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AFM data about C. auris topography, surface biochemistry and nanomechanics are good indicators for the evaluation of the effects of the humanized monoclonal antibody H5K1 alone and in combination with commercially available antifungal drugs.

We report the synthesis, chemical properties, and disulfide bond-reducing performance of a dithiol called NACMEAA, conceived as a hybrid of two biologically relevant thiols: cysteine and cysteamine. NACMEAA is conveniently prepared from inexpensive l-cystine in an efficient manner. As a nonvolatile, highly soluble, and neutral compound at physiological pH with the first thiol p

The SARS-CoV-2 life cycle is strictly dependent on the environmental redox state that influences both virus entry and replication. A reducing environment impairs the binding of the spike protein (S) to the angiotensin-converting enzyme 2 receptor (ACE2), while a highly oxidizing environment is thought to favor S interaction with ACE2. Moreover, SARS-CoV-2 interferes with redox homeostasis in infected cells to promote the oxidative folding of its own proteins. Here we demonstrate that synthetic low molecular weight (LMW) monothiol and dithiol compounds induce a redox switch in the S protein receptor binding domain (RBD) toward a more reduced state. Reactive cysteine residue profiling revealed that all the disulfides present in RBD are targets of the thiol compounds. The reduction of disulfides in RBD decreases the binding to ACE2 in a cell-free system as demonstrated by enzyme-linked immunosorbent and surface plasmon resonance (SPR) assays. Moreover, LMW thiols interfere with protein oxidative folding and the production of newly synthesized polypeptides in HEK293 cells expressing the S1 and RBD domain, respectively. Based on these results, we hypothesize that these thiol compounds impair both the binding of S protein to its cellular receptor during the early stage of viral infection, as well as viral protein folding/maturation and thus the formation of new viral mature particles. Indeed, all the tested molecules, although at different concentrations, efficiently inhibit both SARS-CoV-2 entry and replication in Vero E6 cells. LMW thiols may represent innovative anti-SARS-CoV-2 therapeutics acting directly on viral targets and indirectly by inhibiting cellular functions mandatory for viral replication.

Ataxia Telangiectasia is a rare neurodegenerative disease caused by biallelic mutations in the Ataxia Telangiectasia Mutated gene. No cure is currently available for these patients but positive effects on neurologic features in AT patients have been achieved by dexamethasone administration through autologous erythrocytes (EryDex) in phase II and phase III clinical trials, leading us to explore the molecular mechanisms behind the drug action. During these investigations new ATM variants, which originated from alternative splicing of ATM messenger, were discovered, and detected in vivo in the blood of AT patients treated with EryDex. Some of the new ATM variants, alongside an i n silico designed one, were characterized and examined in AT fibroblast cell lines. ATM variants were capable of rescuing ATM activity in AT cells, particularly in the nuclear role of DNA DSBs recognition and repair, and in the cytoplasmic role of modulating autophagy, antioxidant capacity and mitochondria functionality, all of the features that are compromised in AT but essential for neuron survival. These outcomes are triggered by the kinase and further functional domains of the tested ATM variants, that are useful for restoring cellular functionality. The in silico designed ATM variant eliciting most of the functionality recover may be exploited in gene therapy or gene delivery for the treatment of AT patients.

Truffles in the genus Tuber produce subterranean fruiting bodies that are not able to actively discharge their spores in the environment. For this reason, truffles depend on mycophagous animals for reproduction. Fungus consumption (mycophagy) is a behaviour typical of both vertebrates and invertebrates. Mammals, especially rodents, are the most studied group of mycophagists and have been found to consume a great variety of fungi. Among invertebrates, mycophagy is documented in arthropods, but rarely in molluscs. In our study we assessed the effect on the morphology and mycorrhizal colonization of Tuber aestivum spores after passage through the gut of slugs (Deroceras invadens) and, for comparison, of a house mouse (Mus musculus). Light, scanning electron and atomic force microscopy revealed that the digestion, especially by slugs, freed spores from the asci and modified their morphology. These are believed to be the reasons why we observed an improvement in oak mycorrhization with the slug and rodent ingested spores in comparison to a fresh spore inoculation. We also demonstrated by molecular barcoding that slugs' guts sampled on a Tuber melanosporum truffle ground contain spores from this species and Tuber brumale, further suggesting that some invertebrates are efficient Tuber spore dispersers.

Ataxia-Telangiectasia (A-T) is characterized by cerebellar neurodegeneration and immunodeficiency. Recent studies suggest that very low glucocorticoids (GCs) doses may help improve A-T neurological phenotype in some patients. Interestingly, in GCs studies an unexpected improvement of lymphocytes proliferation in some A-T patients has been observed. GCs are able to upregulate IL-7 Rα expression and rescue it from the recycling. In this study, we compared several immunological functions, including PBMC proliferative responses, cell activation events and IL-7/IL-7 Rα axis functionality, with the neurological behavior during an in-vivo GCs treatment between the most Responder patient to GC and the Non-Responder at all. During in-vivo GC treatment, we observed an increase of lymphocyte proliferation upon stimulation with PHA or IL-7 only in the Responder. This finding paralleled the increase in the surface expression of IL-7 R and up-regulation of the CD69 T-cell activation marker. Internalization and recycling of IL-7 R occurred properly only in the Responder. Microarray analysis revealed a remarkable difference in the DE-genes levels among Responder and Non-Responder, mostly concerning miRNAs and Multiple Complex families. Our findings suggest that the improvement of lymphocyte functionality, which correlates to the neurological behavior, is mediated through an effect of GCs on the IL-7/IL-7 Rα axis.

Leishmaniasis is a vector-borne disease caused by many Leishmania species, which can infect both humans and other mammals. Leishmaniasis is a complex disease, with heterogeneous clinical manifestations ranging from asymptomatic infections to lesions at cutaneous sites (cutaneous leishmaniasis), mucosal sites (mucocutaneous leishmaniasis) or in visceral organs (visceral leishmaniasis), depending on the species and host characteristics. Often, symptoms are inconclusive and leishmaniasis can be confused with other co-endemic diseases. Moreover, co-infections (mainly with HIV in humans) can produce atypical clinical presentations. A correct diagnosis is crucial to apply the appropriate treatment and the use of molecular techniques in diagnosis of leishmaniasis has become increasingly relevant due to their remarkable sensitivity, specificity and possible application to a variety of clinical samples. Among them, real-time PCR (qPCR)-based approaches have become increasingly popular in the last years not only for detection and quantification of Leishmania species but also for species identification. However, despite qPCR-based methods having proven to be very effective in the diagnosis of leishmaniasis, a standardized method does not exist. This review summarizes the qPCR-based methods in the diagnosis of leishmaniasis focusing on the recent developments and applications in this field.

Ceterach officinarum Willd is a plant widespread throughout Europe and used in southern Italy as a diuretic. Beliefs in the benefits of C. officinarum aqueous extract in the treatment of calcium oxalate kidney stones are widely held. Little is known, however, about the actual mechanism of its antilithiatic action. Our results in this in vitro study corroborate C. officinarum aqueous extract as a good source of antioxidants with a high antioxidant effects. Our results also demonstrate a major impact of C. officinarum aqueous extract on in vitro induced calcium oxalate crystallization kinetics and crystal morphology, showing its critical role in kidney stone formation and/or elimination. We show that progressively increasing doses of C. officinarum aqueous extract cause a sequence of effects. A powerful inhibitory action on calcium oxalate monohydrate (COM) growth and aggregation is first observed. C. officinarum aqueous extract also appears highly effective in stimulating nucleation increasing the number and reducing the size of COM crystals, which become progressively thinner, rounded and concave in a dose-dependent manner. These shape-modified COM crystals are known to be less adherent to renal tubular cells and more easily excreted through the urinary tract preventing kidney stone formation. Further, C. officinarum aqueous extract promotes the formation of calcium oxalate dihydrate (COD) rather than the monohydrate so that, at the highest concentrations used, only COD crystals are observed, in significant greater numbers with a clear reduction in their size, in a dose-dependent manner. Furthermore, AFM analyses allowed us to reveal the presence of C. officinarum component(s) on the surfaces of COD and modified COM crystals. The crystal surface adsorbed component(s) are shown to be similarly active as the total aqueous extract, suggesting a trigger factor which may direct crystal modification towards COD forms. In urolithiasis pathogenesis COD crystals are less dangerous than the COM forms due to their lower affinity for renal tubular cells. Our results are important in understanding the mechanisms which guide the modification induced by C. officinarum on the crystallization process. Based on these data, together with no adverse toxic effect being observed on the in vitro model of human intestinal enterocytes, C. officinarum aqueous extract could represent an attractive natural therapy for the treatment of urolithiasis.

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.

Ataxia telangiectasia (A-T) is an incurable and rare hereditary syndrome. In recent times, treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this condition, but the molecular mechanism of action of these analogues remains unknown. Hence, the aim of this study was to gain insight into the molecular mechanism of action of glucocorticoid analogues in the treatment of A-T by investigating the role of Dexamethasone (Dexa) in A-T lymphoblastoid cell lines. We used 2DE and tandem MS to identify proteins that were influenced by the drug in A-T cells but not in healthy cells. Thirty-four proteins were defined out of a total of 746±63. Transcriptome analysis was performed by microarray and showed the differential expression of 599 A-T and 362 wild type (WT) genes and a healthy un-matching between protein abundance and the corresponding gene expression variation. The proteomic and transcriptomic profiles allowed the network pathway analysis to pinpoint the biological and molecular functions affected by Dexamethasone in Dexa-treated cells. The present integrated study provides evidence of the molecular mechanism of action of Dexamethasone in an A-T cellular model but also the broader effects of the drug in other tested cell lines.

Ataxia telangiectasia (AT) is a rare incurable genetic disease caused by biallelic mutations in the Ataxia telangiectasia-mutated gene. Intra-erythrocyte infusion of dexamethasone improves clinical outcomes in AT patients; however, the molecular mechanisms that lead to this improvement remain unknown. Hence, to gain a better understanding of these mechanisms, we assessed the effects of glucocorticoid administration on gene expression in the blood of AT patients. Whole blood was obtained from nine children enrolled in a phase two clinical trial, who were being treated with dexamethasone (AT Dexa), from six untreated AT patients (AT) and from six healthy volunteers (WT). CodeLink Whole Genome Bioarrays were used to assess transcript expression. The reliability of the differentially expressed genes (DEGs) was verified by qRT-PCR analysis. The enriched Gene Ontology (GO) terms and the pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) of DEGs obtained by group comparisons were achieved using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Functional network analyses were computed by Reactome FI. The likely involved transcription factors were revealed by iRegulon. Among the identified DEGs influenced by the pathology and restored by dexamethasone, we detected 522 upregulated probes coding for known proteins, while 22 probes were downregulated, as they were in healthy subjects. These results provide useful information and represent a first step towards gaining a better understanding of the underlying mechanisms of the effects of dexamethasone on AT patients.

In this study, we applied Environmental Scanning Electron Microscopy-Energy Dispersive Spectroscopy (ESEM-EDS) and Atomic Force Microscopy (AFM) analysis to three different cereal caryopses: barley, oat and einkorn wheat. The morphological structures, chemical elemental composition and surface characteristics of the three cereals were described. Regarding the morphology, barley showed the thickest pericarp, providing a strong barrier digestion and absorption of nutrients. The aleurone layer of each cereal type contained protein body globoids within its cells. Large type-A and small type-B starchy granules were revealed in the endosperm of barley and einkorn wheat, whereas irregular starchy granules were found in oats. The starchy granule elemental composition, detected by ESEM-EDS, was rather homogenous in the three cereals, whereas the pericarp and protein body globoids showed heterogeneity. In the protein body globoids, oats showed higher P and K concentrations than barley and einkorn wheat. Regarding the topographic profiles, detected by AFM, einkorn wheat starchy granules showed a surface profile that differed significantly from that of oats and barley, which were quite similar to one another. The present work provides insights into the morphological and chemical makeup of the three grains shedding light on the higher bio-accessibility of einkorn wheat nutrients compared to barley and oats, providing important suggestions for human nutrition and technological standpoints.

Background Ataxia Telangiectasia (AT) is a rare incurable genetic disease, caused by biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this syndrome. Nevertheless, the molecular mechanism underlying the glucocorticoid action in AT patients is not yet understood. Recently, we have demonstrated that Dexamethasone treatment may partly restore ATM activity in AT lymphoblastoid cells by a new ATM transcript, namely ATMdexa1. Results In the present study, the new ATMdexa1 transcript was also identified in vivo, specifically in the PMBCs of AT patients treated with intra-erythrocyte Dexamethasone (EryDex). In these patients it was also possible to isolate new “ATMdexa1 variants” originating from canonical and non-canonical splicing, each containing the coding sequence for the ATM kinase domain. The expression of the ATMdexa1 transcript family was directly related to treatment and higher expression levels of the transcript in patients’ blood correlated with a positive response to Dexamethasone therapy. Neither untreated AT patients nor untreated healthy volunteers possessed detectable levels of the transcripts. ATMdexa1 transcript expression was found to be elevated 8 days after the drug infusion, while it decreased 21 days after treatment. Conclusions For the first time, the expression of ATM splicing variants, similar to those previously observed in vitro, has been found in the PBMCs of patients treated with EryDex. These findings show a correlation between the expression of ATMdexa1 transcripts and the clinical response to low dose dexamethasone administration. Electronic supplementary material The online version of this article (doi:10.1186/s13023-017-0669-2) contains supplementary material, which is available to authorized users.

Ataxia telangiectasia is a rare genetic disease and no therapy is currently available. Glucocorticoid analogues have been shown to improve the neurological symptoms of treated patients. In the present study ataxia telangiectasia and wild type cells were used as a cellular model and treated with dexamethasone. The cells were subsequently investigated for membrane and whole cell mechanical properties by atomic force microscopy. In addition, cytoskeleton protein dynamics and nuclear shapes were assayed by fluorescence microscopy, while western blots were used to assess actin and tubulin content. At the macro level, dexamethasone directly modified the cell shape, Young's modulus and cytoskeleton protein dynamics. At the nano level, the roughness of the cell surface and the local nano-mechanical proprieties were found to be affected by Dexa. Our results show that ataxia telangiectasia and wild type cells are affected by Dexa, although there are dissimilarities in some macro-level and nano-level features between the tested cell lines. The Young's modulus of the cells appears to depend mainly on nuclear shape, with a slight contribution from the tested cytoskeleton proteins. The current study proposes that dexamethasone influences ataxia telangiectasia cell membranes contents, cell components and cell shape.

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.

ObjectiveAtaxia-telangiectasia (AT) is a rare, severe, and ineluctably progressive multisystemic neurodegenerative disease. Variant AT phenotypes have been described in patients with mild- and late-onset neurologic deterioration and atypical features (dystonia and myoclonus). We report on the clinical characteristics and transcriptome profile of patients with a typical AT presentation and genotype who experienced an unexpected favorable course.MethodsA 24-year-old woman developed, by the age of 3 years, all the classic symptoms of AT associated with increased alpha-fetoprotein levels, a compound AT-mutated (ATM) genotype with an inframe deletion c.2250G>A (p.Glu709_Lys750del42) and a missense mutation c.8122G>A (p.Asp2708Gln), and no residual ATM protein expression. By the age of 12 years, ataxia slowly disappeared, and a very mild choreic disorder was the only neurologic feature in adulthood. Brain MRI was normal. The blood transcriptome profile was assessed and compared with that of healthy controls and patients with the classic AT phenotype.ResultsThe atypical clinical course of the patient was associated with a transitional transcriptome profile: while 90% of transcripts were expressed as in patients with the classic AT presentation, 10% of transcripts were expressed as in healthy controls.ConclusionsThe unexpected mild clinical outcome and transcriptome profile of this patient with AT suggest the existence of individual resilience to the altered ATM synthesis. Because of their possible prognostic and therapeutic implications, the identification of modifier factors affecting the phenotype would deserve further studies.

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.

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.

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.

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose-response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.

This study describes a rapid method to identify different truffle species by analysis of their volatile compound fraction using static headspace solid-phase microextraction gas chromatography/mass spectrometry. The volatile organic compounds (VOCs) were extracted using a new 2-cm 50/30 µm DVB/CAR/PDMS fiber placed for 10 min in the headspace of the truffle sample with the vial maintained at 20°C (in a thermostatically controlled analysis room). The mass spectra of the VOC chromatograms were represented as ‘fingerprints’ of the analysed samples. Next, stepwise factorial discriminant analysis afforded a limited number of characteristic fragment ions that allowed a classification of the truffle species studied. This new method provides an effective approach to rapid quality control and identification of truffle species by analysis of their volatile fraction. Moreover, this method offers the advantage of minimizing thermal, mechanical, and chemical modifications of the truffles, thereby reducing the risk of analytical artifacts. Copyright © 2005 John Wiley & Sons, Ltd.

Ectomycorrhizae formation represents one of the most significant steps in the truffle life cycle and is determined by a complex molecular signaling between two symbionts. In order to understand the molecular pathway of ectomycorrhiza development, we focused on the signaling interaction between the ectomycorrhizal fungus Tuber borchii Vittad. and the Tilia americana L. plant roots. The medium of a pre-symbiotic (T. americana-T. borchii) in vitro system was analysed by headspace solid-phase microextraction coupled with gas chromatography and mass spectrometry. In total, 73 volatile organic compounds (VOCs) were identified. Twenty-nine of these VOCs were produced only during the interaction phase between the two partners, leading to a hypothesis that these molecules might act as molecular messengers in order to pilot the ectomycorrhizae formation.

In the last years, nanopore technology has been increasingly exploited for biomolecule detection and analysis. Recently, the main focus of the research has moved from the study of nucleic acids to the analysis of proteins and DNA-protein complexes. In this paper, chemically functionalized solid-state nanopore has been used to recognize Nuclear Factor-kappa B proteins (NF-κB), that are involved in several disorders and inflammation processes, so that their identification is of crucial importance for prognostic applications. In particular, we show that it is possible to electrically detect the specific interaction between p50, a protein belonging to the NF-κB family, and dsLNA probe molecules covalently attached to the surface of a FIB fabricated SiN pore. The obtained results have been compared with those related to BSA protein, which does not interact with the used probes. Finally, the potential of the device has been further tested by analyzing a whole cell extract. In this case, three principal peaks in the distribution of electrical event duration can be identified, corresponding to different interacting NF-κB complexes, so that the methodology appears to be effective also to study biological samples of considerable complexity. Ultimately, the presented data emphasize the selectivity and versatility of the functionalized nanopore device, demonstrating its applicability in bioanalytics and advanced diagnostics.

Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder caused by biallelic mutations in the Ataxia Telangiectasia-mutated gene. A-T shows a complex phenotype ranging from early-onset progressive neurodegeneration to immunodeficiencies, high incidence of infections, and tumors. Unfortunately, no therapy is up to now available for treating this condition. Recently, the short term treatment of ataxia-telangiectasia patients with glucocorticoids was shown to improve their neurological symptoms and possibly reverse cerebellar atrophy. Thus, corticosteroids represent an attractive approach for the treatment of this neurodegenerative disease. However, the molecular mechanism involved in glucocorticoid action in A-T is yet unknown. The aim of our work is to construct cDNA libraries containing those genes which are transactivated by the glucocorticoid analogue, dexamethasone, in A-T human cells. For this purpose, suppression subtractive hybridization has been performed on ATM-null lymphoblastoid cell transcriptome extracted following drug administration. Annotation of whole genes contained in the libraries has been obtained by coupling subtractive hybridization with microarray analysis. Positive transcripts have been validated by quantitative PCR. Through in silico analyses, identified genes have been classified on the basis of the pathway in which they are involved, being able to address signaling required for dexamethasone action. Most of the induced transcripts are involved in metabolic processes and regulation of cellular processes. Our results can help to unravel the mechanism of glucocorticoid action in the reversion of A-T phenotype. Moreover, the induction of a specific region of the ATM transcript has been identified as putative biomarker predictive of dexamethasone efficacy on ataxic patients.

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.

a b s t r a c t Interaction between transcription factors and DNA are essential for regulating gene transcription. The Nuclear factor-B (NF-B) is a ubiquitous transcription factor involved in cell signalling and its failure is a principal cause of several autoimmune and auto-inflammatory disorders. In this paper we have developed an atomic force microscopy (AFM) method to quantitatively charac- terise the interaction force between NF-B and DNA or LNA (locked nucleic acid) double strand molecules containing the NF responsive elements (RE). This process allows the simple testing and selection of LNA based decoy molecules to be used in NF-B modulation decoy strategies. Furthermore the proposed methodology is also suitable for testing drug efficacy on the modulation of NF-B binding to its nucleic acid target sequence. A biological AFM based sensor is therefore considered appropriate for characterising transcription factors and selecting molecules to modulate their activity. © 2011 Elsevier B.V. All rights reserved.

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.

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.

Single-walled carbon nanotubes (SWNTs) due to their unique structural and physicochemical properties, have been proposed as delivery systems for a variety of diagnostic and therapeutic agents. However, SWNTs have proven difficult to solubilize in aqueous solution, limiting their use in biological applications. In an attempt to improve SWNTs' solubility, biocompatibility, and to increase cell penetration we have thoroughly investigated the construction of carbon scaffolds coated with aliphatic carbon chains and phospholipids to obtain micelle-like structures. At first, oxidized SWNTs (2370 ± 30 nmol mg(-1) of SWNTs) were covalently coupled with an alcoholic chain (stearyl alcohol, C(18)H(37)OH; 816 nmol mg(-1) of SWNTs). Subsequently, SWNTs-COOC(18)H(37) derivatives were coated with phosphatidylethanolamine (PE) or -serine (PS) phospholipids obtaining micelle-like structures. We found that cellular uptake of these constructs by phagocytic cells occurs via an endocytotic mechanism for constructs larger than 400 nm while occurs via diffusion through the cell membrane for constructs up to 400 nm. The material that enters the cell by phagocytosis is actively internalized by macrophages and localizes inside endocytotic vesicles. In contrast the material that enters the cells by diffusion is found in the cell cytosol. In conclusion, we have realized new biomimetic constructs based on alkylated SWNTs coated with phospholipids that are efficiently internalized by different cell types only if their size is lower than 400 nm. These constructs are not toxic to the cells and could now be explored as delivery systems for non-permeant cargoes.

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.

It has been established that intracellular ubiquitin pools are subject to regulatory constrains. Less certain is the mechanism by which the pool of conjugated ubiquitin shift in parallel with total ubiquitin, and how this type of regulation affects the flux of substrates through the pathway. In this study we demonstrate that ubiquitin over-expression promotes the destabilization of the ubiquitin protein ligase E6AP, by a mechanism involving self-ubiquitination, and the stabilization of p53. These results represent the very first evidence that the levels of a ubiquitin ligase can be regulated in vivo by ubiquitin abundance, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists. Interestingly, ubiquitin-induced p53 accumulation did not induce cell-cycle arrest, suggesting that although fluctuations of the intracellular ubiquitin content may actively modulate the level of regulatory proteins, this event is not per se sufficient to elicit a cellular response in terms of proliferation.

Background Small 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. Results We 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. Conclusion T. 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.

The small GTPase CDC42 is ubiquitously expressed in eukaryotes, where it participates in the regulation of the cytoskeleton and a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression, apoptosis, and tumorigenesis. As very little is known on the molecular level about mycorrhizal morphogenesis and development and these events depend on a tightly regulated reorganisation of the cytoskeleton network in filamentous fungi, we focused on the molecular characterisation of the cdc42 gene in Tuber borchii Vittad., an ascomycetous hypogeous fungus forming ectomycorrhizae. The entire gene was isolated from a T. borchii cDNA library and Southern blot analyses showed that only one copy of cdc42 is present in the T. borchii genome. The predicted amino acid sequence is very similar to those of other known small GTPases and the similar domain structures suggest a similar function. Real-time PCR analyses revealed an increased expression of Tbcdc42 during the phase preparative to the instauration of symbiosis, in particular after stimulation with root exudate extracts. Immunolocalisation experiments revealed an accumulation of CDC42 in the apical tips of the growing hyphae. When a constitutively active Tbcdc42 mutant was expressed in Saccharomyces cerevisiae, morphological changes typical of pseudohyphal growth were observed. Our results suggest a fundamental role of CDC42 in cell polarity development in T. borchii.

A novel NADP+-dependent d -mannitol dehydrogenase and the corresponding gene from the plant symbiotic ascomycete fungus Tuber borchii was identified and characterized. The enzyme, called TbMDH, is a homotetramer with two zinc atoms per subunit. It catalyzed both d -fructose reduction and d -mannitol oxidation, although it showed the highest substrate specificity and catalytic efficiency for d -fructose. Co-factor specificity was restricted to NADP(H) and the reaction proceeded via a sequential ordered Bi Bi mechanism. The carbon responsive transcriptional pattern showed that Tbmdh is up-regulated when mycelia are transferred to a culture medium containing d -mannitol or d -fructose. The phylogenetic analysis showed TbMDH to be the first example of a fungal d -mannitol-2-dehydrogenase belonging to the medium-chain dehydrogenase/reductases (MDRs). The enzyme identified a new group of proteins, most of them annotated in databases as hypothetical zinc-dependent dehydrogenases, forming a distinct subfamily among the polyol dehydrogenase family.

Here, we report the first evidence of a hexose transporter gene, Tbhxt1, in the ectomycorrhizal ascomycete Tuber borchii Vittadini. The protein encoded by Tbhxt1 functionally complements the hxt-null mutant Saccharomyces cerevisiae EBYVW.4000. TBHXT1 has a strong preference for d -glucose (Km = 38 ± 10 μM) over d -fructose (Km = 16 ± 5 mM) and uncoupling experiments indicate that TBHXT1 catalyzes the transport via a proton-symport mechanism. The investigations on the substrate specificity reveal that TBHXT1 also imports d -mannose, and the use of deoxyglucose analogues shows that the hydroxyl groups at C1, C3 and C4 are important for substrate recognition. Tbhxt1 is not regulated by fructose, but it reaches its highest level of expression at 3 mM glucose and is repressed by very high glucose concentration. Prolonged carbon starvation condition upregulates Tbhxt1, while its expression remains at basal level in the ectomycorrhizal tissue. The mode of regulation of Tbhxt1 is consistent with its role as a high-affinity d -glucose transporter.

A major limitation of highly active antiretroviral therapy is that it fails to eradicate human immunodeficiency virus (HIV) infection due to its limited effects on viral reservoirs carrying replication-competent HIV, including monocytes/macrophages (M/M). Therefore, therapeutic approaches aimed at targeting HIV-infected M/M may prove useful in the clinical management of HIV-infected patients. In previous studies, we have shown that administration of fludarabine-loaded red blood cells (RBC) in vitro selectively induces cell death in HIV-infected M/M via a pSTAT1-dependent pathway. To determine the in vivo efficacy of this novel therapeutic strategy, we treated six naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) with either 9-[2-( R )-(phosphonomethoxy)propyl]adenine (PMPA) only, fludarabine-loaded RBC only, or PMPA in association with fludarabine-loaded RBC. The rationale of this treatment was to target infected M/M with fludarabine-loaded RBC at a time when PMPA is suppressing viral replication taking place in activated CD4 + T cells. In vivo administration of fludarabine-loaded RBC was well tolerated and did not induce any discernible side effect. Importantly, addition of fludarabine-loaded RBC to PMPA delayed the rebound of viral replication after suspension of therapy, thus suggesting a reduction in the size of SIV reservoirs. While administrations of fludarabine-loaded RBC did not induce any change in the CD4 + or CD8 + T-cell compartments, we observed, in chronically SIV-infected SMs, a selective depletion of M/M expressing pSTAT1. This study suggests that therapeutic strategies based on the administration of fludarabine-loaded RBC may be further explored as interventions aimed at reducing the size of the M/M reservoirs during chronic HIV infection.

Mitogen-activated protein kinases (MAPK) are used by organisms to transduce extra cellular signals from the environment in cellular events such as proliferation and differentiation. In the present study, we have characterized the first MAPK from the ectomycorrhizal fungus Tuber borchii (TBMK) which belongs to the YERK1 (yeast extra cellular regulated kinase) subfamily. TBMK is present as a single copy in the genome and the codified protein was phosphorylated during the interaction with the host plant, Tilia americana. Complementation studies showed that TBMK restores pheromone signaling in Saccharomyces cerevisiae and partially restores invasive growth of Fusarium oxysporum that lack the fmk1 gene. This suggests a protein kinase activity and its involvement in the infection processes. Hence, TBMK could play an important role during the pre-symbiotic phase of T. borchii with its host plant in the modulation of genes necessary for the establishment of symbiosis leading to the synthesis of functional ectomycorrhizae.