Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification, and the expression of genes involved in intestinal transport of minerals, osteoblast differentiation, and oxidative stress response.

Currently, the larviculture of many marine fish species with small-sized larvae depends for a short time after hatching, on the supply of high-quality live zooplankton to ensure high survival and growth rates. During the last few decades, the research community has made great efforts to develop artificial diets, which can completely substitute live prey. However, studies aimed at determining optimal levels of minerals in marine larvae compound feeds and the potential of novel delivery vectors for mineral acquisition has only very recently begun. Recently, the agro-food industry has developed several nano-delivery systems, which could be used for animal feed, too. Delivery through nano-encapsulation of minerals and feed additives would protect the bioactive molecules during feed manufacturing and fish feeding and allow an efficient acquisition of active substances into biological system. The idea is that dietary minerals in the form of nanoparticles may enter cells more easily than their larger counterparts enter and thus speed up their assimilation in fish. Accordingly, we evaluated the efficacy of early weaning diets fortified with organic, inorganic, or nanoparticle forms of trace minerals (Se, Zn, and Mn) in gilthead seabream (Sparus aurata) larvae. We tested four experimental diets: a trace mineral-deficient control diet, and three diets supplemented with different forms of trace minerals. At the end of the feeding trial, larvae growth performance and ossification, and the level of expression of six target genes (SLC11A2ß, dmt1, BMP2, OC, SOD, GPX), were evaluated. Our data demonstrated that weaning diets supplemented with Mn, Se, and Zn in amino acid-chelated (organic) or nanoparticle form were more effective than diets supplemented with inorganic form of minerals to promote bone mineralization, and prevent skeletal anomalies in seabream larvae. Furthermore, nanometals markedly improved larval stress resistance in comparison to inorganic minerals and upregulated mRNA copy number of OC gene. The expression of this gene was strongly correlated with mineralization degree, thus confirming its potency as a good marker of bone mineralization in gilthead seabream larvae.

Nitric oxide regulates homeoprotein OTX1 and OTX2 expression in the rat myenteric plexus after intestinal ischemia-reperfusion injury.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by Nω-propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.NEW & NOTEWORTHY Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.

Expression of VEGF-A, Otx homeobox and p53 family genes in proliferative vitreoretinopathy.

INTRODUCTION: Proliferative vitreoretinopathy (PVR) is a severe inflammatory complication of retinal detachment. Pathological epiretinal membranes grow on the retina surface leading to contraction, and surgery fails in 5% to 10% of the cases. We evaluated the expression of VEGF-A, Otx1, Otx2, Otx3, and p53 family members from PVR specimens to correlate their role in inducing or preventing the pathology. METHODS: Twelve retinal samples were taken from patients affected by PVR during therapeutic retinectomies in vitreoretinal surgery. Gene expression was evaluated using quantitative real-time reverse transcriptase PCR analysis and immunohistochemistry, using four healthy human retinae as control. RESULT: Controls showed basal expression of all genes. PVR samples showed little or no expression of Otx1 and variable expression of VEGF-A, Otx2, Otx3, p53, and p63 genes. Significant correlation was found among VEGF-A, Otx2, p53, and p63 and between Otx1 and Otx3. CONCLUSIONS: Otx homeobox, p53 family, and VEGF-A genes are expressed in PVR human retina. We individuated two possible pathways (VEGF-A, Otx2, p53, p63 and Otx1 and Otx3) involved in PVR progression that could influence in different manners the course of the pathology. Individuating the genetic pathways of PVR represents a novel approach to PVR therapies.

Assessing the Effect of Contaminated and Restored Marine Sediments in Different Experimental Mesocosms Using an Integrated Approach and Mytilus galloprovincialis as a Model.

The research presented here was conducted to ascertain the effectiveness of recovery technologies in remediating a compromised marine environment. The multidisciplinary approach aims to integrate traditional chemical-physical analysis and to assess the biological parameters of Mytilus galloprovincialis within different experimental mesocosms (W, G, and B). In particular, this system was designed to reproduce sediment resuspension in a marine environment, which is thought to be one cause of contaminant release. The study combined morphological and ultrastructural observations with DNA damage assessment and mRNA expression of those genes involved in cellular stress responses. The tissues of mussels maintained in the polluted mesocosm showed a higher accumulation of Pb and Hg than in those maintained in restored mesocosm. This observation correlates well with mRNA expression of MT10 and data on DNA damage. The outcome of the biological evaluation consolidates the chemical characterization and supports the concept that the remediation method should be evaluated at an early stage, both to analytically determine the reduction of toxic components and to assess its ultimate impact on the biological system.

Homeoprotein OTX1 and OTX2 involvement in rat myenteric neuron adaptation after DNBS-induced colitis.

BACKGROUND: Inflammatory bowel diseases are associated with remodeling of neuronal circuitries within the enteric nervous system, occurring also at sites distant from the acute site of inflammation and underlying disturbed intestinal functions. Homeoproteins orthodenticle OTX1 and OTX2 are neuronal transcription factors participating to adaptation during inflammation and underlying tumor growth both in the central nervous system and in the periphery. In this study, we evaluated OTX1 and OTX2 expression in the rat small intestine and distal colon myenteric plexus after intrarectal dinitro-benzene sulfonic (DNBS) acid-induced colitis. METHODS: OTX1 and OTX2 distribution was immunohistochemically investigated in longitudinal muscle myenteric plexus (LMMP)-whole mount preparations. mRNAs and protein levels of both OTX1 and OTX2 were evaluated by qRT-PCR and Western blotting in LMMPs. RESULTS: DNBS-treatment induced major gross morphology and histological alterations in the distal colon, while the number of myenteric neurons was significantly reduced both in the small intestine and colon. mRNA levels of the inflammatory markers, TNFα, pro-IL1ß, IL6, HIF1α and VEGFα and myeloperoxidase activity raised in both regions. In both small intestine and colon, an anti-OTX1 antibody labeled a small percentage of myenteric neurons, and prevalently enteric glial cells, as evidenced by co-staining with the glial marker S100ß. OTX2 immunoreactivity was present only in myenteric neurons and was highly co-localized with neuronal nitric oxide synthase. Both in the small intestine and distal colon, the number of OTX1- and OTX2-immunoreactive myenteric neurons significantly increased after DNBS treatment. In these conditions, OTX1 immunostaining was highly superimposable with inducible nitric oxide synthase in both regions. OTX1 and OTX2 mRNA and protein levels significantly enhanced in LMMP preparations of both regions after DNBS treatment. CONCLUSIONS: These data suggest that colitis up-regulates OTX1 and OTX2 in myenteric plexus both on site and distantly from the injury, potentially participating to inflammatory-related myenteric ganglia remodeling processes involving nitrergic transmission.

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas.

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

Mesocosm System to Evaluate BF-MBR Efficacy in Mitigating Oily Wastewater Discharges: an Integrated Study on Mytilus galloprovincialis.

This work presents the results of recovery efficacy of the system "BioFilm Membrane BioReactor" (BF-MBR), in the treatment of oily contaminated seawaters. To this aim, we proposed a multidisciplinary approach that integrates traditional chemical-physical measures together with the assessment on biological sentinel Mytilus galloprovincialis, maintained in a medium-scale artificial system named mesocosm. The setup included: (1) a mesocosm consisting of uncontaminated seawater; (2) a mesocosm composed of an untreated oily wastewater discharge; and (3) a mesocosm receiving the same oily wastewater previously treated by a BF-MBR pilot scale plant. The multidisciplinary approach that included traditional chemical measures on mesocosms together with the evaluation of morphological organization, mRNA expression of those genes involved in cellular stress response, immunohistochemistry and metabolomic analysis on mussel tissues, was able to provide a robust and holistic evidence of how the proposed treatment is able to reduce the overall impact of oily wastewater discharges on the marine ecosystem.

Evaluation of biomarkers in Mytilus galloprovincialis as an integrated measure of biofilm-membrane bioreactor (BF-MBR) system efficiency in mitigating the impact of oily wastewater discharge to marine environment: a microcosm approach.

The large volumes of oily wastewater discharged to marine environment cause heavy impacts on the coastal marine ecosystem. The selection of an appropriate technology to reduce these impacts should be based on the respect of the discharge limits and on the effective assessment and monitoring of its effects on biological organism preservation. To this aim, we set up a controlled microcosm-scale system to compare the effects of a treated and untreated oily wastewater discharge in which the restore process is performed through a Membrane Bio-Reactor. The system is completed by other three microcosms to control and isolate any possible concurrent effect on the Mytilus galloprovincialis, used as sentinel organism. Mytilus galloprovincialis have been kept in all these microcosms, and then mRNA expression and morphology were evaluated on gills and digestive gland. The genes considered in this work are Heat Shock Protein 70 and Metallothionein 10, involved in response to physicochemical sublethal stressors, Superoxide dismutase 1, Catalase, and Cytochrome P450 involved in oxidative stress response. Our results evidenced a significant overexpression, both in gills and digestive gland, of HSP70 in samples maintained in the microcosm receiving the untreated effluent, and of MT10 in those animals kept in microcosm where the effluent was treated. Even though the mRNA modifications are considered "primary" and transient responses which do not always correspond to protein content, the study of these modifications can help to gain insights into the mechanisms of action of xenobiotic exposure. Morphological analysis suggested that, although different, depending on the microcosm, the most serious damages were found in the gill epithelium accompanied with severe haemocyte infiltration, whilst in digestive gland the tissue architecture alterations and the haemocyte infiltration were less pronounced. These observations suggest that the immune system was activated as a general response to stressful stimuli such as the presence of toxic compounds. Moreover, the results indicate that the treatment process is useful. In fact, samples derived from the microcosm receiving the treated effluent, even though presenting some signs of stress, seemed to partially recover the normal structure, although their mRNA expression indicated some cellular suffering.

Magnetic Nanoconjugated Teicoplanin: A Novel Tool for Bacterial Infection Site Targeting.

Nanoconjugated antibiotics can be regarded as next-generation drugs as they possess remarkable potential to overcome multidrug resistance in pathogenic bacteria. Iron oxide nanoparticles (IONPs) have been extensively used in the biomedical field because of their biocompatibility and magnetic properties. More recently, IONPs have been investigated as potential nanocarriers for antibiotics to be magnetically directed to/recovered from infection sites. Here, we conjugated the "last-resort" glycopeptide antibiotic teicoplanin to IONPs after surface functionalization with (3-aminopropyl) triethoxysilane (APTES). Classical microbiological methods and fluorescence and electron microscopy analysis were used to compare antimicrobial activity and surface interactions of naked IONPs, amino-functionalized NPs (NP-APTES), and nanoconjugated teicoplanin (NP-TEICO) with non-conjugated teicoplanin. As bacterial models, differently resistant strains of three Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis) and a Gram-negative representative (Escherichia coli) were used. The results indicated that teicoplanin conjugation conferred a valuable and prolonged antimicrobial activity to IONPs toward Gram-positive bacteria. No antimicrobial activity was detected using NP-TEICO toward the Gram-negative E. coli. Although IONPs and NP-APTES showed only insignificant antimicrobial activity in comparison to NP-TEICO, our data indicate that they might establish diverse interaction patterns at bacterial surfaces. Sensitivity of bacteria to NPs varied according to the surface provided by the bacteria and it was species specific. In addition, conjugation of teicoplanin improved the cytocompatibility of IONPs toward two human cell lines. Finally, NP-TEICO inhibited the formation of S. aureus biofilm, conserving the activity of non-conjugated teicoplanin versus planktonic cells and improving it toward adherent cells.

gDNA qPCR is statistically more reliable than mRNA analysis in detecting leukemic cells to monitor CML.

Chronic Myeloid Leukemia (CML) is a stem cell cancer that arises when t(9;22) translocation occurs in a hematopoietic stem cells. This event results in the expression of the BCR-ABL1 fusion gene, which codes for a constitutively active tyrosine kinase that is responsible for the transformation of a HSC into a CML stem cell, which then gives rise to a clonal myeloproliferative disease. The introduction of Tyrosine Kinase Inhibitors (TKIs) has revolutionized the management of the disease. However, these drugs do not seem to be able to eradicate the malignancy. Indeed, discontinuation trials (STIM; TWISER; DADI) for those patients who achieved a profound molecular response showed 50% relapsing within 12 months. We performed a comparative analysis on 15 CML patients and one B-ALL patient, between the standard quantitative reverse-transcriptase PCR (qRT-PCR) and our genomic DNA patient-specific quantitative PCR assay (gDNA qPCR). Here we demonstrate that gDNA qPCR is better than standard qRT-PCR in disease monitoring after an average follow-up period of 200 days. Specifically, we statistically demonstrated that DNA negativity is more reliable than RNA negativity in indicating when TKIs therapy can be safely stopped.

Identification of reference genes for qPCR analysis during hASC long culture maintenance.

Up to now quantitative PCR based assay is the most common method for characterizing or confirming gene expression patterns and comparing mRNA levels in different sample populations. Since this technique is relative easy and low cost compared to other methods of characterization, e.g. flow cytometry, we used it to typify human adipose-derived stem cells (hASCs). hASCs possess several characteristics that make them attractive for scientific research and clinical applications. Accurate normalization of gene expression relies on good selection of reference genes and the best way to choose them appropriately is to follow the common rule of the "Best 3", at least three reference genes, three different validation software and three sample replicates. Analysis was performed on hASCs cultivated until the eleventh cell confluence using twelve candidate reference genes, initially selected from literature, whose stability was evaluated by the algorithms NormFinder, BestKeeper, RefFinder and IdealRef, a home-made version of GeNorm. The best gene panel (RPL13A, RPS18, GAPDH, B2M, PPIA and ACTB), determined in one patient by IdealRef calculation, was then investigated in other four donors. Although patients demonstrated a certain gene expression variability, we can assert that ACTB is the most unreliable gene whereas ribosomal proteins (RPL13A and RPS18) show minor inconstancy in their mRNA expression. This work underlines the importance of validating reference genes before conducting each experiment and proposes a free software as alternative to those existing.

Effects of Metal Micro and Nano-Particles on hASCs: An In Vitro Model.

As the knowledge about the interferences of nanomaterials on human staminal cells are scarce and contradictory, we undertook a comparative multidisciplinary study based on the size effect of zero-valent iron, cobalt, and nickel microparticles (MPs) and nanoparticles (NPs) using human adipose stem cells (hASCs) as a model, and evaluating cytotoxicity, morphology, cellular uptake, and gene expression. Our results suggested that the medium did not influence the cell sensitivity but, surprisingly, the iron microparticles (FeMPs) resulted in being toxic. These data were supported by modifications in mRNA expression of some genes implicated in the inflammatory response. Microscopic analysis confirmed that NPs, mainly internalized by endocytosis, persist in the vesicles without any apparent cell damage. Conversely, MPs are not internalized, and the effects on hASCs have to be ascribed to the release of ions in the culture medium, or to the reduced oxygen and nutrient exchange efficiency due to the presence of MP agglomerating around the cells. Notwithstanding the results depicting a heterogeneous scene that does not allow drawing a general conclusion, this work reiterates the importance of comparative investigations on MPs, NPs, and corresponding ions, and the need to continue the thorough verification of NP and MP innocuousness to ensure unaffected stem cell physiology and differentiation.

OTX1 and OTX2 as possible molecular markers of sinonasal carcinomas and olfactory neuroblastomas.

OTX Homeobox genes are involved in embryonic morphogenesis and in the development of olfactory epithelium in adult. Mutations occurring in the OTX genes are reported to be associated to tumorigenisis in human. No reports correlate the expression of OTX genes and neoplasms of the nasal cavity. Thus, through immunohistochemical and Real-time PCR analysis we investigated OTX1 and OTX2 expression in the more frequent types of nasal and sinonasal tumours. Variable expression of both genes were found in normal sinonasal mucosa and in tumours. Interestingly, no expression of both OTX genes were detected in sinonasal intestinal-type adenocarcinomas; only OTX1 was found in non-intestinal-type adenocarcinomas and OTX2 was selectively expressed in olfactory neuroblastomas. In conclusion, OTX1 and OTX2 genes might have a role in the pathogenesis of different types of sinonasal neoplasms.

Evaluation of tissue morphology and gene expression as biomarkers of pollution in mussel Mytilus galloprovincialis caging experiment.

The ecosystem is being anthropogenically disturbed, which has serious consequences for the environment and human health, having strong social and economic impacts on the community. One of the most common methods to evaluate the effects of toxic contaminants is based on biomonitoring, e.g., placing Mytilus galloprovincialis in the polluted areas investigated. In this study, we have combined two different methods, transcriptomic and morphological analysis, with the purpose of determining whether cell morphology and the ultrastructural organization of our animal model are related to gene expression in outdoor experiments. The most pronounced changes were observed in mussel gills and digestive gland for mRNA involved in protein machinery (18S, 28S and EF1), while HSP70, MT10, CYP4Y1, SOD1, and CAT mRNAs showed scattered modifications not related to the studied area. In agreement with 18S, 28S, and EF1 mRNA evaluation, optical and electron microscopy demonstrated an initial inflammatory response of the cells that can lead to apoptosis in the caged mussels in all the polluted areas. In conclusion, the application of a multi-disciplinary approach proved to be effective for assessing the biological effects of contaminations on the health of aquatic organisms, and thus suitable to be applied in eco-toxicological studies. Although affected by several uncontrolled environmental variables, the assessment of mRNA can represent a useful endpoint for an integrated estimation of the overall threats to the sea environment within a field research approach.

Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia.

Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy.