Michele Menotta

Geographical traceability of Italian white truffle (Tuber magnatum Pico) by the analysis of volatile organic compounds

Results are presented that were obtained on the geographic traceability of the white truffle Tuber magnatum Pico. Solid-phase microextraction coupled to gas chromatography/mass spectrometry (SPME-GC/MS) was employed to characterize the volatile profile of T. magnatum white truffle produced in seven geographical areas of Italy. The main components of the volatile fraction were identified using SPME-GC/MS. Significant differences in the proportion of volatile constituents from truffles of different geographical areas were detected. The results suggest that, besides genetic factors, environmental conditions influence the formation of volatile organic compounds. The mass spectra of the volatile fraction of the samples were used as fingerprints to characterize the geographical origin. Next, stepwise factorial discriminant analysis afforded a limited number of characteristic fragment ions that allowed a geographical classification of the truffles studied.

Oxidized Ultrashort Nanotubes as Carbon Scaffolds for the Construction of Cell-Penetrating NF-κB Decoy Molecules

Oligonucleotide (ODN) decoys are synthetic ODNs containing the DNA binding sequence of a transcription factor. When delivered to cells, these molecules can compete with endogenous sequences for binding the transcription factor, thus inhibiting its ability to activate the expression of target genes. Modulation of gene expression by decoy ODNs against nuclear factor-kappaB (NF-kappaB), a transcription factor regulating many genes involved in immunity, has been achieved in a variety of immune/inflammatory disorders. However, the successful use of transcription factor decoys depends on an efficient means to bring the synthetic DNA to target cells. It is known that single-walled carbon nanotubes (SWCNTs), under certain conditions, are able to cross the cell membrane. Thus, we have evaluated the possibility to functionalize SWCNTs with decoy ODNs against NF-kappaB in order to improve their intracellular delivery. To couple ODNs to CNTs, we have exploited the carbodiimide chemistry which allows covalent binding of amino-modified ODNs to carboxyl groups introduced onto SWCNTs through oxidation. The effective binding of ODNs to nanotubes has been demonstrated by a combination of microscopic, spectroscopic, and electrophoretic techniques. The uptake and subcellular distribution of ODN decoys bound to SWCNTs was analyzed by fluorescence microscopy. ODNs were internalized into macrophages and accumulated in the cytosol. Moreover, no cytotoxicity associated with SWCNT administration was observed. Finally, NF-kappaB-dependent gene expression was significantly reduced in cells receiving nanomolar concentrations of SWCNT-NF-kappaB decoys compared to cells receiving SWCNTs or SWCNTs functionalized with a nonspecific ODN sequence, demonstrating both efficacy and specificity of the approach.

Dexamethasone partially rescues Ataxia Telangiectasia-Mutated (ATM) deficiency in Ataxia Telangiectasia by promoting a shortened protein variant retaining kinase activity

Background: Ataxia telangiectasia is a genetic disease caused by biallelic mutations in ATM gene. Results: Dexamethasone induces a noncanonical splicing that leads to translation of a shortened ATM variant retaining kinase activity. Conclusion: ATM may be restored by a new molecular mechanism that overcomes most of mutations so far described in ATM gene. Significance: Drug-induced noncanonical splicing may provide new approaches for genetic diseases. Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of nucleotide residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.

High resolution melting (HRM) analysis for the detection of ER22/23EK, BclI, and N363S polymorphisms of the glucocorticoid receptor gene

Polymorphisms in the glucocorticoid receptor (GR) gene have been associated with altered sensitivity to glucocorticoids. We designed a high-resolution melting (HRM) assay to detect, simultaneously, the three most intriguing GR polymorphisms, selected on the bases of clinical relevance and frequencies in caucasian population as described in literature. HRM enables the detection of ER22/23EK and N363S genotypes but fails to discriminate homozygous mutant for the BclI polymorphism from wild-type samples, however a simple spike experiment leads to a clear discrimination between these genotypes. The analyses were performed on a cohort of 70 healthy Caucasian subjects. The method was validated by restriction fragment length polymorphisms; HRM results were found to be in 100% concordance with those observed with the restriction enzymes. We also employed this method on a population of 40 Crohn Disease patients; the analysis demonstrated a significantly higher frequency of the BclI polymorphism in patients than in healthy volunteers. This is, at now, the less expensive and time-and work-saving method to detect GR mutations, providing precision, fast screening and high throughput capabilities.

Electrical characterization of DNA-functionalized solid state nanopores for bio-sensing

We present data concerning the electrical properties of a class of biosensor devices based on bio-functionalized solid state nanopores able to detect different kinds of interactions between probe molecules, chemically attached to the pore surface, and target molecules present in solution and electrophoretically drawn through the nanometric channel. The great potentiality of this approach resides in the fact that the functionalization of a quite large pore (up to 50-60 nm) allows a sufficient diameter reduction for the attainment of a single molecule sensing dimension and selective activation, without the need for further material deposition, such as metal or oxides, or localized surface modification. The results indicate that it will be possible, in the near future, to conceive and design devices for parallel analysis of biological samples made of arrays of nanopores differently functionalized, fabricated by standard lithographic techniques, with important applications in the field of molecular diagnosis.

Dexamethasone improves redox state in ataxia telangiectasia cells by promoting an NRF2‐mediated antioxidant response

Ataxia telangiectasia (A‐T) is a rare incurable neurodegenerative disease caused by biallelic mutations in the gene for ataxia‐telangiectasia mutated (ATM). The lack of a functional ATM kinase leads to a pleiotropic phenotype, and oxidative stress is considered to have a crucial role in the complex physiopathology. Recently, steroids have been shown to reduce the neurological symptoms of the disease, although the molecular mechanism of this effect is largely unknown. In the present study, we have demonstrated that dexamethasone treatment of A‐T lymphoblastoid cells increases the content of two of the most abundant antioxidants [glutathione (GSH) and NADPH] by up to 30%. Dexamethasone promoted the nuclear accumulation of the transcription factor nuclear factor (erythroid‐derived 2)‐like 2 to drive expression of antioxidant pathways involved in GSH synthesis and NADPH production. The latter effect was via glucose 6‐phosphate dehydrogenase activation, as confirmed by increased enzyme activity and enhancement of the pentose phosphate pathway rate. This evidence indicates that glucocorticoids are able to potentiate antioxidant defenses to counteract oxidative stress in ataxia telangiectasia, and also reveals an unexpected role for dexamethasone in redox homeostasis and cellular antioxidant activity.

Label-free quantification of activated NF-κB in biological samples by atomic force microscopy

Nuclear factor-kappaB (NF-kappaB) is a ubiquitous transcription factor involved in the pro-inflammatory response to several factor, and in auto-inflammatory diseases. The usual methods for detection of NF-kappaB DNA binding activity are the electrophoretic mobility shift assay (EMSA), and enzyme-linked immunosorbent assay (ELISA). Here we report a development of a quantitative atomic force microscopy (AFM) based technique, for the analysis of NF-kappaB DNA binding activity. NF-kappaB target sequence DNA has been employed to mica functionalization in order to set up a surface able to capture transcriptionally active NF-kappaB protein complexes from cell lysates, with the aim to detect DNA binding capacity of NF-kappaB from low amount of biological samples such as biopsy. We were able to obtain images of the captured complex on the surface and furthermore we carried out an AFM images quantification. We were able to quantify relative and absolute quantities of NF-kappaB at pico-Molar proteins concentration range from cultured cell samples and from biological fluid cells permitting us to estimate NF-kappaB binding activity. The results obtained by AFM imaging have been compared and validated with EMSA. The present work represents the first quantification approach by AFM analysis. The results and the method may be used toward development of NF-kappaB based bio-diagnostic nano-device.

Molecular and functional characterization of a Rho GDP dissociation inhibitor in the filamentous fungus Tuber borchii

BackgroundSmall GTPases of the Rho family function as tightly regulated molecular switches that govern important cellular functions in eukaryotes. Several families of regulatory proteins control their activation cycle and subcellular localization. Members of the guanine nucleotide dissociation inhibitor (GDI) family sequester Rho GTPases from the plasma membrane and keep them in an inactive form.ResultsWe report on the characterization the RhoGDI homolog of Tuber borchii Vittad., an ascomycetous ectomycorrhizal fungus. The Tbgdi gene is present in two copies in the T. borchii genome. The predicted amino acid sequence shows high similarity to other known RhoGDIs. Real time PCR analyses revealed an increased expression of Tbgdi during the phase preparative to the symbiosis instauration, in particular after stimulation with root exudates extracts, that correlates with expression of Tbcdc42. In a translocation assay TbRhoGDI was able to solubilize TbCdc42 from membranes. Surprisingly, TbRhoGDI appeared not to interact with S. cerevisiae Cdc42, precluding the use of yeast as a surrogate model for functional studies. To study the role of TbRhoGDI we performed complementation experiments using a RhoGDI null strain of Dictyostelium discoideum, a model organism where the roles of Rho signaling pathways are well established. For comparison, complementation with mammalian RhoGDI1 and LyGDI was also studied in the null strain. Although interacting with Rac1 isoforms, TbRhoGDI was not able to revert the defects of the D. discoideum RhoGDI null strain, but displayed an additional negative effect on the cAMP-stimulated actin polymerization response.ConclusionT. borchii expresses a functional RhoGDI homolog that appears as an important modulator of cytoskeleton reorganization during polarized apical growth that antecedes symbiosis instauration. The specificity of TbRhoGDI actions was underscored by its inability to elicit a growth defect in S. cerevisiae or to compensate the loss of a D. discoideum RhoGDI. Knowledge of the cell signaling at the basis of cytoskeleton reorganization of ectomycorrhizal fungi is essential for improvements in the production of mycorrhized plant seedlings used in timberland extension programs and fruit body production.

Effect of the redox state on HIV-1 tat protein multimerization and cell internalization and trafficking

The redox state of the cysteine-rich region of the HIV Tat protein is known to play a crucial role in Tat biological activity. In this article, we show that Tat displays two alternative functional states depending on the presence of either one or three reduced sulphydryl groups in the cysteine-rich region, respectively. Using different approaches, a disulfide pattern has been defined for the Tat protein and a specific DTT-dependent breaking order of disulfide bonds highlighted. The Tat redox state deeply influences macrophage protein uptake. Immunoistochemistry analysis shows that the oxidized protein does not enter cells, whereas partially reduced protein reaches the cytosol and, to a limited extent, the nucleus. Finally electrophoretic analysis shows Tat high-molecular weight multi-aggregation, resulting in the loss of biological activity. This is due to strong electrostatic and metal-binding interactions, whereas Tat dimerization involves metal-binding interactions as well as disulfide bond formation.

Morphological and Molecular Modifications Induced by Different Carbohydrate Sources in Tuber borchii

During the life cycle of mycorrhizal fungi, morphological, genetic and metabolic modifications are induced in the fungus and its symbiotic partner. These changes are influenced by environmental factors: light, gravity, oxygen, temperature, soil type, nutrients, root exudates and the presence of particular bacterial and perhaps fungal and viral populations in the mycorrhizosphere. To determine whether different carbohydrates lead to cell-signalling events and morphofunctional changes in cultured Tuber borchii mycelia, the expression level of genes involved in morphological modifications was investigated using a macroarray technique and real-time RT-PCR. The morphological study showed an increased growth of Tuber mycelia in glucose, while the hyphae were thinner and less branched in sucrose and maltose. This was accompanied by an upregulation of the genes involved in the general cell metabolism, detoxification processes, hyphal growth and cytoskeleton organization. Since glucose is also present in root exudates, the increased expression of these genes might support the hypothesis that glucose can act as a signal for the fungus to indicate the presence of the plant, and to trigger the complex symbiotic process. These mechanisms can lead to morphological modifications, including increased branching of the root which is necessary for the fungus to establish the symbiosis.