Multimethod analysis of large- and low-tapered single file reciprocating instruments: Design, metallurgy, mechanical performance, and irrigation flow.

AIM: To compare eight large- and low-tapered heat-treated reciprocating instruments regarding their design, metallurgy, mechanical properties, and irrigation flow through an in silico model. METHODOLOGY: A total of 472 new 25-mm E-Flex Rex (25/.04 and 25/.06), Excalibur (25/.05), Procodile (25/.06), Reciproc Blue R25 (25/.08v), WaveOne Gold Primary (25/.07v), and Univy Sense (25/.04 and 25/.06) instruments were evaluated regarding their design (stereomicroscopy, scanning electron microscopy, and 3D surface scanning), metallurgy (energy-dispersive X-ray spectroscopy and differential scanning calorimetry), and mechanical performance (cyclic fatigue, torsional resistance, cutting ability, bending and buckling resistance). Computational fluid dynamics assessment was also conducted to determine the irrigation flow pattern, apical pressure, and wall shear stress in simulated canal preparations. Kruskal-Wallis and one-way anova post hoc Tukey tests were used for statistical comparisons (α = 5%). RESULTS: Instruments presented variations in blade numbers, helical angles, and tip designs, with all featuring non-active tips, symmetrical blades, and equiatomic nickel-titanium ratios. Cross-sectional designs exhibited an S-shaped geometry, except for WaveOne Gold. Univy 25/.04 and Reciproc Blue displayed the smallest and largest core diameters at D3. Univy 25/.04 and E-Flex Rec 25/.04 demonstrated the longest time to fracture (p < .05). Reciproc Blue and Univy 25/.04 exhibited the highest and lowest torque to fracture, respectively (p < .05). Univy 25/.04 and Reciproc Blue had the highest rotation angles, whilst E-Flex Rec 25/.06 showed the lowest angle (p < .05). The better cutting ability was observed with E-Flex Rec 25/.06, Procodile, Excalibur, and Reciproc Blue (p > .05). Reciproc R25 and E-Flex Rec showed the highest buckling resistance values (p < .05), with WaveOne Gold being the least flexible instrument. The impact of instruments' size and taper on wall shear stress and apical pressure did not follow a distinct pattern, although Univy 25/.04 and E-Flex Rec 25/.06 yielded the highest and lowest values for both parameters, respectively. CONCLUSIONS: Low-tapered reciprocating instruments exhibit increased flexibility, higher time to fracture, and greater angles of rotation, coupled with reduced maximum bending loads and buckling strength compared to large-tapered instruments. Nevertheless, low-tapered systems also exhibit lower maximum torque to fracture and inferior cutting ability, contributing to a narrower apical canal enlargement that may compromise the penetration of irrigants in that region.

Diagnostic value of cone beam computed tomography for root canal morphology assessment - a micro-CT based comparison.

OBJECTIVES: The aim of this study was to assess cone beam computed tomography (CBCT) as a root canal anatomy diagnostic tool by comparison with micro-CT gold-standard. MATERIALS AND METHODS: 216 two-rooted mandibular molars were first scanned in a CBCT device (200 µm voxel size) and posteriorly in a micro-CT scanner (19.61 µm). The volumes were sequentially screened to classify main root canal anatomy according to Vertucci classification, and for the presence of lateral canals and apical deltas, in both mesial and distal roots. RESULTS: Both methods revealed a higher prevalence of Vertucci Type II and IV in the mesial root, and Vertucci Type I in the distal root. The percentage of agreement for main root canal anatomy classification between CBCT and micro-CT scores was high (85.2%). CONCLUSION: Sensibility to detect both lateral canals and apical deltas with CBCT was low. These results attest to the fact that minor anatomical changes might be difficult to identify with CBCT imaging, hampering its diagnostic value.

MBNL-dependent impaired development within the neuromuscular system in myotonic dystrophy type 1.

AIMS: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1. METHODS: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells. RESULTS: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1. CONCLUSIONS: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays.

Characterization of the file-specific heat-treated ProTaper Ultimate rotary system.

AIM: To compare design, metallurgy and mechanical performance of the ProTaper (PT) Ultimate system with instruments of similar dimensions from the ProGlider, PT Gold and PT Universal systems. METHODOLOGY: New PT Ultimate instruments (n = 248) were compared with instruments of similar dimensions from ProGlider (n = 31), PT Gold (n = 155) and PT Universal (n = 155) systems regarding their number of spirals, helical angle, blade symmetry, tip geometry, surface finishing, nickel/titanium ratio, phase transformation temperatures and mechanical performance. One-way anova and nonparametric Mood's median tests were used for statistical comparison (α = 5%). RESULTS: All instruments had symmetrical blades without radial lands or flat sides, similar surface finishing and an almost equiatomic nickel/titanium ratio, whilst the number of spirals, helical angles and the tip geometry were different. PT Ultimate instruments showed 3 distinct heat treatments that matched with the colour of their metal wire. Slider and ProGlider instruments had similar R-phase start (Rs) and R-phase finish (Rf) temperatures. SX, F1, F2, F3 and Shaper instruments showed equivalent heat treatments (Rs ~45.6°C and Rf ~28.3°C) that were similar to their PT Gold counterparts (Rs ~47.9°C and Rf ~28.2°C), but completely distinct to the PT Universal ones (Rs ~16.2°C and Rf ~-18.2°C). Amongst the PT Ultimate instruments, the lowest maximum torques were observed in the SX (0.44 N cm), Slider (0.45 N cm) and Shaper (0.60 N cm) instruments, whilst the highest was noted in the FXL (4.90 N cm). PT Ultimate Slider and ProGlider had similar torsional (~0.40 N cm) and bending loads (~145.0 gf) (p = 1.000), whilst the other PT Ultimate instruments showed statistically significantly lower maximum torque, higher angle of rotation and lower bending load (higher flexibility) than their counterparts of the PT Universal and PT Gold systems. CONCLUSIONS: The PT Ultimate system comprises instruments with 3 distinct heat treatments that showed similar phase transformation temperatures to their heat-treated analogues. PT Ultimate instruments presented lower torsional strength and superior flexibility than their counterparts, whilst maximum torque, angle of rotation and bending loads progressively increased with their sizes.

Characterization of four heat-treated reciprocating instruments: Design, metallurgy, mechanical performance, and irrigation flow patterns.

AIM: To compare the design, metallurgy, and mechanical properties of four heat-treated reciprocating instruments coupled with the evaluation of the irrigation flow using an in silico model. METHODOLOGY: New EdgeOne Fire Primary, Easy-File Flex Regular 25, WaveOne Gold Primary and Reciproc Blue R25 instruments (n = 124) were initially evaluated regarding their design through stereomicroscopy, scanning electron microscopy and 3D surface scanning. In addition, energy-dispersive X-ray spectroscopy was utilized to determine their elemental composition, and differential scanning calorimetry tests to evaluate their phase transformation temperatures. Their mechanical performance was further assessed through torsional and bending tests. Using scans obtained from a real tooth and the instruments, a computational fluid dynamics assessment was conducted to determine the irrigation flow pattern, apical pressure, and wall shear stress in simulated canal preparation. Mood's median and One-way anova post hoc Tukey tests were used for statistical comparisons (α = 5%). RESULTS: Reciproc Blue exhibited a superior number of blades (n = 8), whereas EdgeOne Fire had the highest overall volume (4.38 mm3 ) and surface area (32.32 mm2 ). At the 3-mm axial level, EdgeOne Fire displayed the lowest core diameter (0.13 mm), while Reciproc Blue had the highest (0.16 mm). All blades were symmetrical, and the tips of the instruments were non-active but differed from each other. The most irregular surfaces were observed in EdgeOne Fire and Easy-File Flex. All instruments were manufactured from nickel-titanium alloys and exhibited distinct phase transformation temperatures. WaveOne Gold and Reciproc Blue demonstrated the highest maximum torque values (1.87 and 1.62 N cm, respectively), while the lowest was observed on EdgeOne Fire (1.21 N cm) (p < .05). The most flexible (p < .05) were EdgeOne Fire (angle of rotation: 602.6°; maximum bending load: 251.4 g.f) and Reciproc Blue (533.2° and 235.6 g.f). There were no significant differences observed in the irrigation flow among the four domains generated by the tested instruments. CONCLUSIONS: Despite observing variations in the design, phase transformation temperatures, and in the torsional and bending test outcomes among the four heat-treated reciprocating instruments, no significant differences were found in the irrigation flow pattern among the different groups in the simulated root canal preparations.

Experimental validation of a computational fluid dynamics model using micro-particle image velocimetry of the irrigation flow in confluent canals.

AIM: This study aimed to experimentally validate a computational fluid dynamics (CFD) model, using micro-particle image velocimetry (micro-PIV) measurements of the irrigation flow velocity field developed in confluent canals during irrigation with a side-vented needle. METHODOLOGY: A microchip with confluent canals, manufactured in polydimethylsiloxane was used in a micro-PIV analysis of the irrigation flow using a side-vented needle placed 3 mm from the end of the confluence of the canals. Velocity fields and profiles were recorded for flow rates of 0.017 and 0.1 ml/s and compared with those predicted in CFD numerical simulations (using a finite volume commercial code - FLUENT) for both laminar and turbulent regimes. RESULTS: The overall flow pattern, isovelocity and vector maps as well as velocity profiles showed a close agreement between the micro-PIV experimental and CFD predicted data. No relevant differences were observed between the results obtained with the laminar and turbulent flow models used. CONCLUSIONS: Results showed that the laminar CFD modelling is reliable to predict the flow in similar domains.

DM1 Transgenic Mice Exhibit Abnormal Neurotransmitter Homeostasis and Synaptic Plasticity in Association with RNA Foci and Mis-Splicing in the Hippocampus.

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease mediated by a toxic gain of function of mutant RNAs. The neuropsychological manifestations affect multiple domains of cognition and behavior, but their etiology remains elusive. Transgenic DMSXL mice carry the DM1 mutation, show behavioral abnormalities, and express low levels of GLT1, a critical regulator of glutamate concentration in the synaptic cleft. However, the impact of glutamate homeostasis on neurotransmission in DM1 remains unknown. We confirmed reduced glutamate uptake in the DMSXL hippocampus. Patch clamp recordings in hippocampal slices revealed increased amplitude of tonic glutamate currents in DMSXL CA1 pyramidal neurons and DG granule cells, likely mediated by higher levels of ambient glutamate. Unexpectedly, extracellular GABA levels and tonic current were also elevated in DMSXL mice. Finally, we found evidence of synaptic dysfunction in DMSXL mice, suggestive of abnormal short-term plasticity, illustrated by an altered LTP time course in DG and in CA1. Synaptic dysfunction was accompanied by RNA foci accumulation in localized areas of the hippocampus and by the mis-splicing of candidate genes with relevant functions in neurotransmission. Molecular and functional changes triggered by toxic RNA may induce synaptic abnormalities in restricted brain areas that favor neuronal dysfunction.

Morphometric analysis of nerve fibers in neural leprosy.

BACKGROUND: The present study assesses the contributions of axonal degeneration and demyelination in leprosy nerve damage. New clinical strategies can emerge from an in-depth understanding of the pathogenesis of neural leprosy (NL). METHODS: Morphometric analysis of myelinated nerve fibers was performed on 44 nerve biopsy samples collected from leprosy patients. Measures of density, diameter distribution, g-ratios, and the counting of axonal ovoids on the myelinated fibers were taken and compared to those in the control group. RESULTS: The proportion of small myelinated fibers increased in the leprosy group while large fiber frequency decreased. Indicative of axonal atrophy, the g-ratio was lower in the leprosy group. The frequency of axonal ovoids was identical to that found in the non-leprosy neuropathies. CONCLUSIONS: Axonal atrophy, Wallerian degeneration, and demyelination coexist in NL. Axonal degeneration predominates over demyelination in the chronic course of the disease; however, this may change during leprosy reactive episodes. This study regards demyelination and axon degeneration as concurrent mechanisms of damage to nerve fibers in leprosy. It also calls into question the view that demyelination is the primary and predominant mechanism in the complex pathogeny of NL.

Squaraine dyes as serum albumins probes: Synthesis, photophysical experiments and molecular docking studies.

Three barbiturate squaraine dyes derived from indolenine or benzothiazole, with different barbituric acid derivatives were prepared, characterized and photophysically evaluated by standard spectroscopic methods. As expectable for squaraines, these dyes showed narrow and intense absorption and emission bands in the Vis/NIR region. The interaction of synthesized dyes with bovine and human serum albumins (BSA and HSA) was also evaluated in phosphate buffer (PB). The results revealed that upon the addition of BSA or HSA the complex dye-protein emit more fluorescence, and the emission intensity is directly proportional to the concentration of protein used (0-3.5 µM). The titration tests allowed to calculate the binding constants, in an order of magnitude of 104-106 M, as well as the limits of detection and quantification in the nanomolar tens range. All dyes showed a good response to the interaction with both proteins, but the most pronounced envisioning their use as protein labeling was observed for the squaraine dye derived from the indolenine with a 1,3-dimethylbarbituric acid moiety. The molecular docking studies revealed the existence of a binding between the compounds and four sites on the HSA molecule, where one of these four locations is a new binding site with which this series of dye interacts.

In vivo and in vitro toxicological evaluations of aqueous extract from Cecropia pachystachya leaves.

CECROPIA PACHYSTACHYA: leaves are popularly used to treat asthma and diabetes. Despite the widespread consumption of this plant, there are few scientific studies regarding its toxicological potential. In order to conduct a thorough study concerning the potential adverse effects, the aim of this study was to assess acute and subacute toxicity tests of crude aqueous extract from C. pachystachya leaves (CAE-Cp) using in vivomodel, as well as in vitro cytotoxicity, genotoxicity and antioxidant activity. In addition, genotoxicity, and cytotoxicity of chlorogenic acid (CGA) and cytotoxicity of isoorientin (ISOO) were also evaluated. The antioxidant activity was verified by DPPH, cytotoxicity using sulforhodamine B (SRB) assay and genotoxicity by comet assay on V79 cells. The phytochemical analysis of CAE-Cp detected flavonoids and tannins, CGA and ISOO as the major compounds utilizing HPLC. The total flavonoid content (6.52 mg/g EQ) and antioxidant activity (EC50 = 62.15 µg/ml) of CAE-Cp were determined. In vitro evaluations with CAE-Cp showed genotoxic effects at 0.31 to 2.5 mg/ml and an expressive cytotoxicity on HT-29 (IC50 = 4.43 µg/ml) cells. CGA was genotoxic against V79 cells at 0.07 mg/ml and cytotoxic against to HT-29 (IC50 = 71.70 µg/ml), OVCAR-3 (IC50 = 80.07 µg/ml), MCF-7 (IC50 = 45.58 µg/ml) and, NCI-H460 (IC50 = 71.89 µg/ml) cancer cell lines. Wistar rats treated with a single dose (2,000 mg/kg) CAE-Cp decreased hemoglobin levels after 14 days, although no significant toxicity was observed in animals after 28 days. In view of the in vitro cytotoxicity and genotoxicity detected, further studies are necessary to establish the safe use of CAE-Cp.

Study of transcranial photobiomodulation at 945-nm wavelength: anxiety and depression.

Transcranial photobiomodulation is an innovative method for the stimulation of neural activity which consists of the exposure of neural tissue to low-level light irradiance. In the present study, light-emitting diodes (LEDs) were used as light source due to their practicality and low cost. The objective was to analyze the effects of transcranial photobiomodulation using 945-nm LED in university students with anxiety and depression. Sample was composed of 22 individuals (17-25 years of age) divided into 2 groups of 11. LED group was treated with 945-nm LEDs for 1 min and 25 s (9.35 J/cm2), while in the placebo group, the device was off when placed in contact with the frontal bone for the same amount of time as in treatment group. Participants were evaluated at baseline and after 30 days with the hospital anxiety and depression scale (HADS), the faces test, the designs test, and the grip strength test. On the HADS for anxiety, the mean PAB, PAA, PhAB, and PhAA were 13.89 ± 3.55, 12.82 ± 3.18, 10.75 ± 2.49, and 6.66 ± 2.50 points, respectively. In the HADS for depression, the mean for the PDB group was 13.89 ± 3.55 points, in the PhDB group 12.82 ± 3.18 points, in the PDA group 10.75 ± 2.49 points, and in the PhDA group 6.66 ± 2.50 points. In the PA and PD groups, mean values of 8.0 ± 1.5 and 8.9 ± 1.26 scores were obtained, but did not reach significance; however, between PA and PhD analysis, a significance level of p = 0.0003 was obtained. The 945-nm LED transcranial photobiomodulation improves brain activity and may clinically decrease anxiety and depression.

Evaluation of the color and translucency of glass-infiltrated zirconia based on the concept of functionally graded materials.

STATEMENT OF PROBLEM: Infiltrated zirconia has promising mechanical properties. However, information about its optical behavior is scarce. PURPOSE: The purpose of this in vitro study was to evaluate the color and translucency of zirconia submitted to infiltration and aging. MATERIAL AND METHODS: Sixty zirconia disks were machined. Ten disks received no treatment (NT group), 10 disks were immersed in a coloring liquid (A2 group), and 10 disks were immersed in a fluorescent liquid (F group). The other 30 disks were submitted to the same treatments plus glass infiltration (NT+I, A2+I, and F+I groups). The coordinates L*, a*, and b* and the Y tristimulus values were obtained to calculate the color (ΔE00), lightness, chroma, and hue differences; the translucency parameter (TP); and the contrast ratio (CR) associated with the specimens. After aging in an autoclave for 4 hours (T1), new measurements were made. Two- and 3-way ANOVAs were used to analyze color differences, TP, and CR. The lightness, chroma, and hue differences were evaluated by a repeated measures ANOVA. Multiple comparisons were made with the Tukey honestly significant difference (HSD) test (α=.05). RESULTS: The greatest color differences were observed in the A2+I group (11.23 ΔE00) (P<.001). Aging affected the chroma of the colored groups (P=.013 and P=.001) but did not affect their translucency (P=.347 for TP and P=.132 for CR). The greatest TP values were found in the NT and NT+I groups (2.54 and 2.34, respectively), whereas the CR was equal to or close to 1 in all groups. CONCLUSIONS: Color differences were observed in the glass-infiltrated groups. The TP and CR were affected by infiltration. Aging did not influence the optical behavior of the specimens.

Fluorescence of natural teeth and restorative materials, methods for analysis and quantification: A literature review.

OBJECTIVE: This review integrates published scientific information about the fluorescence of natural teeth, dental resins and ceramics, and the main methods of analysis and quantification presented in the literature. OVERVIEW: Fluorescence is an emission of light (photons) by a substance that has absorbed light of higher energy. In natural teeth, it is more intense in the dentin than in the enamel and presents a bluish-white color. In dental resins and ceramics, fluorescence is obtained by the incorporation of materials that contain rare-earth luminescence centers (more precisely lanthanide luminescence centers), which allows these artificial materials to simulate natural teeth in a more beautiful and vital-looking way. However, the lack of knowledge about this optical phenomenon on the part of professionals indicates the need for more scientific studies and dissemination on this topic. CONCLUSIONS: Aesthetic materials have variable spectral compositions and fluorescence intensities, which are not always compatible with natural teeth. The fluorescence of teeth and restorative materials can be influenced by several factors, such as aging, temperature, and bleaching. Several devices for fluorescence evaluation and quantification are used in studies under different methodologies, but the small number of studies on the subject make it difficult to compare their results. CLINICAL SIGNIFICANCE: Fluorescence is a fundamental optical property for aesthetic rehabilitations since its presence and intensity in the restorative materials allows achieving an aesthetic result much closer to reality. However, the fluorescent behavior of natural teeth and aesthetic restorative materials is not yet fully understood by researchers and clinicians. Greater understanding of this phenomenon will contribute to the selection, indication, and clinical use of these materials.

The neXtProt knowledgebase on human proteins: current status.

neXtProt (http://www.nextprot.org) is a human protein-centric knowledgebase developed at the SIB Swiss Institute of Bioinformatics. Focused solely on human proteins, neXtProt aims to provide a state of the art resource for the representation of human biology by capturing a wide range of data, precise annotations, fully traceable data provenance and a web interface which enables researchers to find and view information in a comprehensive manner. Since the introductory neXtProt publication, significant advances have been made on three main aspects: the representation of proteomics data, an extended representation of human variants and the development of an advanced search capability built around semantic technologies. These changes are presented in the current neXtProt update.

Transcranial LED therapy on amyloid-ß toxin 25-35 in the hippocampal region of rats.

Excessive Aß deposition in the brain is associated with the formation of senile plaques, and their diffuse distribution is related to Alzheimer's disease. Thirty rats (EG) were irradiated with light-emitting diode (photobiomodulation (PBM)) in the frontal region of the skull after being inoculated with the Aß toxin in the hippocampus; 30 rats were used as the control group (CG). The analysis was conducted at 7, 14, and 21 days after irradiation. We observed a decreased in Aß deposits in treated animals compared with animals in the CG. The behavioral and motor assessment revealed that the EG group covered a larger ground distance and explored the open field than the CG group on days 14 and 21 (p < 0.05). The EG group was statistically significant in the spatial memory test compared to the CG group on day 14. The use of PBM significantly reduced the presence of Aß plaques and improved spatial memory and behavioral and motor skills in treated animals on day 21.

Disease-associated CAG·CTG triplet repeats expand rapidly in non-dividing mouse cells, but cell cycle arrest is insufficient to drive expansion.

Genetically unstable expanded CAG·CTG trinucleotide repeats are causal in a number of human disorders, including Huntington disease and myotonic dystrophy type 1. It is still widely assumed that DNA polymerase slippage during replication plays an important role in the accumulation of expansions. Nevertheless, somatic mosaicism correlates poorly with the proliferative capacity of the tissue and rates of cell turnover, suggesting that expansions can occur in the absence of replication. We monitored CAG·CTG repeat instability in transgenic mouse cells arrested by chemical or genetic manipulation of the cell cycle and generated unequivocal evidence for the continuous accumulation of repeat expansions in non-dividing cells. Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells. These data are consistent with a major role for cell division-independent expansion in generating somatic mosaicism in vivo. Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability. Our data reveal that de novo expansion events are not limited to S-phase and further support a cell division-independent mutational pathway.

Molecular, physiological, and motor performance defects in DMSXL mice carrying >1,000 CTG repeats from the human DM1 locus.

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. DMPK transcripts carrying CUG expansions form nuclear foci and affect splicing regulation of various RNA transcripts. Furthermore, bidirectional transcription over the DMPK gene and non-conventional RNA translation of repeated transcripts have been described in DM1. It is clear now that this disease may involve multiple pathogenic pathways including changes in gene expression, RNA stability and splicing regulation, protein translation, and micro-RNA metabolism. We previously generated transgenic mice with 45-kb of the DM1 locus and >300 CTG repeats (DM300 mice). After successive breeding and a high level of CTG repeat instability, we obtained transgenic mice carrying >1,000 CTG (DMSXL mice). Here we described for the first time the expression pattern of the DMPK sense transcripts in DMSXL and human tissues. Interestingly, we also demonstrate that DMPK antisense transcripts are expressed in various DMSXL and human tissues, and that both sense and antisense transcripts accumulate in independent nuclear foci that do not co-localize together. Molecular features of DM1-associated RNA toxicity in DMSXL mice (such as foci accumulation and mild missplicing), were associated with high mortality, growth retardation, and muscle defects (abnormal histopathology, reduced muscle strength, and lower motor performances). We have found that lower levels of IGFBP-3 may contribute to DMSXL growth retardation, while increased proteasome activity may affect muscle function. These data demonstrate that the human DM1 locus carrying very large expansions induced a variety of molecular and physiological defects in transgenic mice, reflecting DM1 to a certain extent. As a result, DMSXL mice provide an animal tool to decipher various aspects of the disease mechanisms. In addition, these mice can be used to test the preclinical impact of systemic therapeutic strategies on molecular and physiological phenotypes.

RNA toxicity in human disease and animal models: from the uncovering of a new mechanism to the development of promising therapies.

Mutant ribonucleic acid (RNA) molecules can be toxic to the cell, causing human disease through trans-acting dominant mechanisms. RNA toxicity was first described in myotonic dystrophy type 1, a multisystemic disorder caused by the abnormal expansion of a non-coding trinucleotide repeat sequence. The development of multiple and complementary animal models of disease has greatly contributed to clarifying the complex disease pathways mediated by toxic RNA molecules. RNA toxicity is not limited to myotonic dystrophy and spreads to an increasing number of human conditions, which share some unifying pathogenic events mediated by toxic RNA accumulation and disruption of RNA-binding proteins. The remarkable progress in the dissection of disease pathobiology resulted in the rational design of molecular therapies, which have been successfully tested in animal models. Toxic RNA diseases, and in particular myotonic dystrophy, clearly illustrate the critical contribution of animal models of disease in translational research: from gene mutation to disease mechanisms, and ultimately to therapy development. This article is part of a Special Issue entitled: Animal Models of Disease.

Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

Non-ATG-initiated translation directed by microsatellite expansions.

Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.