Graphene and carbon nanotube nanocomposite for gene transfection.

Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

Effect of ethanol on growth of Chrysonilia sitophila ('the red bread mould') and Hyphopichia burtonii ('the chalky mould') in sliced bread.

UNLABELLED: Contamination of food industrial environments and recontamination of finished products by Chrysonilia sitophila and Hyphopichia burtonii have long represented serious problems for the bakery industries. As one of the most common ways to slow down or avoid fungal spoilage on bakery products is the use of ethanol, in the present work the effect of this substance has been assessed on growth of two of the most frequently occurring associated moulds, C. sitophila and H. burtonii, by means of tests on both synthetic media and sliced bread. Test on synthetic media: H. burtonii was less markedly affected in lag-phase duration and radial growth rates by the addition of ethanol to DG18 and the reduction in incubation temperature than C. sitophila that failed to grow at the highest concentrations of ethanol tested (2·0 and 4·0% at 15°C; 4·0% at 25°C). Test on sliced bread: ethanol proved to be effective to prevent spoilage by C. sitophila even at the lowest concentration tested (0·8%, w/w), while higher concentrations (2·0%, w/w) were needed to prevent spoilage by H. burtonii. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that ethanol could represent an effective barrier to prevent spoilage of bakery products by associated moulds such as Chrysonilia sitophila and Hyphopichia burtonii, whose growth on packed and sliced bread was inhibited at very low (0·8%) or medium (2·0%) ethanol concentrations, respectively. The results obtained represent a fundamental point of reference for the bakery industries, as they can apply them in the productive practice to avoid spoilage by C. sitophila and H. burtonii on their products.

Evaluation of Candida albicans adhesion and biofilm formation on a denture base acrylic resin containing silver nanoparticles.

AIM: This study firstly evaluated the activity of a silver nanoparticle (AgNPs) solution against Candida albicans and then the effect of incorporation of AgNPs into a denture base acrylic resin on the material's hydrophobicity, C. albicans adhesion and biofilm formation. METHODS AND RESULTS: The AgNPs solution was synthesized by chemical reduction and characterized. Minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations for planktonic cells and sessile cells (MFCs) of the AgNPs solution against C. albicans were determined. Specimens (n = 360) of silver-incorporated acrylic resin at concentrations of 1000, 750, 500, 250 and 30 ppm were also prepared and stored in PBS for 0, 7, 90 and 180 days. Control was acrylic resin without AgNPs (0 ppm). After the storage periods, contact angles were measured and the specimens were used for C. albicans adherence (37°C; 90 min; n = 9) and biofilm formation (37°C; 48 h; n = 9) by XTT reduction assay. MIC, MFC and MFCs values were 3·98, 15·63 and 1000 ppm, respectively. Incorporation of AgNPs reduced the hydrophobicity of the resin. No effect on adherence and biofilm formation was observed. At 90 and 180 days of storage, there was significant increase in adherence and biofilm formation. CONCLUSIONS: Although the AgNPs solution had antifungal activity, no effect on C. albicans adherence and biofilm formation was observed after its incorporation into a denture base resin. SIGNIFICANCE AND IMPACT OF THE STUDY: The synthesized AgNPs solution is a promising antifungal agent, warranting investigations of more efficient methods of incorporation into denture base resins.

Polyphasic approach for differentiating Penicillium nordicum from Penicillium verrucosum.

The aim of this research was to use a polyphasic approach to differentiate Penicillium verrucosum from Penicillium nordicum, to compare different techniques, and to select the most suitable for industrial use. In particular, (1) a cultural technique with two substrates selective for these species; (2) a molecular diagnostic test recently set up and a RAPD procedure derived from this assay; (3) an RP-HPLC analysis to quantify ochratoxin A (OTA) production and (4) an automated system based on fungal carbon source utilisation (Biolog Microstation™) were used. Thirty strains isolated from meat products and originally identified as P. verrucosum by morphological methods were re-examined by newer cultural tests and by PCR methods. All were found to belong to P. nordicum. Their biochemical and chemical characterisation supported the results obtained by cultural and molecular techniques and showed the varied ability in P. verrucosum and P. nordicum to metabolise carbon-based sources and to produce OTA at different concentrations, respectively.

Spontaneous in-the-bag intraocular lens luxation into the vitreous cavity: last-stage complication of pseudoexfoliative syndrome after phacoemulsification.

PURPOSE: We describe 8 cases of late spontaneous in-the-bag intraocular lens (IOL) luxation into the vitreous cavity, which occurred at the Department of Ophthalmology and Neurosurgery of the University of Siena between January and December 2006. METHODS: In this interventional case series, the medical records of all patients with posterior luxation of in-the-bag IOLs - who had undergone a pars plana vitrectomy with IOL removal and scleral fixation IOL implantation between January and December 2007 at the Department of Ophthalmology and Neurosurgery of Siena, Italy - were retrospectively reviewed. RESULTS: The final post- operative visual acuity was 20/30 or better in 6 patients, while myopic macular degeneration and total retinal detachment limited visual acuity in the remaining 2 patients. CONCLUSION: The high prevalence of pseudoexfoliation (PEX) in the patients who had been operated for cataract phacoemulsification in our department could explain the occurrence of 8 posterior luxations of in-the-bag IOLs in only 1 year. Our study suggests that for the next years we will expect an increase in occurrence of spontaneous in-the- bag IOL luxations in the vitreous cavity. This condition could represent the last stage of PEX syndrome.

Facing the problem of "false positives": re-assessment and improvement of a multiplex RT-PCR procedure for the diagnosis of A. flavus mycotoxin producers.

The aim of our research project was to consolidate a multiplex RT-PCR protocol to detect aflatoxigenic strains of Aspergillus flavus. Several independent A. flavus strains were isolated from corn and flour samples from the North of Italy and from three European countries. Aflatoxin producing/not producing phenotype was assessed by qualitative and quantitative assays at day five of growth in aflatoxin inducing conditions. Expression of 16 genes belonging to the aflatoxin cluster was assayed by multiplex or monomeric RT-PCR. There is a good correlation between gene expression and aflatoxin production. Strains that apparently transcribed all the relevant genes but did not release aflatoxin in the medium ("false positives") were re-assessed for mycotoxin production after extended growth in inducing condition. All the "false positive" strains in actual fact were positive when aflatoxin determination was performed after 10 days of growth. These strains should then be re-classified as "slow aflatoxin accumulators". To optimise the diagnostic procedure, a quintuplex RT-PCR procedure was designed consisting of a primer set directed against four informative aflatoxin cluster genes and the beta-tubulin gene as an internal amplification control. In conclusion we have provided evidence for the robustness and reliability of our RT-PCR protocol in discriminating mycotoxin producer from non-producer strains of A. flavus. and the molecular procedure we devised is a promising tool with which to screen and control the endemic population of A. flavus colonising different areas of the World.

A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus.

AIMS: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing strains of Aspergillus flavus. METHODS AND RESULTS: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression. CONCLUSIONS: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.

Unilateral corneal endothelial dystrophy and anterior keratoconus.

PURPOSE: To describe a case of keratoconus and Fuchs' corneal endothelial dystrophy in the left eye with no corneal disease in the right eye. METHODS: A 64-year-old woman presented with visual impairment in her left eye; computer-assisted topographic analysis and specular microscopy were performed in both eyes and left cornea was histopathologically examined. RESULTS: Keratoconus was diagnosed by slit-lamp examination, keratometry, and computer-assisted topographic analysis. Corneal endothelial dystrophy was diagnosed on the basis of clinical examination and specular microscopy. Histopathologic examination revealed a stromal degeneration typical of keratoconus and a non-guttae form of endothelial dystrophy. CONCLUSIONS: This is a rare case of unilateral corneal endothelial dystrophy and keratoconus.

Coordination properties of a polyamine cryptand with two different binding moieties. A case of a pH-modulated antenna device based on a new Eu(III) cryptate complex.

Protonation and alkali- and alkaline-earth-metal coordination by the dipyridine-containing cryptand L have been studied by means of potentiometric and spectroscopic (UV-vis, (1)H NMR) measurements in aqueous solutions. This ligand is constituted by an aliphatic polyamine chain and a coordinating cleft, delimited by two dipyridine units, where the metal ion is lodged. The resulting complexes are characterized by an unusually high stability. The polyamine chain is not involved, or weakly involved, in metal coordination, and facile protonation can occur on the nitrogen atoms of this moiety. Similar coordination features are found in the Eu(III) complex. A fluorescence emission study reveals that the Eu(III) cryptate shows the characteristic visible emission of the metal, due to the intramolecular energy transfer to the metal ion mainly from the lower energy triplet state of the cryptand. On the other hand, the emission intensity is modulated by pH, giving a maximum at neutral pH and decreasing at both acidic and alkaline pH values.