Analysis and comparisons of gene expression changes in patient- derived neurons from ROHHAD, CCHS, and PWS.

Background: Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) syndrome is an ultra-rare neurocristopathy with no known genetic or environmental etiology. Rapid-onset obesity over a 3-12 month period with onset between ages 1.5-7 years of age is followed by an unfolding constellation of symptoms including severe hypoventilation that can lead to cardiorespiratory arrest in previously healthy children if not identified early and intervention provided. Congenital Central Hypoventilation syndrome (CCHS) and Prader-Willi syndrome (PWS) have overlapping clinical features with ROHHAD and known genetic etiologies. Here we compare patient neurons from three pediatric syndromes (ROHHAD, CCHS, and PWS) and neurotypical control subjects to identify molecular overlap that may explain the clinical similarities. Methods: Dental pulp stem cells (DPSC) from neurotypical control, ROHHAD, and CCHS subjects were differentiated into neuronal cultures for RNA sequencing (RNAseq). Differential expression analysis identified transcripts variably regulated in ROHHAD and CCHS vs. neurotypical control neurons. In addition, we used previously published PWS transcript data to compare both groups to PWS patient-derived DPSC neurons. Enrichment analysis was performed on RNAseq data and downstream protein expression analysis was performed using immunoblotting. Results: We identified three transcripts differentially regulated in all three syndromes vs. neurotypical control subjects. Gene ontology analysis on the ROHHAD dataset revealed enrichments in several molecular pathways that may contribute to disease pathology. Importantly, we found 58 transcripts differentially expressed in both ROHHAD and CCHS patient neurons vs. control neurons. Finally, we validated transcript level changes in expression of ADORA2A, a gene encoding for an adenosine receptor, at the protein level in CCHS neurons and found variable, although significant, changes in ROHHAD neurons. Conclusions: The molecular overlap between CCHS and ROHHAD neurons suggests that the clinical phenotypes in these syndromes likely arise from or affect similar transcriptional pathways. Further, gene ontology analysis identified enrichments in ATPase transmembrane transporters, acetylglucosaminyltransferases, and phagocytic vesicle membrane proteins that may contribute to the ROHHAD phenotype. Finally, our data imply that the rapid-onset obesity seen in both ROHHAD and PWS likely arise from different molecular mechanisms. The data presented here describes important preliminary findings that warrant further validation.

Surface Roughness of Prefabricated Pediatric Zirconia Crowns Following Simulated Toothbrushing.

Purpose: The purpose of this study was to evaluate the surface roughness of three different brands of prefabricated pediatric zirconia crowns (ZRCs) following simulated toothbrushing with a variety of dentifrices. Methods: Ninety-six total maxillary right central incisor prefabricated pediatric ZRCs (n equals 32 ZRCs/brand) were obtained from the manufacturers: Kinder Krowns®, NuSmile®, and Sprig®. ZRCs were equally assigned to dentifrices (n equals eight/dentifrice) with a variety of Relative Dentin Abrasion (RDA) values: Tom's of Maine Children's; Crest Kid's; Prevident 5000; and Crest® Pro-Health. ZRCs were brushed 10,000 strokes with a V-8 Toothbrushing Machine using the assigned dentifrices. Pre- and post-intervention data for the surface roughness of ZRCs, represented in Ra (average roughness) and Rz (mean roughness depth), were obtained using a stylus profilometer. Data were analyzed independently using two-way repeated measures analysis of variance with the Holm- Sidak method (α equals 0.05). Results: Baseline versus brushed Ra Kinder Krowns® with Prevident and Sprig® with Crest Kid's indicated statistically significant differences. Sprig® versus NuSmile® utilizing Crest Kid's were different in change in roughness. Both baseline and brushed NuSmile® dentifrice options were different versus all Sprig® and Kinder® ZRCs. Conclusions: Despite statistical significance, changes in surface roughness were small in scale. Although this study did not address toothbrushing and different dentifrices may affect the mechanical properties, durability, and/or retention properties of ZRCs, the study's results provide confidence to clinicians when using prefabricated pediatric ZRCs as a sustainable treatment option along with other restorative options, such as strip crowns and stainless steel crowns.

Molecular Changes in Prader-Willi Syndrome Neurons Reveals Clues About Increased Autism Susceptibility.

Background: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hormonal dysregulation, obesity, intellectual disability, and behavioral problems. Most PWS cases are caused by paternal interstitial deletions of 15q11.2-q13.1, while a smaller number of cases are caused by chromosome 15 maternal uniparental disomy (PW-UPD). Children with PW-UPD are at higher risk for developing autism spectrum disorder (ASD) than the neurotypical population. In this study, we used expression analysis of PW-UPD neurons to try to identify the molecular cause for increased autism risk. Methods: Dental pulp stem cells (DPSC) from neurotypical control and PWS subjects were differentiated to neurons for mRNA sequencing. Significantly differentially expressed transcripts among all groups were identified. Downstream protein analysis including immunocytochemistry and immunoblots were performed to confirm the transcript level data and pathway enrichment findings. Results: We identified 9 transcripts outside of the PWS critical region (15q11.2-q13.1) that may contribute to core PWS phenotypes. Moreover, we discovered a global reduction in mitochondrial transcripts in the PW-UPD + ASD group. We also found decreased mitochondrial abundance along with mitochondrial aggregates in the cell body and neural projections of +ASD neurons. Conclusion: The 9 transcripts we identified common to all PWS subtypes may reveal PWS specific defects during neurodevelopment. Importantly, we found a global reduction in mitochondrial transcripts in PW-UPD + ASD neurons versus control and other PWS subtypes. We then confirmed mitochondrial defects in neurons from individuals with PWS at the cellular level. Quantification of this phenotype supports our hypothesis that the increased incidence of ASD in PW-UPD subjects may arise from mitochondrial defects in developing neurons.

Significant transcriptional changes in 15q duplication but not Angelman syndrome deletion stem cell-derived neurons.

Background: The inability to analyze gene expression in living neurons from Angelman (AS) and Duplication 15q (Dup15q) syndrome subjects has limited our understanding of these disorders at the molecular level. Method: Here, we use dental pulp stem cells (DPSC) from AS deletion, 15q Duplication, and neurotypical control subjects for whole transcriptome analysis. We identified 20 genes unique to AS neurons, 120 genes unique to 15q duplication, and 3 shared transcripts that were differentially expressed in DPSC neurons vs controls. Results: Copy number correlated with gene expression for most genes across the 15q11.2-q13.1 critical region. Two thirds of the genes differentially expressed in 15q duplication neurons were downregulated compared to controls including several transcription factors, while in AS differential expression was restricted primarily to the 15q region. Here, we show significant downregulation of the transcription factors FOXO1 and HAND2 in neurons from 15q duplication, but not AS deletion subjects suggesting that disruptions in transcriptional regulation may be a driving factor in the autism phenotype in Dup15q syndrome. Downstream analysis revealed downregulation of the ASD associated genes EHPB2 and RORA, both genes with FOXO1 binding sites. Genes upregulated in either Dup15q cortex or idiopathic ASD cortex both overlapped significantly with the most upregulated genes in Dup15q DPSC-derived neurons. Conclusions: Finding a significant increase in both HERC2 and UBE3A in Dup15q neurons and significant decrease in these two genes in AS deletion neurons may explain differences between AS deletion class and UBE3A specific classes of AS mutation where HERC2 is expressed at normal levels. Also, we identified an enrichment for FOXO1-regulated transcripts in Dup15q neurons including ASD-associated genes EHPB2 and RORA indicating a possible connection between this syndromic form of ASD and idiopathic cases.

Clinical Evaluation and Parental Satisfaction with Pediatric Zirconia Anterior Crowns.

PURPOSE: The purpose of this study was to evaluate the clinical success of and parental satisfaction with anterior pediatric zirconia crowns. METHODS: A retrospective analysis of maxillary anterior pediatric zirconia crowns was performed. Crowns were evaluated for retention, gingival health, color match, contour, marginal integrity, and opposing tooth wear. Parental satisfaction regarding the esthetics of the crowns and parental perception of the impact of treatment on the child's appearance and oral health were evaluated by questionnaire. RESULTS: Fifty-seven crowns were evaluated in 18 children. Eight teeth were lost to exfoliation, three were extracted due to pathology, and two crowns debonded, leaving 44 available for examination. The average crown age at time of examination was 20.8 months. Sixteen crowns (36 percent) displayed gingival inflammation and color mismatch. No recurrent caries or opposing tooth wear was noted. Parents reported high satisfaction with the color, size, and shape of the crowns. The majority of parents reported that crowns improved the appearance and oral health of their child (78 percent and 83 percent, respectively). Eight-nine percent of parents reported that they would highly recommend these crowns. CONCLUSIONS: Zirconia crowns are clinically acceptable restorations in the primary maxillary anterior dentition. Parental satisfaction with zirconia crowns is high.

A Questionnaire of Parental Perceptions of Conscious Sedation in Pediatric Dentistry.

PURPOSE: The purpose of this study was to determine the opinions of parents about oral sedation in pediatric dentistry. METHODS: A 21-question questionnaire, administered to parents of children presenting for care in four pediatric dental practices, collected demographic information, media exposure to sedation, and parental knowledge/opinions regarding sedation procedures, such as NPO (nothing by mouth) guidelines, need for restraint, parental presence, and parental acceptance of treatment scenarios. RESULTS: Among 256 questionnaires completed, 235 were usable. Fifty-eight percent of respondents reported using public insurance. Parents agreed/strongly agreed (75 percent) that protective stabilization should not be necessary during sedation, and 87 percent preferred to stay with their child during the appointment. No parents perceived sedation as unsafe. Seventy-three percent of parents were unaware of media coverage of sedation; 82 percent reported it was acceptable for their child to sleep through a sedation appointment, while only 18 percent found it acceptable for the child to be highly reactive. CONCLUSIONS: Parents prefer to remain with their child, believe that sedation is safe and restraint should not be necessary, and are more accepting of the child sleeping during treatment. Most parents are unaware of media coverage of dental sedation for children.

Comparison of mandibular cortical thickness and QCT-derived bone mineral density (BMD) in survivors of childhood acute lymphoblastic leukemia: a retrospective study.

OBJECTIVE: To examine whether panoramic radiograph-determined mandibular cortical thickness correlated with quantitative computed tomography-derived bone mineral density (BMD) in survivors of childhood acute lymphoblastic leukemia (ALL). METHODS: We identified patients treated for ALL at St. Jude Children's Research Hospital, seen in the After Completion of Therapy (ACT) Clinic between January of 2006 and January of 2014 who had QCT-derived BMD and panoramic radiographs obtained within 1 month of each other. Panoramic radiographs were independently scored by a pediatric radiologist, two pediatric dentists, and a general dentist using the Klemetti technique. We used the Spearman's rank correlation test and the multivariate regression model to investigate the effect of evaluator experience on results. RESULTS: The study cohort comprised 181 patients with 320 paired studies: 112 (62%) male, 112 (71%) were white. Median age at ALL diagnosis was 6.4 (range, 0-18.8) years. Median age at study was 11.9 (range, 3.3 to 29.4) years. The median average BMD was 154.6 (range, 0.73-256) mg/cc; median QCT Z-score (age and gender adjusted) was -0.875 (range, -5.04 to 3.2). We found very weak association between panoramic radiograph score and both QCT-BMD average (P = 0.53) and QCT Z-score (P = 0.39). Results were not influenced by level of reader experience. CONCLUSIONS: The Klemetti technique of estimating BMD does not predict BMD deficits in children and young adult survivors of ALL, regardless of reviewer expertise. Alternative methods are needed whereby dental healthcare providers can identify and refer patients at risk for BMD deficits for detailed assessment and intervention.

Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders.

A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSCs) are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSCs that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSCs. We immortalized control DPSCs using human telomerase reverse transcriptase (hTERT) and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSCs share morphological and electrophysiological properties with non-immortalized DPSCs. We also show that differentiation of DPSCs into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NRSF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSCs can be obtained from teeth stored for ≥72 h, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSCs for the study of disease.

Assessment of the Tumorigenic Potential of Spontaneously Immortalized and hTERT-Immortalized Cultured Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) provide an exciting new avenue to study neurogenetic disorders. DPSCs are neural crest-derived cells with the ability to differentiate into numerous tissues including neurons. The therapeutic potential of stem cell-derived lines exposed to culturing ex vivo before reintroduction into patients could be limited if the cultured cells acquired tumorigenic potential. We tested whether DPSCs that spontaneously immortalized in culture acquired features of transformed cells. We analyzed immortalized DPSCs for anchorage-independent growth, genomic instability, and ability to differentiate into neurons. Finally, we tested both spontaneously immortalized and human telomerase reverse transcriptase (hTERT)-immortalized DPSC lines for the ability to form tumors in immunocompromised animals. Although we observed increased colony-forming potential in soft agar for the spontaneously immortalized and hTERT-immortalized DPSC lines relative to low-passage DPSC, no tumors were detected from any of the DPSC lines tested. We noticed some genomic instability in hTERT-immortalized DPSCs but not in the spontaneously immortalized lines tested. We determined that immortalized DPSC lines generated in our laboratory, whether spontaneously or induced, have not acquired the potential to form tumors in mice. These data suggest cultured DPSC lines that can be differentiated into neurons may be safe for future in vivo therapy for neurobiological diseases.

Microleakage of composite and two types of glass ionomer restorations with saliva contamination at different steps.

PURPOSE: This study's purpose was to evaluate microleakage of two types of glass ionomer (GI) and composite restorations with saliva contamination at different stages of restorative procedures. METHODS: Extracted teeth with Class V cavities were restored with a conventional GI, nanofilled RMGI, or total-etched composite. The preparations were contaminated with saliva before the adhesive/primer application or before the restoration placement (N=10). The restored teeth were thermocycled (1000X), stained (basic fuchsin), and sectioned. Microleakage distance was measured and subjected to analysis of variance and Duncan's post-hoc test (P=.05). RESULTS: For the enamel margin, no significant difference was found between the conventional GI and composite restoration, with or without saliva contamination (P>.05). The nanofilled RMGI with contamination before restoration had the highest microleakage. For the cementum margin, composite had significantly more microleakage than both types of GI restorations, regardless of saliva contamination. CONCLUSIONS: Conventional and resin-modified glass ionomer restorations had less cementum microleakage, while the composite had less enamel microleakage. Saliva contamination did not affect microleakage of the conventional GI at either enamel or cementum margins. The nanofilled RMGI system was not sensitive to saliva contamination at the gingival margin, but had increased microleakage at the enamel margin, especially after the primer application.

Dental education in a flat world: advocating for increased global collaboration and standardization.

Globalization is a broad term referring to the increasing connectivity, integration, and interdependence of economies, societies, technologies, cultures, and political and ecological spheres across the world. This position paper was developed by a working group of the 2007 American Dental Education Association (ADEA) Leadership Institute. The authors explore the effect that globalization has had on dentistry and dental education to date and hypothesize what dental education could look like in the years ahead. While the paper is written from a North American perspective, some of the authors bring international expertise and experience to the topic of global dental education in a flat world. Specific issues and barriers addressed in this position paper include variations in accreditation and licensure requirements in dental education throughout the world; the historical development of dental education models (odontology and stomatology) and the need for congruency of these models in the global environment; the competency-based model of education and its relevance to development and implementation of global dental competencies; and the slow adoption of technological advances in dental education for promoting collaborations and encouraging resource sharing among countries. These challenges are discussed as they affect the implementation of a standardized global dental education that can lead to improved access to oral health care services and better oral and overall health for the citizens of the world.

When should children have their first dental visit?

In summary, the goals of infant oral health care are to: Break the cycle of early childhood caries; Disrupt the acquisition of harmful microflora; Manage the risk/benefit of habits; Establish a dental home for health or harm; Impart optimal fluoride protection; and Use anticipatory guidance to arm parents in the therapeutic alliance.