Single molecule fluorescence spectroscopy of pH sensitive oligonucleotide switches.

Several authors demonstrated that an oligonucleotide based pH-sensitive construct can act as a switch between an open and a closed state by changing the pH. To validate this process, specially designed fluorescence dye-quencher substituted oligonucleotide constructs were developed to probe the switching between these two states. This paper reports on bulk and single molecule fluorescence investigations of a duplex-triplex pH sensitive oligonucleotide switch. On the bulk level, only a partial quenching of the fluorescence is observed, similarly to what is observed for other published switches and is supposed to be due to intermolecular interactions between oligonucleotide strands. On the single molecule level, each DNA-based nanometric construct shows a complete switching. These observations suggest the tendency of the DNA construct to associate at high concentration.     

5-Arylidene-2-phenylimino-4-thiazolidinones as PTP1B and LMW-PTP inhibitors

As part of a project aimed at identifying effective low molecular weight nonphosphorus monoanionic inhibitors of PTPs, we have synthesized 4-[(5-arylidene-4-oxo-2-phenyliminothiazolidin-3-yl)methyl]benzoic acids (4) and evaluated their inhibitory activity against human PTP1B and LMW-PTP enzymes. The introduction of a 2-phenylimino moiety onto the 4-thiazolidinone ring was designed to enhance the inhibitor/enzyme affinity by means of further favourable interactions with residues of the active site and the surrounding loops. Some of the compounds (4a–d, f) showed interesting inhibition levels in the low micromolar range. The 5-arylidene moiety of acids 4 proved to markedly influence the potency of these inhibitors. Molecular modeling experiments inside the binding sites of both enzymes were performed.     

Hepatic safety profile of darunavir with low-dose ritonavir (DRV/r) in HIV/HCV coinfected and HIV monoinfected patients

The hepatic safety profile of ART including DRV/r was retrospectively evaluated in antiretroviral-experienced HIV-infected patients (18 HIV/HCV coinfected, group A and 29 infected with HIV alone, group B) during a 72 week study. During the study, liver enzyme values were higher in group A, but in the case of abnormal transaminase levels, the median values did not exceed 1.6xULN. This study showed evidence of long-lasting hepatic safety of ART including PI DRV/r in HIV/HCV coinfected and in HIV monoinfected persons.     

PrPc activation induces neurite outgrowth and differentiation in PC12 cells: role for caveolin-1 in the signal transduction pathway

Cellular prion protein (PrP^c) is a ubiquitous glycoprotein, whose physiological role is poorly characterized. It has been suggested that PrP^c participates in neuritogenesis, neuroprotection, copper metabolism, and signal transduction. In this study we detailed the intracellular events induced by PrP^c antibody-mediated cross-linking in PC12 cells. We found a Fyn-dependent activation of the Ras-Raf pathway, which leads to a rapid and transient phosphorylation of extracellular regulated kinases. In addition, this activation cascade relies on the engagement of integrins, and involves focal adhesion kinase activation. We demonstrated the tyrosine phosphorylation of caveolin-1 as a consequence of PrP^c stimulation, and showed that phosphocaveolin-1 scaffolds and coordinates protein complexes involved in PrP^c-dependent signaling. Moreover, we found that caveolin-1 phosphorylation, is a mechanism for recruiting the C-terminal Src kinase and inactivating Fyn, so as to terminate cell signaling. Furthermore our data support a significant role for PrP^c as a response mediator in neuritogenesis and cell differentiation.     

Resistance of Human Erythrocyte Membranes to Triton X-100 and C<Subscript>12</Subscript>E<Subscript>8</Subscript>

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C₁₂E₈. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C₁₂E₈, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C₁₂E₈ in comparison to intact cells, while the difference in the S values between Triton X-100 and C₁₂E₈ DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C₁₂E₈ detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.     

The occurrence of porosity in reline acrylic resins. Effect of microwave disinfection

Background: Microwave energy has proved to be an effective method for disinfecting acrylic dentures. However, the effect of microwave heating on the porosity of autopolymerising denture reline resins has not been investigated. Objective: The purpose of the study was to determine the effect of microwave disinfection on the porosity of autopolymerised denture reline materials (Kooliner‐K, New Truliner‐NT, Tokuso Rebase Fast‐TR and Ufi Gel Hard‐UGH) and a conventional heat‐polymerised denture base resin (Lucitone 550‐L). Material and methods: Specimens (10 mm × 20 mm × 1 mm) were obtained from the impression surface of the palatal mucosa in a single person and divided into four groups (n = 5). The porosity was evaluated after polymerisation (C1), after two cycles of microwave disinfection (MW2), after seven cycles of microwave disinfection (MW7) and after 7 days storage in water at 37°C (C2). Specimens from group MW7 were exposed to microwave disinfection daily being stored in water at 37°C between exposures. All the replicas were sputter coated with gold and micrographs/digital images were taken of each replica using scanning electron microscopy at magnification × 100. The SEM micrographs were then examined using an image analyser to determine the number of pores. Comparison between materials and groups were made using Kruskal–Wallis tests. Results: MW7 resulted in a significant increase in the number from the pores of material K, but decreased in number in reline material TR and UGH reline resin. The number of pores in materials NT and L remained unaffected following microwave disinfection. Conclusion: Differences in the porosity amongst the materials and for different experimental conditions were observed following microwave disinfection.     

Complex Mutations & Subpopulations of Deletions at Exon 19 of EGFR in NSCLC Revealed by Next Generation Sequencing: Potential Clinical Implications

Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.     

Weight loss and changes in surface roughness of denture base and reline materials after simulated toothbrushing in vitro

doi: 10.1111/j.1741‐2358.2010.00422.x
Weight loss and changes in surface roughness of denture base and reline materials after simulated toothbrushing in vitro Objective: To evaluate the weight loss and the surface roughness of acrylic resins after simulated brushing tests. Material and methods: Ten specimens of each material (Tokuyama Rebase II‐TR, New Truliner‐NT, Ufi Gel Hard‐UH and Lucitone 550‐L) were made. The wear loss (mg) by weight and the surface roughness (Ra μm) of each specimen was determined before and after brushing. The specimens were placed on the brushing machine and a total of 20 000 brushing cycles was performed. The results of weight loss and roughness values were submitted to the anova followed by the Tukey’s test (p = 0.05). Results: The mean weight loss of material L was statistically higher (p < 0.001) than that of the relines TR, UH and NT. No significant differences were found among the roughness values of resins TR, UH and L (p > 0.05). Only for L, toothbrushing increased the surface roughness. After toothbrushing, there was no significant difference between the roughness values of materials L and NT. The highest mean weight loss during the simulated toothbrushing tests was observed for L. Before the toothbrushing tests, the NT exhibited the highest mean roughness. Conclusion: Brushing resulted in increase in roughness only for resin L.     

The sequence and phylogenesis of the α-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion)

In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked ^Iα and ^IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the ^Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.