Publications by authors named "Daniela Uccelletti"

51 Publications

Comparison of analytical approaches for identifying airborne microorganisms in a livestock facility.

Sci Total Environ 2021 Aug 19;783:147044. Epub 2021 Apr 19.

Department of Chemistry, Sapienza University of Rome, 00185 Rome, Italy.

An intensive study, applied to a site characterized by multiple sources of microorganisms, was aimed at understanding the best approach to study bioaerosol. Culture-based, molecular biological, and chemical methods were applied to Particulate Matter (PM) samples collected in a livestock facility, during spring and autumn seasons, in two different outdoor areas. The first one was close to a place where feed was stored and handled and the second next to an open cowshed. Qualitative analysis of bacteria was performed by sequencing techniques applied to DNA extracted from both isolated culturable bacteria and particulate matter samples. Quantification of microorganisms was achieved through three distinct approaches. Microorganism colonies were counted, after incubation at 28 °C, and expressed as colony-forming units (CFU) per m. Chemical method consisted in the identification of individual biomarkers, and their conversion to number of microorganisms per m, using proper conversion factors. Finally, qPCR was applied to DNA extracted from PM samples, and the results were expressed as total amount of bacteria present in the bioaerosol (UG/m). The presence of airborne sterols was also studied to broaden the knowledge of bioaerosol components in atmosphere. Small seasonal differences and major sampling site differences occurred. Obviously, culture-dependent method identified less and different bacteria, than culture-independent approach. The chemical approach and the culture independent metagenomic method were in good agreement. As expected, CFU/m accounted for not more than 0.3% of bacteria calculated as the average of chemical and culture independent metagenomic methods. The complexity of the obtained results shows that the different approaches are complementary to obtain an exhaustive description of bioaresol in terms of concentration, speciation, viability, pathogenicity.
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http://dx.doi.org/10.1016/j.scitotenv.2021.147044DOI Listing
August 2021

Biocompatibility and Antibiofilm Properties of Calcium Silicate-Based Cements: An In Vitro Evaluation and Report of Two Clinical Cases.

Biology (Basel) 2021 May 26;10(6). Epub 2021 May 26.

Department of Oral and Maxillofacial Science, Sapienza University of Rome, Via Caserta 6, 00161 Rome, Italy.

Calcium silicate-based cements have reached excellent levels of performance in endodontics, providing predictable and successful results. To better assess the properties of these bioactive materials, the present study aimed to compare the biocompatibility and antibiofilm properties of ProRoot MTA and Biodentine. Human osteogenic sarcoma (Saos-2) cells were cultured on ProRoot MTA and Biodentine samples or in the presence of both cement extracts. Cell viability assay, measurement of reactive oxygen species (ROS), immunofluorescence analysis, as well as morphological evaluations were conducted. Moreover, was used to assess the biofilm forming ability on ProRoot MTA and Biodentine disks. Finally, both cements were applied in vivo to treat immature permanent teeth affected by reversible pulpitis. Results: Cell viability assay demonstrated that Saos-2 cells had a dose- and time-dependent cytotoxicity to both analyzed cements, although cells exposed to ProRoot MTA showed a better cell vitality than those exposed to Biodentine ( < 0.001). Both cements demonstrated ROS production while this was greater in the case of Biodentine than ProRoot MTA ( < 0.001). Immunofluorescence images of the cytoskeleton and focal adhesions showed no differences in Saos-2 cells grown in the presence of ProRoot MTA eluate; whereas in the Biodentine groups, cells showed a morphology and focal adhesions more similar to that of the control sample, as the eluate concentration decreased. Morphological analysis revealed that Saos-2 cells were more flattened and exhibited better spreading when attached to ProRoot MTA disks than to Biodentine ones. The antibiofilm properties showed a time-dependent powerful inhibition of superficial colonization and an antibiofilm effect of both cements. Clinically, complete root formation of the treated elements was achieved using the two studied cements, showing stable results over time. ProRoot MTA and Biodentine was demonstrated to be biocompatible and to possess antibiofilm properties. Their clinical application in vital pulp therapy provided successful outcomes after 2 years of follow-up.
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http://dx.doi.org/10.3390/biology10060470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226987PMC
May 2021

Assessment of the effects of atmospheric pollutants using the animal model Caenorhabditis elegans.

Environ Res 2020 12 13;191:110209. Epub 2020 Sep 13.

Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, P.le Aldo Moro, 5, 00185, Rome, Italy. Electronic address:

Air pollution is recognized as the world's largest environmental health risk. In this work we evaluated in vivo the effects of three relevant components of atmospheric dusts (brake dust, wood pellet ash and Saharan dust) employing the animal model Caenorhabditis elegans. Main endpoints of C. elegans such as life span, brood size and oxidative stress were addressed by exposing the nematodes to different dust concentrations. Brake dust and pellet ash affected the life span and increased significantly the oxidative stress of exposed nematodes, while Saharan dust showed no effects. Water soluble and insoluble fractions of these dusts were used to investigate the impact of the single fraction on C. elegans. The two fractions of brake dust and pellet ash exerted different effects on C. elegans endpoints in terms of life span and oxidative stress response. These fractions acted in different ways on the worm susceptibility to infection of two human pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) affecting the sek-1 gene expression. In conclusion, our study showed that C. elegans is a valuable tool to investigate in vivo possible effects of atmospheric dusts.
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http://dx.doi.org/10.1016/j.envres.2020.110209DOI Listing
December 2020

In vitro and in vivo lipidomics as a tool for probiotics evaluation.

Appl Microbiol Biotechnol 2020 Oct 2;104(20):8937-8948. Epub 2020 Sep 2.

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome, Italy.

The probiotic bacteria are helpful for nutritional and therapeutic purposes, and they are commercially available in various forms, such as capsules or powders. Increasing pieces of evidence indicate that different growth conditions and variability in manufacturing processes can determine the properties of probiotic products. In recent years, the lipidomic approach has become a useful tool to evaluate the impact that probiotics induce in host physiology. In this work, two probiotic formulations with identical species composition, produced in two different sites, the USA and Italy, were utilized to feed Caenorhabditis elegans, strains and alterations in lipid composition in the host and bacteria were investigated. Indeed, the multicellular organism C. elegans is considered a simple model to study the in vivo effects of probiotics. Nematodes fat metabolism was assessed by gene expression analysis and by mass spectrometry-based lipidomics. Lipid droplet analysis revealed a high accumulation of lipid droplets in worms fed US-made products, correlating with an increased expression of genes involved in the fatty acid synthesis. We also evaluated the lifespan of worms defective in genes involved in the insulin/IGF-1-mediated pathway and monitored the nuclear translocation of DAF-16. These data demonstrated the involvement of the signaling in C. elegans responses to the two diets. Lipidomics analysis of the two formulations was also conducted, and the results indicated differences in phosphatidylglycerol (PG) and phosphatidylcholine (PC) contents that, in turn, could influence nematode host physiology. Results demonstrated that different manufacturing processes could influence probiotics and host properties in terms of lipid composition. KEY POINTS: • Probiotic formulations impact on Caenorhabditis elegans lipid metabolism; • Lipidomic analysis highlighted phospholipid abundance in the two products; • Phosphocholines and phosphatidylglycerols were analyzed in worms fed the two probiotic formulations.
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http://dx.doi.org/10.1007/s00253-020-10864-wDOI Listing
October 2020

Molecular and cellular responses to short exposure to bisphenols A, F, and S and eluates of microplastics in C. elegans.

Environ Sci Pollut Res Int 2021 Jan 21;28(1):805-818. Epub 2020 Aug 21.

Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Piazzale Aldo Moro 1, 00185, Rome, Italy.

Bisphenol F (BPF) and bisphenol S (BPS) have been developed as an alternative to bisphenol A (BPA), a well-known endocrine disruptor, leading to their detection in the aquatic environment. In this work, we used the animal model Caenorhabditis elegans to improve our understanding of their potential effects on the biota and the environment. Our findings demonstrated that, after 24 h exposure, all the bisphenols examined increased the number of apoptotic corpses and the expression of the detoxifying enzymes SOD-3 and GST-4, without affecting the ROS levels, while BPA and BPS significantly enhanced DNA fragmentation. Furthermore, similarly to BPA, BPF and BPS did not alter the lifespan through the activation of SEK-1 and SKN-1 pathways. Thus, this study raises the attention of the risks associated with exposure to BPA alternatives. We also examined the effects of microplastic (MP) eluates on C. elegans. Aqueous extracts of weathered microplastic samples, both at high and low degradation state and pellets, have been evaluated for their effects on lifespan, DNA fragmentation, germline apoptosis, and oxidative stress response. Overall, our findings showed that eluates of low degraded plastics exert a greater toxic effect on the nematode C. elegans compared with the aqueous sample of high degraded plastic fragments and pellets.
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http://dx.doi.org/10.1007/s11356-020-10498-5DOI Listing
January 2021

Antibacterial Activity against of Titanium Surfaces Coated with Graphene Nanoplatelets to Prevent Peri-Implant Diseases. An In-Vitro Pilot Study.

Int J Environ Res Public Health 2020 02 29;17(5). Epub 2020 Feb 29.

Department of Oral and Maxillo-Facial Sciences, Sapienza University of Rome, 00100 Rome, Italy.

Dental implants are one of the most commonly used ways to replace missing teeth. Nevertheless, the close contact with hard and soft oral tissues expose these devices to infectious peri-implant diseases. To prevent such infection, several surface treatments have been developed in the last few years to improve the antimicrobial properties of titanium dental implants. In this in-vitro pilot study, the antimicrobial activity of titanium surfaces coated with different types of graphene nanoplatelets are investigated. Six different colloidal suspensions of graphene nanoplatelets (GNPs) were produced from graphite intercalated compounds, setting the temperature and duration of the thermal shock and varying the number of the exfoliation cycles. Titanium disks with sand-blasted and acid-etched surfaces were sprayed with 2 mL of colloidal GNPs suspensions. The size of the GNPs and the percentage of titanium disk surfaces coated by GNPs were evaluated through a field emission-scanning electron microscope. The antibacterial activity of the specimens against was estimated using a crystal violet assay. The dimension of GNPs decreased progressively after each sonication cycle. The two best mean percentages of titanium disk surfaces coated by GNPs were GNPs and GNPs. The reduction of biofilm development was 14.4% in GNPs, 20.1% in GNPs, 30.3% in GNPs, and 39.2% in GNPs. The results of the study suggested that the surface treatment of titanium disks with GNPs represents a promising solution to improve the antibacterial activity of titanium implants.
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http://dx.doi.org/10.3390/ijerph17051568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084449PMC
February 2020

Antibacterial Effect of Zinc Oxide-Based Nanomaterials on Environmental Biodeteriogens Affecting Historical Buildings.

Nanomaterials (Basel) 2020 Feb 16;10(2). Epub 2020 Feb 16.

Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.

The colonization of microorganisms and their subsequent interaction with stone substrates under different environmental conditions encourage deterioration of materials by multiple mechanisms resulting in changes in the original color, appearance and durability. One of the emerging alternatives to remedy biodeterioration is nanotechnology, thanks to nanoparticle properties such as small size, no-toxicity, high photo-reactivity, and low impact on the environment. This study highlighted the effects of ZnO-based nanomaterials of two bacteria isolated from the Temple of Concordia (Agrigento's Valley of the Temples in Sicily, Italy) that are involved in biodeterioration processes. The antimicrobial activities of ZnO-nanorods (Zn-NRs) and graphene nanoplatelets decorated with Zn-NRs (ZNGs) were evaluated against the Gram positive and two isolates of the Gram negative . ZNGs demonstrated high antibacterial and antibiofilm activities on several substrates such as stones with different porosity. In the case of ZNGs, a marked time- and dose-dependent bactericidal effect was highlighted against all bacterial species. Therefore, these nanomaterials represent a promising tool for developing biocompatible materials that can be exploited for the conservation of cultural heritage. These nanostructures can be successfully applied without releasing toxic compounds, thus spreading their usability.
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http://dx.doi.org/10.3390/nano10020335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075178PMC
February 2020

A Combined Proteomics, Metabolomics and In Vivo Analysis Approach for the Characterization of Probiotics in Large-Scale Production.

Biomolecules 2020 01 18;10(1). Epub 2020 Jan 18.

Department MESVA, University of L'Aquila, 67100 L'Aquila, Italy.

The manufacturing processes of commercial probiotic strains may be affected in different ways in the attempt to optimize yield, costs, functionality, or stability, influencing gene expression, protein patterns, or metabolic output. Aim of this work is to compare different samples of a high concentration (450 billion bacteria) multispecies (8 strains) formulation produced at two different manufacturing sites, United States of America (US) and Italy (IT), by applying a combination of functional proteomics, metabolomics, and in vivo analyses. Several protein-profile differences were detected between IT- and US-made products, with , , and Bifidobacteria being the main affected probiotics/microorganisms. Performing proton nuclear magnetic spectroscopy (H-NMR), some discrepancies in amino acid, lactate, betaine and sucrose concentrations were also reported between the two products. Finally, we investigated the health-promoting and antiaging effects of both products in the model organism . The integration of omics platforms with in vivo analysis has emerged as a powerful tool to assess manufacturing procedures.
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http://dx.doi.org/10.3390/biom10010157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022454PMC
January 2020

Evaluation of Bioaerosol Bacterial Components of a Wastewater Treatment Plant Through an Integrate Approach and In Vivo Assessment.

Int J Environ Res Public Health 2019 12 30;17(1). Epub 2019 Dec 30.

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, 00185 Rome, Italy.

Wastewater carries different pathogenic and non-pathogenic microorganisms that can be dispersed in the surrounding environment. Workers who frequent sewage treatment plants can therefore be exposed to aerosols that contain a high concentration of potentially dangerous biological agents, or they can come into direct contact with contaminated material. This can lead to allergies, infections and occupational health-associated diseases. A characterization of biological risk assessment of bioaerosol exposure is necessary. The aim of this study was to evaluate the application of an interdisciplinary method that combines chemical and biological approaches for the analysis of a bioaerosol derived from a wastewater treatment plant (WWTP) situated in Italy. Sampled filters were analyzed by HPLC-MS/MS spectroscopy that searched for different chemical biomarkers of airborne microorganisms. The analytical quantification was compared to the biological cultural method that revealed an underrated microbial concentration. Furthermore, next generation sequencing analysis was used also to identify the uncultivable species that were not detected by the culture dependent-method. Moreover, the simple animal model was used to evaluate the pathogenicity of two isolates- and that showed multidrug-resistance. This work represents a starting point for the development of a multidisciplinary approach for the validation of bioaerosol exposure on WWTP workplaces.
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http://dx.doi.org/10.3390/ijerph17010273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981557PMC
December 2019

gene affects susceptibility of to infection through glycosylation and stress response pathways' alterations.

Virulence 2019 12;10(1):1013-1025

Department of Biology and Biotechnology "Charles Darwin", University of Rome, Rome, Italy.

Calcium signaling can elicit different pathways involved in an extreme variety of biological processes. Calcium levels must be tightly regulated in a spatial and temporal manner in order to be efficiently and properly utilized in the host physiology. The Ca-ATPase, encoded by gene, was first identified in yeast and localized to the Golgi and it appears to be involved in calcium homeostasis. PMR-1 function is evolutionary conserved from yeast to human, where mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. In this work, we used the model system to gain insight into the downstream response elicited by the loss of gene. We found that knocked down animals not only showed defects in the oligosaccharide structure of glycoproteins at the cell surface but also were characterized by reduced susceptibility to bacterial infection. Although increased resistance to the infection might be related to lack of regular recognition of surface glycoproteins by microbial agents, we provide genetic evidence that interfered nematodes mounted a stronger innate immune response to Gram-positive bacterial infection. Thus, our observations indicate as a candidate gene implicated in mediating the worm's innate immune response.
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http://dx.doi.org/10.1080/21505594.2019.1697118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930020PMC
December 2019

and Probiotics Interactions from a Prolongevity Perspective.

Int J Mol Sci 2019 Oct 10;20(20). Epub 2019 Oct 10.

Research Centre for Food and Nutrition, CREA (Council for Agricultural Research and Economics), 00178 Rome, Italy.

Probiotics exert beneficial effects on host health through different mechanisms of action, such as production of antimicrobial substances, competition with pathogens, enhancement of host mucosal barrier integrity and immunomodulation. In the context of ageing, which is characterized by several physiological alterations leading to a low grade inflammatory status called inflammageing, evidences suggest a potential prolongevity role of probiotics. Unraveling the mechanisms underlying anti-ageing effects requires the use of simple model systems. To this respect, the nematode represents a suitable model organism for the study of both host-microbe interactions and for ageing studies, because of conserved signaling pathways and host defense mechanisms involved in the regulation of its lifespan. Therefore, this review analyses the impact of probiotics on age-related parameters, with particular emphasis on oxidative stress, immunity, inflammation and protection from pathogen infections. The picture emerging from our analysis highlights that several probiotic strains are able to exert anti-ageing effects in nematodes by acting on common molecular pathways, such as insulin/insulin-like growth factor-1 (IIS) and p38 mitogen-activated protein kinase (p38 MAPK). In this perspective, appears to be advantageous for shedding light on key mechanisms involved in host prolongevity in response to probiotics supplementation.
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http://dx.doi.org/10.3390/ijms20205020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834311PMC
October 2019

The Foodborne Strain MBC2 Triggers -Dependent Pro-Longevity Effects in .

Microorganisms 2019 Feb 7;7(2). Epub 2019 Feb 7.

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, 00185 Rome, Italy.

Lactic acid bacteria (LAB) are involved in several food fermentations and many of them provide strain-specific health benefits. Herein, the probiotic potential of the foodborne strain MBC2 was investigated through in vitro and in vivo approaches. was used as an in vivo model to analyze pro-longevity and anti-aging effects. MBC2 showed a high gut colonization capability compared to OP50 (OP50) or L. rhamnosus GG (LGG). Moreover, analysis of pumping rate, lipofuscin accumulation, and body bending showed anti-aging effects in MBC2-fed worms. Studies on PEPT-1 mutants demonstrated that gene was involved in the anti-aging processes mediated by this bacterial strain through DAF-16, whereas the oxidative stress protection was PEPT-1 independent. Moreover, analysis of acid tolerance, bile tolerance, and antibiotic susceptibility were evaluated. MBC2 exerted beneficial effects on nematode lifespan, influencing energy metabolism and oxidative stress resistance, resulted in being tolerant to acidic pH and able to adhere to Caco-2 cells. Overall, these findings provide new insight for application of this strain in the food industry as a newly isolated functional starter. Furthermore, these results will also shed light on molecular players involved in host-microbe interactions.
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http://dx.doi.org/10.3390/microorganisms7020045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406943PMC
February 2019

Enamel remineralization and repair results of Biomimetic Hydroxyapatite toothpaste on deciduous teeth: an effective option to fluoride toothpaste.

J Nanobiotechnology 2019 Jan 25;17(1):17. Epub 2019 Jan 25.

Department of Oral and Maxillo-Facial Sciences, Sapienza University of Rome, Viale Regina Elena 287a, 00161, Rome, Italy.

Background: Dental caries is a recognized worldwide public health problem. Despite being one of the most effective strategies against dental caries, the excessive use of fluorine may result in a potential risk of developing dental fluorosis especially in children under age of six. The purpose of this work is to analyze a fluorine-free toothpaste containing Biomimetic Hydroxyapatite to assess enamel re-mineralizing and repairing properties.

Results: The study was performed in vitro and in vivo, comparing the hydroxyapatite toothpaste with two others toothpaste containing different fluorine concentrations. The coating effect of the micro-structured Hydroxyapatite nanoparticles reintegrates the enamel with a biomimetic film reproducing the structure and the morphology of the biologic Hydroxyapatite of the enamel. As demonstrated, the coating is due to the deposit of a new layer of apatite, which presents fewer particles than the natural enamel, not based on the chemical-physical changes occurring in fluorinated toothpastes. Moreover, it shows resistance to brushing as a consequence of chemical bonds between the synthetic and natural crystals of the enamel.

Conclusions: The use of Biomimetic Hydroxyapatite toothpastes has proven to be a valuable prevention measure against dental caries in primary dentition since it prevents the risk of fluorosis.
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http://dx.doi.org/10.1186/s12951-019-0454-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346538PMC
January 2019

Virulence behavior of uropathogenic Escherichia coli strains in the host model Caenorhabditis elegans.

Microbiologyopen 2019 06 31;8(6):e00756. Epub 2018 Oct 31.

Department of Public Health and Infectious Diseases, Microbiology Section, Sapienza University, Rome, Italy.

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Although a number of bacteria can cause UTIs, most cases are due to infection by uropathogenic Escherichia coli (UPEC). UPEC are a genetically heterogeneous group that exhibit several virulence factors associated with colonization and persistence of bacteria in the urinary tract. Caenorhabditis elegans is a tiny, free-living nematode found worldwide. Because many biological pathways are conserved in C. elegans and humans, the nematode has been increasingly used as a model organism to study virulence mechanisms of microbial infections and innate immunity. The virulence of UPEC strains, characterized for antimicrobial resistance, pathogenicity-related genes associated with virulence and phylogenetic group belonging was evaluated by measuring the survival of C. elegans exposed to pure cultures of these strains. Our results showed that urinary strains can kill the nematode and that the clinical isolate ECP110 was able to efficiently colonize the gut and to inhibit the host oxidative response to infection. Our data support that C. elegans, a free-living nematode found worldwide, could serve as an in vivo model to distinguish, among uropathogenic E. coli, different virulence behavior.
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http://dx.doi.org/10.1002/mbo3.756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562141PMC
June 2019

Anti-Candidal Activity and In Vitro Cytotoxicity Assessment of Graphene Nanoplatelets Decorated with Zinc Oxide Nanorods.

Nanomaterials (Basel) 2018 Sep 21;8(10). Epub 2018 Sep 21.

Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy.

is the most common pathogenic fungus that is isolated in nosocomial infections in medically and immune-compromised patients. The ability of to convert its form from yeast to hyphal morphology contributes to biofilm development that effectively shelters against the action of antifungals molecules. In the last years, nanocomposites are the most promising solutions against drug-resistant microorganisms. The aim of this study was to investigate the antifungal activity of graphene nanoplateles decorated with zinc oxide nanorods (ZNGs) against the human pathogen . We observed that ZNGs were able to induce a significant mortality in fungal cells, as well as to affect the main virulence factors of this fungus or rather the hyphal development and biofilm formation. Reactive Oxygen Species (ROS) formation in yeast cells resulted one of the mechanisms of ZNGs to induce mortality. Finally, the toxicity of this nanomaterial was tested also on human keratinocyte cell line HaCaT. Our data indicated that ZNGs resulted not toxic when their aggregation state decreased by adding glycerol as emulsifier to ZNGs suspensions or when HaCaT cells were grown on ZNGs-coated glasses. Overall, the results that were obtained indicated that ZNGs could be exploited as an antifungal nanomaterial with a high degree of biocompatibility on human cells.
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http://dx.doi.org/10.3390/nano8100752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215111PMC
September 2018

Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease.

Int J Mol Sci 2018 Jun 20;19(6). Epub 2018 Jun 20.

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, 00185 Rome, Italy.

The term orthodisease defines human disorders in which the pathogenic gene has orthologs in model organism genomes. Yeasts have been instrumental for gaining insights into the molecular basis of many human disorders, particularly those resulting from impaired cellular metabolism. We and others have used yeasts as a model system to study the molecular basis of Hailey-Hailey disease (HHD), a human blistering skin disorder caused by haploinsufficiency of the gene the orthologous of the yeast gene . We observed that cells defective for gene share several biological similarities with HHD derived keratinocytes. Based on the conservation of ATP2C1/PMR1 function from yeast to human, here we used a yeast-based assay to screen for molecules able to influence the pleiotropy associated with deletion. We identified six compounds, Kaempferol, Indirubin, Lappaconite, Cyclocytidine, Azomycin and Nalidixic Acid that induced different major shape phenotypes in . These include mitochondrial and the cell-wall morphology-related phenotypes. Interestingly, a secondary assay in mammalian cells confirmed activity for Kaempferol. Indeed, this compound was also active on human keratinocytes depleted of ATP2C1 function by siRNA-treatment used as an in-vitro model of HHD. We found that Kaempferol was a potent NRF2 regulator, strongly inducing its expression and its downstream target . In addition, Kaempferol could decrease oxidative stress of ATP2C1 defective keratinocytes, characterized by reduced NRF2-expression. Our results indicated that the activation of these pathways might provide protection to the HHD-skin cells. As oxidative stress plays pivotal roles in promoting the skin lesions of Hailey-Hailey, the NRF2 pathway could be a viable therapeutic target for HHD.
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http://dx.doi.org/10.3390/ijms19061814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032253PMC
June 2018

and Selection of Potentially Probiotic Lactobacilli From Nocellara del Belice Table Olives.

Front Microbiol 2018 28;9:595. Epub 2018 Mar 28.

Food & Nutrition Research Centre, Council for Agricultural Research and Economics, Rome, Italy.

Table olives are increasingly recognized as a vehicle as well as a source of probiotic bacteria, especially those fermented with traditional procedures based on the activity of indigenous microbial consortia, originating from local environments. In the present study, we report characterization at the species level of 49 Lactic Acid Bacteria (LAB) strains deriving from Nocellara del Belice table olives fermented with the Spanish or Castelvetrano methods, recently isolated in our previous work. Ribosomal 16S DNA analysis allowed identification of 4 , 3 , 14 , 19 , 7 , and 2 . The and strains were subjected to further screening to evaluate their probiotic potential, using a combination of and approaches. The majority of them showed high survival rates under simulated gastro-intestinal conditions, and positive antimicrobial activity against serovar Typhimurium, and enterotoxigenic (ETEC) pathogens. Evaluation of antibiotic resistance to ampicillin, tetracycline, chloramphenicol, or erythromycin was also performed for all selected strains. Three strains were selected as very good performers in the initial testing screens, they were antibiotic susceptible, as well as capable of inhibiting pathogen growth . Parallel screening employing the simplified model organism , fed the strains as a food source, revealed that one and one strains significantly induced prolongevity effects and protection from pathogen-mediated infection. Moreover, both strains displayed adhesion to human intestinal epithelial Caco-2 cells and were able to outcompete foodborne pathogens for cell adhesion. Overall, these results are suggestive of beneficial features for novel LAB strains, which renders them promising candidates as starters for the manufacturing of fermented table olives with probiotic added value.
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http://dx.doi.org/10.3389/fmicb.2018.00595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882814PMC
March 2018

Graphene-based dental adhesive with anti-biofilm activity.

J Nanobiotechnology 2017 Dec 12;15(1):89. Epub 2017 Dec 12.

Research Center for Nanotechnology Applied to Engineering of Sapienza University (CNIS), Sapienza University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy.

Background: Secondary caries are considered the main cause of dental restoration failure. In this context, anti-biofilm and bactericidal properties are desired in dental materials against pathogens such as Streptococcus mutans. To this purpose, graphene based materials can be used as fillers of polymer dental adhesives. In this work, we investigated the possibility to use as filler of dental adhesives, graphene nanoplatelets (GNP), a non toxic hydrophobic nanomaterial with antimicrobial and anti-biofilm properties.

Results: Graphene nanoplatelets have been produced starting from graphite intercalated compounds through a process consisting of thermal expansion and liquid exfoliation. Then, a dental adhesive filled with GNPs at different volume fractions has been produced through a solvent evaporation method. The rheological properties of the new experimental adhesives have been assessed experimentally. The adhesive properties have been tested using microtensile bond strength measurements (µ-TBS). Biocidal activity has been studied using the colony forming units count (CFU) method. The anti-biofilm properties have been demonstrated through FE-SEM imaging of the biofilm development after 3 and 24 h of growth.

Conclusions: A significantly lower vitality of S. mutans cells has been demonstrated when in contact with the GNP filled dental adhesives. Biofilm growth on adhesive-covered dentine tissues demonstrated anti-adhesion properties of the produced materials. µ-TBS results demonstrated no significant difference in µ-TBS between the experimental and the control adhesive. The rheology tests highlighted the necessity to avoid low shear rate regimes during adhesive processing and application in clinical protocol, and confirmed that the adhesive containing the 0.2%wt of GNPs possess mechanical properties comparable with the ones of the control adhesive.
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http://dx.doi.org/10.1186/s12951-017-0322-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728064PMC
December 2017

Evaluation of the antibacterial power and biocompatibility of zinc oxide nanorods decorated graphene nanoplatelets: new perspectives for antibiodeteriorative approaches.

J Nanobiotechnology 2017 Aug 1;15(1):57. Epub 2017 Aug 1.

Department of Biology and Biotechnology C. Darwin, Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, Italy.

Background: Nanotechnologies are currently revolutionizing the world around us, improving the quality of our lives thanks to a multitude of applications in several areas including the environmental preservation, with the biodeterioration phenomenon representing one of the major concerns.

Results: In this study, an innovative nanomaterial consisting of graphene nanoplatelets decorated by zinc oxide nanorods (ZNGs) was tested for the ability to inhibit two different pathogens belonging to bacterial genera frequently associated with nosocomial infections as well as biodeterioration phenomenon: the Gram-positive Staphylococcus aureus and the Gram-negative Pseudomonas aeruginosa. A time- and dose-dependent bactericidal effect in cell viability was highlighted against both bacteria, demonstrating a strong antimicrobial potential of ZNGs. Furthermore, the analysis of bacterial surfaces through Field emission scanning electron microscopy (FESEM) revealed ZNGs mechanical interaction at cell wall level. ZNGs induced in those bacteria deep physical damages not compatible with life as a result of nanoneedle-like action of this nanomaterial together with its nanoblade effect. Cell injuries were confirmed by Fourier transform infrared spectroscopy, revealing that ZNGs antimicrobial effect was related to protein and phospholipid changes as well as a decrease in extracellular polymeric substances; this was also supported by a reduction in biofilm formation of both bacteria. The antibacterial properties of ZNGs applied on building-related materials make them a promising tool for the conservation of indoor/outdoor surfaces. Finally, ZNGs nanotoxicity was assessed in vivo by exploiting the soil free living nematode Caenorhabditis elegans. Notably, no harmful effects of ZNGs on larval development, lifespan, fertility as well as neuromuscular functionality were highlighted in this excellent model for environmental nanotoxicology.

Conclusions: Overall, ZNGs represent a promising candidate for developing biocompatible materials that can be exploitable in antimicrobial applications without releasing toxic compounds, harmful to the environment.
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http://dx.doi.org/10.1186/s12951-017-0291-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539890PMC
August 2017

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Subspecies and Strains While Testing in the Model Organism Highlighted Probiotic Properties.

Front Microbiol 2017 28;8:1206. Epub 2017 Jun 28.

Department of Chemistry, Sapienza University of RomeRome, Italy.

represents a technologically relevant member of lactic acid bacteria, since the two subspecies and are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to subspecies , and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism , with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to subspecies . Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that subsp. diet exerted beneficial effects on nematodes. On the other hand, both subsp. strains affected lifespan and larval development. We have characterized three strains belonging to subspecies and highlighting their divergent origin. In particular, the two closely related isolates subspecies display different galactose metabolic capabilities. Moreover, the subspecies strain demonstrated potential probiotic features. Combination of platforms coupled with screening in the simple model organism is a powerful tool to characterize industrially relevant bacterial isolates.
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http://dx.doi.org/10.3389/fmicb.2017.01206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487477PMC
June 2017

Muramic and dipicolinic acids in atmospheric particulate matter as biomarkers of bacteria and bacterial spores.

Anal Bioanal Chem 2017 Feb 7;409(6):1657-1666. Epub 2016 Dec 7.

Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy.

Airborne bacteria are components of the atmospheric aerosol particles and can be responsible of allergic disease, regardless of their viability. In this paper, we report a method for the determination of total (viable and nonviable) bacterial content in airborne particles, using muramic and dipicolinic acids as biomarkers of bacteria and bacterial spores, respectively. The analytical procedure was optimized with bacteria and spores of Bacillus subtilis. After extraction and purification, the two biomarkers were analyzed by HPLC-ESI-MS/MS and their percentage was evaluated to be used as conversion factor. The present method for the determination of the total bacterial content was then applied to environmental samples, after a proper collection in an urban site. Thanks to the use of a low pressure impactor, capable of fractionating particles into the range of 0.03-10 μm, it was also possible to study the bacterial content in ultrafine, fine, and coarse particulate matter. The results from this study showed that muramic and dipicolinic acids can be determined together in one chromatographic run in reversed phase ion pair chromatography. Bacteria were more abundant than bacterial spores in the urban atmosphere, both showing a higher concentration in the coarse fraction of particles, although bacteria and bacterial spore amounts per unit mass of ultrafine particles were higher than in fine and coarse particles.
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http://dx.doi.org/10.1007/s00216-016-0111-yDOI Listing
February 2017

Characterization of the transcription factor encoding gene, KlADR1: metabolic role in Kluyveromyces lactis and expression in Saccharomyces cerevisiae.

Microbiology (Reading) 2016 11 15;162(11):1933-1944. Epub 2016 Sep 15.

Department of Biology and Biotechnology 'C. Darwin', Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.

In Saccharomyces cerevisiae, Adr1 is a zinc-finger transcription factor involved in the transcriptional activation of ADH2. Deletion of KlADR1, its putative ortholog in Kluyveromyces lactis, led to reduced growth in glycerol, oleate and yeast extract-peptone medium suggesting, as in S. cerevisiae, its requirement for glycerol, fatty acid and nitrogen utilization. Moreover, growth comparison on yeast extract and peptone plates showed in K. lactis a KlAdr1-dependent growth trait not present in S. cerevisiae, indicating different metabolic roles of the two factors in their environmental niches. KlADR1 is required for growth under respiratory and fermentative conditions like KlADH, alcohol dehydrogenase genes necessary for metabolic adaptation during the growth transition. Using in-gel native alcohol dehydrogenase assay, we showed that this factor affected the Adh pattern by altering the balance between these activities. Since the activity most affected by KlAdr1 is KlAdh3, a deletion analysis of the KlADH3 promoter allowed the isolation of a DNA fragment through which KlAdr1 modulated its expression. The expression of the KlADR1-GFP gene allowed the intracellular localization of the factor in K. lactis and S. cerevisiae, suggesting in the two yeasts a common mechanism of KlAdr1 translocation under fermentative and respiratory conditions. Finally, the chimeric Kl/ScADR1 gene encoding the zinc-finger domains of KlAdr1 fused to the transactivating domains of the S. cerevisiae factor activated in Scadr1Δ the transcription of ADH2 in a ScAdr1-dependent fashion.
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http://dx.doi.org/10.1099/mic.0.000374DOI Listing
November 2016

The loss of ATP2C1 impairs the DNA damage response and induces altered skin homeostasis: Consequences for epidermal biology in Hailey-Hailey disease.

Sci Rep 2016 08 16;6:31567. Epub 2016 Aug 16.

Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.

Mutation of the Golgi Ca(2+)-ATPase ATP2C1 is associated with deregulated calcium homeostasis and altered skin function. ATP2C1 mutations have been identified as having a causative role in Hailey-Hailey disease, an autosomal-dominant skin disorder. Here, we identified ATP2C1 as a crucial regulator of epidermal homeostasis through the regulation of oxidative stress. Upon ATP2C1 inactivation, oxidative stress and Notch1 activation were increased in cultured human keratinocytes. Using RNA-seq experiments, we found that the DNA damage response (DDR) was consistently down-regulated in keratinocytes derived from the lesions of patients with Hailey-Hailey disease. Although oxidative stress activates the DDR, ATP2C1 inactivation down-regulates DDR gene expression. We showed that the DDR response was a major target of oxidative stress-induced Notch1 activation. Here, we show that this activation is functionally important because early Notch1 activation in keratinocytes induces keratinocyte differentiation and represses the DDR. These results indicate that an ATP2C1/NOTCH1 axis might be critical for keratinocyte function and cutaneous homeostasis, suggesting a plausible model for the pathological features of Hailey-Hailey disease.
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http://dx.doi.org/10.1038/srep31567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985699PMC
August 2016

Zinc Oxide Nanorods-Decorated Graphene Nanoplatelets: A Promising Antimicrobial Agent against the Cariogenic Bacterium Streptococcus mutans.

Nanomaterials (Basel) 2016 Sep 29;6(10). Epub 2016 Sep 29.

BBCD, Department of Biology and Biotechnology, Sapienza University of Rome, Rome 00185, Italy.

Nanomaterials are revolutionizing the field of medicine to improve the quality of life due to the myriad of applications stemming from their unique properties, including the antimicrobial activity against pathogens. In this study, the antimicrobial and antibiofilm properties of a novel nanomaterial composed by zinc oxide nanorods-decorated graphene nanoplatelets (ZNGs) are investigated. ZNGs were produced by hydrothermal method and characterized through field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) techniques. The antimicrobial activity of ZNGs was evaluated against , the main bacteriological agent in the etiology of dental caries. Cell viability assay demonstrated that ZNGs exerted a strikingly high killing effect on cells in a dose-dependent manner. Moreover, FE-SEM analysis revealed relevant mechanical damages exerted by ZNGs at the cell surface of this dental pathogen rather than reactive oxygen species (ROS) generation. In addition, inductively coupled plasma mass spectrometry (ICP-MS) measurements showed negligible zinc dissolution, demonstrating that zinc ion release in the suspension is not associated with the high cell mortality rate. Finally, our data indicated that also biofilm formation was affected by the presence of graphene-zinc oxide (ZnO) based material, as witnessed by the safranin staining and growth curve analysis. Therefore, ZNGs can be a remarkable nanobactericide against one of the main dental pathogens. The potential applications in dental care and therapy are very promising.
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http://dx.doi.org/10.3390/nano6100179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245199PMC
September 2016

Impact of a Complex Food Microbiota on Energy Metabolism in the Model Organism Caenorhabditis elegans.

Biomed Res Int 2015 19;2015:621709. Epub 2015 Apr 19.

Food & Nutrition Research Center (CRA-NUT), Agricultural Research Council, Via Ardeatina 546, 00178 Rome, Italy.

The nematode Caenorhabditis elegans is widely used as a model system for research on aging, development, and host-pathogen interactions. Little is currently known about the mechanisms underlying the effects exerted by foodborne microbes. We took advantage of C. elegans to evaluate the impact of foodborne microbiota on well characterized physiological features of the worms. Foodborne lactic acid bacteria (LAB) consortium was used to feed nematodes and its composition was evaluated by 16S rDNA analysis and strain typing before and after colonization of the nematode gut. Lactobacillus delbrueckii, L. fermentum, and Leuconostoc lactis were identified as the main species and shown to display different worm gut colonization capacities. LAB supplementation appeared to decrease nematode lifespan compared to the animals fed with the conventional Escherichia coli nutrient source or a probiotic bacterial strain. Reduced brood size was also observed in microbiota-fed nematodes. Moreover, massive accumulation of lipid droplets was revealed by BODIPY staining. Altered expression of nhr-49, pept-1, and tub-1 genes, associated with obesity phenotypes, was demonstrated by RT-qPCR. Since several pathways are evolutionarily conserved in C. elegans, our results highlight the nematode as a valuable model system to investigate the effects of a complex microbial consortium on host energy metabolism.
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http://dx.doi.org/10.1155/2015/621709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417589PMC
February 2016

ER stress induced by the OCH1 mutation triggers changes in lipid homeostasis in Kluyveromyces lactis.

Res Microbiol 2015 Feb-Mar;166(2):84-92. Epub 2015 Jan 7.

Department of Biology and Biotechnology, Sapienza-University of Rome, Rome, Italy. Electronic address:

In Kluyveromyces lactis yeast, OCH1 encodes for the α-1,6-mannosyltrasferase that adds the initial α-1,6-mannose to the outer-chains of N-glycoproteins. Kloch1-1 mutant cells showed altered calcium homeostasis and endoplasmic reticulum (ER) stress. Since ER plays a major role in lipid biosynthesis and lipid droplet (LD) formation, herein the impact of Och1p depletion on lipid homeostasis was investigated. Transcriptional profiles of genes involved in biosynthesis of fatty acids, their amount and composition changed in mutant cells. An increased amount of ergosterol was determined in these cells. Enhanced transcription of genes involved in both synthesis and mobilization of LDs was also found in Kloch1-1 cells, accompanied by a reduced amount of LDs. We provide evidence that ER alterations, determined by protein misfolding as a result of reduced N-glycosylation, induced altered lipid homeostasis in Kloch1-1 cells. Chemical chaperone 4-phenyl butyrate (4-PBA) slightly alleviated the LD phenotype in cells depleted of Och1p. Remarkably, complete suppression of ER stress, via increased expression of plasma membrane calcium channel subunit Mid1, fully restored lipid homeostasis in mutant cells. To further reinforce this finding, low numbers of LDs were observed in wild type cells when ER stress was triggered by DTT treatment.
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http://dx.doi.org/10.1016/j.resmic.2014.12.011DOI Listing
August 2015

Loss of Notch1-dependent p21(Waf1/Cip1) expression influences the Notch1 outcome in tumorigenesis.

Cell Cycle 2014 6;13(13):2046-55. Epub 2014 May 6.

Department of Molecular Medicine; Sapienza University of Rome; Rome, Italy.

Notch signaling plays a complex role in carcinogenesis, and its signaling pathway has both tumor-suppressor and oncogenic components. In this study we investigated the effects of reactive oxygen species (ROS) on Notch1 signaling outcome in keratinocyte biology. We demonstrate that Notch1 function contributes to the arsenic-induced keratinocyte transformation. We found that acute exposure to arsenite increases oxidative stress and inhibits proliferation of keratinocyte cells by upregulation of p21(waf1/Cip1). The necessity of p21(waf1/Cip1) for arsenite-induced cell death was demonstrated by targeted downregulation of p21(waf1/Cip1) by using RNA interference. We further demonstrated that on acute exposure to arsenite, p21(waf1/Cip1) is upregulated and Notch1 downmodulated, whereas on chronic exposure to arsenite, malignant progression of arsenite-treated keratinocytes cells was accompanied by regained expression and activity of Notch1. Notch1 activity in arsenite-transformed keratinocytes inhibits arsenite-induced upregulation of p21(waf1/Cip1) by sustaining c-myc expression. We further demonstrated that c-myc collaborates with Nrf2, a key regulator for the maintenance of redox homeostasis, to promote metabolic activities that support cell proliferation and cytoprotection. Therefore, Notch1-mediated repression of p21(waf1/Cip1) expression results in the inhibition of cell death and keratinocytes transformation. Our results not only demonstrate that sustained Notch1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect, but also may provide mechanistic insights into the molecular aspects that determine whether Notch signaling will be either oncogenic or tumor suppressive.
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http://dx.doi.org/10.4161/cc.29079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111696PMC
January 2015

Anti-Candida activity of 1-18 fragment of the frog skin peptide esculentin-1b: in vitro and in vivo studies in a Caenorhabditis elegans infection model.

Cell Mol Life Sci 2014 Jul 10;71(13):2535-46. Epub 2013 Nov 10.

Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185, Rome, Italy.

Candida albicans represents one of the most prevalent species causing life-threatening fungal infections. Current treatments to defeat Candida albicans have become quite difficult, due to their toxic side effects and the emergence of resistant strains. Antimicrobial peptides (AMPs) are fascinating molecules with a potential role as novel anti-infective agents. However, only a few studies have been performed on their efficacy towards the most virulent hyphal phenotype of this pathogen. The purpose of this work is to evaluate the anti-Candida activity of the N-terminal 1-18 fragment of the frog skin AMP esculentin-1b, Esc(1-18), under both in vitro and in vivo conditions using Caenorhabditis elegans as a simple host model for microbial infections. Our results demonstrate that Esc(1-18) caused a rapid reduction in the number of viable yeast cells and killing of the hyphal population. Esc(1-18) revealed a membrane perturbing effect which is likely the basis of its mode of action. To the best of our knowledge, this is the first report showing the ability of a frog skin AMP-derived peptide (1) to kill both growing stages of Candida; (2) to promote survival of Candida-infected living organisms and (3) to inhibit transition of these fungal cells from the roundish yeast shape to the more dangerous hyphal form at sub-inhibitory concentrations.
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http://dx.doi.org/10.1007/s00018-013-1500-4DOI Listing
July 2014

Inhibition of microbial growth by carbon nanotube networks.

Nanoscale 2013 Oct 9;5(19):9023-9. Epub 2013 Aug 9.

Dept. Biology and Biotechnology, Sapienza-University of Rome, Rome, Italy.

In the last years carbon nanotubes have attracted increasing attention for their potential applications in the biomedical field as diagnostic and therapeutic nano tools. Here we investigate the antimicrobial activity of different fully characterized carbon nanotube types (single walled, double walled and multi walled) on representative pathogen species: Gram-positive Staphylococcus aureus, Gram-negative Pseudomonas aeruginosa and the opportunistic fungus Candida albicans. Our results show that all the carbon nanotube types possess a highly significant antimicrobial capacity, even though they have a colony forming unit capacity and induction of oxidative stress in all the microbial species to a different extent. Moreover, scanning electron microscopy analysis revealed that the microbial cells were wrapped or entrapped by carbon nanotube networks. Our data taken together suggest that the reduced capacity of microbial cells to forming colonies and their oxidative response could be related to the cellular stress induced by the interactions of pathogens with the CNT network.
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http://dx.doi.org/10.1039/c3nr02091fDOI Listing
October 2013
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