Affinity Purification-Mass Spectrometry and Single Fiber Physiology/Proteomics Reveals Mechanistic Insights of C18ORF25.

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise in humans. Phosphorylation was shown to improve the ex vivo skeletal muscle contractile function in mice, but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein/protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, which revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localized to the nucleus and the contractile apparatus in the skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled a detailed assessment of various steps in the excitation-contraction pathway including SR calcium handling and force generation, followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein/phenotype associations and 36 fiber-type specific differences, following loss of C18Orf25. Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.

Long-term effects produced by early treatment of Class III malocclusion with rapid maxillary expansion and facemask followed by fixed appliances: A multicentre retro-prospective controlled study.

OBJECTIVE: To assess the short- and long-term dentoskeletal effects of early Class III treatment with rapid maxillary expansion and facemask (RME/FM) followed by fixed appliances. MATERIALS AND METHODS: A total of 44 patients (27 females, 17 males) treated consecutively with RME/FM were included from the archives of 3 centres. Three lateral cephalograms were available: T0 (before the start of RME/FM therapy, mean age 8.1 ± 1.8 years), T1 (immediately after RME/FM, mean age 9.8 ± 1.6 years), and T2 (long-term observation, mean age 19.5 ± 1.6 years). A control group of 17 untreated Class III subjects (12 females and 5 males) also was selected. Between-group statistical comparisons were performed with ANCOVA. RESULTS: No statistically significant differences for any of the cephalometric variables were found at T0. In the short term, the treated group showed significant improvements in ANB (+2.9°), Wits appraisal (+2.7 mm), SNA (+1.8°) and SNB (-1.1°). A significant closure of CoGoMe angle (-1.3°) associated with smaller increments along Co-Gn (-2.4 mm) also was found together with a significant increase in intermaxillary divergence (+1.3°). In the long-term, significant improvements in ANB (+2.6°), Wits appraisal (+2.7 mm) and SNB (-1.7°) were recorded together with a significant closure of the CoGoMe angle (-2.9°). No significant long-term changes in vertical skeletal relationships were found. CONCLUSIONS: RME/FM therapy was effective in improving Class III dentoskeletal relationships in the short term. These changes remained stable in the long-term due mainly to favourable mandibular changes.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction.

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2-/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. Small-angle X-ray diffraction of C2-/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2-/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

A fluorogenic micrococcal nuclease-based probe for fast detection and optical imaging of Staphylococcus aureus in prosthetic joint and fracture-related infections.

PURPOSE: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the 'smart-activatable' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus. METHODS: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria. RESULTS: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min. CONCLUSION: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials.

The benefit of diagnostic whole genome sequencing in schizophrenia and other psychotic disorders.

Schizophrenia has a multifactorial etiology, involving a polygenic architecture. The potential benefit of whole genome sequencing (WGS) in schizophrenia and other psychotic disorders is not well studied. We investigated the yield of clinical WGS analysis in 251 families with a proband diagnosed with schizophrenia (N = 190), schizoaffective disorder (N = 49), or other conditions involving psychosis (N = 48). Participants were recruited in Israel and USA, mainly of Jewish, Arab, and other European ancestries. Trio (parents and proband) WGS was performed for 228 families (90.8%); in the other families, WGS included parents and at least two affected siblings. In the secondary analyses, we evaluated the contribution of rare variant enrichment in particular gene sets, and calculated polygenic risk score (PRS) for schizophrenia. For the primary outcome, diagnostic rate was 6.4%; we found clinically significant, single nucleotide variants (SNVs) or small insertions or deletions (indels) in 14 probands (5.6%), and copy number variants (CNVs) in 2 (0.8%). Significant enrichment of rare loss-of-function variants was observed in a gene set of top schizophrenia candidate genes in affected individuals, compared with population controls (N = 6,840). The PRS for schizophrenia was significantly increased in the affected individuals group, compared to their unaffected relatives. Last, we were also able to provide pharmacogenomics information based on CYP2D6 genotype data for most participants, and determine their antipsychotic metabolizer status. In conclusion, our findings suggest that WGS may have a role in the setting of both research and genetic counseling for individuals with schizophrenia and other psychotic disorders and their families.

Three-dimensional comparison of bone-borne and tooth-bone-borne maxillary expansion in young adults with maxillary skeletal deficiency.

OBJECTIVE: To compare the transverse dental and skeletal changes in patients treated with bone-anchored palatal expander (bone-borne, BB) compared to patients treated with tooth and bone-anchored palatal expanders (tooth-bone-borne, TBB) using cone-beam computer tomography (CBCT) and 3D image analysis. METHODS: The sample comprised 30 patients with transverse maxillary discrepancy treated with two different types of appliances: bone-borne (Group BB) and tooth-bone-borne (Group TBB) expanders. CBCT scans were acquired before (T1) and after completion of maxillary expansion (T2); the interval was 5.4 ± 3.4 and 6.2 ± 2.1 months between the T1 and the T2 scans of Group TBB (tooth-bone-borne) and Group BB (bone-borne), respectively. Transverse, anteroposterior and vertical linear and angular three-dimensional dentoskeletal changes were assessed after cranial base superimposition. RESULTS: Both groups displayed marked transverse skeletal expansion with a greater ratio of skeletal to dental changes. Greater changes at the nasal cavity, zygoma and orbital levels were found in Group BB. A relatively parallel sutural opening in an anterior-posterior direction was observed in Group TBB; however, the Group BB presented a somewhat triangular (V-shaped) opening of the suture that was wider anteriorly. Small downward-forward displacements were observed in both groups. Asymmetric expansion occurred in approximately 50% of the patients in both groups. CONCLUSION: Greater skeletal vs dental expansion ratio and expansion of the circummaxillary regions were found in Group BB, the group in which a bone-borne expander was used. Both groups presented skeletal and dental changes, with a similar amount of posterior palate expansion. Asymmetric expansion was observed in both groups.

Three-dimensional Evaluation of the Carriere Motion 3D Appliance in the treatment of Class II malocclusion.

INTRODUCTION: This study aimed to quantify the outcomes of adolescent patients with Class II malocclusion treated with the Carriere Motion 3D Appliance (CMA) combined with full fixed appliances. METHODS: Cone-beam computed tomography scans of 22 patients were available before orthodontic treatment (T1), at removal of the CMA (T2), and posttreatment (T3). The average age of the patients was 13.5 ± 1.6 years at T1, 14.1 ± 0.2 years at T2, and 15.6 ± 0.5 years at T3. The 3-dimensional image analysis procedures were performed using ITK-SNAP (version 3.6.0; www.itksnap.org, Hatfield, Pa) and SlicerCMF (version 4.11.0; http://www.slicer.org, Cambridge, Mass); skeletal and dentoalveolar changes relative to cranial base, maxillary, and mandibular regional superimpositions were evaluated. RESULTS: Changes were analyzed with 1 sample t tests using the mean differences during the CMA phase (T1 to T2) and total treatment time (T1 to T3). Significant skeletal changes included a slight reduction of ANB from T1 to T3, mandibular growth (Co-Gn increment of 1.2 mm and 3.3 mm from T1 to T2 and T1 to T3, respectively), inferior displacement of point A, and anterior and inferior displacement of point B. The mandibular plane did not change significantly during treatment. During the CMA treatment, posterior tipping and distal rotation of the maxillary molars, tip back and inferior displacement of the maxillary canines, significant mesial rotation, and superior displacement of the mandibular molars were observed. These movements rebounded during the full fixed appliance phase except for the molar and canine vertical displacements. Clinically significant dental changes during treatment included a reduction in overjet and overbite, Class II correction of the molar and canine relationship, and proclination of the mandibular incisors. CONCLUSIONS: The CMA is an effective treatment modality for Class II correction in growing patients because of a combination of mesial movement of the mandibular molar, distal rotation of the maxillary molar, and anterior displacement of the mandible.

Cnksr2 Loss in Mice Leads to Increased Neural Activity and Behavioral Phenotypes of Epilepsy-Aphasia Syndrome.

Epilepsy Aphasia Syndromes (EAS) are a spectrum of childhood epileptic, cognitive, and language disorders of unknown etiology. CNKSR2 is a strong X-linked candidate gene implicated in EAS; however, there have been no studies of genetic models to dissect how its absence may lead to EAS. Here we develop a novel Cnksr2 KO mouse line and show that male mice exhibit increased neural activity and have spontaneous electrographic seizures. Cnksr2 KO mice also display significantly increased anxiety, impaired learning and memory, and a progressive and dramatic loss of ultrasonic vocalizations. We find that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at both excitatory and inhibitory postsynapses. Proteomics analysis reveals Cnksr2 anchors key binding partners at synapses, and its loss results in significant alterations of the synaptic proteome, including proteins implicated in epilepsy disorders. Our results validate that loss of CNKSR2 leads to EAS and highlights the roles of Cnksr2 in synaptic organization and neuronal network activity.SIGNIFICANCE STATEMENT Epilepsy Aphasia Syndromes (EAS) are at the severe end of a spectrum of cognitive-behavioral symptoms seen in childhood epilepsies, and they remain an inadequately understood disorder. The prognosis of EAS is frequently poor, and patients have life-long language and cognitive disturbances. Here we describe a genetic mouse model of EAS, based on the KO of the EAS risk gene Cnksr2 We show that these mice exhibit electrophysiological and behavioral phenotypes similar to those of patients, providing an important new model for future studies of EAS. We also provide insights into the molecular disturbances downstream of Cnksr2 loss by using in vivo quantitative proteomics tools.

Mutations in myosin S2 alter cardiac myosin-binding protein-C interaction in hypertrophic cardiomyopathy in a phosphorylation-dependent manner.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disorder primarily caused by mutations in the ß-myosin heavy-chain gene. The proximal subfragment 2 region (S2), 126 amino acids of myosin, binds with the C0-C2 region of cardiac myosin-binding protein-C to regulate cardiac muscle contractility in a manner dependent on PKA-mediated phosphorylation. However, it is unknown if HCM-associated mutations within S2 dysregulate actomyosin dynamics by disrupting its interaction with C0-C2, ultimately leading to HCM. Herein, we study three S2 mutations known to cause HCM: R870H, E924K, and E930Δ. First, experiments using recombinant proteins, solid-phase binding, and isothermal titrating calorimetry assays independently revealed that mutant S2 proteins displayed significantly reduced binding with C0-C2. In addition, CD revealed greater instability of the coiled-coil structure in mutant S2 proteins compared with S2Wt proteins. Second, mutant S2 exhibited 5-fold greater affinity for PKA-treated C0-C2 proteins. Third, skinned papillary muscle fibers treated with mutant S2 proteins showed no change in the rate of force redevelopment as a measure of actin-myosin cross-bridge kinetics, whereas S2Wt showed increased the rate of force redevelopment. In summary, S2 and C0-C2 interaction mediated by phosphorylation is altered by mutations in S2, which augment the speed and force of contraction observed in HCM. Modulating this interaction could be a potential strategy to treat HCM in the future.

Rho GTPase complementation underlies BDNF-dependent homo- and heterosynaptic plasticity.

The Rho GTPase proteins Rac1, RhoA and Cdc42 have a central role in regulating the actin cytoskeleton in dendritic spines, thereby exerting control over the structural and functional plasticity of spines and, ultimately, learning and memory. Although previous work has shown that precise spatiotemporal coordination of these GTPases is crucial for some forms of cell morphogenesis, the nature of such coordination during structural spine plasticity is unclear. Here we describe a three-molecule model of structural long-term potentiation (sLTP) of murine dendritic spines, implicating the localized, coincident activation of Rac1, RhoA and Cdc42 as a causal signal of sLTP. This model posits that complete tripartite signal overlap in spines confers sLTP, but that partial overlap primes spines for structural plasticity. By monitoring the spatiotemporal activation patterns of these GTPases during sLTP, we find that such spatiotemporal signal complementation simultaneously explains three integral features of plasticity: the facilitation of plasticity by brain-derived neurotrophic factor (BDNF), the postsynaptic source of which activates Cdc42 and Rac1, but not RhoA; heterosynaptic facilitation of sLTP, which is conveyed by diffusive Rac1 and RhoA activity; and input specificity, which is afforded by spine-restricted Cdc42 activity. Thus, we present a form of biochemical computation in dendrites involving the controlled complementation of three molecules that simultaneously ensures signal specificity and primes the system for plasticity.

Autocrine BDNF-TrkB signalling within a single dendritic spine.

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are crucial for many forms of neuronal plasticity, including structural long-term potentiation (sLTP), which is a correlate of an animal's learning. However, it is unknown whether BDNF release and TrkB activation occur during sLTP, and if so, when and where. Here, using a fluorescence resonance energy transfer-based sensor for TrkB and two-photon fluorescence lifetime imaging microscopy, we monitor TrkB activity in single dendritic spines of CA1 pyramidal neurons in cultured murine hippocampal slices. In response to sLTP induction, we find fast (onset < 1 min) and sustained (>20 min) activation of TrkB in the stimulated spine that depends on NMDAR (N-methyl-d-aspartate receptor) and CaMKII signalling and on postsynaptically synthesized BDNF. We confirm the presence of postsynaptic BDNF using electron microscopy to localize endogenous BDNF to dendrites and spines of hippocampal CA1 pyramidal neurons. Consistent with these findings, we also show rapid, glutamate-uncaging-evoked, time-locked BDNF release from single dendritic spines using BDNF fused to superecliptic pHluorin. We demonstrate that this postsynaptic BDNF-TrkB signalling pathway is necessary for both structural and functional LTP. Together, these findings reveal a spine-autonomous, autocrine signalling mechanism involving NMDAR-CaMKII-dependent BDNF release from stimulated dendritic spines and subsequent TrkB activation on these same spines that is crucial for structural and functional plasticity.

Variations in maxillary second molar position of untreated subjects with normal occlusions: A long-term observational study.

INTRODUCTION: The purpose of the present study was to evaluate the long-term variations in maxillary second molar position in untreated subjects with normal occlusion. SETTING AND SAMPLE POPULATION: A sample of 39 subjects (18 females and 21 males) selected from the University of Michigan Growth Study (UMGS) was followed longitudinally with digital dental casts at 3 observation times: T1, when the maxillary permanent second molars were fully erupted, T2, last observation available in the longitudinal series (38 subjects), and T3, at least 20 years after T2 (12 subjects). MATERIALS AND METHODS: Digital measurements were recorded with an open-source software. Outcome variables were sagittal and transverse inclinations of the upper second molars. Two mixed-effect models were performed. RESULTS: The maxillary second molars had a distolingual inclination at T1, T2 and T3. Sagittal and transverse inclination showed progressive significant uprighting from T1 through T3 (P < .001). From T1 to T2, the adjusted difference in sagittal crown inclination was 8.0° (95% CI from 6.5° to 9.6°; P < .001). From T2 to T3, the adjusted difference was 5.5° (95% CI from 3.0° to 8.1°; P < .001). From T1 to T2, the adjusted difference in transverse crown inclination was 1.9° (95% CI from 0.4° to 3.5°; P = .011). From T2 to T3, the adjusted difference was 6.0° (95% CI from 3.4° to 8.5°; P < .001). CONCLUSIONS: Along with age, maxillary second molars showed a progressive significant uprighting with a decrease in the distal and lingual inclinations.

Cardiac myosin binding protein-C phosphorylation regulates the super-relaxed state of myosin.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) accelerates cardiac contractility. However, the mechanisms by which cMyBP-C phosphorylation increases contractile kinetics have not been fully elucidated. In this study, we tested the hypothesis that phosphorylation of cMyBP-C releases myosin heads from the inhibited super-relaxed state (SRX), thereby determining the fraction of myosin available for contraction. Mice with various alanine (A) or aspartic acid (D) substitutions of the three main phosphorylatable serines of cMyBP-C (serines 273, 282, and 302) were used to address the association between cMyBP-C phosphorylation and SRX. Single-nucleotide turnover in skinned ventricular preparations demonstrated that phosphomimetic cMyBP-C destabilized SRX, whereas phospho-ablated cMyBP-C had a stabilizing effect on SRX. Strikingly, phosphorylation at serine 282 site was found to play a critical role in regulating the SRX. Treatment of WT preparations with protein kinase A (PKA) reduced the SRX, whereas, in nonphosphorylatable cMyBP-C preparations, PKA had no detectable effect. Mice with stable SRX exhibited reduced force production. Phosphomimetic cMyBP-C with reduced SRX exhibited increased rates of tension redevelopment and reduced binding to myosin. We also used recombinant myosin subfragment-2 to disrupt the endogenous interaction between cMyBP-C and myosin and observed a significant reduction in the population of SRX myosin. This peptide also increased force generation and rate of tension redevelopment in skinned fibers. Taken together, this study demonstrates that the phosphorylation-dependent interaction between cMyBP-C and myosin is a determinant of the fraction of myosin available for contraction. Furthermore, the binding between cMyBP-C and myosin may be targeted to improve contractile function.

Skeletal MyBP-C isoforms tune the molecular contractility of divergent skeletal muscle systems.

Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein, localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle type-specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, 4 MyBP-C isoforms (1 fast-type MyBP-C and 3 N-terminally spliced slow-type MyBP-C) were expressed in EDL, but only the 3 slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that, only in the C-zone, MyBP-C Ca2+ sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-C's N terminus as N-terminal proteolysis attenuated MyBP-C's functional capacities. To determine each MyBP-C isoform's contribution to thin filament Ca2+ sensitization and slowing in the C-zone, we used a combination of in vitro motility assays using expressed recombinant N-terminal fragments and in silico mechanistic modeling. Our results suggest that each skeletal MyBP-C isoform's N terminus is functionally distinct and has modulatory capacities that depend on the muscle type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.

Three-dimensional evaluation of skeletal and dental effects of treatment with maxillary skeletal expansion.

INTRODUCTION: The objective was to determine the skeletal and dental changes with microimplant assisted rapid palatal expansion (MARPE) appliances in growing (GR) and nongrowing (NG) patients using cone-beam computed tomography and 3-dimensional imaging analysis. METHODS: The sample consisted of 25 patients with transverse maxillary discrepancy treated with a maxillary skeletal expander, a type of MARPE appliance. Cone-beam computed tomography scans were taken before and after maxillary expansion; the interval was 6.0 ± 4.3 months. The sample was divided into GR and NG groups using cervical vertebral and midpalatal suture maturation. Linear and angular 3-dimensional dentoskeletal changes were assessed after cranial base superimposition. Groups were compared with independent-samples t test (P <0.05). RESULTS: Both groups displayed marked transverse changes with a similar ratio of skeletal to dental transverse changes and parallel sutural opening from the posterior nasal spine-anterior nasal spine; a similar amount of expansion occurred in the anterior and the posterior regions of the maxilla. The maxilla expanded skeletally without rotational displacements in both groups. The small downward-forward displacements were similar in both groups, except that the GR group had a significantly greater vertical displacement of the canines (GR, 1.7 ±1.0 mm; NG, 0.6 ± 0.8 mm; P = 0.02) and anterior nasal spine (GR, 1.1 ± 0.6 mm; NG, 0.5 ± 0.5 mm; P = 0.004). CONCLUSIONS: Treatment of patients with MARPE appliance is effective in GR and NG patients. Although greater skeletal and dental changes were observed in GR patients, a similar ratio of skeletal to dental transverse changes was observed in both groups.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Predicting mandibular growth based on CVM stage and gender and with chronological age as a curvilinear variable.

OBJECTIVE: The aim of this study was to develop a prediction model that combines the information derived from chronological age (analysed as a curvilinear variable), gender and the CVM method to predict mandibular growth. SETTINGS AND SAMPLE POPULATION: 50 participants (29 females, 21 males) were selected from the AAOF Craniofacial Growth Legacy Collection, the Michigan Growth Study and the Denver Child Growth study. MATERIALS AND METHODS: In this investigation, 456 lateral cephalograms were analysed by applying a mixed effect model. The outcome variable was the annualized increment in total mandibular length (Co-Gn) during the year following the lateral cephalogram on which the cervical stage and chronological age were evaluated. The predictive variables were chronological age up to the fifth order, gender, stage of cervical vertebral maturation, as well as interactions between age and gender, age and cervical stage, and gender and cervical stage. RESULTS: Cervical stage, chronological age up to the fourth order, gender, and the interaction between age and gender were significant predictors of annualized increments in mandibular length. The annualized increment in Co-Gn was significantly greater for CS 3 when compared to all other cervical stages. Further, annualized increments in Co-Gn for CS 1 and CS 2 were significantly greater when compared to CS 5. CONCLUSIONS: Cervical stage, chronological age and gender can be used jointly to predict the annualized increment in mandibular growth. Cervical stage 3 exhibited the greatest annualized increase in mandibular length.

Prognostic approach to Class III malocclusion through case-based reasoning.

OBJECTIVE: This investigation evaluates the evidence of case-based reasoning (CBR) in providing additional information on the prediction of future Class III craniofacial growth. SETTINGS AND SAMPLE POPULATION: The craniofacial characteristics of 104 untreated Class III subjects (7-17 years of age), monitored with two lateral cephalograms obtained during the growth process, were evaluated. MATERIALS AND METHODS: Data were compared with the skeletal characteristics of subjects who showed a high degree of skeletal imbalance ('prototypes') obtained from a large data set of 1263 Class III cross-sectional subjects (7-17 years of age). RESULTS: The degree of similarity of longitudinal subjects with the most unbalanced prototypes allowed the identification of subjects who would develop a subsequent unfavourable skeletal growth (accuracy: 81%). The angle between the palatal plane and the sella-nasion line (PP-SN angle) and the Wits appraisal were two additional craniofacial features involved in the early prediction of the adverse progression of the Class III skeletal imbalance. CONCLUSIONS: Case-based reasoning methodology, which uses a personalized inference method, may bring additional information to approximate the skeletal progression of Class III malocclusion.

Three-dimensional mandibular dental changes with aging.

INTRODUCTION: This study aimed to evaluate the 3-dimensional (3D) mandibular dental changes over 42 years using the registration of digital models. METHODS: The sample comprised digital dental models of 8 untreated subjects (4 males and 4 females) with normal occlusion measured longitudinally at ages 17 years (T1) and 60 years (T2). Using 13 landmarks placed on the mucogingival junction, we registered the T2 model on the T1 model. Three-dimensional changes in the position of the landmarks on the buccal cusp tip of the posterior teeth and incisal edge of the central incisors were measured by 2 examiners. Registration and measurements were performed using SlicerCMF (version 3.1; http://www.slicer.org) software. Intra- and interrater agreements were evaluated using intraclass correlation coefficients and the Bland-Altman method. One-sample t tests were used for evaluating interphase 3D dental changes (P <0.05). RESULTS: Adequate intra- and interrater reproducibility was found. From T1 to T2, the mandibular teeth showed significant 3D positional changes. A significant dental eruption relative to the mucogingival junction was observed for the anterior and posterior teeth. Anteroposterior movements of mandibular teeth were not significant except for the right molar that drifted mesially. Transverse movements included slight lingual tipping at canines and premolars regions. CONCLUSIONS: Dental changes in untreated normal occlusion were very slight from early to mature adulthood. The eruption of the mandibular teeth was the most consistent finding. A tendency for mesial movement of molars and lingual movement of first premolars and canines was observed in the mandible during the aging process.

TrkB-Shc Signaling Protects against Hippocampal Injury Following Status Epilepticus.

Temporal lobe epilepsy (TLE) is a common and commonly devastating form of human epilepsy for which only symptomatic therapy is available. One cause of TLE is an episode of de novo prolonged seizures [status epilepticus (SE)]. Understanding the molecular signaling mechanisms by which SE transforms a brain from normal to epileptic may reveal novel targets for preventive and disease-modifying therapies. SE-induced activation of the BDNF receptor tyrosine kinase, TrkB, is one signaling pathway by which SE induces TLE. Although activation of TrkB signaling promotes development of epilepsy in this context, it also reduces SE-induced neuronal death. This led us to hypothesize that distinct signaling pathways downstream of TrkB mediate the desirable (neuroprotective) and undesirable (epileptogenesis) consequences. We subsequently demonstrated that TrkB-mediated activation of phospholipase Cγ1 is required for epileptogenesis. Here we tested the hypothesis that the TrkB-Shc-Akt signaling pathway mediates the neuroprotective consequences of TrkB activation following SE. We studied measures of molecular signaling and cell death in a model of SE in mice of both sexes, including wild-type and TrkBShc/Shc mutant mice in which a point mutation (Y515F) of TrkB prevents the binding of Shc to activated TrkB kinase. Genetic disruption of TrkB-Shc signaling had no effect on severity of SE yet partially inhibited activation of the prosurvival adaptor protein Akt. Importantly, genetic disruption of TrkB-Shc signaling exacerbated hippocampal neuronal death induced by SE. We conclude that therapies targeting TrkB signaling for preventing epilepsy should spare TrkB-Shc-Akt signaling and thereby preserve the neuroprotective benefits.SIGNIFICANCE STATEMENT Temporal lobe epilepsy (TLE) is a common and devastating form of human epilepsy that lacks preventive therapies. Understanding the molecular signaling mechanisms underlying the development of TLE may identify novel therapeutic targets. BDNF signaling thru TrkB receptor tyrosine kinase is one molecular mechanism promoting TLE. We previously discovered that TrkB-mediated activation of phospholipase Cγ1 promotes epileptogenesis. Here we reveal that TrkB-mediated activation of Akt protects against hippocampal neuronal death in vivo following status epilepticus. These findings strengthen the evidence that desirable and undesirable consequences of status epilepticus-induced TrkB activation are mediated by distinct signaling pathways downstream of this receptor. These results provide a strong rationale for a novel therapeutic strategy selectively targeting individual signaling pathways downstream of TrkB for preventing epilepsy.