CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Quantifying cell-generated mechanical forces within living embryonic tissues.

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN µm(-2)) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles.

'Love and trust, you can be blinded': HIV risk within relationships among Latina women in Miami, Florida.

OBJECTIVES: Latina women are disproportionately affected by HIV in the US, and account for 30% of all HIV infections in Miami-Dade County, Florida. The main risk for Latina women is heterosexual contact. Little is known about the relational and cultural factors that may impact women's HIV risk perception. This study aims to describe Latina women's perception of their HIV risk within a relational, cultural, and linguistic context. DESIGN: Eight focus groups of Latina women (n = 28), four English speaking groups and four Spanish speaking groups, were conducted between December 2013 and May 2014. Women were recruited from a diversion program for criminal justice clients and by word of mouth. Eligibility criteria included the following: self-identify as Hispanic/Latino, 18-49 years of age, and self-identify as heterosexual. A two-level open coding analytic approach was conducted to identify themes across groups. RESULTS: Most participants were foreign-born (61%) and represented the following countries: Cuba (47%), Honduras (17.5%), Mexico (12%), as well as Nicaragua, Puerto Rico, Colombia, and Venezuela (15%). Participant ages ranged between 18 and 49, with a mean age of 32 years. Relationship factors were important in perceiving HIV risk including male infidelity, women's trust in their male partners, relationship type, and getting caught up in the heat of the moment. For women in the English speaking groups, drug use and trading sex for drugs were also reasons cited for putting them at risk for HIV. English speaking women also reported that women should take more responsibility regarding condom use. CONCLUSION: Findings emphasize the importance of taking relational and cultural context into account when developing HIV prevention programs for Latina women. Interventions targeting English speaking Latina women should focus on women being more proactive in their sexual health; interventions focused on Spanish speaking women might target their prevention messages to either men or couples.

Alberta reconstructive technique (ART): An innovative approach using digital surgical design and simulation in advanced jaw reconstruction with occlusion-based prefabricated vascularized fibular flaps and primary osseointegrated implant installation.

The Alberta reconstructive technique (ART) is an innovative surgical procedure performed on patients undergoing primary jaw resection and reconstruction. The ART procedure was developed in collaboration with the Institute for Reconstructive Sciences in Medicine and the Division of Otolaryngology-Head and Neck Surgery, University of Alberta.

Network-based inference from complex proteomic mixtures using SNIPE.

MOTIVATION: Proteomics presents the opportunity to provide novel insights about the global biochemical state of a tissue. However, a significant problem with current methods is that shotgun proteomics has limited success at detecting many low abundance proteins, such as transcription factors from complex mixtures of cells and tissues. The ability to assay for these proteins in the context of the entire proteome would be useful in many areas of experimental biology. RESULTS: We used network-based inference in an approach named SNIPE (Software for Network Inference of Proteomics Experiments) that selectively highlights proteins that are more likely to be active but are otherwise undetectable in a shotgun proteomic sample. SNIPE integrates spectral counts from paired case-control samples over a network neighbourhood and assesses the statistical likelihood of enrichment by a permutation test. As an initial application, SNIPE was able to select several proteins required for early murine tooth development. Multiple lines of additional experimental evidence confirm that SNIPE can uncover previously unreported transcription factors in this system. We conclude that SNIPE can enhance the utility of shotgun proteomics data to facilitate the study of poorly detected proteins in complex mixtures. AVAILABILITY AND IMPLEMENTATION: An implementation for the R statistical computing environment named snipeR has been made freely available at http://genetics.bwh.harvard.edu/snipe/. CONTACT: ssunyaev@rics.bwh.harvard.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Temporomandibular joint formation requires two distinct hedgehog-dependent steps.

We conducted a genetic analysis of the developing temporo-mandibular or temporomandi-bular joint (TMJ), a highly specialized synovial joint that permits movement and function of the mammalian jaw. First, we used laser capture microdissection to perform a genome-wide expression analysis of each of its developing components. The expression patterns of genes identified in this screen were examined in the TMJ and compared with those of other synovial joints, including the shoulder and the hip joints. Striking differences were noted, indicating that the TMJ forms via a distinct molecular program. Several components of the hedgehog (Hh) signaling pathway are among the genes identified in the screen, including Gli2, which is expressed specifically in the condyle and in the disk of the developing TMJ. We found that mice deficient in Gli2 display aberrant TMJ development such that the condyle loses its growth-plate-like cellular organization and no disk is formed. In addition, we used a conditional strategy to remove Smo, a positive effector of the Hh signaling pathway, from chondrocyte progenitors. This cell autonomous loss of Hh signaling allows for disk formation, but the resulting structure fails to separate from the condyle. Thus, these experiments establish that Hh signaling acts at two distinct steps in disk morphogenesis, condyle initiation, and disk-condyle separation and provide a molecular framework for future studies of the TMJ.

The Modified Alberta Reconstructive Technique: A prospective cohort study.

INTRODUCTION: Jaw defect reconstructions have been transformed by the development of free tissue transfer using vascularized bone incorporating osseointegrated dental implants. We recently developed a modification our method this procedure and termed it the Modified Alberta Reconstruction Technique (MART). The objective of this study aimed to assess the soft tissue component and outcomes of the MART as compared to the Alberta Reconstructive Technique (ART) or conventional (BDD) reconstructions. PATIENTS AND METHODS: This was a prospective cohort study of adult patients who underwent jaw reconstruction with dental implant rehabilitation between 2000 and 2019 in Edmonton, Alberta. Patients were aged-matched and placed into a cohort based on the type of reconstruction they received. Outcomes were compared between the groups. Expert and aesthetic analyses were performed. Statistical analysis was conducted to determine significance. RESULTS: A total of 46 patients (15 BDD, 15 ART and 16 MART) were included. Demographics were similar between groups. There was no difference in complications. The soft tissue component of the MART cohort was more favourable to work with as judged by the occlusal reconstructive experts. The MART was rated as more aesthetically appealing in comparison to the BDD and ART (p = 0.049). CONCLUSIONS: The MART is a safe, effective, and aesthetically appealing procedure. It yields a good functional result and a clinically better soft tissue component for occlusal reconstructions. For a select group of patients requiring jaw reconstruction, the MART is an ideal reconstructive option as the modification provides good control of the soft tissue around the implants.

Development of a Patient-Centered Functional Outcomes Questionnaire in Head and Neck Cancer.

Importance: Incorporation of patient perspectives, or patient-reported outcomes, in functional outcome measures has been gaining prominence in the literature on reconstructive surgery. Objective: To create and validate an instrument for measuring the main functional areas of concern for patients with head and neck cancer. Design, Setting, and Participants: This 4-phase mixed-methods qualitative study was conducted from July 1, 2013, to June 30, 2016, in a quaternary head and neck oncology center in Edmonton, Alberta, Canada. Patients were recruited from 3 Head and Neck Research Network sites: University of Alberta (Edmonton, Canada), Mount Sinai Health Network (New York, New York), and University of Turku Hospital (Turku, Finland). The inclusion criteria included 18 years of age or older, diagnosis of squamous cell carcinoma involving the subsites of the head and neck (ie, oral cavity, oropharynx, hypopharynx, and larynx), and at least 1 year since treatment completion. Those patients who were undergoing additional active treatment or with evidence of disease recurrence were excluded. Data were analyzed from July 1, 2013, to June 30, 2016. Main Outcomes and Measures: The primary outcome measures were the clinical correlation of the Edmonton-33 instrument scores with swallowing, speech, dry mouth, and chewing assessment outcomes. Results: In total, 10 patients with head and neck cancer (mean age, 59.6 years; 6 men [60%]) were included in phase 1 of the study, 5 patients (mean age, 55.2 years) were included in phase 2, 10 patients were included in phase 3, and 25 patients with head and neck cancer (mean age, 62.6 years; 14 men [56%]) participated in the phase 4 validation. The Edmonton-33 instrument scores correlated strongly with the swallowing scores of the MD Anderson Dysphagia Inventory (r = 0.77; 95% CI, 0.49-1.0), the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Head and Neck 35 (EORTC QLQ-H&N35) (r = -0.73; 95% CI, -1.0 to -0.44), and the modified barium swallow test (r = -0.60; 95% CI, -0.94 to -0.25). The instrument scores were also strongly correlated with the Speech Handicap Index scores (r = -0.64; 95% CI, -0.97 to -0.31), word intelligibility scores (r = 0.61; 95% CI, 0.27-0.95), and sentence intelligibility scores (r = 0.55; 95% CI, 0.19-0.91). A moderate to strong correlation was observed between the Edmonton-33 instrument and the EORTC QLQ-H&N35 scores in the dry mouth (r = -0.54; 95% CI, -0.91 to -0.18) and chewing (r = -0.45; 95% CI, -0.84 to -0.06) domains. The factor loading values for the domains of swallowing, speech, dry mouth, and chewing were all greater than 0.3. The mean factor loading values for the items related to swallowing were 0.71 (95% CI, 0.62-0.80) and for the items related to speech were 0.76 (95% CI, 0.72-0.80). The mean factor loading values for the items related to dry mouth were 0.71 (95% CI, 0.59-0.83) and for those related to chewing were 0.77 (95% CI, 0.69-0.85). Conclusions and Relevance: The Edmonton-33 appears to be a validated instrument that will allow patients with head and neck cancer to assess and report their own functional outcomes. It could serve as a single comprehensive measure for functional outcomes.

Beavertail modification of the radial forearm free flap in total oral glossectomy reconstruction: Technique and functional outcomes.

OBJECTIVE: The total oral tongue (anterior 2/3 glossectomy) defect is seldom addressed in the literature. This is the first series to describe a consistent technique for its reconstruction. The aim of the study is to describe the use of the beavertail modified radial forearm free flap (BTRFFF) to reconstruct a total oral tongue defect and the functional and quality of life outcomes associated with it. STUDY DESIGN: Retrospective review of prospectively collected data from 2000 to 2010. METHODS: All patients at the University of Alberta undergoing head and neck free flap surgery are enrolled in a prospective functional outcomes program. Pre-operatively and at set post-operative time points patients complete videofluoroscopic swallowing studies (VFSS), speech evaluations and quality of life questionnaires (EORTC H&N-35). Peri-operative outcomes were also measured. RESULTS: 17 consecutive patients were included. All were gastrostomy tube free at 12 months post-operatively and tolerating a full soft diet with aspiration scores of 0. Swallowing transit times increased by a mean of 0.4 s (p = 0.32). Speech intelligibility remained high with mean sentence intelligibility at 75% and single word intelligibility at 62%. Quality of life scores returned to baseline and remained satisfactory. Complications related to the BTRFFF were limited to scarring. CONCLUSIONS: The BTRFFF provides a robust reconstructive option for the total oral tongue defect with excellent long term functional outcomes and quality of life.

The Alberta Reconstructive Technique: An Occlusion-Driven and Digitally Based Jaw Reconstruction.

OBJECTIVES/HYPOTHESIS: The free flap reconstructive protocols of the jaws have been refined over the years and presently are based on bone-driven approaches that generally use the lower border of the mandible or the anterior surface of the maxilla as the templates for reconstruction because these contours are deemed important to the eventual cosmetic outcomes of patients. The ultimate goal of functional jaw reconstruction, however, is the reconstruction of the dental occlusion and oral rehabilitation. The purpose of the present study was to evaluate the Alberta reconstructive technique (ART), which is a new approach of occlusion-driven jaw reconstruction with digitally planned immediate osseointegrated implant installation. STUDY DESIGN: Prospective cohort study. METHODS: This research study considers the ART's safety, effectiveness, accuracy, timeliness of reconstruction, aesthetic appeal, and cost-effectiveness in comparison with the standard bone-driven and delayed osseointegrated implant installation (BDD) protocol. RESULTS: The ART procedures were as safe and more effective at achieving full occlusal reconstruction and oral rehabilitation. The ART cohort of patients achieved oral rehabilitation in 21.4 month as compared to 73.1 months for the BDD cohort. There were no differences in the aesthetic appeal the two groups. The ART cost an average of $22,004 less than BDD and we calculated the quality adjusted life years gain to be between 2.14 and 4.04 in favour of ART. CONCLUSIONS: The ART is a good option for patients with jaw defects. It provides a safe, effective, accurate, aesthetic, and cost-effective reconstruction that restores form and function in a timely manner. LEVEL OF EVIDENCE: 2b Laryngoscope, 129:S1-S14, 2019.

Multi-dimensional analysis of oral cavity and oropharyngeal defects following cancer extirpation surgery, a cadaveric study.

BACKGROUND: Defects following resection of tumors in the head and neck region are complex; more detailed and defect-specific reconstruction would likely result in better functional and cosmetic outcomes. The objectives of our study were: 1) to improve the understanding of the two- and three-dimensional nature of oral cavity and oropharyngeal defects following oncological resection and 2) to assess the geometric dimensions and the shapes of fasciocutaneous free flaps and locoregional tissue flaps required for reconstruction of these defects. METHODS: This study was an anatomic cadaveric study which involved creating defects in the oral cavity and oropharynx in two cadaveric specimens. Specifically, partial and total glossectomies, floor of mouth excisions, and base of tongue excisions were carried out. These subsites were subsequently geometrically analyzed and their volumes measured. The two-dimensional (2D) assessment of these three-dimensional (3D) structures included measures of surface area and assessment of tissue contours and shapes. RESULTS: The resected specimens all demonstrated unique dimensional geometry for the various anatomic sites. Using 2D analysis, hemiglossectomy defects revealed right triangle geometry, whereas total glossectomy geometry was a square. Finally, the base of tongue defects exhibited a trapezoid shape. CONCLUSIONS: Customizing the geometry and dimensions of fasciocutaneous free flaps so that they are specific to the confronted head and neck defects will likely result in better functional and cosmetic outcomes.

Transoral robotic surgery with radial forearm free flap reconstruction: case control analysis.

BACKGROUND: The resection of large oropharyngeal tumors traditionally involves a lip-splitting mandibulotomy for adequate margin visualization and free flap reconstruction of the surgical defect. Transoral robotic surgery (TORS) has emerged as a technique that can resect large and complex oropharyngeal tumors, avoiding a lip-splitting approach. The aim of this study is to compare the lip-splitting mandibulotomy approach versus TORS for the management of advanced stage oropharyngeal carcinomas. METHODS: Prospectively collected data from 18 patients with advanced stage oropharyngeal squamous cell carcinoma (OPSCC) who received TORS with radial forearm free flap reconstruction (RFFF) was compared to a matched cohort of 39 patients who received a lip-splitting mandibulotomy and RFFF. Patients were matched for stage, p16 positivity, smoking, age and gender. Length of hospital stay (LOHS), tracheostomy decanulation time, operative time, surgical margin status, and post-operative complications were compared between groups. RESULTS: Patients who received TORS with RFFF had a significantly lower mean LOHS, compared to patients who were treated by lip-splitting mandibulotomy and RFFF (14.4 vs 19.7 days, p = 0.03). No significant differences were seen between groups in terms of operative time, tracheostomy decannulation time, margin positivity and post-operative complications. CONCLUSION: TORS with radial forearm free flap reconstruction is a safe, effective and cost-saving alternative to the lip-splitting mandibulotomy approach for the treatment of advanced stage OPSCC.

Functional and quality of life outcomes after partial glossectomy: a multi-institutional longitudinal study of the head and neck research network.

BACKGROUND: While aggressive treatment for oral cancer may optimize survival, decrements in speech and swallowing function and quality of life often result. This exploratory study investigated how patients recover their communicative function, swallowing ability, and quality of life after primary surgery [with or without adjuvant (chemo)radiation therapy] for tongue cancer over the course of the first year post-operation. METHODS: Patients treated for oral cancer at three institutions (University of Alberta Hospital, Mount Sinai Beth Israel Medical Center, and Turku University Hospital) were administered patient-reported outcomes assessing speech [Speech Handicap Index (SHI)], swallowing [(M.D. Anderson Dysphagia Inventory (MDADI)] and quality of life [European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Head and Neck Module (EORTC-H&N35)]. Outcome measures were completed pre-operatively and at 1-, 6-, and 12-months post-operatively. RESULTS: One hundred and seventeen patients undergoing partial glossectomy with reconstruction participated in this study. Results indicated no significant differences in swallowing function (MDADI and EORTC-H&N35 subscales) between baseline and 6 months post-surgery and no significant differences in speech function (SHI subscales) between baseline and 1 year post-surgery. Most quality of life domains (EORTC-H&N35 subscales) returned to baseline levels by 1 year post-operation, while difficulties with dry mouth and sticky saliva persisted. A clear time trend of adjuvant (chemo)radiation therapy negatively affecting dry mouth scores over time was identified in this study, while negative independent effects of chemoradiation on MDADI swallowing, and EORTC-H&N35 swallowing, eating, and opening mouth subscales were found. CONCLUSIONS: Assessment time influenced patient-reported speech, swallowing, and quality of life outcomes, while treatment (by time) effects were found for only swallowing and quality of life outcomes. Results of the present study will help guide clinical care and will be useful for patient counseling on expected short and long-term functional and quality of life outcomes of surgical and adjuvant treatment for oral cavity cancer.

Bone-impacted fibular free flap: Long-term dental implant success and complications compared to traditional fibular free tissue transfer.

BACKGROUND: The purpose of this study was to compare complications and dental implant success between the bone-impacted fibula free flap (BIFFF) and the traditional fibular free flap used in mandibular and midface reconstruction. METHODS: Retrospective review of all patients undergoing BIFFF or traditional fibular free flap reconstruction from 2001 to 2009 was undertaken. Complications related to the BIFFF and traditional fibular free flap site of reconstruction were compared. Dental implant success rates for each type of flap were compared at 1-year intervals for 5 years. RESULTS: One hundred fourteen patients underwent 81 BIFFFs and 35 traditional fibular free flaps. No significant difference in complications between BIFFF (20.9%) and traditional fibular free flap (25.7%) reconstruction was observed. Logistic regression analysis revealed only the site as a predictor of both single and multiple complications. At 5 years postimplantation, dental implant success rates were 95.5% and 77.1% for BIFFF and traditional fibular free flap, respectively (p = .006). CONCLUSION: BIFFF reconstruction is a novel surgical technique that may improve long-term dental implant success rates with no additional risk of complications. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1783-E1787, 2016.

Predictors of Failed and Delayed Decannulation after Head and Neck Surgery.

OBJECTIVE: To determine the variables that are predictive of failed decannulation (FD), delayed decannulation (DD), and days to decannulation in patients who underwent head and neck cancer resection with free tissue transfer reconstruction for head and neck squamous cell carcinoma. DESIGN: Case series with chart review. SETTING: Tertiary care otolaryngology-head and neck surgery referral center. SUBJECT AND METHODS: Patients (N = 108) were included who underwent head and neck cancer resection with free tissue transfer reconstruction and tracheostomy between 2011 and June 2014. Patients with laryngectomy, previous tracheostomy, and other airway pathology necessitating tracheotomy were excluded. Preoperative patient variables and cancer site/staging variables were analyzed, as well as extent of structures resected and type of reconstruction. Univariate and multivariate binary logistic and Cox regression analyses were used to determine predictors of FD and DD. Cox regression analysis was used to determine predictors of days to decannulation. RESULTS: Of the 108 included patients, 16 had FD, and 26 had DD. Univariate analysis demonstrated that advanced stage (r = 0.233, P = .021), total glossectomy (r = 0.924, P < .001), anterolateral thigh flap reconstruction (r = 0.906, P < .001), smoking at time of surgery (r = 0.319, P = .002), and pack years (r = 0.322, P = .001) were associated with FD. Cox regression analysis showed that total glossectomy, exp(B) = 15.837 (95% confidence interval [95% CI]: 1.949-128.679); anterolateral thigh flap reconstruction, exp(B) = 8.439 (95% CI: 2.435-29.620); and smoking status, exp(B) = 2.970 (95% CI: 1.617-5.456) were independent predictors of days to decannulation and FD. CONCLUSIONS: Patients with total glossectomy defects and those who continue to smoke are at increased risk for FD and DD. Aggressive smoking cessation programs may decrease the risk of FD and DD. Patients should be counseled about their risk profiles.

Anatomical examination of the fibula: digital imaging study for osseointegrated implant installation.

BACKGROUND: Free vascularized fibular flaps are commonly used in jaw reconstruction. CT scan images of the fibula are used in digital planning of jaw reconstruction. In order to fully describe the anatomy of the fibula, an imaging study of the fibula was undertaken. The purpose of the present study was to examine the anatomical structure of the fibula using patient CT images. METHODS: The CT scan images of fibulae of 20 patients were used for the study. The results of the analysis showed that, of the widths, the anterior border of the fibula to the posterior surface was the largest dimension. The shape type analysis showed that the triangular type was most prominent near the head of the fibula, and the irregular type was most prominent towards the lateral malleolus. RESULTS: The results of height and width related to the long axis of implant installation showed that the width of the central section was the largest. With respect to the length of available bone volume, the length near the lateral malleolus was larger than that near the head of the fibula. The results showed that there were significant differences in size between male and female fibulae. CONCLUSION: The present study provides a CT scan based analysis of the anatomy of the fibula. Important information for the optimal site of installation of osseointegrated implants in fibular free flap reconstructions is also provided.

A Wnt-bmp feedback circuit controls intertissue signaling dynamics in tooth organogenesis.

Many vertebrate organs form through the sequential and reciprocal exchange of signaling molecules between juxtaposed epithelial and mesenchymal tissues. We undertook a systems biology approach that combined the generation and analysis of large-scale spatiotemporal gene expression data with mouse genetic experiments to gain insight into the mechanisms that control epithelial-mesenchymal signaling interactions in the developing mouse molar tooth. We showed that the shift in instructive signaling potential from dental epithelium to dental mesenchyme was accompanied by temporally coordinated genome-wide changes in gene expression in both compartments. To identify the mechanism responsible, we developed a probabilistic technique that integrates regulatory evidence from gene expression data and from the literature to reconstruct a gene regulatory network for the epithelial and mesenchymal compartments in early tooth development. By integrating these epithelial and mesenchymal gene regulatory networks through the action of diffusible extracellular signaling molecules, we identified a key epithelial-mesenchymal intertissue Wnt-Bmp (bone morphogenetic protein) feedback circuit. We then validated this circuit in vivo with compound genetic mutations in mice that disrupted this circuit. Moreover, mathematical modeling demonstrated that the structure of the circuit accounted for the observed reciprocal signaling dynamics. Thus, we have identified a critical signaling circuit that controls the coordinated genome-wide expression changes and reciprocal signaling molecule dynamics that occur in interacting epithelial and mesenchymal compartments during organogenesis.

An evolutionarily conserved enhancer regulates Bmp4 expression in developing incisor and limb bud.

To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs). These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.

Reconstruction of the midface and maxilla.

PURPOSE OF REVIEW: To review all pertinent topics related to midface and maxillary reconstruction including current classification schemes and reconstructive techniques. RECENT FINDINGS: The maxilla represents the functional and aesthetic cornerstone of the midface region. Defects in this area represent a unique challenge to the reconstructive surgeon as the complex anatomy of this area can be challenging to repair and reconstruct. The current indications as well as advantages and disadvantages of different reconstructive approaches are reviewed here in order to provide reconstructive surgeons with optimal information for surgical and clinical decision making in this challenging patient population. Recent developments in midface reconstruction options including near total facial transplant as well as tissue engineering techniques are also reviewed. SUMMARY: Multiple reconstructive pathways can be followed in restoring maxillary and midface defects. The surgeon and rest of the reconstructive team must make individualized decisions based on the nature of the defect as well as the specific needs and concerns of the patient when selecting the optimal reconstructive approach for each patient.

The lip-splitting mandibulotomy: aesthetic and functional outcomes.

To determine the aesthetic and functional outcomes of the most invasive approach to oral cavity/oropharyngeal lesions, the lip-splitting mandibulotomy approach (LSMA), versus the least invasive, the trans-oral approach (TOA). Retrospective paired-cohort study. Thirty-six patients with oral/oropharyngeal cancers treated with primary surgical extirpation, bilateral neck dissections and adjuvant radiation therapy were enrolled. Half underwent LSMA and half TOA. Patients were paired into gender, age and follow-up time matched cohorts. The primary outcome measure was overall patient satisfaction with scar cosmesis assessed with a 10-point Likert scale. Clinician and naïve viewer ratings of disfigurement in addition to the validated Vancouver scar scale (VSS) and patient and observer scar assessment scale (POSAS) were completed. Functional outcomes included lower-lip sensation and movement as well as oral continence assessment. LSMA patients had very high satisfaction and low perceptions of disfigurement with no statistically significant differences between cohorts (p>.05). VSS and POSAS results failed to demonstrate significant differences between groups (p>.05). Naïve observers, however, found the LSMA to be more disfiguring than TOA scars (p=.03). No significant differences in lower-lip sensation to touch, two-point discrimination or temperature distinction were found (p>.05). House-Brackman and movement symmetry scores were significantly indifferent between cohorts (p>.05). Video-fluoroscopic swallowing studies showed no difference in oral continence between cohorts (p>.05). The LSMA provides satisfactory scarring and low self-perception of disfigurement for patients. Moreover, the LSMA does not impact lower-lip sensation, movement or oral continence.