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The aim of the current study was to identify which subgroups of children with Attention Deficit Hyperactivity Disorder (ADHD) benefitted the most from playing a Serious Game (SG) intervention shown in a randomized trial to improve behavioral outcomes.Pre-intervention characteristics [i.e., gender, age, intellectual level of functioning, medication use, computer experience, ADHD subtype, severity of inattention problems, severity of hyperactivity/impulsivity problems, comorbid Oppositional Defiant Disorder (ODD) and Conduct Disorder (CD) symptoms] were explored as potential moderators in a Virtual Twins (VT) analysis to identify subgroups for whom the SG intervention was most effective. Primary outcome measures were parent-reported time management, planning/organizing and cooperation skills.Two subgroups were identified. Girls (n = 26) were identified as the subgroup that was most likely to show greater improvements in planning/organizing skills as compared to the estimated treatment effect of the total group of participants. Furthermore, among the boys, those (n = 47) with lower baseline levels of hyperactivity and higher levels of CD symptoms showed more improvements in their planning/organizing skills when they played the SG intervention as compared to the estimated treatment effect of the total group of participants.Using a VT analysis two subgroups of children with ADHD, girls, and boys with both higher levels of CD and lower levels of hyperactivity, were identified. These subgroups mostly benefit from playing the SG intervention developed to improve ADHD related behavioral problems. Our results imply that these subgroups have a higher chance of treatment success.

Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.

Two experiments examined the nature of visuo-spatial mental imagery generation and maintenance in 4-, 6-, 8-, 10-year old children and adults (N = 211). The key questions were how image generation and maintenance develop (Experiment 1) and how accurately children and adults coordinate mental and visually perceived images (Experiment 2). Experiment 1 indicated that basic image generation and maintenance abilities are present at 4 years of age but the precision with which images are generated and maintained improves particularly between 4 and 8 years. In addition to increased precision, Experiment 2 demonstrated that generated and maintained mental images become increasingly similar to visually perceived objects. Altogether, findings suggest that for simple tasks demanding image generation and maintenance, children attain adult-like precision younger than previously reported. This research also sheds new light on the ability to coordinate mental images with visual images in children and adults.

Currently available therapies for candidiasis are based on antifungal drugs belonging to azole and echinocandin families that interfere with different aspects of fungal metabolism. These drugs, beyond their specific effects, elicit also a cellular stress including an unbalance of redox state that is counteracted not only utilizing antioxidant species but also increasing the outcome export by transporters to detoxify the internal environment. These cellular actions are both based on the cytosolic concentration of reduced glutathione (GSH). In this paper we investigated the effects of two antifungal drugs fluconazole and micafungin on the redox state of the cell in C. albicans to understand if the resistance to these drugs is accompanied by variation of glutathione metabolism. Analyses of resistant strains showed a marked difference in glutathione contents in strains resistant to fluconazole (CO23RFLC) or micafungin (CO23RFK). In CO23RFLC, the total amount of glutathione was more than doubled with respect to CO23RFK thanks to the increased activity of γ-glutamilcysteine synthetase, the key enzyme involved in GSH synthesis. We demonstrated that the GSH increase in CO23RFLC conferred to this strain a clear advantage in counteracting oxidative toxic agents while assignment of other roles, such as a more efficient elimination of the drug from the cell, should be considered more speculative. As far as MCFG resistance is concerned, from our data a role of glutathione metabolism in supporting this condition is not evident. Overall our data indicate that glutathione metabolism is differently affected in the two resistant strains and that glutathione system may play an important role in the global organization of C.albicans cells for resistance to fluconazole. Such scenario may pave the way to hypothesize the use of oxidant drugs or inhibitors able to deplete reduced glutathione level as a novel approach, for counteracting the resistance to this specific antifungal drug.

Amyloid beta peptide (Aβ) causes neurodegeneration by several mechanisms including oxidative stress, which is known to induce DNA damage with the consequent activation of poly (ADP-ribose) polymerase (PARP-1). To elucidate the role of PARP-1 in the neurodegenerative process, SH-SY5Y neuroblastoma cells were treated with Aβ25-35 fragment in the presence or absence of MC2050, a new PARP-1 inhibitor. Aβ25-35 induces an enhancement of PARP activity which is prevented by cell pre-treatment with MC2050. These data were confirmed by measuring PARP-1 activity in CHO cells transfected with amylod precursor protein and in vivo in brains specimens of TgCRND8 transgenic mice overproducing the amyloid peptide. Following Aβ25-35 exposure a significant increase in intracellular ROS was observed. These data were supported by the finding that Aβ25-35 induces DNA damage which in turn activates PARP-1. Challenge with Aβ25-35 is also able to activate NF-kB via PARP-1, as demonstrated by NF-kB impairment upon MC2050 treatment. Moreover, Aβ25-35 via PARP-1 induces a significant increase in the p53 protein level and a parallel decrease in the anti-apoptotic Bcl-2 protein. These overall data support the hypothesis of PARP-1 involvment in cellular responses induced by Aβ and hence a possible rationale for the implication of PARP-1 in neurodegeneration is discussed.

BACKGROUND:Human Papillomavirus (HPV)-16 is a paradigm for "high-risk" HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation. METHODOLOGY/PRINCIPAL FINDINGS:By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7. CONCLUSIONS/SIGNIFICANCE:This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.

Anti-beta-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective beta-glucan epitope(s) and protection mechanisms, we studied two anti-beta-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the beta1,3-beta1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to beta1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to beta1,6- and beta1,4-linked glucose sequences in addition to beta1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-beta-glucan antibodies may affect details of the beta-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize beta-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

Currently available therapies for candidiasis are based on antifungal drugs belonging to azole and echinocandin families that interfere with different aspects of fungal metabolism. These drugs, beyond their specific effects, elicit also a cellular stress including an unbalance of redox state that is counteracted not only utilizing antioxidant species but also increasing the outcome export by transporters to detoxify the internal environment. These cellular actions are both based on the cytosolic concentration of reduced glutathione (GSH). In this paper we investigated the effects of two antifungal drugs fluconazole and micafungin on the redox state of the cell in C. albicans to understand if the resistance to these drugs is accompanied by variation of glutathione metabolism. Analyses of resistant strains showed a marked difference in glutathione contents in strains resistant to fluconazole (CO23RFLC) or micafungin (CO23RFK). In CO23RFLC, the total amount of glutathione was more than doubled with respect to CO23RFK thanks to the increased activity of γ-glutamilcysteine synthetase, the key enzyme involved in GSH synthesis. We demonstrated that the GSH increase in CO23RFLC conferred to this strain a clear advantage in counteracting oxidative toxic agents while assignment of other roles, such as a more efficient elimination of the drug from the cell, should be considered more speculative. As far as MCFG resistance is concerned, from our data a role of glutathione metabolism in supporting this condition is not evident. Overall our data indicate that glutathione metabolism is differently affected in the two resistant strains and that glutathione system may play an important role in the global organization of C.albicans cells for resistance to fluconazole. Such scenario may pave the way to hypothesize the use of oxidant drugs or inhibitors able to deplete reduced glutathione level as a novel approach, for counteracting the resistance to this specific antifungal drug. [ABSTRACT FROM AUTHOR]

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model. Both mAbs bound to fungal cell surface and to the β1,3-β1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to β1,3-linked (laminarin-like) glucose sequences whereas the nonprotective IgM bound to β1,6- and β1,4-linked glucose sequences in addition to β1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells. Our study demonstrates that the isotype of anti-β-glucan antibodies may affect details of the β-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize β-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells. [ABSTRACT FROM AUTHOR]

C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host's immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host's environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality.

The pathological form of prion protein (PrP(Sc)), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc) extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP₉₀₋₂₃₁) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc). In the present study we demonstrate that hPrP₉₀₋₂₃₁, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP₉₀₋₂₃₁ bearing pathogenic mutations (D202N and E200K). We also report that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP₉₀₋₂₃₁ cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.