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Bacteria use quorum sensing (QS) to communicate with each other via secreted small autoinducers produced by individuals. QS allows bacteria to display a unified response that benefits the species during adaptation to environment, colonization, and defense against competitors. In oral streptococci, the CSP-ComDE QS is an inducible DNA damage repair system that is pivotal for bacterial survival. In the oral pathogen Streptococcus mutans, the QS system positively influences the formation of antibiotic persisters, cells that can survive antibiotic attack by entering a non-proliferative state. We recently identified a novel gene, pep299, that is activated in the persister cell fraction induced by QS. In this study, we focused our investigation on the role of pep299, a gene encoding a bacteriocin-like peptide, in the formation of antibiotic persisters. Mutant Δ299, unable to produce Pep299, showed a dramatic reduction in the number of stress-induced persisters. Using a co-culture assay, we showed that cells overproducing pep299 induced the formation of persisters in the mutant, suggesting that Pep299 was actively secreted and detected by neighboring cells. Cells exposed to DNA damage conditions activated the gene expression of pep299. Interestingly, our results suggested that the pep299 gene was also involved in the regulation of a QS-inducible toxin–antitoxin system. Our study suggests that the pep299 gene is at the core of the triggered persistence phenotype in S. mutans, allowing cells to transition into a state of reduced metabolic activity and antibiotic tolerance.

An important factor in the assessment of caries risk is the presence of specific oral microflora, especially Streptococcus mutans. Some S. mutans strains possess proteins capable of binding collagen, such as the Cnm and Cbm proteins. The aim is to determine the presence of S. mutans strains carrying collagen binding proteins in a group of subjects with severe early childhood caries (S-ECC). S. mutans strains isolated from 15 S-ECC children were analyzed for collagen binding domains (cbd) of the cnm (cbd/cnm) and cbm (cbd/cbm) genes and their ability to bind to collagen. S. mutans strains positive for cbd/cnm or cbd/cbm were only found in 3 subjects with the most severe caries profile, with one subject having both cbd/cnm and cbd/cbm, and the other two with one of each. cnm/cbm-positive S. mutans strains bound to collagen substrate more avidly compared with negative S. mutans strains from each of the three groups. Our findings of an association between the presence of the collagen binding domains of the cnm/cbm genes in plaque S. mutans and the most aggressive form of caries profile in children offer a potential strategy to identify an individual’s risk for caries progression. Our study should be replicated in other settings and communities in longitudinal and longer-term studies. Our data offer a potential tool in the caries risk management and assessment in children.

Objective The objective of this study was to assess the shear bond strength (SBS) of orthodontic brackets bonded to uncut enamel with universal self-etch 1-step adhesive systems. Methods Extracted uncut premolars (n = 160) were randomly divided into 4 groups for treatment with Scotchbond Universal Adhesive (SU), All-Bond Universal (BU), Clearfil Universal Bond (CU) or the control, Adper Scotchbond Multi-Purpose Adhesive. Following bonding of brackets on tooth surfaces, teeth were stored in distilled water for 24 h and 6 months, and brackets were tested for SBS. The adhesive remnant index (ARI) and quantitative percentage of remaining resin (%RR) were recorded. Scanning electron microscopy was used to analyze debonded surfaces qualitatively. SBS and %RR data were analyzed by 2-way ANOVA followed by the Tukey test (α = 0.05). Results At neither time did these universal adhesives achieve satisfactory SBS for orthodontic treatment. The control group had the highest SBS, ARI score and mean %RR (and these differences were significant), while the BU group had the lowest SBS. SBS mean values and ARI scores decreased over time for SU and BU, but remained stable for CU. There was no difference in %RR among the universal adhesives tested. Conclusion None of the universal adhesives used in self-etch mode achieved SBS values (at 24 h and 6 months) that were satisfactory for orthodontic treatment.

Bacteriocins are ribosomally synthesized proteinaceous antibacterial peptides. They selectively interfere with the growth of other bacteria. The production and secretion of bacteriocins confer a distinct ecological advantage to the producer in competing against other bacteria that are present in the same ecological niche. Streptococcus mutans, a significant contributor to the development of dental caries, is one of the most prolific producers of bacteriocins, known as mutacins in S. mutans. In this study, we characterized the locus encoding mutacin B-Ny266, a lantibiotic with a broad spectrum of activity. The chromosomal locus is composed of six predicted operon structures encoding proteins involved in regulation, antimicrobial activity, biosynthesis, modification, transport, and immunity. Mutacin B-Ny266 was purified from semisolid cultures, and two inhibitory peptides, LanA and LanA′, were detected. Both peptides were highly modified. Such modifications include dehydration of serine and threonine and the formation of a C-terminal aminovinyl-cysteine (AviCys) ring. While LanA peptide alone is absolutely required for antimicrobial activity, the presence of LanA′ enhanced the activity of LanA, suggesting that B-Ny266 may function as a two-peptide lantibiotic. The activation of lanAA′ expression is most likely controlled by the conserved two-component system NsrRS, which is activated by LanA peptide but not by LanA′. The chromosomal locus encoding mutacin B-Ny266 was not universally conserved in all sequenced S. mutans genomes. Intriguingly, the genes encoding LanAA′ peptides were restricted to the most invasive serotypes of S. mutans. IMPORTANCE Although dental caries is largely preventable, it remains the most common and costly infectious disease worldwide. Caries is initiated by the presence of dental plaque biofilm that contains Streptococcus mutans, a species extensively characterized by its role in caries development and formation. S. mutans deploys an arsenal of strategies to establish itself within the oral cavity. One of them is the production of bacteriocins that confer a competitive advantage by targeting and killing closely related competitors. In this work, we found that mutacin B-Ny266 is a potent lantibiotic that is effective at killing a wide array of oral streptococci, including nearly all S. mutans strains tested. Lantibiotics produced by oral bacteria could represent a promising strategy to target caries pathogens embedded in dental plaque biofilm.

The oral pathogen Streptococcus mutans communicates using a canonical Gram-positive quorum sensing system, CSP-ComDE. The CSP pheromone already known to be involved in the development of genetic competence positively influences the formation of persisters, dormant variants of regular cells that are highly tolerant to antimicrobial therapy. It is now believed that the persistence phenotype is the end result of a stochastic switch in the expression of toxin-antitoxin (TA) modules. TAs consist of a pair of genes that encode two components, a stable toxin and its cognate labile antitoxin. Transcription analyses revealed that three core genes encoding a putative TA system, called SmuATR, were members of the S. mutans CSP regulon. We hypothesized that S. mutans is using its CSP-ComDE system as a deterministic mechanism for persister formation through the activation of smuATR locus. We showed here that the SmuATR system constitutes a novel tripartite type II TA system in which the smuA and smuT genes encode an antitoxin and a toxin, respectively, while SmuR is a transcriptional repressor involved in the autoregulation of the operon. Ectopic expression of SmuA - SmuT is associated with the CSP-inducible persistence phenotype. In contrast, overexpression of SmuT alone is bactericidal and causes membrane permeabilization. To our knowledge, SmuATR is the first functional chromosomal tripartite TA system shown to be induced by the bacterial quorum sensing system and involved in persister formation.

Introduction Mixed outcomes have been found in animal and clinical studies with regard to the use of low-level laser therapy (LLLT) as a modality to accelerate orthodontic tooth movement (OTM). One major reason for the variable findings is the different methodologies and protocols for laser therapy use. Objective The aim of this study was to determine whether orthodontically moved molars exposed to two different wattages at the same energy density of LLLT exhibited differences in the amount of tooth movement and molecular and histological changes in the adjacent periodontal areas. Methods An orthodontic force was applied to rat upper first molars exposed to 500 mW (EX-500) and 1000 mW (EX-1000) of laser application, with a control group (CT) with no laser application. Gene expression in the periodontal ligament (PDL) and histology of the palatal gingiva of the molars were analyzed. Results There was a statistically significant difference for OTM between EX-500 but not between EX-1000 and CT groups. RANKL and MMP-13 expression levels in the PDL of orthodontically moved molars, however, were increased significantly in laser-exposed groups compared to CT. Early signs of dysplasia were observed in over half of the animals in the EX-1000 group. Conclusions Our results provide evidence for molecular changes and the potential dysplastic effects of laser on the surrounding soft tissues. Further studies are needed to better identify an optimum laser protocol to maximize the desired effect.

Bacteriocins are small ribosomally synthesized antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB), a group of Gram-positive bacteria (cocci, rods) expressing high tolerance for low pH. Bacteriocins kill bacteria rapidly and are biologically active at very low concentrations. Bacteriocins produced by LAB are primarily active against closely related bacterial species. Many bacteriocins have been investigated with respect to their potential use in promoting human, plant, and animal health, and as food biopreservatives. Bacteriocins produced by LAB are particularly interesting since several LAB have been granted GRAS (Generally Recognized as Safe) status. Because it is not always possible to extract active bacteriocins secreted from cells grown in liquid medium, we developed a simple and inexpensive peptide extraction procedure using a semi-solid nutrient-rich agar medium. We hereby present a detailed procedure that leads to the rapid extraction of secreted bioactive bacteriocin peptides from the oral species Streptococcus mutans, a prolific bacteriocin-producing species, and its potential application for bacteriocin extraction from other LAB (e.g., Streptococcus, Lactococcus, Enterococcus). We also present a simple method for the detection of bacteriocin activity from the purified extracellular peptide extract.

The aims of this study were to investigate if Toll-like receptor 4 (TLR4) is expressed in the medullary dorsal horn (MDH) and if medullary application of a TLR4 antagonist (lipopolysaccharides from Rhodobacter sphaeroides, LPS-RS) can attenuate changes in nociceptive sensorimotor responses or TLR4 expression that might be evoked by mustard oil (MO) application to the right maxillary first molar tooth pulp. Of 41 adult male Sprague-Dawley rats used in the study, 23 received intrathecal application of the TLR4 antagonist LPS-RS (25 μg/10 μl; LPS-RS group) or isotonic saline (10 μl; vehicle control group) 10 min before pulpal application of MO (95%; 0.2 μl). Bilateral electromyographic (EMG) activities of the anterior digastric and masseter muscles were recorded continuously before and until 15 min after the MO application to the pulp. In 6 of these 23 rats and an additional 18 rats, the caudal medulla containing the ipsilateral and contralateral MDH was removed after euthanasia for subsequent Western Blot analysis of TLR4 expression in LPS-RS (n = 8) and vehicle (n = 8) groups and a naïve group (n = 8). The % change from baseline in the MO-evoked EMG activities within the anterior digastric muscles were significantly smaller in the LPS-RS group than the control group (two-way ANOVA, post hoc Bonferroni, P < 0.0001). Western Blot analysis revealed similar levels of TLR4 expression in the caudal medulla of the naïve, vehicle and LPS-RS groups. These novel findings suggest that TLR4 signaling in the caudal medulla may mediate MO-induced acute dental inflammatory pain in rats.

Streptococcus salivarius strain LAB813 was isolated from the dental plaque biofilm of a caries-free child with healthy oral tissues. We report here the complete genome sequence of S. salivarius strain LAB813. This genome consists of a chromosome of 2.2 Mb and a megaplasmid, pSAL813, of 183 kb.

Introduction Management of mandibular condylar fractures is difficult in children with their inherently dynamic and unstable deciduous and mixed dentitions. We present a variation of the conservative fixed orthodontic approach that was used as an adjunct to aid in the reduction of a bilateral condylar fracture in a pediatric patient. Methods A boy, aged 10 years 9 months, came with clinical signs and symptoms of mandibular fracture after being involved in a motor vehicle accident. A computed tomography scan showed a vertical fracture on the left condylar head, a displaced fracture of the right condylar neck, and a mandibular symphysis fracture. The patient was treated with an orthodontic fixed appliance instead of an arch bar splint, followed by elastic traction to achieve a proper occlusion and condylar remodeling. Follow-up appointments were made 2 weeks and 1, 2, 20, 37, and 49 months after treatment. Results Clinical recovery was observed 2 months after treatment. At the follow-up appointments at 20, 37, and 49 months, jaw function and occlusal relationship remained stable, and no ankylosis was observed. The computed tomography scans showed that the right condyle had remodeled, and the left condyle exhibited a slight curve in the head at 49 months posttreatment. The patient's satisfaction with these treatment results was high. Conclusions Conservative treatment of a mandibular fracture by fixed orthodontic means is a viable treatment option that is relatively straightforward and cost-effective and has a high level of patient acceptance and comfort.

Streptococcus mutans LAB761 has been isolated from dental plaque collected from a child with severe caries. We report here the complete genome sequence of S. mutans strain LAB761, which has a chromosome of 2.0 Mb., Streptococcus mutans LAB761 has been isolated from dental plaque collected from a child with severe caries. We report here the complete genome sequence of S. mutans strain LAB761, which has a chromosome of 2.0 Mb. The genome sequence reported herein contains several loci encoding double-glycine-motif peptides and lantibiotic and nonlantibiotic bacteriocins.

Photobiostimulation refers to the alterations, produced by relatively low levels of irradiation, in chemical, physical, and metabolic processes in target tissues with little or no temperature changes. A number of investigations have focused attention on the use of two photobiostimulation modalities, low-level laser therapy (LLLT) and light-emitting diodes (LED), to accelerate orthodontic tooth movement (OTM). Many of these studies have been performed on animal models and patients, often with mixed results. The increase in tooth movement in studies that showed positive effects of laser therapy was about 0.5mm/month in humans. However, no statistically significant changes in tooth movement rates were observed in some other studies. Conflicting results from different studies are attributed to the variability in parameters and lack of rigorous research design and statistical analyses. We conclude, upon review of the available literature, that the apparent increase in tooth movement obtained from use of LLLT can be of significant benefit in the clinic provided that more rigorous research, in both animal models and humans, is conducted to improve the consistency and predictability of laser and LED therapy usage.

Nitric oxide (NO) is involved in bone remodelling and has been shown to play a role in regulating the rate of orthodontic tooth movement (OTM) in rat models. In humans, however, the role of NO in OTM remains less clear. In this study, NO concentration in gingival crevicular fluid (GCF) was measured in patients undergoing orthodontic treatment. Thirteen male participants (ages 11-18 years) planned for non-extraction fixed orthodontic therapy were recruited. Samples of GCF were collected from each maxillary central incisor and first and second molar immediately before (T0), 1h after (T1), and 3-4 days after (T2) application of light orthodontic forces. The maxillary second molars were not included in the appliance and served as controls. Measureable NO levels were consistently obtained from all sampled sites. Total NO levels showed significantly higher NO levels (p0.05) at T1 at the buccal surfaces of the central incisors when compared to the first and second molars. The results indicate a possible role for NO in OTM at the pressure sites of incisors at early time points. Further studies are required to determine whether NO levels in the periodontal ligament tissues of human teeth during OTM are affected by a force gradient and the magnitude of the applied force.

The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases.

Expression studies have implicated FLRT2 in cranial neural crest cell migration and prechondrogenic cell condensation during craniofacial skeletogenesis. We aimed to determine whether FLRT2 was involved in mediating cell-matrix interactions in the ATDC5 chondroprogenitor cell line. Immunolocalization experiments of ATDC5 cells revealed that FLRT2 was present on the cell membrane as well as extracellularly, where it colocalized with Fibronectin (Fn). After cell extraction of the matrix, FLRT2 was identified in the ATDC5-derived extracellular matrix (ECM) and was further found to be associated with Fn-coated beads in cell cultures. Blockage of Fn fibril formation via a blocking peptide resulted in a concomitant decrease in extracellular FLRT2 accumulation. Over a 7-day period following the replenishment of the Fn blocking peptide to the cultures, there was a partial rebound in Fn fibril formation that was accompanied by a concomitant reappearance of FLRT2 co-expression. Co-immunoprecipitation confirmed that FLRT2 and Fn interacted, either directly or indirectly. Immunoprecipitation and Western blot analyses with antibodies recognizing epitopes located on the extra- and intracellular domains of FLRT2 further revealed the presence of different sized bands, suggesting that FLRT2 may exist in both membrane-bound and shed forms. Our data therefore provide evidence that FLRT2 and/or its cleavage products may be cooperating with Fn and other ECM proteins to regulate critical cellular events. Further studies will be necessary in delineate more precisely the roles of FLRT2 in mediating cell- and cell-matrix interactions during normal development.

Aim Caffeine is one of the most widely consumed behaviorally active substances in the world. Although its effects on the central nervous system and bone metabolism have been documented, as yet there is no report on its effect on tissues in the oral cavity. In this study we analyzed the viability of human gingival fibroblasts (HGF) and alkaline phosphatase (ALP) enzyme activity after exposure to different concentrations of caffeine for different exposure time periods. Methods The HGF were cultured with different concentrations of caffeine. Viability of cells exposed to caffeine was analyzed by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay to assess mitochondrial dehydrogenase activity. The activity of ALP was analyzed at specific time intervals after caffeine addition. Results Our results showed that caffeine of concentrations

The shape and size of tooth roots are genetically and phylogenetically predetermined. Clinical defects in root formation can manifest in the form of shortened roots caused by either root agenesis or root resorption. We report on a patient who came at age 7 years for space management. In the 2-year period after the initial visit, maxillary arch expansion was performed, followed by serial extractions of all 4 first premolars. A radiograph taken about 18 months after the serial extraction showed that although the crowns of all 4 second premolars had erupted fully into the arch, the roots were only about half of their normal length. With a family history of 1 sibling with a missing second premolar and the symmetrical distribution and pattern of the teeth in the 4 dental quadrants, we speculated that the arrested root development was due most likely to a genetic predisposition. Arrested root development is difficult to predict, but a potential warning sign is a family history of malformed or missing teeth. Proper, adequate, and accurate records continue to remain critical for both medical and legal purposes in the treatment of patients with potential problems in root agenesis.

Failure of the primary lip and palate to fuse leads to clefts of the lip, a birth defect with an incidence of 1 for every 500 in some races. Epithelial cells lining the facial processes of the primary lip and palate, the lateral and medial nasal processes (LNP and MNP), must first make contact to go through a series of highly regulated and coordinated sequence of events to form the normal midface. As yet, many of the basic mechanisms underlying the fusion events of the epithelial-lined surfaces are not known. This is due in part to the difficulty associated with the isolation of the epithelial cells for further study and analysis. The objective of this study was to test the use of laser capture microdissection to collect clean populations of primary lip and palate epithelial cells destined to fuse. Fusing and nonfusing epithelial cell populations of the MNP and LNP were isolated by laser capture microdissection and assayed for gene expression of Bmp-4, Tgfβ-2, and their type 1 receptors, Alk-3 and Alk-5, respectively. Transcripts of Bmp-4/Alk-3 and Tgfβ-2/Alk-5 were restricted to the epithelial seam of the fusion site, and the epithelium of the prefusion site, in patterns previously reported. Data indicated our ability to isolate clean populations of epithelial and mesenchymal cells around the primary palate fusion site, allowing precise analysis of tissue and site-specific gene expression at high resolution. This study provides the basis of further analysis of the potential molecular players of MNP and LNP fusion and nonfusion of epithelial cells.

Objective: To understand the molecular basis of early orthodontic tooth movement by looking at the expression of KI-67, runt-related transcription factor 2 (Runx2), and tumor necrosis factor ligand superfamily member 11 (RANKL) proteins. Materials and Methods: We employed a rat model of early orthodontic tooth movement using a split-mouth design (where contralateral side serves as a control) and performed immunohistochemical staining to map the spatial expression patterns of three proteins at 3 and 24 hours after appliance insertion. Results: We observed increased expression of KI-67, a proliferation marker, and RANKL, a molecule associated with osteoclastic differentiation, in the compression sites of the periodontal ligament subjected to 3 hours of force. In contrast, there was increased expression of KI-67 and Runx2, a marker of osteoblast precursors, in tension areas after 24 hours of force. Decreased KI-67 expression in the mesial and distal regions of the periodontal ligament was observed at the midpoint of the tooth root. Conclusions: The early RANKL expression indicates that at this early stage cells are involved in osteoclast precursor signaling. Also, decreased KI-67 expression found near the midpoint of the tooth root is believed to represent the center of rotation, providing a molecular means of visualizing mechanical loading patterns.

Craniosynostosis is a relatively common birth defect characterized by the premature fusion of one or more cranial sutures. Examples of craniosynostosis syndromes include Crouzon (CS), Pfeiffer (PS), and Apert (AS) syndrome, with clinical characteristics such as midface hypoplasia, hypertelorism, and in some cases, limb defects. Mutations in Fibroblast Growth Factor Receptor-2 comprise the majority of known mutations in syndromic forms of craniosynostosis. A number of clinical reports of FGFR-associated craniosynostosis patients and mouse mutants have been linked to gastrointestinal tract (GIT) disorders, leading to the hypothesis of a direct link between FGFR-associated craniosynostosis syndromes and GIT malformations. We conducted an investigation to determine GIT symptoms in a sample of FGFR-associated craniosynostosis syndrome patients and a mouse model of CS containing a mutation (W290R) in Fgfr2. We found that, compared to the general population, the incidence of intestinal/bowel malrotation (IM) was present at a higher level in our sample population of patients with FGFR-associated craniosynostosis syndromes. We also showed that the mouse model of CS had an increased incidence of cecal displacement, suggestive of IM. These findings suggest a direct relationship between FGFR-related craniosynostosis syndromes and GIT malformations. Our study may shed further light on the potential widespread impact FGFR mutations on different developmental systems. Based on reports of GIT malformations in children with craniosynostosis syndromes and substantiation with our animal model, GIT malformations should be considered in any child with an FGFR2-associated craniosynostosis syndrome. © 2016 Wiley Periodicals, Inc.

Increasing the rate of orthodontic tooth movement (OTM) can reduce risks such as periodontal disease and caries.This split-mouth randomized controlled clinical trial investigated whether light emitting diode (LED) phototherapy could accelerate the rate of OTM.The study was conducted at the Graduate Orthodontics Clinic at the University of Toronto, Faculty of Dentistry.17 dental arches from 11 orthodontic participants with bilaterally symmetrical extraction of premolars were included. During space closure of single tooth extraction sites, LED phototherapy was applied to one side of the dental arch for a specified time and the contralateral side acted as the control. Space closure was measured immediately prior to, during and later in space closure.All participants were compliant with LED application. Our results revealed no significant changes in the rate of OTM with LED phototherapy over 3 months of extraction space closure.Our findings were contrary to previous findings of the effect of laser phototherapy on regulating the rate of OTM. Further investigations are warranted to analyse whether the duration or method of LED delivery would have an effect on the rate of OTM. Toronto. This trial was registered at clinicaltrials.gov (NCT01490385).

Mutations in FgfR2 cause several craniosynostosis syndromes, including Crouzon Syndrome (CS), but knowledge of the specific mechanisms by which FGF effects morphogenesis is lacking. The gene CRISPL...

The molecular mechanisms by which the primordia of the midface grow and fuse to form the primary palate are not well characterized. This is in spite of the fact that failure of growth and/or fusion of these facial primordia leads to the common human craniofacial birth defects, clefts of the lip with or without clefts of the palate. Members of the transforming growth factor beta (Tgfbeta) superfamily have been shown to play critical roles during craniofacial development. Specifically, the role of Tgfbeta-3 in mediating the fusion of the embryonic secondary palatal shelves is well documented. In a screen for genes expressed during fusion of the murine midfacial processes, Tgfbeta2 was identified as a gene differentially expressed during fusion of the lateral and medial nasal processes. The objective of our study was to analyze the spatial and temporal expression of Tgfbeta2 during critical stages of midfacial morphogenesis at both the transcript and protein levels. We also compared the pattern of expression of Tgfbeta2 with that of Bmp4, a gene shown previously to be involved in mediating the fusion process in the midface. Our results showed Tgfbeta2 expression in a very restrictive area of the epithelial layer along the borders of the midfacial primordia, in a pattern very similar to that of Bmp4. The highly restrictive and spatial and temporal pattern of expression of Tgfbeta2 implicates its role in mediating the fusion of the midfacial processes, possibly through interacting with Bmp4 in the regulation of apoptosis and/or epithelial-mesenchymal transformation. A greater understanding of the role of this gene will clarify how the normal midface grows and the mechanisms behind cleft development.

The molecular mechanisms by which the primordia of the midface grow and fuse to form the primary palate portion of the craniofacial region are not well characterized. This is in spite of the fact that failure of growth and/or fusion of these primordia leads to the most common craniofacial birth defect in humans (i.e. clefts of the lip and/or palate). Bmp4 plays a critical role during early embryonic development and has previously been shown to play a role in epithelial-mesenchymal interactions in the craniofacial region of chicks. We analyze the expression of bmp4 in mouse as the midfacial processes undergo fusion to form the primary palate. We show that bmp4 is expressed in a very distinct manner in the three midfacial processes (lateral nasal, LNP, medial nasal, MNP, and maxillary processes, MxP) that ultimately fuse to form the midface. Prior to fusion of the midfacial processes, bmp4 is expressed in the ectoderm of the LNP, MNP, and MxP in a distinct spatial and temporal manner near and at the site of fusion of the midface. Bmp4 appears to demarcate the cells in the LNP and MNP that will eventually contact and fuse with each other. As fusion of the three prominences proceeds, some bmp4 expressing cells are trapped in the fusion line. Later, the expression of bmp4 switches to the mesenchyme of the midface underlying its initial expression in the ectoderm. The switch occurs soon after fusion of the three processes. The pattern of expression in the midfacial region implicates the important role of bmp4 in mediating the fusion process, possibly through apoptosis of cells in the putative site of fusion, during midfacial morphogenesis.

Application of a force to a tooth initiates a series of histological, cellular, biological, and molecular changes in the tension and compression sites in the periodontium of the tooth. During the early stages of orthodontic tooth movement (OTM), little actual movement is evident. However, a number of molecular changes reflective of important biological events such as inflammation, extracellular remodeling, and mechanosensing by osteocytes are occurring in the periodontium, mediated by members of the Interleukin and Tumor Necrosis Factor family of proteins, prostaglandins, and metalloproteinases. Bone resorption and deposition predominate in the later stages of tooth movement, and a number of proteins in the RANK/RANKL/osteoprotegerin and the RUNX2 signaling pathways have been shown to be expressed in the periodontium of teeth subject to different magnitude and duration of orthodontic forces. So far, all genes shown to be involved in OTM are those documented to play important roles in similar processes in the body, i.e., there is no unique gene that regulates OTM. The challenge in the field of OTM studies lies in being able to integrate the numerous signaling networks in the diverse parts of the periodontium during OTM. This challenge can only be met by creative approaches and ideas in a multi-disciplinary setting.

The sense of smell is perceived by olfactory receptor neurons (ORN) present in the olfactory epithelium located in the posterosuperior aspect of the nasal cavity. The axons of these ORN migrate to the olfactory bulb (OB), forming a nervous layer on the outermost part of the bulb, and finally synapse in glomerular structures in the OB. The ORN are unique in that they are constantly being renewed throughout life. We characterized the defects in the nasal cavity and olfactory nervous supply of Twirler (Tw) mice by histological and immunohistochemical means. Tw homozygotes have previously been shown to present with midfacial abnormalities in the form of clefts of the lip and palate (Lyon, 1958; Gong et al., 2000). We found that in the Tw homozygotes, the OB was abnormally shaped, the skeletal framework underlying the OB was disrupted, and the morphology of the nasal cavity was altered with poorly defined nasal turbinates. Immunohistochemical staining with antibodies that marked nerves in general (PGP 9.5) and mature ORN (omp) in the olfactory epithelium at two different embryonic stages and in newborn mice revealed the stratification of the olfactory epithelium in Tw homozygotes, albeit slightly thinner compared to wildtype. A striking difference in the olfactory epithelium was the lack of differentiation of the ORN in Tw homozygotes and the reduced axonal input to the OB. In Tw homozygotes at 14.5 days of embryonic development, the presence of many mature ORN found randomly in the mesenchyme suggests the loss of olfactory pathfinding cues to the OB. It is believed that the lack of appropriate pathfinding cues observed in the Tw homozygotes was responsible for the OB not having the appropriate trophic effect on the development and maturation of the ORN as had been observed in partially bulbectomized animals. The defects in the Twirler may prove to be a valuable system to analyze problems in olfactory pathfinding and maturation.

Objective: The development of the secondary palate of Twirler (Tw) mice was characterized, and a quantitative coronal plane evaluation of the width and length of the craniofacial regions of homozygous mutant embryos was conducted. Results and Conclusions: The secondary palatal shelves were retarded in growth and the timing of elevation from a vertical to horizontal position. The homozygous Tw embryos, when compared with their wild-type littermates, also had a significantly wider midface. It is not surprising, therefore, that the short palatal shelves in a wide midface resulted in cleftings of the secondary palate observed in all homozygous Tw mice. These findings are consistent with similar studies in humans and other murine models.

Tumorhead (TH) is a novel maternal gene product from Xenopus laevis containing several basic domains and a weak coiled-coil. Overexpression of wild-type TH resulted in increased proliferation of neural plate cells, causing expansion of the neural field followed by neural tube and craniofacial abnormalities. Overexpressed TH protein repressed neural differentiation and neural crest markers, but did not inhibit the neural inducers, pan-neural markers or mesodermal markers. Loss of function by injection of anti-TH antibody inhibited cell proliferation. Our data are consistent with a model in which tumorhead functions in regulating differentiation of the neural tissues but not neural induction or determination through its effect on cell proliferation.

Objective:Since its first description, the A strain of mice have been utilized extensively as models to study the processes involved in clefting of the midfacial region. Of the A substrains, the A/WySn has a spontaneous rate of clefting of the lip of about 20% to 30%. The A/WySn mouse model was utilized in this study to analyze and compare the phenotypic and molecular changes in the midfacial region of embryos with and without cleft.Results:Scanning electron microscopy and skeletal and cartilage preparations of newborn A/WySn pups showed the presence of bilateral and unilateral clefts of the lips and the disruption of the skeletal and cartilaginous components of the mice with clefts of the lip. The expression of the msx1 homeobox gene was analyzed by whole mount in situ hybridization of A/WySn embryos at different stages of midfacial development. The results showed that there was misregulation of the expression of the msx1 gene in embryos with cleft, with a persistence of expression in the distal growing tips of the midfacial processes and in areas that have fused in normal embryos without cleft.Conclusions:Although the genetic defect in A/WySn mice is not known, a possible candidate gene has been mapped to a corresponding human chromosome carrying retinoic acid receptor α, and there exists a possibility that msx1 is in the same genetic pathway affected by the mutation of the gene in A/WySn.

Twirler ( Tw ) is a semidominant mutation in the mouse affecting the embryonic development of the midfacial region. Most heterozygous Tw mice, +/−, become obese at adulthood with a concomitant decrease in fertility. Homozygous mice have clefts of the midfacial region and a disrupted nasal cavity. Midfacial clefts included clefts of the palate combined with either unilateral or bilateral clefts of the lip. The clefts of the lip were either complete or incomplete. The palatal shelves in Tw / Tw were very much reduced. Apart from these defects, homozygous Tw looked normal, and were born alive, although they reportedly die within 24 h after birth. It is proposed that the Twirler model can be used to improve understanding of the genetic mechanisms involved in the normal development of the midfacial region.

Expression studies have implicated FLRT2 in cranial neural crest cell migration and prechondrogenic cell condensation during craniofacial skeletogenesis. We aimed to determine whether FLRT2 was involved in mediating cell-matrix interactions in the ATDC5 chondroprogenitor cell line. Immunolocalization experiments of ATDC5 cells revealed that FLRT2 was present on the cell membrane as well as extracellularly, where it colocalized with Fibronectin (Fn). After cell extraction of the matrix, FLRT2 was identified in the ATDC5-derived extracellular matrix (ECM) and was further found to be associated with Fn-coated beads in cell cultures. Blockage of Fn fibril formation via a blocking peptide resulted in a concomitant decrease in extracellular FLRT2 accumulation. Over a 7-day period following the replenishment of the Fn blocking peptide to the cultures, there was a partial rebound in Fn fibril formation that was accompanied by a concomitant reappearance of FLRT2 co-expression. Co-immunoprecipitation confirmed that FLRT2 and Fn interacted, either directly or indirectly. Immunoprecipitation and Western blot analyses with antibodies recognizing epitopes located on the extra- and intracellular domains of FLRT2 further revealed the presence of different sized bands, suggesting that FLRT2 may exist in both membrane-bound and shed forms. Our data therefore provide evidence that FLRT2 and/or its cleavage products may be cooperating with Fn and other ECM proteins to regulate critical cellular events. Further studies will be necessary in delineate more precisely the roles of FLRT2 in mediating cell- and cell-matrix interactions during normal development.

Defects of the head and neck region account for a substantial portion of all human birth disorders. The high incidence of malformations in this region may be attributed in part to the intricate means by which the facial region is assembled during embryonic development. The starting constituent for the majority of skeletal and connective tissues in the face is a pluripotent population of cells, the cranial neural crest (CNC) cells. This population of cells exhibit remarkable migratory abilities and diversity of potential cell types. This review draws on extensive research that has been done in the field, focusing specifically on findings generated in the last decade on cell behavior and the gene regulatory networks of migratory CNC cells. In the later part of this review, the importance of the CNC cells in the overall development of the craniofacial region will be illustrated with a discussion of a craniofacial birth defect, the Treacher Collins syndrome. The next decade will most likely herald in an era of greater understanding of the integrative molecular networks at different stages of the development of the CNC cells. Such new information is essential towards a better understanding the etiology and pathogenesis of the many craniofacial birth defects and will ultimately lead to new therapeutic modalities.

The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases.

BACKGROUND Mutations in Fibroblastic Growth Factor Receptors (FGFR) have been associated with human craniosynostotic birth defects like Crouzon syndrome. Several anecdotes and case reports have indicated higher incidence of gastrointestinal tract disorders in FGFR-associated craniosynostotic birth defects. Our objective was to characterize esophageal defects in a mouse model of human Crouzon syndrome, with a mutation in codon 290 of FGFR2. METHODS Dissected esophagi of Fgfr2W290R postnatal heterozygous (HET) and wild-type mice were analyzed by histological staining, immunohistochemically with cell proliferation marker, and functionally by strain gauge measures of electrically evoked contractile force. RESULTS The esophagi of HETs were noticeably smaller but with wider lumen than those of wild-type littermates. The HET esophagi showed a decrease in proliferation and an increase in expression of Sonic Hedgehog as compared to wild-type esophagi. Histological investigations revealed reduced amounts and disorganization of collagen in muscle layers. Functional analysis revealed altered contractile properties in HET with reduced peak amplitude and prolonged duration of evoked contractile force response and lower stimulation threshold. CONCLUSION The defects observed in the esophagus of the mutant may explain some of the clinical symptoms observed in humans, for example, recurrent vomiting, gastroesophageal reflux, and esophageal strictures. Taken together, our results provide evidence for the importance of Fibroblastic Growth Factor signaling in the growth and patterning of the esophagus, providing a possible scientific basis for the gastrointestinal tract clinical findings in craniosynostotic patients. Furthermore, the findings also provide a sound scientific rationale for any changes in the clinical management of gastrointestinal tract problems in patients with craniosynostotic defects. Birth Defects Research (Part A) 97:578–586, 2013. © 2013 Wiley Periodicals, Inc.

Mutations in Fibroblastic Growth Factor Receptors (FGFR) have been associated with human craniosynostotic birth defects like Crouzon syndrome. Several anecdotes and case reports have indicated higher incidence of gastrointestinal tract disorders in FGFR-associated craniosynostotic birth defects. Our objective was to characterize esophageal defects in a mouse model of human Crouzon syndrome, with a mutation in codon 290 of FGFR2.Dissected esophagi of Fgfr2(W290R) postnatal heterozygous (HET) and wild-type mice were analyzed by histological staining, immunohistochemically with cell proliferation marker, and functionally by strain gauge measures of electrically evoked contractile force.The esophagi of HETs were noticeably smaller but with wider lumen than those of wild-type littermates. The HET esophagi showed a decrease in proliferation and an increase in expression of Sonic Hedgehog as compared to wild-type esophagi. Histological investigations revealed reduced amounts and disorganization of collagen in muscle layers. Functional analysis revealed altered contractile properties in HET with reduced peak amplitude and prolonged duration of evoked contractile force response and lower stimulation threshold.The defects observed in the esophagus of the mutant may explain some of the clinical symptoms observed in humans, for example, recurrent vomiting, gastroesophageal reflux, and esophageal strictures. Taken together, our results provide evidence for the importance of Fibroblastic Growth Factor signaling in the growth and patterning of the esophagus, providing a possible scientific basis for the gastrointestinal tract clinical findings in craniosynostotic patients. Furthermore, the findings also provide a sound scientific rationale for any changes in the clinical management of gastrointestinal tract problems in patients with craniosynostotic defects.

Few three-dimensional (3-D) models exist to study the cellular aspects and molecular regulation of orthodontic tooth movement (OTM). The aim of this study was to develop a 3-D in vitro model to study mechanical loading of human periodontal ligament (PDL) fibroblasts (hPDLF). hPDLF were seeded within collagen gels to form a PDLF analogue. Characterisation of the seeded collagen gels revealed that the gels supported cell proliferation, viability and the emergence of a possible contractile phenotype, replicating the constrained condition of the human PDL in vivo. We next developed a 3-D model that incorporated a seeded collagen gel interlocked mechanically at two ends to movable end plates. The movable end plates allowed for static tensile or compressive loading of the hPDLF-seeded collagen gels. Preliminary testings showed that this 3-D model mimicked PDL strains similar to those observed during OTM. Our 3-D model of OTM therefore offers promise for use as a model system in future studies to improve our understanding of the effects of OTM on PDLF.

In situ hybridization (ISH) is a technique that offers the ability to detect, and assay for alterations in, the spatial distribution of gene transcripts in an organism and is thus an invaluable tool to understanding the molecular basis during various developmental processes. Changes at the molecular levels consequent to toxicological perturbations and characterization of the expression of new or uncharacterized genes can be performed at the spatial and temporal levels with relative ease, speed, and specificity. Knowledge of expression pattern of specific genes allows one to formulate hypotheses of possible functions, molecular partners, and signaling pathways. We describe a procedure for ISH analysis of developing whole mouse embryos with the use of nonradioactive in vitro transcribed antisense RNA (riboprobes) and detected indirectly by a colorimetric reaction.

In situ hybridization (ISH) is a technique that offers the ability to detect, and assay for alterations in, the spatial distribution of gene transcripts in an organism and is thus an invaluable tool to understanding the molecular basis during various developmental processes. Changes at the molecular levels consequent to toxicological perturbations and characterization of the expression of new or uncharacterized genes can be performed at the spatial and temporal levels with relative ease, speed, and specificity. Knowledge of expression pattern of specific genes allows one to formulate hypotheses of possible functions, molecular partners, and signaling pathways. We describe a procedure for ISH analysis of developing whole mouse embryos with the use of nonradioactive in vitro transcribed antisense RNA (riboprobes) and detected indirectly by a colorimetric reaction.

OBJECTIVE: To test the use of macrophage colony-stimulating factor (M-CSF), an early osteoclast recruitment/differentiation factor, in increasing the rate of osteoclastic recruitment and differentiation as a means of accelerating tooth movement. MATERIALS AND METHODS: The distribution of osteoclasts and their precursors in the periodontal ligament (PDL) of teeth was initially characterized in a mouse model by immunohistochemical expression analyses of markers of osteoclast differentiation. We next administered two different dosages of M-CSF in the PDL of molars subject to force. Tooth movement was measured and correlated with changes in expression of M-CSF downstream genes in the PDL. RESULTS: We found that monocytes may have differentiated into preosteoclasts before being recruited to the PDL during the lag phase of tooth movement, and an influx of multinucleated osteoclasts occurred after 6 days. The lower dose of M-CSF was found to be most effective in increasing the amount of tooth movement and expression of M-CSF downstream genes and TRAP, an osteoclast marker. In contrast, administration of a higher dose of M-CSF resulted in a decrease in the expression of one gene downstream of M-CSF and possible inhibition of osteoclast formation. CONCLUSIONS: Exogenous administration of optimal dosages of M-CSF to orthodontically moved teeth provides potential for clinical studies in accelerating tooth movement.

Introduction Rapid palatal expanders (RPEs) have attachments cemented to the teeth and a screw that covers the palate. Because of their position and relative size, RPEs can affect speech. Our objective was to assess speech perturbation and adaptation related to RPE appliances over time. Methods RPEs were planned for the treatment of 22 patients in the orthodontic clinic at the University of Toronto in Canada. Speech recordings were made at 6 time points: before RPE placement, after placement, during expansion, during retention, after removal, and 4 weeks after removal. The speech recordings consisted of 35 sentences, from which 3 sentences were chosen for analysis. Speech acceptability was assessed perceptually by 10 listeners who rated each sentence on an equal-appearing interval scale. The vowel formants for /i/ and the fricative spectra for /s/ and /∫/ were measured with speech analysis software. Repeated-measures analysis of variance with post-hoc paired t tests was used for statistical analysis. Results When the appliance was placed, speech acceptability deteriorated. Over time, the ratings improved and returned to baseline when the appliance was removed. For the vowel /i/, the first formant increased, and the second formant decreased in frequency, indicating centralization of the vowel. The formants returned to the pretreatment levels during treatment. For the fricatives (/s/ and /∫/), low-to-high frequency ratios indicated that the fricatives were distorted when the appliance was placed. The ratios returned to baseline levels once the appliance was removed. The results for the spectral moments indicated that spectral mean decreased and skewness became more positive. Repeated-measures analysis of variance showed significant effects for time for all acoustic measures. Conclusions Speech was altered and distorted when the appliance was first placed. The patients’ speech gradually improved over time and returned to baseline once the appliance was removed. The results from the study will be useful for pretreatment counseling of patients and their families.

Introduction: Clefts of the lip and palate result when the midfacial processes fail to fuse during embryonic development. Many of the cellular and molecular events underlying the fusion of these midfacial processes are unknown. There is evidence from our laboratory and others that Bmp-4 is a signal that regulates the process of fusion in the midface. RNA interference (RNAi) is a relatively new technique used to knock down select RNA levels—and ultimately protein levels—by using short RNA strands complementary to target mRNA in the cytoplasm. This was the first investigation to use RNAi to study the developing wild-type murine midface in vivo. This study used RNAi technology to decrease levels of Bmp-4 RNA during critical stages of midfacial fusion with the hypothesis that correct temporal and spatial expression of Bmp-4 is critical for normal fusion and formation of the midface. Methods: The sample included 19 timed pregnant wild-type (C57B1/6J strain) mice and their litters. Pregnant mice received tail-vein injections of plasmids coding for an epifluorescent label (DsRed) with or without the Bmp-4 RNAi sequence 3 days before typical embryonic midface fusion. Embryos were harvested before, during, and after the typical date of midface fusion, and delivery of the plasmids was confirmed via epifluoroscopy and reverse transcriptase polymerase chain reaction (RT-PCR) for the DsRed label. Results: Alterations in morphology were observed by gross examination and by sectioning the embryos. Levels of Bmp-4 mRNA and protein in the embryonic midface were examined with RT-PCR and immunohistochemistry, respectively. Injections of RNA plasmids via the maternal tail-vein to pregnant mothers reliably delivered plasmids to the developing embryonic midface with an average 46% (± 12.2) knockdown in Bmp-4 RNA levels. Conclusions: Reduced BMP-4 levels caused prefusion delays in the outgrowth of the lateral and medial nasal processes. These processes later recovered, producing no detectable long-term morphological differences when compared with the control embryos. Ectopic Bmp-4 protein expression was observed in the lateral and medial nasal processes mesenchyme in Bmp-4 knockdown embryos superior to the region of primary palatal fusion. These findings implicated Bmp-4 signaling in the outgrowth and development of the midfacial processes. This study introduced the use of RNAi for the study of protein signaling in the developing wild-type murine midface in vivo.

Dentinogenesis imperfecta (DGI) and dentin dysplasia (DD) are allelic disorders that primarily affect the formation of tooth dentin. Both conditions are autosomal-dominant and can be caused by mutations in the dentin sialophosphoprotein gene ( DSPP, 4q21.3). We recruited 23 members of a four-generation kindred, including ten persons with dentin defects, and tested the hypothesis that these defects are linked to DSPP. The primary dentition showed amber discoloration, pulp obliteration, and severe attrition. The secondary dentition showed either pulp obliteration with bulbous crowns and gray discoloration or thistle-tube pulp configurations, normal crowns, and mild gray discoloration. Haplotype analyses showed no recombination between three 4q21-q24 markers and the disease locus. Mutational analyses identified no coding or intron junction sequence variations associated with affection status in DMP1, MEPE, or the DSP portion of DSPP. The defects in the permanent dentition were typically mild and consistent with a diagnosis of DD-II, but some dental features associated with DGI-II were also present. We conclude that DD-II and DGI-II are milder and more severe forms, respectively, of the same disease.