A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

In vitro evaluation of a silane containing self-adhesive resin luting agent.

OBJECTIVES: To investigate the setting characteristics, wettability and bonding capacity with a lithium disilicate ceramic of a silane containing self-adhesive resin luting agent (Panavia SA Universal-PU). METHODS: The degree of conversion (DC %) and extent of acid neutralization (SY %) of PU were measured on dual- (DC) and self-cured (SC) specimens after 10, 30 and 60 min storage by ATR-FTIR spectroscopy, whereas the presence of silanols was traced by curve-fitting the 60 min spectra, using the silane-free analog (Panavia SA Plus-PS) as a control. The role of a dedicated adhesive (Clearfil Universal Bond Quick-CU) in assisting the early DC % in PU-SC was investigated on 10 min-stored specimens. The water contact angles on polished and HF acid-etched lithium disilicate surfaces (IPS e.max Press), were assessed before and after silanization by unset PU or a silane primer (Ultradent Silane-SL). Finally, the shear strength of PU-DC specimens bonded to the acid-etched ceramic surfaces was determined before and after SL treatment. RESULTS: The DC % was higher in DC than SC (PU, PS; all time intervals), in PU-SC than PS-SC (30, 60 min) and in the CU assisted PU-SC group. The SY % was lower in DC than SC (PU, PS) and higher in PS-SC than PU-SC groups. Silanols were found only in unset PU and PU-DC groups. SL treatment provided higher water contact angles on polished and acid-etched ceramic surfaces and higher shear bond strength on acid-etched ceramic surfaces than PU (p < 0.05 for all comparisons). SIGNIFICANCE: Although the degree of conversion of the silane containing luting agent was improved in the self-curing mode, especially in the adhesive assisted group, it was still inferior to light-curing. Acid-neutralization and presence of silanols were affected by the setting modes. The use of a silane primer enhanced the hydrophobicity and bond strength of the silane containing luting agent with the etched ceramic substrate.

Autophagy in spinal muscular atrophy: from pathogenic mechanisms to therapeutic approaches.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder caused by the depletion of the ubiquitously expressed survival motor neuron (SMN) protein. While the genetic cause of SMA has been well documented, the exact mechanism(s) by which SMN depletion results in disease progression remain elusive. A wide body of evidence has highlighted the involvement and dysregulation of autophagy in SMA. Autophagy is a highly conserved lysosomal degradation process which is necessary for cellular homeostasis; defects in the autophagic machinery have been linked with a wide range of neurodegenerative disorders, including amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. The pathway is particularly known to prevent neurodegeneration and has been suggested to act as a neuroprotective factor, thus presenting an attractive target for novel therapies for SMA patients. In this review, (a) we provide for the first time a comprehensive summary of the perturbations in the autophagic networks that characterize SMA development, (b) highlight the autophagic regulators which may play a key role in SMA pathogenesis and (c) propose decreased autophagic flux as the causative agent underlying the autophagic dysregulation observed in these patients.

Mitochondrial Targeting and Imaging with Small Organic Conjugated Fluorophores: A Review.

The last decade has seen an increasingly large number of studies reporting on the development of novel small organic conjugated systems for mitochondrial imaging exploiting optical signal transduction pathways. Mitochondria are known to play a critical role in a number of key biological processes, including cellular metabolism. Importantly, irregularities on their working function are nowadays understood to be intimately linked to a range of clinical conditions, highlighting the importance of targeting mitochondria for therapeutic benefits. In this work we carry out an in-depth evaluation on the progress to date in the field to pave the way for the realization of superior alternatives to those currently existing. The manuscript is structured by commonly used chemical scaffolds and comprehensively covers key aspects factored in design strategies such as synthetic approaches as well as photophysical and biological characterization, to foster collaborative work among organic and physical chemists as well as cell biologists.

The effect of cooling procedures on monomer elution from heat-cured polymethyl methacrylate denture base materials.

OBJECTIVE: To evaluate the amount of methyl methacrylate (MMA) released in water from heat-cured polymethyl methacrylate (PMMA) denture base materials subjected to different cooling procedures. METHODOLOGY: Disk-shaped specimens (Ø:17 mm, h:2 mm) were fabricated from Paladon 65 (PA), ProBase Hot (PB), Stellon QC-20 (QC) and Vertex Rapid Simplified (VE) denture materials using five different cooling procedures (n=3/procedure): A) Bench-cooling for 10 min and then under running water for 15 min; B) Cooling in water-bath until room temperature; C) Cooling under running water for 15 min; D) Bench-cooling, and E) Bench-cooling for 30 min and under running water for 15 min. A, B, D, E procedures were proposed by the manufacturers, while the C was selected as the fastest one. Control specimens (n=3/material) were fabricated using a long polymerization cycle and bench-cooling. After deflasking, the specimens were ground, polished and stored in individual containers with 10 ml of distilled water for seven days (37oC). The amount of water-eluted MMA was measured per container using isocratic ultra-fast liquid chromatography (UFLC). Data were analyzed using Student's and Welch's t-test (α=0.05). RESULTS: MMA values below the lower quantification limit (LoQ=5.9 ppm) were registered in B, C, E (PA); E (PB) and B, D, E (QC) procedures, whereas values below the detection limit (LoD=1.96 ppm) were registered in A, D (PA); A, B, C, D (PB); C, D, E (VE) and in all specimens of the control group. A, B (VE) and A, C (QC) procedures yielded values ranging from 6.4 to 13.2 ppm with insignificant differences in material and procedure factors (p>0.05). CONCLUSIONS: The cooling procedures may affect the monomer elution from denture base materials. The Ε procedure may be considered a universal cooling procedure compared to the ones proposed by the manufacturers, with the lowest residual monomer elution in water.

The effect of cooling procedures on monomer elution from heat-cured polymethyl methacrylate denture base materials

Abstract Objective To evaluate the amount of methyl methacrylate (MMA) released in water from heat-cured polymethyl methacrylate (PMMA) denture base materials subjected to different cooling procedures. Methodology Disk-shaped specimens (Ø:17 mm, h:2 mm) were fabricated from Paladon 65 (PA), ProBase Hot (PB), Stellon QC-20 (QC) and Vertex Rapid Simplified (VE) denture materials using five different cooling procedures (n=3/procedure): A) Bench-cooling for 10 min and then under running water for 15 min; B) Cooling in water-bath until room temperature; C) Cooling under running water for 15 min; D) Bench-cooling, and E) Bench-cooling for 30 min and under running water for 15 min. A, B, D, E procedures were proposed by the manufacturers, while the C was selected as the fastest one. Control specimens (n=3/material) were fabricated using a long polymerization cycle and bench-cooling. After deflasking, the specimens were ground, polished and stored in individual containers with 10 ml of distilled water for seven days (37oC). The amount of water-eluted MMA was measured per container using isocratic ultra-fast liquid chromatography (UFLC). Data were analyzed using Student's and Welch's t-test (α=0.05). Results MMA values below the lower quantification limit (LoQ=5.9 ppm) were registered in B, C, E (PA); E (PB) and B, D, E (QC) procedures, whereas values below the detection limit (LoD=1.96 ppm) were registered in A, D (PA); A, B, C, D (PB); C, D, E (VE) and in all specimens of the control group. A, B (VE) and A, C (QC) procedures yielded values ranging from 6.4 to 13.2 ppm with insignificant differences in material and procedure factors (p>0.05). Conclusions The cooling procedures may affect the monomer elution from denture base materials. The Ε procedure may be considered a universal cooling procedure compared to the ones proposed by the manufacturers, with the lowest residual monomer elution in water.

The Effect of Touch-Cure Polymerization on the Conversion and Hardness of Core Build-Up Resin Composites: A Laboratory Study.

To improve the self-curing capacity and interfacial strength with dentine of dual-cured composite materials, touch-cure activators have been introduced. The aim of the study was to evaluate the effect of these activators on the hardness and conversion of dual-cured resin composite core build-up restoratives. The materials tested were Clearfil DC Core Plus (CF) and Gradia Core (GC) with the corresponding adhesives Clearfil S3 Bond Plus (for CF) and G-Premio Bond/G-Premio DCA activator (for GC). Disk-shaped specimens (n = 6/group) were prepared for the following groups: dual-cured, self-cured and self-cured in contact with the adhesive activators at the bottom surface. After a 3-week storage period (dark/dry/37 °C) the Martens hardness (HM) and degree of conversion (DC%) were determined for the previously mentioned groups and the top surfaces of groups in contact with the adhesives. A statistical analysis was performed by a one-way ANOVA and Holm-Sidak test per material and a Pearson's correlation analysis (HM vs. DC%) at an α = 0.05. The self-cured specimens resulted in significantly lower HM and DC% values from the dual-cured group, as expected. However, in the presence of the adhesives with touch-cure activators, the conversion of the self-cured groups showed insignificant differences in HM and DC% from the dual-cured in both composite materials. The improvements on the bottom composite surfaces in contact with the adhesives did not extend to the entire specimen length. Nevertheless, improved interfacial curing may improve interfacial durability.

Effect of Water Storage on Hardness and Interfacial Strength of Resin Composite Luting Agents Bonded to Surface-Treated Monolithic Zirconia.

BACKGROUND: Durable bonding between resin composite luting agents (CLA) and zirconia is still a matter of controversy. The purpose of this study was to evaluate the effect of water storage on hardness and interfacial strength of three CLA, a non-adhesive (Multilink Automix/ML), an adhesive (Panavia F 2.0/PF) and a self-adhesive (PermaCem 2.0/PC), bonded to polished (CL) and grit-blasted (AL: 50 µm alumina, SJ: Sil-Jet + Monobond Plus silane) monolithic zirconia surfaces. METHODS: CLA specimens (n = 5/cement, condition) were prepared, stored under dry conditions or immersed in water, and Vickers hardness (VH) measurements were obtained at 1 h, 24 h, 1 week and 3 weeks intervals. Optical profilometry was used to determine the roughness parameters (Sa, Sz, Sdr, Sci) of zirconia surfaces (n = 5/treatment). A shear strength test (SBS, n = 10 × 2/cement) was performed to assess the strength and fractography of the cements bonded to zirconia after isothermal water storage and thermal-cycling (TC). RESULTS: PF demonstrated significantly lower VHN after water storage at all time intervals, PC at 1 w, 3 w and ML at 3 w. SJ and AL showed significantly higher values from CL in all roughness parameters. Weibull analysis revealed the following significance in σο ranking within the same material: AL, SJ, ALTC > SJTC, CL > CLTC (PF); SJ, SJTC, AL, ALTC > CL, CLTC (PC) and SJ, SJTC > AL > ALTC > CL, CLTC (ML). Within the same surface treatment subgroups, the significance in σo ranking was PC, ML > PF (before/after TC) for SJ; PC > PF > ML (before TC), PC, PF > ML (after TC) for AL, and PC > PF > ML (before/after TC) for CL. For the m ranking, the only significant difference within each material group was found in PC (AL > ALTC) and for the same surface treatment in AL (PC > ML). CONCLUSION: There are significant differences in the water plasticization susceptibility of the CLA tested; the materials with adhesive monomers were the most affected. Tribo-chemical silica coating combined with a silane coupling agent was the most efficient bonding treatment for the non-adhesive and the self-adhesive materials. The adhesive CLA performed better on alumina-blasted than on tribo-chemically coated surfaces.

Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy.

BACKGROUND: Understanding the genetic modifiers of neurodegenerative diseases can provide insight into the mechanisms underlying these disorders. Here, we examine the relationship between the motor neuron disease spinal muscular atrophy (SMA), which is caused by reduced levels of the survival of motor neuron (SMN) protein, and the actin-bundling protein Plastin 3 (PLS3). Increased PLS3 levels suppress symptoms in a subset of SMA patients and ameliorate defects in SMA disease models, but the functional connection between PLS3 and SMN is poorly understood. RESULTS: We provide immunohistochemical and biochemical evidence for large protein complexes localized in vertebrate motor neuron processes that contain PLS3, SMN, and members of the hnRNP F/H family of proteins. Using a Caenorhabditis elegans (C. elegans) SMA model, we determine that overexpression of PLS3 or loss of the C. elegans hnRNP F/H ortholog SYM-2 enhances endocytic function and ameliorates neuromuscular defects caused by decreased SMN-1 levels. Furthermore, either increasing PLS3 or decreasing SYM-2 levels suppresses defects in a C. elegans ALS model. CONCLUSIONS: We propose that hnRNP F/H act in the same protein complex as PLS3 and SMN and that the function of this complex is critical for endocytic pathways, suggesting that hnRNP F/H proteins could be potential targets for therapy development.

Surface Characteristics and Color Stability of Gingiva-Colored Resin Composites.

The purpose of this study was to investigate the surface characteristics and color stability of gingiva-colored composite restorative materials (Anaxgum-ANG, Ceramage-CMG and Gradia Gum-GRG). The microstructure, composition, degree of conversion (DC %) and 3D roughness (Sa, Sz, Sdr, Sc) were examined by LV-SEM/EDS, ATR-FTIR and optical profilometry, respectively. For the color stability (CIE L*, a*, b* system) and hardness (HV), measurements were performed at baseline and after 30 days storage in distilled water, coffee and red wine. The ANG and GRG contain prepolymerized particles in aromatic and aliphatic resin matrices, respectively, whereas CMG contains inorganic zirconia silicate/silica particles, in an aromatic resin matrix, with a smaller particle size and a higher surface area fraction. Urethane monomers were mainly identified in CMG and GRG. The DC% showed statistically insignificant differences between the materials. The same applied for the roughness parameters, except for the greatest Sdr in CMG. ANG showed a color difference (ΔE) of > 3.3 after immersion in all media, CMG in coffee and wine and GRG only in coffee. Sc was the only roughness parameter demonstrating correlations with the ΔL*, Δb* and ΔE*. The HV values showed insignificant differences between the storage conditions per material. There are important differences in the color stability of the materials tested, which were mostly affected by the roughness parameters due to variations in their microstructure.

Self-Etch Silane Primer: Reactivity and Bonding with a Lithium Disilicate Ceramic.

The aim of the study was to evaluate the stability, reactivity, and bond strength with a lithium disilicate ceramic of a self-etch silane primer (Monobond Etch and Prime/MEP). The stability was evaluated by 1H-,31P-NMR spectroscopy (before/after aging), and the reactivity by micro MIR-FTIR spectroscopy on Ge surfaces (0, 1, 24 h) using a prehydrolyzed silane primer (Calibra Silane Coupling Agent/CLB), as a control. The effect of MEP vs. 5% HF-etching on ceramic roughness was assessed by optical profilometry. The shear bond strength (SBS) of a resin composite bonded to polished ceramic surfaces treated with MEP, HF without silane (HF+NS), HF+CLB, and HF+MEP (n = 20) was evaluated after storage in water (A: 37 °C/1 week, B: 5000×/5-55 °C and C: 100 °C/24 h). Aging did not affect the silanol groups of MEP, but only the phosphate co-monomer. Silanols were reactive forming siloxanes, but exhibited lower consumption rate than CLB. HF-etching induced significantly higher values than MEP, in all the roughness parameters tested (Sa, Sz, Sdr, Sc, Sv), with the greatest differences found in Sdr and Sv. For SBS, MEP was inferior to all treatments/storage conditions, except of HF+NS in A, where the values were similar. However, on a HF-etched substrate, MEP provided highest strength and reliability.

The effect of different cooling procedures on mechanical properties of denture base materials measured by instrumented indentation testing.

PURPOSE: To evaluate the different cooling procedures on the mechanical properties of five heat-cured polymethyl methacrylate (PMMA) denture materials. METHODS: 250 specimens were made equally from Meliodent (ME), Paladon 65 (PA), Probase Hot (PB), Stellon QC-20 (QC) and Vertex Rapid Simplified (VE) implementing five different cooling procedures (n=10/procedure): A) removal from water bath, bench-cooling (10min) and cooling under water (15min), B) remain in water bath till room temperature, C) removal from water bath and cooling in water for 15min, D) removal from water bath and bench cooling till room temperature and E) removal from water bath, bench cooling for 30min and cooling under water for 15min. The specimens were immersed in distilled water (15 days/37ºC) and then subjected to Instrumented Indentation Testing for Martens Hardness (HM), indentation modulus (EIT) and elastic index (ηΙΤ). Results were statistically analyzed by two- and one-way Analysis of variance (ANOVA) plus Tukey post hoc tests (α=0.05). RESULTS: The highest values for HM were recorded for QC, PA, VE with B cooling procedure, PB with A and ME with E, for EΙΤ for QC, PB with A, for PA, VE with B and ME with E, and for ηIT for QC, PB with B, PB with E, ME with C and VE with D. CONCLUSIONS: The cooling procedures recommended for PB resulted in the lowest mechanical properties. A and B may be considered as universal short- and long-cooling procedures respectively providing the highest mechanical properties for the materials tested.

Reactivity and Bond Strength of Universal Dental Adhesives with Co-Cr Alloy and Zirconia.

The aim of this study was to evaluate (a) the reactivity of six universal dental adhesives with polished cobalt-chrome (Co-Cr) alloy and zirconia (3Y-TZP) surfaces; and (b) to assess the shear bond strength (SBS) of a resin composite with polished and alumina-blasted surfaces as mediated by these adhesives. The products tested were Adhese Universal (AD), All-Bond Universal (AB), Clearfill Universal Bond (CB), G-Premio Bond (GP), Prelude One (PO) and Scotchbond Universal (SB). The reactivity on polished substrates was evaluated by reflection infrared microscopy (RFTIRM). The roughness parameters of polished and 50 µm alumina grit-blasted surfaces were assessed by optical profilometry. The SBS of the composite bonded to the substrates treated with each adhesive (n = 10/product) was evaluated after 1 week of storage (H2O/37 °C) by Weibull statistics. Evidence of phosphate interaction with polished substrates was obtained by FTIRM, with higher peaks on the alloy. Alumina-blasting increased all roughness parameters with higher values on the alloy. AD, CB were the strongest (σ0) treatments on alloy surfaces and AD, CB, AB, SB on zirconia. GP was the weakest on both substrates and the least reliable (ß) on alloy. On polished alloy GP, PO performed better (σ0), whereas on zirconia there were no significant differences. All adhesives showed more prominent reaction with the Co-Cr alloy than with 3Y-TZP.

Silane reactivity and resin bond strength to lithium disilicate ceramic surfaces.

OBJECTIVE: To evaluate the silane status in commercially available products and their bonding capacity with polished glass-ceramic surfaces before and after hydrofluoric (HF) acid-etching. METHODS: The products tested were Calibra Silane Coupling Agent/CS, G-Multi Primer/GM, Kerr Silane Primer/KS, Monobond Plus/MB and Scotchbond Universal Adhesive/SB. The silane status was studied by 13C nuclear magnetic resonance spectroscopy (13C-NMR). The roughness parameters of polished (group A) and HF acid-etched (group B) lithium disilicate glass-ceramic surfaces were measured by optical profilometry (n = 5/group). The interaction of the products with group A and B ceramic surfaces was examined by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The shear strength (SBS) of a flowable composite bonded to the ceramic surfaces (groups A, B) was assessed before (NS) and after silane treatment (n = 20/group, product). RESULTS: The NMR analysis showed the presence of silanol monomers only in CS. Methoxylated-siloxane adducts were found in GM, silanol-siloxane adducts in MB, SB, and siloxane polymers in KS. Acid-etching greatly increased Sa, Sz, Sdr, Sc and Sv parameters (p < 0.001) and ATR-FTIR analysis demonstrated evidence of bonding with the substrate in CS. Weibull analysis of SBS revealed the following rankings in characteristic life (p < 0.05): CS > SB,KS,MB > GM > NS (group A) and CS > GM > SB,KS,MB,NS (group B). The most reliable treatment in both groups was CS. For the same silane treatment, the SBS of group B were significantly higher from group A. Failures were mainly of adhesive type, except of several partial resin cohesive failures found in group B. SIGNIFICANCE: The chemical bonding capacity of the silanes was highest in products with silanol monomers. Acid-etching increased bond strength to a level that neutralized the silane contribution in products with silanol-siloxane adducts and siloxane polymers, providing thus bond strength values similar to silane-free treatments.

Universal Adhesives: Setting Characteristics and Reactivity with Dentin.

The aim of the study was to evaluate the performance of six commercially available universal dental adhesives: Adhese Universal (ADU), All-Bond Universal (ABU), Clearfil Universal Bond Quick (CBQ), G-Premio Bond (GPB), Prelude One (PRO) and Scotchbond Universal (SBU). The properties tested were: (a) degree of C=C conversion (DC%); (b) Vickers micro-hardness (VHN); (c) extent of oxygen inhibition (OI/µm), all related with the adhesive film properties; (d) extent of dentin demineralisation (DM%), insoluble salt formation (AS%); and (e) shear bond strength (SBS, self-etch mode) related to the adhesive-dentin interactions. Statistical analysis (α = 0.05) was performed by one-way ANOVA and Tukey's test (DC%, VHN, OI, DM% AS%) and Weibull analysis (SBS, σ0-ß). The DC ranged from 67.2-82.5% (all >GPB), OI from 5.6-18.6 µm (SBU > ADU, GPB, ABU > CBQ > PRO), microhardness from 1.1-6.6 VHN (SBU > ADU > ABU > CBQ > PRO > GPB: not measurable), DM from 69.3% (GPB) to 16-12.5% (CBQ, SBU, ADU) and 13.2-10.6% (ABU, ADU, PRO), in homogeneous groups and AS from 26-15.9% (ABU, CBQ > GPB, PRO, ADU, SBU). For SBS the σ0 (characteristic life) ranged from 29.3-16.6 MPa (CBQ, ADU, ABU, SBU > PRO > GPB), the ß (reliability) from 5.1-9.7 (p > 0.05). All failure modes were of mixed type (adhesive and composite cohesive). Although all these adhesives were based on the 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) adhesive monomer, the different co-monomers, solvents and catalysts led to variations in their film properties, reactivity and bonding capacity with dentin.

Single copy/knock-in models of ALS SOD1 in C. elegans suggest loss and gain of function have different contributions to cholinergic and glutamatergic neurodegeneration.

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) lead to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that disproportionately affects glutamatergic and cholinergic motor neurons. Previous work with SOD1 overexpression models supports a role for SOD1 toxic gain of function in ALS pathogenesis. However, the impact of SOD1 loss of function in ALS cannot be directly examined in overexpression models. In addition, overexpression may obscure the contribution of SOD1 loss of function in the degeneration of different neuronal populations. Here, we report the first single-copy, ALS knock-in models in C. elegans generated by transposon- or CRISPR/Cas9- mediated genome editing of the endogenous sod-1 gene. Introduction of ALS patient amino acid changes A4V, H71Y, L84V, G85R or G93A into the C. elegans sod-1 gene yielded single-copy/knock-in ALS SOD1 models. These differ from previously reported overexpression models in multiple assays. In single-copy/knock-in models, we observed differential impact of sod-1 ALS alleles on glutamatergic and cholinergic neurodegeneration. A4V, H71Y, G85R, and G93A animals showed increased SOD1 protein accumulation and oxidative stress induced degeneration, consistent with a toxic gain of function in cholinergic motor neurons. By contrast, H71Y, L84V, and G85R lead to glutamatergic neuron degeneration due to sod-1 loss of function after oxidative stress. However, dopaminergic and serotonergic neuronal populations were spared in single-copy ALS models, suggesting a neuronal-subtype specificity previously not reported in invertebrate ALS SOD1 models. Combined, these results suggest that knock-in models may reproduce the neurotransmitter-type specificity of ALS and that both SOD1 loss and gain of toxic function differentially contribute to ALS pathogenesis in different neuronal populations.

Stability and reactivity of γ-ΜPTMS silane in some commercial primer and adhesive formulations.

OBJECTIVES: To evaluate the stability and reactivity of γ-methacryloxypropyl trimethoxysilane (MPTMS) in commercially available primers and adhesives. METHODS: Four representative primer formulations [Calibra Silane Coupling Agent/Dentsply (CLB), G-Multi Primer/GC (GMP), Kerr Silane Primer/Kerr (KSP), Monobond Plus/Ivoclar Vivadent (MBP)] and a universal adhesive [Scotchbond Universal/3M ESPE (SBU)] containing MPTMS were analyzed spectroscopically. For the stability study, the silanol content was evaluated in bulk solutions as received (reference-RE) and after aging (AG, 48°C/1month) by 1H, 13C, 31P NMR and in fresh films by transmission FTIR analysis (TIR, films applied on Ge windows after solvent evaporation). The reactivity, as expressed by the siloxane formation capacity of the RE products, was evaluated by micro-multiple internal reflectance FTIR analysis (MIR, films applied on Ge crystals) after drying and ethanol rinsing (t0) and following 1 (t1) and 24h (t24) storage (air/37°C). RESULTS: NMR and TIR showed ∼60% MPTMS silanol groups in RE-CLB, with the other (∼40%) groups being methylated or ethoxylated. In AG-CLB, the silanol peaks further decreased, while ethoxylation and siloxane derivatization increased. In all other products and aging conditions no silanols were traced and formation of small- and large-size MPTMS derivatives was evident. Apart from the 10-MDP molecule, phosphorous impurities were identified in all RE specimens (2-5%), which after AG reached a maximum value of 15% (MBP). MIR analysis showed siloxane formation in all products, regardless the presence of free silanols (t1), which further increased at t24 especially in CLB, GMP and MBP. SIGNIFICANCE: MPTMS silanols are very sensitive to mild thermal aging. Incorporation of MPTMS in the same vials with adhesive and conventional methacrylate monomers, results in derivatization with no detectable silanols, even in fresh materials. The condensates formed may induce additional siloxane formation due to residual activity, which greatly varies among the materials tested. These may have a detrimental effect on MPTMS silanol chemisorption and bonding capacity.

Effect of tribochemical treatments and silane reactivity on resin bonding to zirconia.

OBJECTIVE: The aim of the study was to assess the roughness, structure and bond strength with zirconia of four grit-blasting treatments combined with three silane types, the reactivity of which was evaluated, as well. METHODS: The grit-blasted treatments performed on zirconia (Lava) were alumina (ALU), CoJet (COJ), SilJet (SLJ) and SilJet Plus (SJP, with silica-encapsulated silane). The other two silanes selected were the S-Bond (SB, prehydrolyzed) and Clearfil Ceramic Primer Plus (CP, prehydrolyzed with 10-MDP). The activity of the silanols in the silanes was evaluated by FTIR spectroscopy. Optical profilometry and Raman microspectroscopy were used for the assessment of roughness (Sa, Sz, Sdr parameters) and structure (monoclinic volume-Vm) of zirconia, before (REF) and after grit-blasting, and a shear bond strength (SBS) with a flowable resin composite, for the investigation of the bonding capacity of the treatments. RESULTS: Only SB demonstrated reactive silanols. CP and the SJP silanes were mostly in a polymerized siloxane state. Roughness was increased after grit-blasting as follows: ALU>SLJ,SJP>COJ>REF (Sa,Sz) and ALU>SLJ,COJ,SJP>REF (Sdr). ALU demonstrated the highest Vm (7.52%) from all other treatments (4.16-4.81%) and the REF (0%). COJ and SLJ showed the highest SBS (14-15.94MPa) regardless of the silane type used. SJP showed no significant differences from SLJ-SB and COJ-SB. Weibull analysis showed a reliability (ß) ranking of COJ, SJP, SLJ, ALU-CP>ALU-SB>REF and a characteristic life (η) ranking of COJ, SLJ, ≥SLJ-SB, SJP, ALU≥ALU-SB,REF-CP>REF-SB. SIGNIFICANCE: The reactivity of the silanes used showed great variations to support a predictable effect in all treatments. CP with deactivated silanols demonstrated a) the most reliable and strongest treatment with a silica-rich powder (COJ), despite the lowest Sa,Sz substrate values and b) high strength with a low-silica powder (SLJ) with higher Sa,Sz substrate values. Therefore, it may be concluded that 10-MDP greatly contributes to the bonding mechanism of the silane containing primers.

PARK2 Depletion Connects Energy and Oxidative Stress to PI3K/Akt Activation via PTEN S-Nitrosylation.

PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely unknown. Here we show that PARK2 is altered in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitric oxide synthase (eNOS), enhanced levels of reactive oxygen species, and a concomitant increase in oxidized nitric oxide levels, thereby promoting the inhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylation in supporting cell survival and proliferation under conditions of energy deprivation.