A New In Vivo Model System to Assess the Toxicity of Semiconductor Nanocrystals

In the emerging area of nanotechnology, a key issue is related to the potential impacts of the novel nanomaterials on the environment and human health, so that this technology can be used with minimal risk. Specifically designed to combine on a single structure multipurpose tags and properties, smart nanomaterials need a comprehensive characterization of both chemicophysical properties and adequate toxicological evaluation, which is a challenging endeavour; the in vitro toxicity assays that are often employed for nanotoxicity assessments do not accurately predict in vivo response. To overcome these limitations and to evaluate toxicity characteristics of cadmium telluride quantum dots in relation to surface coatings, we have employed the freshwater polyp Hydra vulgaris as a model system. We assessed in vivo acute and sublethal toxicity by scoring for alteration of morphological traits, population growth rates, and influence on the regenerative capabilities providing new investigation clues for nanotoxicology purposes

Hymyc1 Downregulation Promotes Stem Cell Proliferation in Hydra vulgaris

Hydra is a unique model for studying the mechanisms underlying stem cell biology. The activity of the three stem cell lineages structuring its body constantly replenishes mature cells lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. In vertebrates, one of many genes that participate in regulating stem cell homeostasis is the protooncogene c-myc, which has been recently identified also in Hydra, and found expressed in the interstitial stem cell lineage. In the present paper, by developing a novel strategy of RNA interference-mediated gene silencing (RNAi) based on an enhanced uptake of small interfering RNAi (siRNA), we provide molecular and biological evidence for an unexpected function of the Hydra myc gene (Hymyc1) in the homeostasis of the interstitial stem cell lineage. We found that Hymyc1 inhibition impairs the balance between stem cell self renewal/differentiation, as shown by the accumulation of stem cell intermediate and terminal differentiation products in genetically interfered animals. The identical phenotype induced by the 10058-F4 inhibitor, a disruptor of c-Myc/Max dimerization, demonstrates the specificity of the RNAi approach. We show the kinetic and the reversible feature of Hymyc1 RNAi, together with the effects displayed on regenerating animals. Our results show the involvement of Hymyc1 in the control of interstitial stem cell dynamics, provide new clues to decipher the molecular control of the cell and tissue plasticity in Hydra, and also provide further insights into the complex myc network in higher organisms. The ability of Hydra cells to uptake double stranded RNA and to trigger a RNAi response lays the foundations of a comprehensive analysis of the RNAi response in Hydra allowing us to track back in the evolution and the origin of this process

Strumenti di genomica funzionale per l’identificazione e la caratterizzazione del gene rgga nella risposta allo stress idrico in patata ed Arabidopsis.

Il deficit idrico e la presenza di alte concentrazioni di sale nel suolo limitano enormemente la produttività di specie agrarie; si stima che circa il 28% dei suoli coltivabili non abbia una disponibilità adeguata di acqua per la produzione di specie di interesse. La risposta delle piante a stress abiotici è un fenomeno complesso che coinvolge un ampio network di geni. La comprensione dei meccanismi molecolari alla base della risposta allo stress e, soprattutto, dell’adattamento a condizioni ambientali avverse è di fondamentale importanza per l’ottenimento di varietà delle maggiori specie vegetali che garantiscano stabilità di produzione anche in condizioni ambientali sub-ottimali e promuovendo, inoltre, lo sviluppo di un’agricoltura eco-sostenibile. Il presente progetto di ricerca ha avuto come obiettivo l’identificazione e la caratterizzazione di geni la cui espressione è regolata in risposta a carenza idrica in patata (Solanum tuberosum). Allo scopo di identificare geni chiave nella risposta al deficit idrico a breve termine (condizioni di shock) e in quella a lungo termine (condizioni di adattamento), è stato effettuato, inizialmente, uno studio del trascrittoma di colture cellulari di patata in risposta a stress idrico, indotto da polietilene glicole (PEG 20%), attraverso un’analisi microarray. L’analisi dei dati microarray ha permesso l’identificazione di 1055 geni differenzialmente espressi di cui 130 e 337 geni sovraespressi nelle cellule stressate e adattate, rispettivamente. Una dettagliata caratterizzazione dei profili di espressione in cellule di patata sottoposte a stress idrico è stata effettuata, inoltre, mediante quantitative Real Time PCR (qRT-PCR). Lo studio di espressione condotto su un set di 25 geni selezionati ha permesso di confermare e validare i dati ottenuti mediante microarray. Inoltre, i geni selezionati sono risultati regolati da stress anche in foglie e radici provenienti da piante di patata sottoposte a prolungato deficit idrico, confermando il loro potenziale coinvolgimento nei meccanismi di graduale adattamento a condizioni di carenza idrica prolungata. E’ stato effettuato, quindi, un dettagliato studio molecolare e funzionale del gene codificante per una proteina RNA-binding (rgga), la cui espressione è risultata specificamente indotta durante condizioni di adattamento allo stress in cellule ed in piante di patata. La sequenza codificante del gene rgga di S. tuberosum è stata isolata e ulteriormente caratterizzata mediante un approccio bioinformatico, evidenziando elevata similitudine con proteine RNA binding di altre specie vegetali. Una più approfondita analisi di rgga è stata effettuata in Arabidopsis thaliana, l’organismo vegetale modello maggiormente caratterizzato e per il quale sono disponibili validi strumenti di analisi di genomica strutturale e funzionale. In particolare è stato evidenziato mediante qRT-PCR e Northern blotting che l’espressione del gene rgga è indotta in cellule di Arabidopsis in risposta ad alte concentrazioni di NaCl, PEG ed ABA (Acido Abscissico) e in giovani piantine sottoposte a stress osmotico di lunga durata. L’ espressione in vivo di rgga è stata studiata mediante localizzazione del gene reporter GUS clonato a valle del promotore del gene. Il saggio istochimico ha rivelato che rgga è espresso costitutivamente in piante di Arabidopsis; in particolare una forte attività di GUS è stata ritrovata nella radice, nei fasci vascolari, negli stomi e nel polline. L’analisi al microscopio confocale di piante transgeniche esprimenti la proteina di fusione RGGA-YFP (Yellow Fluorescent Protein), ha evidenziato una localizzazione prevalentemente citoplasmatica della proteina di interesse. Al fine di comprendere i meccanismi di risposta e tolleranza in condizioni di carenza idrica, è stato condotto, inoltre, un approccio genetico di “gain and loss of function” mediante l’uso di mutanti knockout (rgga ko) e l’ottenimento di piante transgeniche di Arabidopsis sovraesprimenti rgga (rgga202). Le analisi fenotipiche e fisiologiche in condizioni controllo ed in condizioni di stress hanno evidenziato una scarsa capacità di germinazione nel mutante knockout, rispetto alle piante controllo, in presenza di 120 mM di NaCl. Piante rgga202, invece, sono risultate maggiormente tolleranti rispetto al controllo ed al knockout in condizioni di stress imposte da trattamenti con PEG e NaCl. I risultati ottenuti suggeriscono che rgga riveste un ruolo cruciale nella complessa risposta della pianta a stress osmotico e, in particolare, nei meccanismi cellulari coinvolti nell’adattamento e nella tolleranza

Methodological approaches for nanotoxicology using cnidarian models

The remarkable amenability of aquatic invertebrates to laboratory manipulation has already made a few species belonging to the phylum Cnidaria as attracting systems for exploring animal development. The proliferation of molecular and genomic tools, including the whole genomic sequence of the freshwater polyp Hydra vulgaris and the starlet sea anemone Nematostella vectensis, further enhances the promise of these species to investigate the evolution of key aspects of development biology. In addition, the facility with which cnidarian population can be investigated within their natural ecological context suggests that these models may be profitably expanded to address important questions in ecology and toxicology. In this review, we explore the traits that make Hydra and Nematostella exceptionally attractive model organisms in context of nanotoxicology, and highlight a number of methods and developments likely to further increase that utility in the near future. © 2013 Informa UK Ltd All rights reserved

Nanotechnology in plant science: to make a long story short

This mini-review aims at gaining knowledge on basic aspects of plant nanotechnology. While in recent years the enormous progress of nanotechnology in biomedical sciences has revolutionized therapeutic and diagnostic approaches, the comprehension of nanoparticle-plant interactions, including uptake, mobilization and accumulation, is still in its infancy. Deeper studies are needed to establish the impact of nanomaterials (NMs) on plant growth and agro-ecosystems and to develop smart nanotechnology applications in crop improvement. Herein we provide a short overview of NMs employed in plant science and concisely describe key NM-plant interactions in terms of uptake, mobilization mechanisms, and biological effects. The major current applications in plants are reviewed also discussing the potential use of polymeric soft NMs which may open new and safer opportunities for smart delivery of biomolecules and for new strategies in plant genetic engineering, with the final aim to enhance plant defense and/or stimulate plant growth and development and, ultimately, crop production. Finally, we envisage that multidisciplinary collaborative approaches will be central to fill the knowledge gap in plant nanotechnology and push toward the use of NMs in agriculture and, more in general, in plant science research.</p

Nanotechnology in plant science: to make a long story short

This mini-review aims at gaining knowledge on basic aspects of plant nanotechnology. While in recent years the enormous progress of nanotechnology in biomedical sciences has revolutionized therapeutic and diagnostic approaches, the comprehension of nanoparticle-plant interactions, including uptake, mobilization and accumulation, is still in its infancy. Deeper studies are needed to establish the impact of nanomaterials (NMs) on plant growth and agro-ecosystems and to develop smart nanotechnology applications in crop improvement. Herein we provide a short overview of NMs employed in plant science and concisely describe key NM-plant interactions in terms of uptake, mobilization mechanisms, and biological effects. The major current applications in plants are reviewed also discussing the potential use of polymeric soft NMs which may open new and safer opportunities for smart delivery of biomolecules and for new strategies in plant genetic engineering, with the final aim to enhance plant defense and/or stimulate plant growth and development and, ultimately, crop production. Finally, we envisage that multidisciplinary collaborative approaches will be central to fill the knowledge gap in plant nanotechnology and push toward the use of NMs in agriculture and, more in general, in plant science research.</p

Beyond transcription: RNA‐binding proteins as emerging regulators of plant response to environmental constraints.

RNA-binding proteins (RBPs) govern many aspects of RNA metabolism, including pre-mRNA processing, transport, stability/decay and translation. Although relatively few plant RNA-binding proteins have been characterized genetically and biochemically, more than 200 RBP genes have been predicted in Arabidopsis and rice genomes, suggesting that they might serve specific plant functions. Besides their role in normal cellular functions, RBPs are emerging also as an interesting class of proteins involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions. Here, we review the most recent results and evidence on the functional role of RBPs in plant adaptation to various unfavourable environmental conditions and their contribution to enhance plant tolerance to abiotic stresses, with special emphasis on osmotic and temperature stress

Nanotoxicology using the sea anemone Nematostella vectensis: from developmental toxicity to genotoxicology

Concomitant with the fast-growing advances in the synthesis and engineering of colloidal nanocrystals, an urgent evaluation of their toxicity on human beings and environment is strongly encouraged by public health organisations. Despite the in vitro approaches employed for toxicological screening of hazardous compounds, the use of simple and cost-effective living organisms may enormously contribute to solve unanswered questions related to embryotoxic and teratogenic effects of nanomaterials. Here, the sea anemone Nematostella vectensis (Cnidaria, Anthozoa) is presented as a novel model organism to profile bio/non-bio interactions and to show a comprehensive toxicological analysis performed on embryos, larvae and adults treated with fluorescent cadmium-based nanocrystals. Spanning from in vivo biodistribution to molecular investigations, different behaviours and effects depending on the composition and surface coatings are showed. Rod-shaped cadmium selenide/cadmium sulfide (CdSe/CdS) nanocrystals resulted in excellent imaging probes to track N. vectensis development with negligible adverse effects, while spherical CdTe nanocrystals severely impaired embryogenesis, resulting in aberrant phenotypes and deregulation of developmental genes, which raise severe worries for a safe use of this type of nanoparticles for human purposes and environmental contamination. © 2014 Informa UK, Ltd

Bridging the fields of nanoscience and toxicology: nanoparticle impact on biological models

In the emerging area of nanotechnology a key issue is related to the potential impacts of the novel nanomaterials on the environment and human health so that this technology can be used with minimal risk. Specifically designed to combine on a single structure multipurpose tags and properties, nanomaterials need a comprehensive characterization of both chemicophysical properties and toxicological evaluation, which is a challenging endeavor: the in vitro toxicity assays that are employed for nanotoxicity assessments do not accurately predict in vivo response. To overcome these limitations and gain a deeper understanding of nanoparticle-cell interactions, we have employed cnidarian models, in particular the freshwater polyp Hydra vulgaris, not opposed to more complex and evoluted systems, but to add valuable information, at an intermediate level between prokaryotes and vertebrates, on both cytoxicity and on pollution affecting the environment. By testing CdSe/CdS core shell nanocrystals in vivo, at whole animal level, we investigated the impact of their properties on uptake, accumulation, biodistribution, elicitation of behavioural responses. Spanning from animal to cell biology, we provide an analysis on metal based and semiconductor NC, discussing the crucial role played by the synthesis route and chemical surface on the toxicity for living organisms