Assembly of Biologically Functional Structures by Nucleic Acid Templating: Implementation of a Strategy to Overcome Inhibition by Template Excess.

Delivery of therapeutic molecules to pathogenic cells is often hampered by unintended toxicity to normal cells. In principle, this problem can be circumvented if the therapeutic effector molecule is split into two inactive components, and only assembled on or within the target cell itself. Such an in situ process can be realized by exploiting target-specific molecules as templates to direct proximity-enhanced assembly. Modified nucleic acids carrying inert precursor fragments can be designed to co-hybridize on a target-specific template nucleic acid, such that the enforced proximity accelerates assembly of a functional molecule for antibody recognition. We demonstrate the in vitro feasibility of this adaptation of nucleic acid-templated synthesis (NATS) using oligonucleotides bearing modified peptides ("haplomers"), for templated assembly of a mimotope recognized by the therapeutic antibody trastuzumab. Enforced proximity promotes mimotope assembly via traceless native chemical ligation. Nevertheless, titration of participating haplomers through template excess is a potential limitation of trimolecular NATS. In order to overcome this problem, we devised a strategy where haplomer hybridization can only occur in the presence of target, without being subject to titration effects. This generalizable NATS modification may find future applications in enabling directed targeting of pathological cells.

Nox2 Inhibition Regulates Stress Response and Mitigates Skeletal Muscle Fiber Atrophy during Simulated Microgravity.

Insufficient stress response and elevated oxidative stress can contribute to skeletal muscle atrophy during mechanical unloading (e.g., spaceflight and bedrest). Perturbations in heat shock proteins (e.g., HSP70), antioxidant enzymes, and sarcolemmal neuronal nitric oxidase synthase (nNOS) have been linked to unloading-induced atrophy. We recently discovered that the sarcolemmal NADPH oxidase-2 complex (Nox2) is elevated during unloading, downstream of angiotensin II receptor 1, and concomitant with atrophy. Here, we hypothesized that peptidyl inhibition of Nox2 would attenuate disruption of HSP70, MnSOD, and sarcolemmal nNOS during unloading, and thus muscle fiber atrophy. F344 rats were divided into control (CON), hindlimb unloaded (HU), and hindlimb unloaded +7.5 mg/kg/day gp91ds-tat (HUG) groups. Unloading-induced elevation of the Nox2 subunit p67phox-positive staining was mitigated by gp91ds-tat. HSP70 protein abundance was significantly lower in HU muscles, but not HUG. MnSOD decreased with unloading; however, MnSOD was not rescued by gp91ds-tat. In contrast, Nox2 inhibition protected against unloading suppression of the antioxidant transcription factor Nrf2. nNOS bioactivity was reduced by HU, an effect abrogated by Nox2 inhibition. Unloading-induced soleus fiber atrophy was significantly attenuated by gp91ds-tat. These data establish a causal role for Nox2 in unloading-induced muscle atrophy, linked to preservation of HSP70, Nrf2, and sarcolemmal nNOS.

Nox2 signaling and muscle fiber remodeling are attenuated by losartan administration during skeletal muscle unloading.

Reduced mechanical loading results in atrophy of skeletal muscle fibers. Increased reactive oxygen species (ROS) are causal in sarcolemmal dislocation of nNOS and FoxO3a activation. The Nox2 isoform of NADPH oxidase and mitochondria release ROS during disuse in skeletal muscle. Activation of the angiotensin II type 1 receptor (AT1R) can elicit Nox2 complex formation. The AT1R blocker losartan was used to test the hypothesis that AT1R activation drives Nox2 assembly, nNOS dislocation, FoxO3a activation, and thus alterations in morphology in the unloaded rat soleus. Male Fischer 344 rats were divided into four groups: ambulatory control (CON), ambulatory + losartan (40 mg kg-1  day-1 ) (CONL), 7 days of tail-traction hindlimb unloading (HU), and HU + losartan (HUL). Losartan attenuated unloading-induced loss of muscle fiber cross-sectional area (CSA) and fiber-type shift. Losartan mitigated unloading-induced elevation of ROS levels and upregulation of Nox2. Furthermore, AT1R blockade abrogated nNOS dislocation away from the sarcolemma and elevation of nuclear FoxO3a. We conclude that AT1R blockade attenuates disuse remodeling by inhibiting Nox2, thereby lessening nNOS dislocation and activation of FoxO3a.

Can Patients With Isolated Mandibular Fractures Be Treated as Outpatients?

PURPOSE: Treating mandibular fractures urgently is controversial. The purpose of this study was to estimate and compare the rates of postoperative inflammatory complications (POICs) in patients with isolated mandibular fractures treated in a nonurgent manner by an outpatient protocol versus a traditional, urgent inpatient protocol. PATIENTS AND METHODS: We implemented a retrospective cohort study and enrolled a sample of patients with isolated mandibular fractures treated with open reduction-internal fixation (ORIF). The primary predictor variable was the treatment protocol: outpatient (elective) or inpatient (urgent). The outpatient group was treated with closed reduction and intermaxillary fixation, discharged, and scheduled for definitive treatment as outpatients. The inpatient group was admitted to the hospital, and the fracture was treated with ORIF as soon as possible. The primary outcome variable was POIC (present or absent). Descriptive, bivariate, and multiple logistic regression statistics were computed to measure the association between the treatment protocol and POICs, with statistical significance set at P < .05. RESULTS: The study sample was composed of 193 patients, with 82 in the outpatient group and 111 in the inpatient group. The frequency of POICs was 17.1% and 18.9% in the outpatient and inpatient groups, respectively (P = .13; relative risk, 0.80; 95% confidence interval [CI], 0.62 to 1.0). The time to ORIF was not significantly associated with POICs (P = .71). After adjustment for treatment group, fracture location, and time to fracture stabilization, smoking (P = .04, odds ratio, 2.3; 95% CI, 1.0 to 5.1) and intraoral incision with a transbuccal trocar (P = .02, odds ratio, 3.4; 95% CI, 1.2 to 9.8) were associated with an increased risk of POICs. Length of stay was 0.6 ± 0.8 days in the outpatient group compared with 2.7 ± 2.0 days in the inpatient group (P < .0001). CONCLUSIONS: An outpatient model to treat isolated mandibular fractures was not associated with an increased risk of POICs. This outpatient care model reduced the hospital length of stay without increasing the risk of POICs.

Effect of combined fish oil & Curcumin on murine skeletal muscle morphology and stress response proteins during mechanical unloading.

Skeletal muscle is a highly adaptable tissue capable of remodeling when dynamic stress is altered, including changes in mechanical loading and stretch. When muscle is subjected to an unloaded state (e.g., bedrest, immobilization, spaceflight) the resulting loss of muscle cross sectional area (CSA) impairs force production. In addition, muscle fiber-type shifts from slow to fast-twitch fibers. Unloading also results in a downregulation of heat shock proteins (e.g., HSP70) and anabolic signaling, which further exacerbate these morphological changes. Our lab recently showed reactive oxygen species (ROS) are causal in unloading-induced alterations in Akt and FoxO3a phosphorylation, muscle fiber atrophy, and fiber-type shift. Nutritional supplements such as fish oil and curcumin enhance anabolic signaling, glutathione levels, and heat shock proteins. We hypothesized that fish oil, rich in omega-3-fatty acids, combined with the polyphenol curcumin would enhance stress protective proteins and anabolic signaling in the rat soleus muscle, concomitant with synergistic protection of morphology. C57BL/6 mice were assigned to 3 groups (n = 6/group): ambulatory controls (CON), hindlimb unloading (HU), and hindlimb unloading with 5% fish oil, 1% curcumin in diet (FOC). FOC treatments began 10 days prior to HU and tissues were harvested following 7 days of HU. FOC mitigated the unloading induced decrease in CSA. FOC also enhanced abundance of HSP70 and anabolic signaling (Akt phosphorylation, p70S6K phosphorylation), while reducing Nox2, a source of oxidative stress. Therefore, we concluded that the combination of fish oil and curcumin prevents skeletal muscle atrophy due to a boost of heat shock proteins and anabolic signaling in an unloaded state.

Primary intraosseous mucoepidermoid carcinoma of the mandible: radiographic evolution and clinicopathological features.

Mucoepidermoid carcinoma (MEC) can be rarely found as a primarily intraosseous lesion and mistaken for other intraosseous or odontogenic pathology. A 65-year-old man had a poorly defined radiolucency distal to the left mandibular second molar root. Periapical radiographs demonstrated a minor radiolucency from 2.5 years prior. An oral and maxillofacial surgeon felt the radiolucency represented periodontal disease, extracting tooth #18. The differential diagnosis of mixed radiolucent/radio-opaque mandibular lesions includes: (1) fibro-osseous lesion, (2) odontogenic and non-odontogenic cyst, (3) infection and inflammatory lesion, or (4) benign or malignant neoplasm (odontogenic, non-odontogenic, or metastatic). Histological analysis revealed low-grade MEC. A composite resection was performed with a 1 cm margin from first molar to ascending ramus. A buccal fat pad advancement flap covered the defect with an iliac crest bone graft placed later for a resulting osseous defect. Careful examination and diagnostic work-up for odontogenic cysts should be provided as they may harbour malignant tumours.

Effect of Eukarion-134 on Akt-mTOR signalling in the rat soleus during 7 days of mechanical unloading.

NEW FINDINGS: What is the central question of this study? Translocation of nNOSµ initiates catabolic signalling via FoxO3a and skeletal muscle atrophy during mechanical unloading. Recent evidence suggests that unloading-induced muscle atrophy and FoxO3a activation are redox sensitive. Will a mimetic of superoxide dismutase and catalase (i.e. Eukarion-134) also mitigate suppression of the Akt-mTOR pathway? What is the main finding and its importance? Eukarion-134 rescued Akt-mTOR signalling and sarcolemmal nNOSµ, which were linked to protection against the unloading phenotype, muscle fibre atrophy and partial fibre-type shift from slow to fast twitch. The loss of nNOSµ from the sarcolemma appears crucial to Akt phosphorylation and is redox sensitive, although the mechanisms remain unresolved. ABSTRACT: Mechanical unloading stimulates rapid changes in skeletal muscle morphology, characterized by atrophy of muscle fibre cross-sectional area and a partial fibre-type shift from slow to fast twitch. Recent studies revealed that oxidative stress contributes to activation of forkhead box O3a (FoxO3a), proteolytic signalling and unloading-induced muscle atrophy via translocation of the µ-splice variant of neuronal nitric oxide synthase (nNOSµ) and activation of FoxO3a. There is limited understanding of the role of reactive oxygen species in the Akt-mammalian target of rapamycin (mTOR) pathway signalling during unloading. We hypothesized that Eukarion-134 (EUK-134), a mimetic of the antioxidant enzymes superoxide dismutase and catalase, would protect Akt-mTOR signalling in the unloaded rat soleus. Male Fischer 344 rats were separated into the following three study groups: ambulatory control (n = 11); 7 days of hindlimb unloading + saline injections (HU, n = 11); or 7 days of HU + EUK-134; (HU + EUK-134, n = 9). EUK-134 mitigated unloading-induced dephosphorylation of Akt, as well as FoxO3a, in the soleus. Phosphorylation of mTOR in the EUK-treated HU rats was not different from that in control animals. However, EUK-134 did not significantly rescue p70S6K phosphorylation. EUK-134 attenuated translocation of nNOSµ from the membrane to the cytosol, reduced nitration of tyrosine residues and suppressed upregulation of caveolin-3 and dysferlin. EUK-134 ameliorated HU-induced remodelling, atrophy of muscle fibres and the 12% increase in type II myosin heavy chain-positive fibres. Attenuation of the unloaded muscle phenotype was associated with decreased reactive oxygen species, as assessed by ethidium-positive nuclei. We conclude that oxidative stress affects Akt-mTOR signalling in unloaded skeletal muscle. Direct linkage of abrogation of nNOSµ translocation with Akt-mTOR signalling during unloading is the subject of future investigation.

Geometry of anterior open bite correction.

Correction of anterior open bite is a frequently encountered and challenging problem for the craniomaxillofacial surgeon and orthodontist. Accurate clinical evaluation, including cephalometric assessment, is paramount for establishing the diagnosis and appropriate treatment plan. The purposes of this technical note were to discuss the basic geometric principles involved in the surgical correction of skeletal anterior open bites and to offer a simple mathematical model for predicting the amount of posterior maxillary impaction with concomitant mandibular rotation required to establish an adequate overbite. Using standard geometric principles, a mathematical model was created to demonstrate the relationship between the magnitude of the open bite and the magnitude of the rotational movements required for correction. This model was then validated using a clinical case. In summary, the amount of open bite closure for a given amount of posterior maxillary impaction depends on anatomic variables, which can be obtained from a lateral cephalogram. The clinical implication of this relationship is as follows: patients with small mandibles and steep mandibular occlusal planes will require greater amounts of posterior impaction.

Skeletal and soft tissue response to automated, continuous, curvilinear distraction osteogenesis.

PURPOSE: To document the bone formation and soft tissue changes in response to automated, continuous, curvilinear distraction osteogenesis (DO) at rates greater than 1 mm/day in a minipig model. MATERIALS AND METHODS: Two groups of Yucatan minipigs underwent automated, continuous, curvilinear DO of the right mandible: group A, 1.5 mm/day (n = 5); and group B, 3.0 mm/day (n = 5). Each minipig underwent 12 mm of distraction followed by 24 days of fixation. The distracted and contralateral mandibles were harvested at the end of fixation. The percentage of surface area (PSA) of the regenerate occupied by bone, fibrous tissue, cartilage, and hematoma was determined using computerized histomorphometric analysis. The control groups consisted of DO wounds distracted discontinuously at 1 mm/day and the nonoperated contralateral mandible. The ipsilateral and contralateral digastric muscles were harvested and stained for proliferating cell nuclear antigen (PCNA), myogenic differentiation-1 (MyoD), and paired Box 7 protein (PAX7). RESULTS: All 10 minipigs completed the distraction and fixation period. The PSA occupied by bone was similar for groups A (PSA 64.36% ± 5.87%) and B (PSA 63.83% ± 3.37%) and the control group (1 mm/day; PSA 64.89% ± 0.56%) but was less than that on the nonoperated side (PSA 84.67% ± 0.86%). The PSA occupied by cartilage and hematoma in all groups was minimal (<1.1%). The digastric muscles had no abnormal tissue or inflammation, and PAX7, MyoD, and PCNA expression had returned to the baseline levels. CONCLUSIONS: The results of the present study indicate that bone formation in response to automated, continuous, and curvilinear DO at a rate of 1.5 and 3.0 mm/day is nearly identical to that with discontinuous DO at 1 mm/day. In addition, no deleterious effects were found on the digastric muscles.

Effects of mandibular distraction osteogenesis on three-dimensional airway anatomy in children with congenital micrognathia.

PURPOSE: To assess the 3-dimensional (3D) computed tomography (CT) changes in airway size and shape in children with congenital micrognathia treated by mandibular distraction osteogenesis (DO). PATIENTS AND METHODS: This was a retrospective study of patients with congenital micrognathia, treated by mandibular DO, who had pre- and postoperative 3D maxillofacial CT scans from the hard palate to the hyoid bone. Digital 3D-CT reconstructions were made before and after distraction. Demographic (age, gender, and diagnosis) and anatomic (airway size and shape) variables were recorded and analyzed. The pre-distraction measures of size and shape were compared with the post-distraction measures. P≤.05 was considered significant. RESULTS: During the study period (1999 to 2010), 17 children with congenital micrognathia underwent mandibular DO. Of these patients, 11 (3 females) met the inclusion criteria. The mean age was 6.8 years (range 1.3 to 20.6). All subjects had first and second pharyngeal arch deformities. Nine were tracheostomy dependent before distraction. Postdistraction increases in the anteroposterior diameter (153%), lateral airway diameter (70%), airway volume (76%), minimal retroglossal (162%) and retropalatal (77%), and minimal cross-sectional areas (282%) were obtained. The mean airway length decreased after distraction by 4 mm DO also affected the airway shape: the airway surface area and airway compactness increased after distraction. Six subjects were decannulated or had their tracheostomies capped. Three subjects remained tracheostomy dependent after distraction. The reasons for continued tracheostomy included copious secretions, muscle hypotonia, hypopharnygeal stenosis, and mandibular hypomobility. CONCLUSIONS: DO for congenital micrognathia increases airway size, decreases airway length, and alters the shape as measured using 3D-CT.

Serial histologic and immunohistochemical changes in anterior digastric myocytes in response to distraction osteogenesis.

PURPOSE: To document histologic and immunohistochemical changes in the anterior digastric muscle during distraction osteogenesis (DO). MATERIALS AND METHODS: Nineteen Yucatan minipigs with mixed dentition were used for these experiments. Group A (n = 16) underwent unilateral mandibular distraction at a rate of 1 mm/day (no latency) for 12 days. Animals were killed at mid-DO (n = 5), end-DO (n = 5), mid-fixation (n = 4), and end-fixation (n = 2). Group B (n = 2) underwent acute 12-mm advancement, and group C (n = 1) dissection and osteotomy. Animals from groups B and C were killed at the end-DO time point. Digastric muscles from treatment and contralateral sides of all animals were harvested and embedded in paraffin. Specimens were stained with hematoxylin/eosin or immunohistochemically for proliferating cell nuclear antigen (PCNA; total cell proliferation), paired Box-7 gene protein (Pax7; satellite cells), or myogenic differentiation 1 protein (MyoD; differentiating myoblasts). Descriptive and bivariate statistics were computed to compare groups (P ≤ .05 statistically significant). RESULTS: All animals survived the operation and observation period; there were no device failures. Two animals (1 at mid-DO, 1 at mid-fixation) were eliminated from the study because of postoperative infection. There was minimal digastric inflammation, fibrosis, and muscle fiber size variability during active DO. Immunohistochemical analysis showed statistically significant increases in PCNA (cellular proliferation), Pax7 (satellite cells), and MyoD (differentiating myoblasts) positive nuclei in digastrics at mid-DO and end-DO. CONCLUSIONS: Results of this study indicate that there are minimal pathologic changes but significant increases in PCNA, Pax7, and MyoD positive nuclei during active distraction. This supports the hypothesis that the digastric muscle response to DO consists of proliferation and hypertrophy.

Mandibular fracture severity and patient health status are associated with postoperative inflammatory complications.

PURPOSE: To identify risk factors associated with postoperative inflammatory complications (POICs) after treatment of mandibular fractures. PATIENTS AND METHODS: The investigators designed a case-control study and enrolled a sample of patients treated for mandibular fractures at Massachusetts General Hospital between August 2004 and January 2010. Subjects who developed POICs after fracture management were categorized as cases. A POIC was defined as 1) recurrent swelling, fever, increased pain, or trismus; 2) wound dehiscence with purulent drainage; 3) exposed or infected hardware; 4) abscess formation; 5) radiographic evidence of osteomyelitis; and/or 6) presence of a fistula. Controls had no complications. For each case, 2 controls were selected. Predictor variables were categorized into the following sets: demographic, fracture-specific, and perioperative. Bivariate and multiple logistic regression analyses were used to identify factors associated with POICs. RESULTS: During the study interval, 575 subjects with mandible fractures were evaluated and treated. The study sample consisted of 44 cases and 88 controls. In the multiple regression model, an increasing Mandibular Injury Severity Score (odds ratio = 1.4; 95% confidence interval 1.2-1.6) and a positive medical history (odds ratio = 1.4; 95% confidence interval 1.03-1.8) were significantly (P < .05) associated with an increased risk for a POIC. CONCLUSION: Fracture severity assessed using the Mandibular Injury Severity Score and pre-existing medical problems were associated with increased risk for postoperative inflammatory complications following treatment of mandibular fractures.

Three-dimensional computed tomographic airway analysis of patients with obstructive sleep apnea treated by maxillomandibular advancement.

PURPOSE: To evaluate changes in airway size and shape in patients with obstructive sleep apnea (OSA) after maxillomandibular advancement (MMA) and genial tubercle advancement (GTA). MATERIALS AND METHODS: This was a retrospective cohort study, enrolling a sample of adults with polysomnography-confirmed OSA who underwent MMA + GTA. All subjects who had preoperative and postoperative 3-dimensional computed tomography (CT) scans to evaluate changes in airway size and shape after MMA + GTA were included. Preoperative and postoperative sleep- and breathing-related symptoms were recorded. Descriptive and bivariate statistics were computed. For all analyses, P < .05 was considered statistically significant. RESULTS: During the study period, 13 patients underwent MMA + GTA, of whom 11 (84.6%) met the inclusion criteria. There were 9 men and 2 women with a mean age of 39 years. The mean body mass index was 26.3; mean respiratory disturbance index (RDI), 48.8; and mean lowest oxygen saturation, 80.5%. After MMA + GTA, there were significant increases in lateral and anteroposterior airway diameters (P < .01), volume (P = .02), surface area (P < .01), and cross-sectional areas at multiple sites (P < .04). Airway length decreased (P < .01) and airway shape (P = .04) became more uniform. The mean change in RDI was -60%. CONCLUSIONS: Results of this preliminary study indicate that MMA + GTA appears to produce significant changes in airway size and shape that correlate with a decrease in RDI.

Histomorphometric analysis of the porcine mandibular distraction wound.

PURPOSE: To analyze the sequence of histomorphometric changes in the regenerate during distraction osteogenesis (DO) of the minipig mandible. MATERIALS AND METHODS: A total of 16 minipigs underwent unilateral mandibular DO using a protocol of 0-day latency and a 1-mm/day rate for 12 days, and 24 days of fixation. The mandibles were harvested at mid-DO, end-DO, mid-fixation, and end-fixation. An additional 2 minipigs underwent acute lengthening, and 1 sham control was included. Serial gross examinations and plain radiographs were performed before paraffin embedding. The sections were stained with hematoxylin-eosin or hematoxylin/alcian blue/sirius red stain. Histomorphometric analysis was performed to determine the percentage of surface area (PSA) occupied by hematoma, fibrous tissue, cartilage, and bone. RESULTS: All 19 minipigs survived the operation, and 17 survived the observation period; 2 were killed because of infection (mid-DO, n = 1 and end-fixation, n = 1). No device failures occurred. Of the 17 specimens, 4 were at mid-DO, 4 at end-DO, 4 at mid-fixation, and 2 at end-fixation; 2 were in the acute lengthening group, and 1 was the sham control. Hematoma was present only at mid-DO (16.61 +/- 8.07 PSA) and end-DO (1.17 +/- 2.33 PSA). Fibrous tissue decreased from mid-DO (53.12 +/- 8.59 PSA) to end-fixation (25.00 +/- 0.83 PSA). Cartilage was present in end-DO (1.72 +/- 2.71 PSA), mid-fixation (5.82 +/- 6.64 PSA), and acute lengthening (1.43 +/- 0.95 PSA). Bone increased from mid-DO (25.18 +/- 0.99 PSA) to end-fixation (64.89 +/- 0.79 PSA) and occurred earlier in the superior and middle thirds of the wounds. Periosteal bone formation predominated over endosteal bone formation early in distraction. CONCLUSION: The results of the present study indicate that bone formation in this model consists of both intramembranous and endochondral components, with intramembranous osteogenesis predominating. Bone formation occurred earlier in the superior/middle portions of the wound, possibly owing to osteoinductive properties of developing tooth buds and the inferior alveolar nerve, respectively.