Mek/ERK1/2-MAPK and PI3K/Akt/mTOR signaling plays both independent and cooperative roles in Schwann cell differentiation, myelination and dysmyelination.

Multiple signals are involved in the regulation of developmental myelination by Schwann cells and in the maintenance of a normal myelin homeostasis throughout adult life, preserving the integrity of the axons in the PNS. Recent studies suggest that Mek/ERK1/2-MAPK and PI3K/Akt/mTOR intracellular signaling pathways play important, often overlapping roles in the regulation of myelination in the PNS. In addition, hyperactivation of these signaling pathways in Schwann cells leads to a late onset of various pathological changes in the sciatic nerves. However, it remains poorly understood whether these pathways function independently or sequentially or converge using a common mechanism to facilitate Schwann cell differentiation and myelin growth during development and in causing pathological changes in the adult animals. To address these questions, we analyzed multiple genetically modified mice using simultaneous loss- and constitutive gain-of-function approaches. We found that during development, the Mek/ERK1/2-MAPK pathway plays a primary role in Schwann cell differentiation, distinct from mTOR. However, during active myelination, ERK1/2 is dependent on mTOR signaling to drive the growth of the myelin sheath and regulate its thickness. Finally, our data suggest that peripheral nerve pathology during adulthood caused by hyperactivation of Mek/ERK1/2-MAPK or PI3K is likely to be independent or dependent on mTOR-signaling in different contexts. Thus, this study highlights the complexities in the roles played by two major intracellular signaling pathways in Schwann cells that affect their differentiation, myelination, and later PNS pathology and predicts that potential therapeutic modulation of these pathways in PNS neuropathies could be a complex process.

Developmental stage-specific role of Frs adapters as mediators of FGF receptor signaling in the oligodendrocyte lineage cells.

FGF signaling is important for numerous cellular processes and produces diverse cellular responses. Our recent studies using mice conditionally lacking FGF-Receptor-1 (Fgfr1) or Fgfr2 during different stages of myelinogenesis revealed that Fgfr signaling is first required embryonically for the specification of oligodendrocyte progenitors (OPCs) and then later postnatally for the growth of the myelin sheath during active myelination but not for OPC proliferation, differentiation, or ensheathment of axons. What intracellular signal transduction pathways are recruited immediately downstream of Fgfrs and mediate these distinct developmentally regulated stage-specific responses remain unclear. The adapter protein Fibroblast-Growth-Factor-Receptor-Substrate-2 (Frs2) is considered a key immediate downstream target of Fgfrs. Therefore, here, we investigated the in vivo role of Frs adapters in the oligodendrocyte lineage cells, using a novel genetic approach where mice were engineered to disrupt binding of Frs2 to Fgfr1 or Fgfr2, thus specifically uncoupling Frs2 and Fgfr signaling. In addition, we used conditional mutants with complete ablation of Frs2 and Frs3. We found that Frs2 is required for specification of OPCs in the embryonic telencephalon downstream of Fgfr1. In contrast, Frs2 is largely dispensable for transducing Fgfr2-mediated signals for the growth of the myelin sheath during postnatal myelination, implying the potential involvement of other adapters downstream of Fgfr2 for this function. Together, our data demonstrate a developmental stage-specific function of Frs2 in the oligodendrocyte lineage cells. This contextual requirement of adapter proteins, downstream of Fgfrs, could partly explain the distinct responses elicited by the activation of Fgfrs during different stages of myelinogenesis.

Morphological measurements of anatomical landmarks in human maxillary first molar pulp chambers and evaluation of number of pulp canal orifices using spiral computed tomography: An in vitro study.

CONTEXT: Comprehensive understanding of the anatomic position of pulp canal orifices and the measurements of the molar pulp space may maintain the pulp health during conservative tooth preparation and minimize the possibility of mishaps during endodontic therapy. AIMS: The idea of the present study was to analyze the morphological measurements of anatomical landmarks in human maxillary first molar pulp chambers and evaluation of number of pulp canal orifices using three-dimensional spiral computed tomography (SCT). SUBJECTS AND METHODS: One hundred and thirty extracted intact human adult maxillary first molars were chosen from the North Indian population and were analyzed using SCT in axial and coronal sections. STATISTICAL ANALYSIS USED: Standard deviation, mean, and coefficient of variance were calculated. Interobserver reliability was evaluated using kappa value to avoid any bias. RESULTS: The results from our study showed that 69.23% of the sample teeth had four canal orifices, the mesial and distal pulp horns were present at an average distance of 0.80 ± 0.36 mm and 0.41 ± 0.34 mm, respectively, above the cementoenamel junction (CEJ), and the mean distance from the central groove of central fossa to furcation and the central groove of central fossa to the pulp chamber's roof was 8.37 ± 0.33 mm and 3.87 ± 0.29 mm, respectively. The average distance of the pulp chamber's floor from the furcation was found to be 2.47 ± 0.11 mm. The highest degree of variance was observed in case of relation of CEJ to pulp horns, i.e., 44.85% and 82.60%. CONCLUSIONS: The dimensions observed in this study and its resemblance to the various studies reported in literature shift the fundamental anatomic approach to a more systemic quantifiable approach to the endodontic maxillary first molar access preparation.

Racial predilection of C-shaped canal configuration in the mandibular second molar.

AIM: The purpose of this article is to determine the racial predilection of C-shaped canal configuration in a mandibular second molar. BACKGROUND: Unusual root canal anatomy always poses a diagnostic and treatment challenge. Identification of such variation is important for the success of root canal treatment outcome. C-shaped canal configuration is such an aberrant morphology of molar teeth that vary in different population and is commonly seen in a mandibular second molar. Thus, knowledge of racial predilection of C-shaped canal configuration in different population for early diagnosis is obligatory. MATERIALS AND METHODS: An exhaustive search was undertaken to identify published research articles related to C-shaped canal configuration in mandibular second molars. Forty-three research articles were analyzed which included 12,481 mandibular second molars. Chi-square test using value of P < 0.05 was performed to assess the statistical significance of this anomalous anatomic variation among the different population. RESULTS: Statistical test revealed a significant variation between the Asian and nonAsian population. The highest incidence of racial predilection was observed in China (Asia) with 93.1%, and the minimum was observed in America with 2.7%. CONCLUSION: This research reported that racial predilection of C-shaped canal configuration in mandibular second molar varies significantly.

Reattachment of complex fractures; a reality by advances in self-etch bonding systems.

An 18-year-old female patient reported to the Department of Conservative and Endodontics with the chief complaint of fractured tooth with respect to 21 and increased pain and mobility tooth with respect to 22. Intraoral periapical radiograph of 21 revealed coronal loss of tooth structure involving enamel, dentin, and pulp, suggestive of split tooth with respect to 21. Intraoral examination revealed a fracture of coronal structure of 22 and increased mobility in the coronal aspect, suggestive of horizontal crown-root fracture. For management of 21, after endodontic phase, placement of fiberpost, and coronal buildup, intentional reimplantation was done to expose and reattach the vertically fractured root fragment. For management of 22, after endodontic phase, crown lengthening was done, and the fractured fragment was reattached by making it a Natural Richmond's Crown. Radiographs revealed a complete sealing of the fractured fragment and proper positioning of the tooth.

Independent and cooperative roles of the Mek/ERK1/2-MAPK and PI3K/Akt/mTOR pathways during developmental myelination and in adulthood.

Multiple extracellular and intracellular signals regulate the functions of oligodendrocytes as they progress through the complex process of developmental myelination and then maintain a functionally intact myelin sheath throughout adult life, preserving the integrity of the axons. Recent studies suggest that Mek/ERK1/2-MAPK and PI3K/Akt/mTOR intracellular signaling pathways play important, often overlapping roles in the regulation of myelination. However, it remains poorly understood whether they function independently, sequentially, or converge using a common mechanism to facilitate oligodendrocyte differentiation, myelin growth, and maintenance. To address these questions, we analyzed multiple genetically modified mice and asked whether the deficits due to the conditional loss-of-function of ERK1/2 or mTOR could be abrogated by simultaneous constitutive activation of PI3K/Akt or Mek, respectively. From these studies, we concluded that while PI3K/Akt, not Mek/ERK1/2, plays a key role in promoting oligodendrocyte differentiation and timely initiation of myelination through mTORC1 signaling, Mek/ERK1/2-MAPK functions largely independently of mTORC1 to preserve the integrity of the myelinated axons during adulthood. However, to promote the efficient growth of the myelin sheath, these two pathways cooperate with each other converging at the level of mTORC1, both in the context of normal developmental myelination or following forced reactivation of the myelination program during adulthood. Thus, Mek/ERK1/2-MAPK and the PI3K/Akt/mTOR signaling pathways work both independently and cooperatively to maintain a finely tuned, temporally regulated balance as oligodendrocytes progress through different phases of developmental myelination into adulthood. Therapeutic strategies aimed at targeting remyelination in demyelinating diseases are expected to benefit from these findings.

Neuregulin 1 type III improves peripheral nerve myelination in a mouse model of congenital hypomyelinating neuropathy.

Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 (NRG1) type III regulate Schwann cell fate and myelination. Here we ask if modulating NRG1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we improved the myelination defects by early overexpression of NRG1 type III. Surprisingly, the improvement was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 and oligodendrocyte myelin glycoprotein. We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial NRG1 type III signaling has beneficial effects and improves myelination defects during development in a model of CHN.

Morphology and prevalence of middle canals in the mandibular molars: A systematic review.

OBJECTIVES: The purpose of this paper is to systematically review the various studies and case reports on the morphology and prevalence of middle canals in the mandibular molars. METHODOLOGY: Electronic databases such as MEDLINE, PubMed, EBSCOhost, ScienceDirect and various journals were screened to identify published literature till March 2017 and earlier for articles related to middle canals in the human permanent mandibular molars. Obtained articles were categorized as original researches, case reports and review articles. Well-defined review questions were developed using the patient population, intervention, comparison and outcome framework to summarize the objectives: "Does middle canal vary in morphology and anatomic location? What is the prevalence of middle canals in mandibular molars? Does ethnicity affect the prevalence of middle canals in mandibular molars?" Morphology was studied and prevalence rates were determined from the evaluation of data extracted from the articles. RESULTS: The search strategy resulted in 87 articles, of which 36 were original research papers and 51 were case reports. The prevalence of middle canals in the various populations ranged from 0.26% to 53.8%. Middle canals were reported in Europeans, Asians, Africans and South and North American populations. The prevalence of middle mesial canal and middle distal canal in various races was reported as up to 53.8% and 10%, respectively. The orifice of middle canal exists below a dentinal projection in the groove between the two main canals. They were observed in fin, confluent and independent configuration. Out of these, confluent configuration was more prevalent. CONCLUSION: Middle canal varies in morphology and anatomic location. Ethnicity affects the prevalence of middle canals in the mandibular molars.

Proteomics Analysis of Myelin Composition.

The identification and functional characterization of the repertoire of myelin proteins provides a valuable foundation for understanding molecular mechanisms of myelination and the pathogenesis of human myelin disease. Here we provide a procedure for the purification of myelin from rodent or human brains and a large-scale analysis of the myelin proteome, using the shotgun approach of one-dimensional PAGE and liquid chromatography (LC)/tandem mass spectrometry (MS).

Comparison of canal transportation and centering ability of Twisted Files, HyFlex controlled memory, and Wave One using computed tomography scan: An in vitro study.

AIM: The aim of the present study was to compare the canal transportation and centering ability of three rotary nickel-titanium (NiTi) systems (Twisted Files [TF], HyFlex controlled memory [CM], and Wave One [WO]) in curved root canals using computed tomography (CT). MATERIALS AND METHODS: Sixty freshly extracted single-rooted teeth having curved root canals with at least 25-35 degrees of curvature were selected. The teeth were randomly divided into three experimental groups of twenty each. After preparation with TF, HyFlex CM, and WO, all teeth were scanned using CT to determine the root canal shape. Pre- and post-instrumentation images were obtained at three levels, 3 mm apical, 9 mm middle, and 15 mm coronal above the apical foramen were compared using CT software. Amount of transportation and centering ability were assessed. The three groups were statistically compared with analysis of variance and post hoc Tukey's honestly significant difference test. RESULTS: Least apical transportation and higher centering ability were seen in HyFlex CM file system in all the three sections followed by TF. WO file system showed maximum transportation. CONCLUSIONS: The canal preparation with HyFlex CM file system showed lesser transportation and better centering ability than TF, WO file system.

Effect of Different Composite Restorations on the Cuspal Deflection of Premolars Restored with Different Insertion Techniques- An In vitro Study.

INTRODUCTION: This study was conducted to assess the effect of different composite materials on the cuspal deflection of premolars restored with bulk placement of resin composite in comparison to horizontal incremental placement and modified tangential incremental placement. AIM: The aim of this study was to evaluate the cuspal deflection caused by different composite materials when different insertion techniques were used. MATERIALS AND METHODS: Two different composite materials were used that is Tetric N Ceram (Ivoclar Vivadent marketing, India) and SonicFillTM (Kerr Sybron Dental). Forty standardized Mesio-Occluso-Distal (MOD) preparations were prepared on maxillary first premolars. Each group was divided according to composite insertion technique (n=10), as follows: Group I - bulk insertion using Tetric N Ceram, Group II - Horizontal incremental insertion technique using Tetric N Ceram, Group III- Modified tangential incremental technique using Tetric N Ceram, and Group IV- bulk insertion using SonicFillTM. Preparations were acid-etched, and bonded with adhesive resin to provide micro mechanical attachment before restoration using a uniform etching and bonding protocol in all the groups. All groups received the same total photo-polymerization time. Cuspal deflection was measured during the restorative procedure using customized digital micrometer assembly. One-way ANOVA test was applied for the analysis of significant difference between the groups, p-value less than 0.05 was considered statistically significant. RESULTS: The average cuspal deflections for the different groups were as follows: Group I 0.045±0.018, Group II 0.029±0.009, Group III 0.018±0.005 and Group IV 0.017±0.004. The intergroup comparison revealed statistically significant difference. CONCLUSION: A measurable amount of cuspal deflection was present in all the four studied groups. In general, bulkfill restoration technique with conventional composite showed significantly highest cusp deflection. There were no significant differences in cuspal deflection among sonicFillTM and modified tangential incremental insertion techniques.

A conservative management of iatrogenically damaged distal root of the mandibular second molar.

Trauma to the adjacent hard and soft tissue is the most common iatrogenic injury during extraction of the mandibular third molar. As every functional component of the dental arch is of prime importance in contemporary dental practice, the major concern must be in conserving the tooth and its structure as much as possible. The present case discusses the application of this conservative approach for management of iatrogenically damaged distal root of the mandibular second molar during extraction of impacted third molar, in which excessive guttering of alveolar bone and fractured apical third of distal root of 37 was observed radiographically. A conservative and noninvasive approach was successfully achieved to restore the damaged root by the bioactive material. Sealing of the remaining root with mineral trioxide aggregate allowed regeneration of soft and hard tissue around it.

Sustained activation of ERK1/2 MAPK in Schwann cells causes corneal neurofibroma.

Recent studies have shown that constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in Schwann cells (SCs) increases myelin thickness in transgenic mice. In this secondary analysis, we report that these transgenic mice develop a postnatal corneal neurofibroma with the loss of corneal transparency by age six months. We show that expansion of non-myelinating SCs, under the control of activated ERK1/2, also drive myofibroblast differentiation that derives from both SC precursors and resident corneal keratocytes. Further, these mice also harbor activated mast cells in the central cornea, which contributes to pathological corneal neovascularization and fibrosis. This breach of corneal avascularity and immune status is associated with the growth of the tumor pannus, resulting in a corneal stroma that is nearly four times its normal size. In corneas with advanced disease, some axons became ectopically myelinated, and the disruption of Remak bundles is evident. To determine whether myofibroblast differentiation was linked to vimentin, we examined the levels and phosphorylation status of this fibrotic biomarker. Concomitant with the early upregulation of vimentin, a serine 38-phosphorylated isoform of vimentin (pSer38vim) increased in SCs, which was attributed primarily to the soluble fraction of protein-not the cytoskeletal portion. However, the overexpressed pSer38vim became predominantly cytoskeletal with the growth of the corneal tumor. Our findings demonstrate an unrecognized function of ERK1/2 in the maintenance of corneal homeostasis, wherein its over-activation in SCs promotes corneal neurofibromas. This study is also the first report of a genetically engineered mouse that spontaneously develops a corneal tumor.

Signaling by FGF Receptor 2, Not FGF Receptor 1, Regulates Myelin Thickness through Activation of ERK1/2-MAPK, Which Promotes mTORC1 Activity in an Akt-Independent Manner.

FGF signaling has emerged as a significant "late-stage" regulator of myelin thickness in the CNS, independent of oligodendrocyte differentiation. Therefore, it is critically important to identify the specific FGF receptor type and its downstream signaling molecules in oligodendrocytes to obtain better insights into the regulatory mechanisms of myelin growth. Here, we show that FGF receptor type 2 (FGFR2) is highly enriched at the paranodal loops of myelin. Conditional ablation of this receptor-type, but not FGF receptor type 1 (FGFR1), resulted in attenuation of myelin growth, expression of major myelin genes, key transcription factor Myrf and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) activity. This was rescued by upregulating ERK1/2 activity in these mice, strongly suggesting that ERK1/2 are key transducers of FGFR2 signals for myelin growth. However, given that the PI3K/Akt/mechanistic target of rapamycin (mTOR) pathway is also known to regulate myelin thickness, we examined FGFR2-deficient mice for the expression of key signaling molecules in this pathway. A significant downregulation of p-mTOR, p-Raptor, and p-S6RP was observed, which was restored to normal by elevating ERK1/2 activity in these mice. Similar downregulation of these molecules was observed in ERK1/2 knock-out mice. Interestingly, since p-Akt levels remained largely unchanged in these mice, it suggests a mechanism of mTORC1 activation by ERK1/2 in an Akt-independent manner in oligodendrocytes. Taken together, these data support a model in which FGFs, possibly from axons, activate FGFR2 in the oligodendrocyte/myelin compartment to increase ERK1/2 activation, which ultimately targets Myrf, as well as converges with the PI3K/Akt/mTOR pathway at the level of mTORC1, working together to drive the growth of the myelin sheath, thus increasing myelin thickness.SIGNIFICANCE STATEMENT It is well accepted that myelin is a biologically active membrane in active communication with the axons. However, the axonal signals, the receptors on myelin, and the integration of intracellular signaling pathways emanating downstream from these receptors that drive the growth of the myelin sheath remain poorly understood in the CNS. This study brings up the intriguing possibility that FGF receptor 2, in the oligodendrocyte/myelin compartment, may be one such signal. Importantly, it provides compelling evidence linking FGFR2 with the ERK1/2-MAPK pathway, which converges with the PI3K/Akt/mTOR (mechanistic target of rapamycin) pathway at the level of mTORC1 and also regulates the transcription factor Myrf, together providing a mechanistic framework for regulating both the transcriptional and translational machinery required for the proper growth of the myelin sheath.

Conditional knockout of TOG results in CNS hypomyelination.

The tumor overexpressed gene (TOG) protein is present in RNA granules that transport myelin basic protein (MBP) mRNA in oligodendrocyte processes to the myelin compartment. Its role was investigated by conditionally knocking it out (KO) in myelinating glia in vivo. TOG KO mice have severe motor deficits that are already apparent at the time of weaning. This phenotype correlates with a paucity of myelin in several CNS regions, the most severe being in the spinal cord. In the TOG KO optic nerve <30% of axons are myelinated. The number of oligodendrocytes in the corpus callosum, cerebellum, and cervical spinal cord is normal. In the absence of TOG, the most patent biochemical change is a large reduction in MBP content, yet normal amounts of MBP transcripts are found in the brain of affected animals. MBP transcripts are largely confined to the cell body of the oligodendrocytes in the TOG KO in contrast to the situation in wild type mice where they are found in the processes of the oligodendrocytes and in the myelin compartment. These findings indicate that MBP gene expression involves a post-transcriptional TOG-dependent step. TOG may be necessary for MBP mRNA assembly into translation permissive granules, and/or for transport to preferred sites of translation. GLIA 2017;65:489-501.

Splinting of Longitudinal Fracture: An Innovative Approach.

Trauma may result in craze lines on the enamel surface, one or more fractured cusps of posterior teeth, cracked tooth syndrome, splitting of posterior teeth, and vertical fracture of root. Out of these, management of some fractures is of great challenge and such teeth are generally recommended for extraction. Literature search reveals attempts to manage such fractures by full cast crown, orthodontic wires, and so forth, in which consideration was given to extracoronal splinting only. However, due to advancement in materials and technologies, intracoronal splinting can be achieved as well. In this case report, longitudinal fractures in tooth #27, tooth #37, and tooth #46 had occurred. In #27, fracture line was running mesiodistally involving the pulpal floor resulting in a split tooth. In teeth 37 and 46, fractures of the mesiobuccal cusp and mesiolingual cusp were observed, respectively. They were restored with cast gold inlay and full cast crown, respectively. Longitudinal fracture of 27 was treated with an innovative approach using intracanal reinforced composite with Ribbond, external reinforcement with an orthodontic band, and full cast metal crown to splint the split tooth.

Strength of ERK1/2 MAPK Activation Determines Its Effect on Myelin and Axonal Integrity in the Adult CNS.

UNLABELLED: Myelin growth is a tightly regulated process driven by multiple signals. ERK1/2-MAPK signaling is an important regulator of myelin thickness. Because, in demyelinating diseases, the myelin formed during remyelination fails to achieve normal thickness, increasing ERK1/2 activity in oligodendrocytes is of obvious therapeutic potential for promoting efficient remyelination. However, other studies have suggested that increased levels of ERK1/2 activity could, in fact, have detrimental effects on myelinating cells. Because the strength, duration, or timing of ERK1/2 activation may alter the biological outcomes of cellular responses markedly, here, we investigated the effect of modulating ERK1/2 activity in myelinating cells using transgenic mouse lines in which ERK1/2 activation was upregulated conditionally in a graded manner. We found enhanced myelin gene expression and myelin growth in the adult CNS at both moderate and hyperactivated levels of ERK1/2 when upregulation commenced during developmental myelination or was induced later during adulthood in quiescent preexisting oligodendrocytes, after active myelination is largely terminated. However, a late onset of demyelination and axonal degeneration occurred at hyperelevated, but not moderately elevated, levels regardless of the timing of the upregulation. Similarly, myelin and axonal pathology occurred with elevated ERK1/2 activity in Schwann cells. We conclude that a fine tuning of ERK1/2 signaling strength is critically important for normal oligodendrocyte and Schwann cell function and that disturbance of this balance has negative consequences for myelin and axonal integrity in the long term. Therefore, therapeutic modulation of ERK1/2 activity in demyelinating disease or peripheral neuropathies must be approached with caution. SIGNIFICANCE STATEMENT: ERK1/2-MAPK activation in oligodendrocytes and Schwann cells is an important signal for promoting myelin growth during developmental myelination. Here, we show that, when ERK1/2 are activated in mature quiescent oligodendrocytes during adulthood, new myelin growth is reinitiated even after active myelination is terminated, which has implications for understanding the mechanism underlying plasticity of myelin in adult life. Paradoxically, simply increasing the "strength" of ERK1/2 activation changed the biological outcome from beneficial to detrimental, adversely affecting myelin and axonal integrity in both the CNS and PNS. Therefore, this study highlights the complexity of ERK1/2-MAPK signaling in the context of oligodendrocyte and Schwann cell function in the adult animal and emphasizes the need to approach potential therapeutic modulation of ERK1/2 activity with caution.

Management of Grossly Decayed Mandibular Molar with Different Designs of Split Cast Post and Core.

Mandibular molar with extensive loss of tooth structure, especially where no cavity wall is remaining, and insertion of posts in both the roots appear necessary so as to achieve proper retention for the core material. A single unit metal casting with two posts, one in the mesial root and the other in the distal divergent root, is difficult to fabricate due to difference in the path of insertion of the two posts. Multisection post and core or single cast post and core with auxiliary post can be an effective design to manage grossly decayed mandibular molars.