Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.

Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT.

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.

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.

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.

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.

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.

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis.

Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca2+-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.

Decreased function of survival motor neuron protein impairs endocytic pathways.

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.

The neuroprotective drug riluzole acts via small conductance Ca2+-activated K+ channels to ameliorate defects in spinal muscular atrophy models.

Spinal muscular atrophy (SMA), a recessive neuromuscular disorder, is caused by diminished function of the Survival Motor Neuron (SMN) protein. To define the cellular processes pertinent to SMA, parallel genetic screens were undertaken in Drosophila and Caenorhabditis elegans SMA models to identify modifiers of the SMN loss of function phenotypes. One class of such genetic modifiers was the small conductance, Ca(2+)-activated K(+) (SK) channels. SK channels allow efflux of potassium ions when intracellular calcium increases and can be activated by the neuroprotective drug riluzole. The latter is the only drug with proven, albeit modest, efficacy in the treatment of amyotrophic lateral sclerosis. It is unclear if riluzole can extend life span or ameliorate symptoms in SMA patients as previous studies were limited and of insufficient power to draw any conclusions. The critical biochemical target of riluzole in motor neuron disease is not known, but the pharmacological targets of riluzole include SK channels. We examine here the impact of riluzole in two different SMA models. In vertebrate neurons, riluzole treatment restored axon outgrowth caused by diminished SMN. Additionally, riluzole ameliorated the neuromuscular defects in a C. elegans SMA model and SK channel function was required for this beneficial effect. We propose that riluzole improves motor neuron function by acting on SK channels and suggest that SK channels may be important therapeutic targets for SMA patients.

PARK2 deletions occur frequently in sporadic colorectal cancer and accelerate adenoma development in Apc mutant mice.

In 100 primary colorectal carcinomas, we demonstrate by array comparative genomic hybridization (aCGH) that 33% show DNA copy number (DCN) loss involving PARK2, the gene encoding PARKIN, the E3 ubiquitin ligase whose deficiency is responsible for a form of autosomal recessive juvenile parkinsonism. PARK2 is located on chromosome 6 (at 6q25-27), a chromosome with one of the lowest overall frequencies of DNA copy number alterations recorded in colorectal cancers. The PARK2 deletions are mostly focal (31% approximately 0.5 Mb on average), heterozygous, and show maximum incidence in exons 3 and 4. As PARK2 lies within FRA6E, a large common fragile site, it has been argued that the observed DCN losses in PARK2 in cancer may represent merely the result of enforced replication of locally vulnerable DNA. However, we show that deficiency in expression of PARK2 is significantly associated with adenomatous polyposis coli (APC) deficiency in human colorectal cancer. Evidence of some PARK2 mutations and promoter hypermethylation is described. PARK2 overexpression inhibits cell proliferation in vitro. Moreover, interbreeding of Park2 heterozygous knockout mice with Apc(Min) mice resulted in a dramatic acceleration of intestinal adenoma development and increased polyp multiplicity. We conclude that PARK2 is a tumor suppressor gene whose haploinsufficiency cooperates with mutant APC in colorectal carcinogenesis.

Conserved genes act as modifiers of invertebrate SMN loss of function defects.

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

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.

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.

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 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.

Neurodegenerative disorders: insights from the nematode Caenorhabditis elegans.

Neurodegenerative diseases impose a burden on society, yet for the most part, the mechanisms underlying neuronal dysfunction and death in these disorders remain unclear despite the identification of relevant disease genes. Given the molecular conservation in neuronal signaling pathways across vertebrate and invertebrate species, many researchers have turned to the nematode Caenorhabditis elegans to identify the mechanisms underlying neurodegenerative disease pathology. C. elegans can be engineered to express human proteins associated with neurodegeneration; additionally, the function of C. elegans orthologs of human neurodegenerative disease genes can be dissected. Herein, we examine major C. elegans neurodegeneration models that recapitulate many aspects of human neurodegenerative disease and we survey the screens that have identified modifier genes. This review highlights how the C. elegans community has used this versatile organism to model several aspects of human neurodegeneration and how these studies have contributed to our understanding of human disease.

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.

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.