally high expression of CD90, CD44, and CD105, while CD146 expression was more restricted to ∼30% of cells. No expression was observed for CD31, CD34, and CD45 Ags. Two 3D tissue- and cell-based ex vivo models of the hDF supported the long-term maintenance of hDF-specific cell phenotypes and their ability to recapitulate typical cellular differentiation states. As such, these ex vivo models could be used to study the physiopathology of human odontogenesis. In addition, in a therapeutic context, they could be used to examine the role of specific chemical signals (e.g., new therapeutic agents) in the processes of dental tissue repair and regeneration.

3D-dynamic culture models represent an invaluable tool for a better comprehension of tumor biology and drug response, as they accurately re-create/preserve the complex multicellular organization and the dynamic interactions of the parental microenvironment, which can affect tumor fate and drug sensitivity. Hence, development of models that recapitulate tumor within its embedding microenvironment is an imperative need. This is particularly true for multiple myeloma (MM), which survives almost exclusively in the bone marrow (BM). To meet this need, we have previously exploited and validated an innovative 3D-dynamic culture technology, based on the use of the Rotary Cell Culture System (RCCS ™) bioreactor . Here, we describe, step by step, the procedures we have employed to establish two human MM ex vivo models, i.e., the culture of human BM-derived isolated cells and of MM tissues from patients.

U94, the latency gene of human herpesvirus 6, was found to inhibit migration, invasion and proliferation of vascular endothelial cells (ECs). Because of its potent anti-migratory activity on ECs, we tested the capability of U94 to interfere with the individual steps of the metastatic cascade. We examined the U94 biological activity on the human breast cancer cell line MDA-MB 231, as a model of highly aggressive cancer cell. Here we show that the expression of U94 delivered by an HSV-1-based amplicon promoted down-modulation of Src and downstream molecules linked to cell motility and proliferation. Indeed, U94 expression strongly inhibited cell migration, invasiveness and clonogenicity. We investigated the effects of U94 in a three-dimensional rotary cell-culture system and observed the ability of U94 to modify tumor cell morphology by inducing a partial mesenchymal-to-epithelial transition. In fact, despite U94 did not induce any expression of the epithelial marker E-cadherin, it down-modulated different mesenchymal markers as β-catenin, Vimentin, TWIST, Snail1, and MMP2. In vivo data on the tumorigenicity of MDA-MB 231 displayed the capability of U94 to control tumor growth, invasiveness and metastasis, as well as tumor-driven angiogenesis. The antitumor U94 activity was also confirmed on the human cervical cancer cell line HeLa. The ability of U94 to inhibit cell growth, invasion and metastasis opens the way to a promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.

Densitometry is considered to be the gold standard in bone quality assessment. However, since its introduction, the medical community has been aware that mineral density is only one of the factors that influence the bone risk of fracture, which also depends on the bone's trabecular arrangement and, in particular, on the trabecular architecture's load bearing capabilities. At the University of Trieste, in recent years, a test has been developed that simulates the application of compressive loads on trabecular architecture's reconstructions extracted from digital radiographs. In this work, the test is described, and the results obtained by applying the appraisal in a particular case of severe osteoporosis of the hand, complicating a Complex Regional Pain Syndrome (CRPS) type II, are presented. The test was able to quantify the pathological alterations of bone micro-architecture by means of a Structural Index (SI), which was absolutely significant and relevant to the clinical situation. Important research and clinical opportunities of application of the test include accurate evaluation of osteoporotic bone diseases, careful clinical follow-up and monitoring of responses to therapeutic approaches, and, prospectively, reliable quantification of biological damage (forensic field).

This is a post-print versión of the article: Gonzalez-Gil, L., Papa, M., Feretti, D., Ceretti, E., Mazzoleni, G., Steimberg, N., ... & Carballa, M. (2016). Is anaerobic digestion effective for the removal of organic micropollutants and biological activities from sewage sludge?. Water research, 102, 211-220. https://doi.org/10.1016/j.watres.2016.06.025 The occurrence of emerging organic micropollutants (OMPs) in sewage sludge has been widely reported; nevertheless, their fate during sludge treatment remains unclear. The objective of this work was to study the fate of OMPs during mesophilic and thermophilic anaerobic digestion (AD), the most common processes used for sludge stabilization, by using raw sewage sludge without spiking OMPs. Moreover, the results of analytical chemistry were complemented with biological assays in order to verify the possible adverse effects (estrogenic and genotoxic) on the environment and human health in view of an agricultural (re)use of digested sludge. Musk fragrances (AHTN, HHCB), ibuprofen (IBP) and triclosan (TCS) were the most abundant compounds detected in sewage sludge. In general, the efficiency of the AD process was not dependent on operational parameters but compound-specific: some OMPs were highly biotransformed (e.g. sulfamethoxazole and naproxen), while others were only slightly affected (e.g. IBP and TCS) or even unaltered (e.g. AHTN and HHCB). The MCF-7 assay evidenced that estrogenicity removal was driven by temperature. The Ames test did not show point mutation in Salmonella typhimurium while the Comet test exhibited a genotoxic effect on human leukocytes attenuated by AD. This study highlights the importance of combining chemical analysis and biological activities in order to establish appropriate operational strategies for a safer disposal of sewage sludge. Actually, it was demonstrated that temperature has an insignificant effect on the disappearance of the parent compounds while it is crucial to decrease estrogenicity This work was funded by Xunta de Galicia through MicroDAN project (EM 2012/087) and I2C Predoctoral Grant Program (PRE/2013/111) and by the Spanish Ministry of Education through FPU Grant (FPU13/01255) and the Ramón y Cajal contract (RYC-2012-10397). The authors belong to CRETUS (AGRUP2015/02) and to the Galician Competitive Research Group (GRC 2013-032). Part of this work was carried out within a Short Term Scientific Mission (STSM) of the Water2020 Cost Action ES1202: Conceiving Wastewater Treatment in 2020 | Energetic, environmental and economic challenges SI

Gap-junctional intercellular communication (GJIC) in 20 primary human liver tumors with different degrees of malignancy has been studied at the functional and molecular levels. When GJIC capacity was determined by dye-transfer assay performed directly with freshly removed tumor tissue, significant reduction was found in all samples, regardless of their morphology. In addition, a selective lack of GJIC between tumor and surrounding non-tumorous cells was observed in some cases, probably due to the physical separation between them resulting from encapsulation of tumors. There was, however, no essential change in the level of expression of the major liver gap-junction protein, connexin (cx) 32, in liver tumors as measured by Northern and Western blot analyses. Immunohistochemical study revealed aberrant localization of cx 32 in the majority of malignant liver tumors. Instead of cytoplasmic membrane localization at intercellular contacts, cx 32 was detected mainly either intracytoplasmically or in plasma membrane free from contact with other cells. We did not detect any mutation in the coding sequence of the cx 32 gene from any of the human liver tumors we tested. Thus it is likely that the aberrant localization of cx 32 in tumor cells is due to disruption of the mechanisms for establishment of this protein into gap-junction plaques, rather than to structural abnormality of the cx 32 protein itself. Another member of the connexin family, cx 43, not detectable in non-tumorigenic hepatocytes, was expressed in several tumors, especially in invasive areas, but was detected in only a few tumor cells and was localized intracytoplasmically, suggesting that cx 43 protein is not involved in GJIC in the tumors.

On May 19, 2015, a meeting was held on the use of alternative methods for the evaluation of hepatotoxicity. It was chaired by Prof. Francesca Caloni and Prof. Giovanna Mazzoleni, and organized by CELLTOX, the Italian Association of in vitro Toxicology, in partnership with the Italian Reference Center for Alternative Methods, Welfare and Care of Laboratory Animal and MISTRAL (Integrated Models for Prevention and Protection in Environmental and Occupational Health) Research Center, University of Brescia, Italy. The meeting was hosted by the Istituto Zooprofilattico Sperimentale della Lombardia e Emilia Romagna, Brescia. The conference was introduced by Dr Maura Ferrari, who reported on the activity of the Italian Reference Center for Alternative Methods, Welfare and Care of Laboratory Animals, indicating the need for alternative liver models.

We propose a human-derived neuro-/glial cell three-dimensionalin vitromodel to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool forin vitrostudies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

The rapid development of three-dimensional (3D) culture systems and engineered cell-based tissue models gave rise to an increasing need of new techniques, allowing the microscopic observation of cell behavior/morphology in tissue-like structures, as clearly signalled by several authors during the last decennium. With samples consisting of small aggregates of isolated cells grown in suspension, it is often difficult to produce an optimal embedded preparation that can be further successfully processed for classical histochemical investigations. In this work, we describe a new, easy to use, efficient method that enables to embed an enriched "preparation" of isolated cells/small 3D cell aggregates, without any cell stress or damage. As for after tissue-embedding procedures, the cellular blocks can be further suitably processed for efficient histochemical as well as immunohistochemical analyses, rendering more informative-and attractive-studies onto 3D cell-based culture of neo-tissues.

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.

A major source of the wide presence of EDCs (Endocrine Disrupting Compounds) in water bodies is represented by direct/indirect discharge of sewage. Recent scientific literature reports data about their trace concentration in water, sediments and aquatic organisms, as well as removal efficiencies of different wastewater treatment schemes. Despite the availability of a huge amount of data, some doubts still persist due to the difficulty in evaluating synergistic effects of trace pollutants in complex matrices. In this paper, an integrated assessment procedure was used, based on chemical and biological analyses, in order to compare the performance of two full scale biological wastewater treatment plants (either equipped with conventional settling tanks or with an ultrafiltration membrane unit) and tertiary ozonation (pilot scale). Nonylphenol and bisphenol A were chosen as model EDCs, together with the parent compounds mono- and di-ethoxylated nonylphenol (quantified by means of GC–MS). Water estrogenic activity was evaluated by applying the human breast cancer MCF-7 based reporter gene assay. Process parameters (e.g., sludge age, temperature) and conventional pollutants (e.g., COD, suspended solids) were also measured during monitoring campaigns. Conventional activated sludge achieved satisfactory removal of both analytes and estrogenicity. A further reduction of biological activity was exerted by MBR (Membrane Biological Reactor) as well as ozonation; the latter contributed also to decrease EDC concentrations.

Major hepatic resection in cirrhotic patients is associated with impaired liver regeneration and failure, leading to high peri-operative mortality. In this work, the causes of defective regeneration in cirrhotic liver and the utility of IL-6 treatment were investigated in an experimental model combining cirrhosis and partial hepatectomy in the rat. Relative to normal controls, decompensated cirrhotic animals showed decreased survival, while compensated cirrhotic animals showed similar survival but reduced hepatic DNA synthesis and newly regenerated liver mass amount. Defective liver regeneration was associated with a decrease in STAT3 and NF-kB activation, consistent with an increased accumulation of their respective inhibitors PIAS3 and IkBalpha, and with a decreased induction of Bcl-xL. Treatment with recombinant IL-6 enhanced survival of decompensated cirrhotic animals, while it did not affect survival of compensated cirrhotic animals but sustained liver regeneration, by restoring STAT3 and NF-kB activation and Bcl-xL induction to the levels found in normal controls. The pro-growth effects exerted by IL-6 treatment in cirrhotic liver were attained also at low, pharmacologically acceptable doses. In conclusion, our results suggest that IL-6 treatment may be therapeutic in major resection of cirrhotic liver.

Manganese (Mn) is both an essential nutrient and a toxicant, with specific effects on liver and kidney (acute exposure) and on central nervous system (CNS) (chronic exposure). Mn neurotoxicity includes neurobehavioral disorders and extra-pyramidal motor dysfunctions ( manganism ), possibly due to focal injuries to the basal ganglia. Even if widely investigated, the molecular mechanisms responsible for Mn toxicity remain to be clarified. Aim of this study was to identify suitable in vitro models to investigate these molecular pathways. To this purpose we compared the effect of manganese chloride on four cell lines, representative of the main target organs of Mn toxicity in vivo . HepG2 and MDCK cell lines were selected for liver and kidney, respectively; glial GL15 and neuronal SHSY5Y cells were used as models of CNS components. To complete the “motor system” model, skeletal muscle C2C12 cells were also included. Our results demonstrate that hepatic, renal, glial and neuronal cell types differently react to Mn, mirroring the specific in vivo response of the tissue they represent. This confirms their value as suitable in vitro models to study Mn-related toxic events. Interestingly, also muscle C2C12 cells showed a noticeable sensitivity to Mn, preferential targets being differentiated myotubes.

Tubular epithelium represents the primary target of mercuric ions (Hg(2+)) nephrotoxicity. Although widely investigated, the mechanisms of Hg(2+) cell uptake, accumulation and excretion all along the nephron remain largely unknown. In the present study, native distal tubular-derived Madin-Darby canine kidney (MDCK) cells exposed to subcytotoxic (micromolar) HgCl(2) concentrations were used for investigating specific mechanisms involved in the tubular response to toxic metals. Inductively coupled plasma-mass spectrometry (ICP-MS) was firstly used for assessing HgCl(2) solubility and then for quantifying Hg(2+) cell uptake. Exposed to HgCl(2), MDCK cells showed a rapid, but transient, Hg(2+) accumulation. The metallic cation was found to affect cell density and morphology, being these effects related to the dose and the time of exposure. In parallel, an Hg(2+)-induced up-regulation of endogenous MRP1 and MRP2 export pumps, a significant HgCl(2)-dependent induction of protective cellular thiols and an increase in the glutathione conjugates metabolism were also observed. The functional suppression of MRPs activity, obtained by MK-571 treatment, increased the Hg(2+) cell content and the sensitivity of MDCK cells to HgCl(2). Our results demonstrate that, in MDCK cells, inorganic Hg(2+) promotes the activation of specific detoxifying pathways that may, at least partly, depend on the activity of MRP transporters.

Gap junction intercellular communication (GJIC) plays an essential role in the control of growth, differentiation, and functions of different tissues. The expression of connexins (Cxs), the structural proteins of gap junctions, is developmentally regulated and tissue-specific. In vivo hepatocytes express Cx32 and Cx26. Most currently available in vitro hepatic cell systems express Cx43 instead of the expected Cxs. This work analyzes the GJIC competence and Cx expression of the highly differentiated and polarized hepatoma-derived hybrid cell lines, WIF 12-1 and WIF-B. It shows (using two dye transfer assays) that both lines communicate efficiently and that the acquisition of GJIC competence precedes the formation of bile canaliculi. Interestingly, these cells communicate via Cx32 expression, whereas Cx26 and Cx43 are not expressed, as demonstrated by Western and Northern blotting, immunocytochemistry, and confocal microscopy. The human fibroblast W138 parent communicates via Cx43, whereas the rat hepatoma parent Fao and the subclone WIF 12-1 TGdelta, that has lost the human X chromosome, do not communicate, the expression of Cx32 being restricted to the mRNA in these two lines. The GJIC competence of WIF cells could thus result from the activation of the human X chromosome-linked Cx32 gene.

Gap-junctional intercellular communication is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in vivo tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homotypic Junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual Junctional permeabilities, differentially affected by the chemicals; they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms.

On the basis of in vivo toxicological long-term tests performed on rodents, the herbicide Linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] has been classified by the U.S. Environmental Protection Agency (EPA) (1988) as a class C carcinogen (possibly carcinogenic to humans). However, when Linuron was analyzed for genotoxicity, negative results were obtained. An epigenetic, tumor-promoting potential was hence suspected to be responsible for the oncogenic activity of the molecule. In the absence of in vivo data concerning the tumor-promoting activity of the herbicide and being well established that tumor promoters interfere with the cell growth rate and gap-junctional permeability, the effects of technical grade Linuron and of its trade formulation (Linuron 50) on these parameters were investigated. This is especially important in the case of the formulated preparation for a correct estimate of the health hazard to humans. Cytotoxicity and gap-junctional intercellular communication (GJIC) assays were performed on the endothelial cell line F-BAE GM 7373, an in vitro cell system known to be responsive to the biological effects of tumor promoters. A time- and dose-related cytotoxic effect was found for both Linuron and Linuron 50, the latter being the far more cytotoxic of the two. However, when tested at noncytotoxic concentrations over a period of 48 h, neither Linuron nor Linuron 50 altered the capacity of F-BAE GM 7373 cells to intercommunicate. On the basis of the results obtained, the possibility that Linuron and Linuron 50 are endowed with tumor-promoting activity is discussed. In vivo studies on tumor-promoting and genotoxic activity are in progress to complement the information available on the toxicological properties of Linuron and its trade preparation.