MiR-124 synergism with ELAVL3 enhances target gene expression to promote neuronal maturity.

Neuron-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), direct cell fate switching of human fibroblasts to neurons when ectopically expressed by repressing antineurogenic genes. How these miRNAs function after the repression of fibroblast genes for neuronal fate remains unclear. Here, we identified targets of miR-9/9*-124 as reprogramming cells activate the neuronal program and reveal the role of miR-124 that directly promotes the expression of its target genes associated with neuronal development and function. The mode of miR-124 as a positive regulator is determined by the binding of both AGO and a neuron-enriched RNA-binding protein, ELAVL3, to target transcripts. Although existing literature indicates that miRNA-ELAVL family protein interaction can result in either target gene up-regulation or down-regulation in a context-dependent manner, we specifically identified neuronal ELAVL3 as the driver for miR-124 target gene up-regulation in neurons. In primary human neurons, repressing miR-124 and ELAVL3 led to the down-regulation of genes involved in neuronal function and process outgrowth and cellular phenotypes of reduced inward currents and neurite outgrowth. Our results highlight the synergistic role between miR-124 and RNA-binding proteins to promote target gene regulation and neuronal function.

Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity.

The mammalian imprinted Dlk1-Dio3 locus produces multiple long non-coding RNAs (lncRNAs) from the maternally inherited allele, including Meg3 (i.e., Gtl2) in the mammalian genome. Although this locus has well-characterized functions in stem cell and tumor contexts, its role during neural development is unknown. By profiling cell types at each stage of embryonic stem cell-derived motor neurons (ESC~MNs) that recapitulate spinal cord development, we uncovered that lncRNAs expressed from the Dlk1-Dio3 locus are predominantly and gradually enriched in rostral motor neurons (MNs). Mechanistically, Meg3 and other Dlk1-Dio3 locus-derived lncRNAs facilitate Ezh2/Jarid2 interactions. Loss of these lncRNAs compromises the H3K27me3 landscape, leading to aberrant expression of progenitor and caudal Hox genes in postmitotic MNs. Our data thus illustrate that these lncRNAs in the Dlk1-Dio3 locus, particularly Meg3, play a critical role in maintaining postmitotic MN cell fate by repressing progenitor genes and they shape MN subtype identity by regulating Hox genes.

When a gene is active, its DNA sequence is 'transcribed' to form a molecule of RNA. Many of these RNAs act as templates for making proteins. But for some genes, the protein molecules are not their final destinations. Their RNA molecules instead help to control gene activity, which can alter the behaviour or the identity of a cell. For example, experiments performed in individual cells suggest that so-called long non-coding RNAs (or lncRNAs for short) guide how stem cells develop into different types of mature cells. However, it is not clear whether lncRNAs play the same critical role in embryos.Yen et al. used embryonic stem cells to model how motor neurons develop in the spinal cord of mouse embryos. This revealed that motor neurons produce large amounts of a specific group of lncRNAs, particularly one called Meg3. Further experiments showed that motor neurons in mouse embryos that lack Meg3 do not correctly silence a set of genes called the Hox genes, which are crucial for laying out the body plans of many different animal embryos. These neurons also incorrectly continue to express genes that are normally active in an early phase of the stem-like cells that make motor neurons.There is wide interest in how lncRNAs help to regulate embryonic development. With this new knowledge of how Meg3 regulates the activity of Hox genes in motor neurons, research could now be directed toward investigating whether lncRNAs help other tissues to develop in a similar way.

Mechanistic Insights Into MicroRNA-Induced Neuronal Reprogramming of Human Adult Fibroblasts.

The use of transcriptional factors as cell fate regulators are often the primary focus in the direct reprogramming of somatic cells into neurons. However, in human adult fibroblasts, deriving functionally mature neurons with high efficiency requires additional neurogenic factors such as microRNAs (miRNAs) to evoke a neuronal state permissive to transcription factors to exert their reprogramming activities. As such, increasing evidence suggests brain-enriched miRNAs, miR-9/9∗ and miR-124, as potent neurogenic molecules through simultaneously targeting of anti-neurogenic effectors while allowing additional transcription factors to generate specific subtypes of human neurons. In this review, we will focus on methods that utilize neuronal miRNAs and provide mechanistic insights by which neuronal miRNAs, in synergism with brain-region specific transcription factors, drive the conversion of human fibroblasts into clinically relevant subtypes of neurons. Furthermore, we will provide insights into the age signature of directly converted neurons and how the converted human neurons can be utilized to model late-onset neurodegenerative disorders.

MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts.

The ability to convert human somatic cells efficiently to neurons facilitates the utility of patient-derived neurons for studying neurological disorders. As such, ectopic expression of neuronal microRNAs (miRNAs), miR-9/9∗ and miR-124 (miR-9/9∗-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. However, how miR-9/9∗-124 break the cell fate barrier to activate the neuronal program remains to be defined. Here, we identified an anti-neurogenic function of EZH2 in fibroblasts that acts outside its role as a subunit of Polycomb Repressive Complex 2 to directly methylate and stabilize REST, a transcriptional repressor of neuronal genes. During neuronal conversion, miR-9/9∗-124 induced the repression of the EZH2-REST axis by downregulating USP14, accounting for the opening of chromatin regions harboring REST binding sites. Our findings underscore the interplay between miRNAs and protein stability cascade underlying the activation of neuronal program.

MicroRNA filters Hox temporal transcription noise to confer boundary formation in the spinal cord.

The initial rostrocaudal patterning of the neural tube leads to differential expression of Hox genes that contribute to the specification of motor neuron (MN) subtype identity. Although several 3' Hox mRNAs are expressed in progenitors in a noisy manner, these Hox proteins are not expressed in the progenitors and only become detectable in postmitotic MNs. MicroRNA biogenesis impairment leads to precocious expression and propagates the noise of Hoxa5 at the protein level, resulting in an imprecise Hoxa5-Hoxc8 boundary. Here we uncover, using in silico simulation, two feed-forward Hox-miRNA loops accounting for the precocious and noisy Hoxa5 expression, as well as an ill-defined boundary phenotype in Dicer mutants. Finally, we identify mir-27 as a major regulator coordinating the temporal delay and spatial boundary of Hox protein expression. Our results provide a novel trans Hox-miRNA circuit filtering transcription noise and controlling the timing of protein expression to confer robust individual MN identity.

Defining functional classes of Barth syndrome mutation in humans.

The X-linked disease Barth syndrome (BTHS) is caused by mutations in TAZ; TAZ is the main determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, cardiolipin. To date, a detailed characterization of endogenous TAZ has only been performed in yeast. Further, why a given BTHS-associated missense mutation impairs TAZ function has only been determined in a yeast model of this human disease. Presently, the detailed characterization of yeast tafazzin harboring individual BTHS mutations at evolutionarily conserved residues has identified seven distinct loss-of-function mechanisms caused by patient-associated missense alleles. However, whether the biochemical consequences associated with individual mutations also occur in the context of human TAZ in a validated mammalian model has not been demonstrated. Here, utilizing newly established monoclonal antibodies capable of detecting endogenous TAZ, we demonstrate that mammalian TAZ, like its yeast counterpart, is localized to the mitochondrion where it adopts an extremely protease-resistant fold, associates non-integrally with intermembrane space-facing membranes and assembles in a range of complexes. Even though multiple isoforms are expressed at the mRNA level, only a single polypeptide that co-migrates with the human isoform lacking exon 5 is expressed in human skin fibroblasts, HEK293 cells, and murine heart and liver mitochondria. Finally, using a new genome-edited mammalian BTHS cell culture model, we demonstrate that the loss-of-function mechanisms for two BTHS alleles that represent two of the seven functional classes of BTHS mutation as originally defined in yeast, are the same when modeled in human TAZ.

Mir-17∼92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN.

Motor neurons (MNs) are unique because they project their axons outside of the CNS to innervate the peripheral muscles. Limb-innervating lateral motor column MNs (LMC-MNs) travel substantially to innervate distal limb mesenchyme. How LMC-MNs fine-tune the balance between survival and apoptosis while wiring the sensorimotor circuit en route remains unclear. Here, we show that the mir-17∼92 cluster is enriched in embryonic stem cell (ESC)-derived LMC-MNs and that conditional mir-17∼92 deletion in MNs results in the death of LMC-MNs in vitro and in vivo. mir-17∼92 overexpression rescues MNs from apoptosis, which occurs spontaneously during embryonic development. PTEN is a primary target of mir-17∼92 responsible for LMC-MN degeneration. Additionally, mir-17∼92 directly targets components of E3 ubiquitin ligases, affecting PTEN subcellular localization through monoubiquitination. This miRNA-mediated regulation modulates both target expression and target subcellular localization, providing LMC-MNs with an intricate defensive mechanism that controls their survival.

Effect of occlusal splints for the management of patients with myofascial pain: a randomized, controlled, double-blind study.

BACKGROUND: Occlusal splints have been the preferred modalities in the management of myofascial temporomandibular disorders (TMDs), but now controversy exists in reporting whether they are successful for TMDs treatments. The aim of this study was to give objective evidence to the assessment of treatment effect of occlusal splints for myofascial TMDs patients by clinical assessments and surface electromyography (sEMG) measurements of masseter muscles (MM). METHODS: Thirty-six patients (12 males and 24 females) aged 16 - 57 (38 ± 11) years participated in the study. All participants diagnosed with myofascial TMD were randomized into two groups (18 of each). Patients in the first group (A) were treated with occlusal splints for 1 month, while patients in the second group (B) were treated with placebo (non-occluding palatal) splints. Clinical assessments were performed at the beginning of the study and 1 month after treatment. sEMG measurements for MM were performed at mandibular postural position (MPP) and maximum intercuspal contacted position (ICP) 1 month after the treatment. The root mean square (RMS) and the median frequency (MF) as linear indices of sEMG data were used to demonstrate muscle activity and muscle fatigue. Data were analyzed by ANOVA and post hoc SNK test. The differences were considered significant at P < 0.05. RESULTS: It was found that 89% of group A either completely recovered (39%) or clinically improved (50%), while only 22% of group B had a spontaneous improvement. sEMG analysis showed that at MPP, the mean of RMS value of MM in group A was lower than that of group B, which shows statistical differences (P < 0.01). At ICP, the RMS value of MM in group A was higher than that of group B, which shows statistical differences (P < 0.01). At MPP, MF value of MM in group A was higher than that of group B (P < 0.05). At ICP, MF value of MM was lower than that of group B (P < 0.01). CONCLUSIONS: Occlusal splint could eliminate or improve the signs and symptoms of TMD patients with myofascial pain. sEMG analysis indicates that the wearing of occlusal splints may reduce the degree of fatigue of the masticatory muscles. The splint therapy outcome has a correlation with the electromyographic changes in the masticatory muscles.

Receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression in chronic apical periodontitis: possible association with inflammatory cells.

BACKGROUND: Receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators (RANKL and OPG) and inflammatory cell infiltration in chronic apical periodontitis. METHODS: The samples of chronic periapical lesions (n = 40) and healthy periapical tissues (n = 10) were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analyzed for the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68. RESULTS: The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues (P < 0.001). The number of RANKL-positive cells was higher in lesions with severe inflammatory infiltration than in those with light inflammatory infiltration (P < 0.05). Significantly increased RANKL expression was found with T lymphocytes (CD3(+)), macrophages (CD68(+)) and B lymphocytes (CD20(+)) infiltration (P < 0.05). No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration. CONCLUSIONS: RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.

[Colonization of Tannerella forsythus and Prevotella intermedia in dental plaque samples from children of Changchun Ziqiang primary school].

OBJECTIVE: To detect the presence of Tannerella forsythus (Tf) and Prevotella intermedia (Pi) using polymerase chain reaction (PCR) in the oral plaque samples from children and investigate the relationship between bacteria and clinical parameters. METHODS: A total of 151 children aged 7 to 12 years were selected from Changchun primary school. The supragingival plaque sample was collected from the mesiobuccal and labial surfaces of the right maxillary central incisor (FDI1) and the right maxillary first molar (FDI6). Extracted DNA from plaque samples was used for PCR analysis. Intraoral examination, probing depth (PD) and bleeding on probing (BOP) were performed and recorded. RESULTS: The detection rate for Tf was 40.3% (118/293) and Pi was 46.4% (136/293) in supragingival plaque. The detection rates for Tf and Pi in molars were much higher than those in incisors (P < 0.01). The detection rate of Tf and Pi was positively related to BOP+ and PD. The detection rate for Pi decreased gradually with age, and the detection rate for Tf was highest in the group aged 7 to 8 and the detection rates for Tf and Pi were higher in the gingiva with BOP+ than that with BOP- (P > 0.05). The detection rates for Tf increased remarkably with BOP+ and especially when PD was greater than 4 mm. CONCLUSIONS: Detection rates of putative periodontal pathogens from healthy children of 7 to 12 years of age were high. The detection rates for Tf and Pi in molars were much higher than those in incisors, and the presence of Tf and Pi in supragingival plaque was related to periodontal parameters.

Stress area of the mandibular alveolar mucosa under complete denture with linear occlusion at lateral excursion.

BACKGROUND: The rocking and instability of a loaded complete denture (CD) during lateral excursion reduce the bearing area under the denture base, causing localized high stress concentrations. This can lead to mucosal tenderness, ulceration, and alveolar bone resorption, and the linear occlusion design was to decrease the lateral force exerted on the denture and to ensure denture stability. But it is not known how the bearing areas of linear occlusal CDs (LOCDs) and anatomic occlusal CDs (AOCDs) differ. The purpose of this study was to analyze and compare the distributions of the high and low vertical stress-bearing areas in the mandibular alveolar mucosa under LOCDs and AOCDs at lateral excursion. METHODS: Computerized tomography (CT) and finite element analysis were used to establish three-dimensional models of an edentulous maxilla and mandible with severe residual ridge resorption. These models were composed of maxillary and mandibular bone structure, mucosa, and the LOCD or AOCD. Lateral excursion movements of the mandible were simulated and the vertical stress-bearing areas in the mucosa under both mandibular CDs were analyzed using ANSYS 7.0. RESULTS: On the working side, the high stress-bearing (-0.07 to -0.1 MPa) area under the LOCD during lateral excursion was smaller than that under the AOCD, while the medium stress-bearing (-0.03 to -0.07 MPa) area under the LOCD was 1.33-fold that under the AOCD. The medium stress-bearing area on the non-working side under the LOCD was 2.4-fold that under the AOCD. Therefore, the overall medium vertical stress-bearing area under the LOCD was 20% larger than that under the AOCD. CONCLUSIONS: During lateral excursion, the medium vertical stress-bearing area under a mandibular LOCD was larger and the high vertical stress-bearing area was smaller than that under an AOCD. Thus, the vertical stress under the LOCD was distributed more evenly and over a wider area than that under the AOCD, thereby improving denture stability.

[Distribution of periodontal pathogens in dental plaque samples from 7 to 12-years-old children of Changchun Ziqiang primary school].

OBJECTIVE: To detect the presence of Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) using polymerase chain reaction (PCR) in the oral plaque samples from children and investigate the relationship between bacteria and clinical parameters. METHODS: A total of 151 children aged 7 to 12 years were selected from Changchun Ziqiang primary school. The supragingival plaque sample was collected from the mesiobuccal and labial surfaces of the right maxillary central incisor and the right maxillary first molar. Extracted DNA from plaque samples was used for PCR analysis. Intraoral examination, probing depth (PD) and bleeding on probing (BOP) were performed and recorded. RESULTS: The detection rate for Pg was 27.6% and Aa 54.3% in supragingival plaque. The detection rates for Pg in molars were much higher than those in incisors (P < 0.01). The detection rate of Pg was positively related to BOP+ and PD. The detection rate for Pg increased gradually with aging, and the detection rate for Aa was highest in the group aged 11 to 12 and the detection rates for Pg and Aa were higher in the gingiva with BOP+ than that with BOP- (P < 0.05). The detection rates for Pg increased remarkably with BOP+ and especially when PD was greater than 4 mm. CONCLUSIONS: Detection rates of putative periodontal pathogens from healthy children of 7 to 12 years of age were high. The detection rates for Pg in molars were much higher than those in incisors,and the presence of Pg and Aa in supragingival plaque was related to periodontal parameters.

Bond strength of different adhesives to normal and caries-affected dentins.

BACKGROUND: Currently, several systems of dentin substrate-reacting adhesives are available for use in the restorative treatment against caries. However, the bond effectiveness and property of different adhesive systems to caries-affected dentin are not fully understood. The objective of this study was to evaluate the bond strength of different adhesives to both normal dentin (ND) and caries-affected dentin (CAD) and to analyze the dentin/adhesive interfacial characteristics. METHODS: Twenty eight extracted human molars with coronal medium carious lesions were randomly assigned to four groups according to adhesives used. ND and CAD were bonded with etch-and-rinse adhesive Adper Single Bond 2 (SB2) or self-etching adhesives Clearfil SE Bond (CSE), Clearfil S(3) Bond (CS3), iBond GI (IB). Rectangular sticks of resin-dentin bonded interfaces 0.9 mm(2) were obtained. The specimens were subjected to microtensile bond strength (microTBS) testing at a crosshead speed of 1 mm/min. Mean microTBS was statistically analyzed with analysis of variance (ANOVA) and Student-Newman-Keuls tests. Interfacial morphologies were analyzed by Scanning Electron Microscopy (SEM). RESULTS: Etch-and-rinse adhesive Adper(TM) Single Bond 2 yielded high bond strength when applied to both normal and caries-affected dentin. The two-step self-etching adhesive Clearfil SE Bond generated the highest bond strength to ND among all adhesives tested but a significantly reduced strength when applied to CAD. For the one-step self-etching adhesives, Clearfil S(3) Bond and iBond GI, the bond strength was relatively low regardless of the dentin type. SEM interfacial analysis revealed that hybrid layers were thicker with poorer resin tag formation and less resin-filled lateral branches in the CAD than in the ND for all the adhesives tested. CONCLUSION: The etch-and-rinse adhesive performed more effectively to caries-affected dentin than the self-etching adhesives.

[Stress distribution in alveolar bone around implants under implant supported overdenture with linear occlusion at lateral occlusion].

OBJECTIVE: To analyze stress distribution in alveolar bone around implants of implant supported overdentures (ISO) with linear occlusion and with anatomic occlusion at lateral mandibular position, and to justify the possibility of decreased injurious force around implants in ISO with linear occlusion. METHODS: Computerized tomography scan and finite element analysis (FEA) were used to set up two 3-D FEA models of maxillae and mandible with severe residual ridge resorption. The mucosa, linear and anatomic occlusal ISO with bar attachments, and two implants inserted between mandibular foramina were also established in the models. With the condition of imitating the loading of masseter muscles, these models were loaded to simulate the stress distributions in alveolar bone around implants under ISO at lateral occlusion position. RESULTS: At lateral occlusion, the stress distributions in alveolar bone around implants under ISO with anatomic occlusion were mainly on the lingual and distal sides of the working side implants. However, stress distributions under ISO with linear occlusion were on the distal sides of bilateral implants. Both the stress peaks of ISOs with linear occlusion and with the anatomic one appeared in the working side. In anatomic occlusion model, sigma(z): -6.47 MPa and 6.81 MPa, sigma(1): -4.20 MPa and 7.20 MPa (negative value: compressive stress, positive value: tensile stress); in linear occlusion model, sigma(z): -4.86 MPa and 3.04 MPa, sigma(1): -3.48 MPa and 5.33 MPa. CONCLUSIONS: At lateral occlusion, when comparing the ISO with two different occlusion schemes, stress peak in alveolar bone around implants in the linear occlusion model was lower than that in the anatomic occlusion model at equal loading situation. Stress in the alveolar bone under ISO with linear occlusion distributed more evenly than that under ISO with anatomic occlusion.

[Primary clinical application of linear occlusion in immediate loading and implant-supported mandibular overdenture].

OBJECTIVE: To investigate clinical feasibility and technical features of immediate loading with linear occlusion on 2-implant-supported overdenture and to evaluate short-term effect of the treatment METHODS: Six edentulous patients with severe residual ridge resorption were enrolled. Two interforaminal implants were inserted for each patient and then immediate impressions were taken. Implant-supported bar-retained overdentures were restored for the patients within 24 hours. Clinical and radiographic examinations were conducted post-operative 1 week and 1, 3, 6, and 12 months. Thereafter at every 6 months the stability of implants, tissue situations around implants, radiographs and satisfaction level of patients were examined for each patient. RESULTS: Six cases and 12 implants were followed from 9 to 30 months. No implant was loosened or dropped. Sulculus bleeding index was 0-1 and probing depth of sulcus was less than 2 mm. The gingival tissues around the implants were healthy. Radiographic examination showed that bone resorption was less than 1 mm in the first year. The alveolar bone around the implants hasn't show obvious resorption stable height. Patients were satisfied with the implants. CONCLUSIONS: The results of this study indicate that immediate loading using linear occlusion on implant-supported bar-retained overdenture is predictable in some cases and can achieve satisfaction in the short-term service. Further study is needed to assess the long-term results.