Calling differentially methylated regions from whole genome bisulphite sequencing with DMRcate.

Whole genome bisulphite sequencing (WGBS) permits the genome-wide study of single molecule methylation patterns. One of the key goals of mammalian cell-type identity studies, in both normal differentiation and disease, is to locate differential methylation patterns across the genome. We discuss the most desirable characteristics for DML (differentially methylated locus) and DMR (differentially methylated region) detection tools in a genome-wide context and choose a set of statistical methods that fully or partially satisfy these considerations to compare for benchmarking. Our data simulation strategy is both biologically informed-employing distribution parameters derived from large-scale consortium datasets-and thorough. We report DML detection ability with respect to coverage, group methylation difference, sample size, variability and covariate size, both marginally and jointly, and exhaustively with respect to parameter combination. We also benchmark these methods on FDR control and computational time. We use this result to backend and introduce an expanded version of DMRcate: an existing DMR detection tool for microarray data that we have extended to now call DMRs from WGBS data. We compare DMRcate to a set of alternative DMR callers using a similarly realistic simulation strategy. We find DMRcate and RADmeth are the best predictors of DMRs, and conclusively find DMRcate the fastest.

Gene expression allelic imbalance in ovine brown adipose tissue impacts energy homeostasis.

Heritable trait variation within a population of organisms is largely governed by DNA variations that impact gene transcription and protein function. Identifying genetic variants that affect complex functional traits is a primary aim of population genetics studies, especially in the context of human disease and agricultural production traits. The identification of alleles directly altering mRNA expression and thereby biological function is challenging due to difficulty in isolating direct effects of cis-acting genetic variations from indirect trans-acting genetic effects. Allele specific gene expression or allelic imbalance in gene expression (AI) occurring at heterozygous loci provides an opportunity to identify genes directly impacted by cis-acting genetic variants as indirect trans-acting effects equally impact the expression of both alleles. However, the identification of genes showing AI in the context of the expression of all genes remains a challenge due to a variety of technical and statistical issues. The current study focuses on the discovery of genes showing AI using single nucleotide polymorphisms as allelic reporters. By developing a computational and statistical process that addressed multiple analytical challenges, we ranked 5,809 genes for evidence of AI using RNA-Seq data derived from brown adipose tissue samples from a cohort of late gestation fetal lambs and then identified a conservative subgroup of 1,293 genes. Thus, AI was extensive, representing approximately 25% of the tested genes. Genes associated with AI were enriched for multiple Gene Ontology (GO) terms relating to lipid metabolism, mitochondrial function and the extracellular matrix. These functions suggest that cis-acting genetic variations causing AI in the population are preferentially impacting genes involved in energy homeostasis and tissue remodelling. These functions may contribute to production traits likely to be under genetic selection in the population.

Unexpected features of the dark proteome.

We surveyed the "dark" proteome-that is, regions of proteins never observed by experimental structure determination and inaccessible to homology modeling. For 546,000 Swiss-Prot proteins, we found that 44-54% of the proteome in eukaryotes and viruses was dark, compared with only ∼14% in archaea and bacteria. Surprisingly, most of the dark proteome could not be accounted for by conventional explanations, such as intrinsic disorder or transmembrane regions. Nearly half of the dark proteome comprised dark proteins, in which the entire sequence lacked similarity to any known structure. Dark proteins fulfill a wide variety of functions, but a subset showed distinct and largely unexpected features, such as association with secretion, specific tissues, the endoplasmic reticulum, disulfide bonding, and proteolytic cleavage. Dark proteins also had short sequence length, low evolutionary reuse, and few known interactions with other proteins. These results suggest new research directions in structural and computational biology.

Coherence analysis discriminates between retroviral integration patterns in CD34(+) cells transduced under differing clinical trial conditions.

Unequivocal demonstration of the therapeutic utility of γ-retroviral vectors for gene therapy applications targeting the hematopoietic system was accompanied by instances of insertional mutagenesis. These events stimulated the ongoing development of putatively safer integrating vector systems and analysis methods to characterize and compare integration site (IS) biosafety profiles. Continuing advances in next-generation sequencing technologies are driving the generation of ever-more complex IS datasets. Available bioinformatic tools to compare such datasets focus on the association of integration sites (ISs) with selected genomic and epigenetic features, and the choice of these features determines the ability to discriminate between datasets. We describe the scalable application of point-process coherence analysis (CA) to compare patterns produced by vector ISs across genomic intervals, uncoupled from association with genomic features. To explore the utility of CA in the context of an unresolved question, we asked whether the differing transduction conditions used in the initial Paris and London SCID-X1 gene therapy trials result in divergent genome-wide integration profiles. We tested a transduction carried out under each condition, and showed that CA could indeed resolve differences in IS distributions. Existence of these differences was confirmed by the application of established methods to compare integration datasets.

De novo identification of differentially methylated regions in the human genome.

BACKGROUND: The identification and characterisation of differentially methylated regions (DMRs) between phenotypes in the human genome is of prime interest in epigenetics. We present a novel method, DMRcate, that fits replicated methylation measurements from the Illumina HM450K BeadChip (or 450K array) spatially across the genome using a Gaussian kernel. DMRcate identifies and ranks the most differentially methylated regions across the genome based on tunable kernel smoothing of the differential methylation (DM) signal. The method is agnostic to both genomic annotation and local change in the direction of the DM signal, removes the bias incurred from irregularly spaced methylation sites, and assigns significance to each DMR called via comparison to a null model. RESULTS: We show that, for both simulated and real data, the predictive performance of DMRcate is superior to those of Bumphunter and Probe Lasso, and commensurate with that of comb-p. For the real data, we validate all array-derived DMRs from the candidate methods on a suite of DMRs derived from whole-genome bisulfite sequencing called from the same DNA samples, using two separate phenotype comparisons. CONCLUSIONS: The agglomeration of genomically localised individual methylation sites into discrete DMRs is currently best served by a combination of DM-signal smoothing and subsequent threshold specification. The findings also suggest the design of the 450K array shows preference for CpG sites that are more likely to be differentially methylated, but its overall coverage does not adequately reflect the depth and complexity of methylation signatures afforded by sequencing. For the convenience of the research community we have created a user-friendly R software package called DMRcate, downloadable from Bioconductor and compatible with existing preprocessing packages, which allows others to apply the same DMR-finding method on 450K array data.

Blood-based protein biomarker panel for the detection of colorectal cancer.

BACKGROUND: The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test. PRINCIPAL FINDINGS: In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2). CONCLUSIONS: Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.

A panel of genes methylated with high frequency in colorectal cancer.

BACKGROUND: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. METHODS: Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). RESULTS: Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. CONCLUSIONS: This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Novel systems for dynamically assessing insulin action in live cells reveals heterogeneity in the insulin response.

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach - each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard-wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre-programmed within each cell and is not the result of intracellular stochastic events.

Adhesive use in oral and maxillofacial surgery.

Presently, tissue adhesives and sealants have limited use in oral and maxillofacial surgical procedures. Skin closure occurs regularly with cyanoacrylate adhesives. Sealing of dural tears in conjunction with dural closure has been shown to be very successful. With the development of more head and neck reconstructive procedures and cosmetic procedures, demand will increase for better surgical adhesives. Clinical trials are beginning for newly developed adhesives with the chemical characterizations, the safe reabsorptive profile, and the adhesive strength necessary to benefit oral and maxillofacial surgery patients in the near future. Adhesives for bone fixation, while in early development, also show a promising chemical profile and will be of significant benefit to oral and maxillofacial surgical patients.

A-366833: a novel nicotinonitrile-substituted 3,6-diazabicyclo[3.2.0]-heptane alpha4beta2 nicotinic acetylcholine receptor selective agonist: Synthesis, analgesic efficacy and tolerability profile in animal models.

5-[(1R,5S)-3,6-Diazabicyclo[3.2.0]heptan-6-yl]nicotinonitrile (A-366833) is a novel nicotinic acetylcholine receptor (nAChR) ligand that binds to the agonist-binding site ([3H]-cytisine) with Ki value of 3.1 nM and exhibits agonist selectivity at alpha4beta2 nAChR relative to the alpha3beta4 nAChR subtype. The analgesic effects of A-366833 were examined across a variety of animal models including the mouse model of writhing pain (abdominal constriction), the rat models of acute thermal (hot box), persistent chemical (formalin) and neuropathic (spinal nerve ligation, SNL) pain. In the abdominal constriction model, A-366833 was effective at doses ranging from 0.062 to 0.62 micromol/kg (i.p.). In addition, A-366833 demonstrated significant effects in acute thermal pain (6.2-19.0 micromol/kg, i.p.), formalin (1.9-19 micromol/kg i.p.) and SNL (1.9-19 micromol/kg i.p.) models. The systemic effects of A-366833 were attenuated by pretreatment with mecamylamine (5 micromol/kg i.p.) in both the formalin and SNL models, suggesting that the analgesic effects of A-366833 in models of persistent nociceptive and neuropathic pain are mediated by activation of nAChRs. Pharmacokinetic investigations of A-366833 in rat revealed moderate brain:plasma distribution, half-life of 1.5h and excellent oral bioavailability of 73%. Comparison of peak plasma levels at the minimal effective doses across rat models of acute thermal pain, formalin and SNL with the maximal exposure that does not evoke emesis in ferret revealed therapeutic margins ranging from 6- to 22-fold. These studies indicate that compounds like A-366833 with improved agonist selectivity at alpha4beta2 vs. alpha3beta4 nAChR can elicit a broad spectrum of analgesic efficacy without concurrent adverse effects.

Novel heterocyclic-substituted benzofuran histamine H3 receptor antagonists: in vitro properties, drug-likeness, and behavioral activity.

Three novel heterocyclic benzofurans A-688057 (1), A-687136 (2), and A-698418 (3) were profiled for their in vitro and in vivo properties as a new series of histamine H(3) receptor antagonists. The compounds were all found to have nanomolar potency in vitro at histamine H(3) receptors, and when profiled in vivo for CNS activity, all were found active in an animal behavioral model of attention. The compound with the most benign profile versus CNS side effects was selected for greater scrutiny of its in vitro properties and overall drug-likeness. This compound, A-688057, in addition to its potent and robust efficacy in two rodent behavioral models at blood levels ranging 0.2-19 nM, possessed other favorable features, including high selectivity for H(3) receptors (H(3), K(i)=1.5 nM) versus off-target receptors and channels (including the hERG K(+) channel, K(i)>9000 nM), low molecular weight (295), high solubility, moderate lipophilicity (logD(pH7.4)=2.05), and good CNS penetration (blood/brain 3.4x). In vitro toxicological tests indicated low potential for phospholipidosis, genotoxicity, and CYP(450) inhibition. Even though pharmacokinetic testing uncovered only moderate to poor oral bioavailability in rat (26%), dog (30%), and monkey (8%), and only moderate blood half-lives after i.v. administration (t(1/2) in rat of 2.9h, 1.7h in dog, 1.8h in monkey), suggesting poor human pharmacokinetics, the data overall indicated that A-688057 has an excellent profile for use as a pharmacological tool compound.

Differential effects of ciproxifan and nicotine on impulsivity and attention measures in the 5-choice serial reaction time test.

Deficits in attention and response inhibition are apparent across several neurodegenerative and neuropsychiatric disorders for which current pharmacotherapy is inadequate. While it is difficult to model such executive processes in animals, the 5-choice serial reaction time test (5-CSRTT), which originated from the continuous performance test (CPT) in humans, may serve as a useful translational assay for efficacy in these key behavioral domains. At Wyeth and Abbott, we recently investigated the utility of employing the 5-CSRTT in adult rats. This involved training and testing groups of rats over an extended period of several months and required the animals to learn to nose-poke into one of five apertures following presentation of a brief visual stimulus in that aperture in order to obtain a food reward. When the stimulus duration was short, the rat had to pay close attention to make a correct choice--a nose-poke into the aperture with the brief visual stimulus. We evaluated nicotine and the histamine H(3) receptor antagonist, ciproxifan, since compounds targeting both nicotinic and histaminergic neurotransmission are currently under investigation for treating cognitive dysfunction in ADHD, AD and schizophrenia. After approximately 12 weeks of training, rats were tested with drug when they had achieved stable performance. Nicotine (0.2, 0.4 mg/kg s.c.) significantly improved accuracy and reduced errors of omission (reflecting improved attention and vigilance) when baseline performance was <90% correct. In contrast, nicotine tended to worsen accuracy when baseline performance was >90% correct. Using the same test paradigm, ciproxifan (3mg/kg i.p.) reduced premature responding, a measure of impulsivity. Under conditions of variable stimulus duration, ciproxifan also improved accuracy and decreased impulsivity. In summary, we have replicated previous findings by others of positive effects of nicotine on attention, but also showed that this is dependent on baseline performance. We also expanded on previous positive findings by others with ciproxifan on attention and both Wyeth and Abbott demonstrate for the first time decreased impulsivity with this mechanism.

Synthesis and structure-activity relationships of 3,8-diazabicyclo[4.2.0]octane ligands, potent nicotinic acetylcholine receptor agonists.

A series of potent neuronal nicotinic acetylcholine receptor (nAChR) ligands based on a 3,8-diazabicyclo[4.2.0]octane core have been synthesized and evaluated for affinity and agonist efficacy at the human high affinity nicotine recognition site (halpha4beta2) and in a rat model of persistent nociceptive pain (formalin model). Numerous analogs in this series exhibit picomolar affinity in radioligand binding assays and nanomolar agonist potency in functional assays, placing them among the most potent nAChR ligands known for the halpha4beta2 receptor. Several of the compounds reported in this study (i.e., 24, 25, 28, 30, 32, and 47) exhibit equivalent or greater affinity for the halpha4beta2 receptor relative to epibatidine, and like epibatidine, many exhibit robust analgesic efficacy in the rat formalin model of persistent pain.

Effects of histamine H3 receptor antagonists in two models of spatial learning.

Despite the well-described attention and short-term memory enhancing effects of H3 receptor antagonists, and evidence to suggest a close relationship between central histaminergic and cholinergic systems, there is a paucity of evidence for a role for H3 receptor blockade in spatial learning. To address this, we investigated two H3 receptor antagonists in a visual discrimination water maze in rats, and in a Barnes circular maze in mice. Thioperamide and ciproxifan significantly attenuated a scopolamine-induced deficit in the water maze task, while only ciproxifan showed a modest attenuation in the Barnes maze. Taken together, these data suggest a role for H3 receptors in spatial learning that appears to be task-dependent.

Pharmacological properties of ABT-239 [4-(2-{2-[(2R)-2-Methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile]: II. Neurophysiological characterization and broad preclinical efficacy in cognition and schizophrenia of a potent and selective histamine H3 receptor antagonist.

Acute pharmacological blockade of central histamine H3 receptors (H3Rs) enhances arousal/attention in rodents. However, there is little information available for other behavioral domains or for repeated administration using selective compounds. ABT-239 [4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile] exemplifies such a selective, nonimidazole H3R antagonist with high affinity for rat (pK(i) = 8.9) and human (pK(i) = 9.5) H3Rs. Acute functional blockade of central H3Rs was demonstrated by blocking the dipsogenia response to the selective H3R agonist (R)-alpha-methylhistamine in mice. In cognition studies, acquisition of a five-trial, inhibitory avoidance test in rat pups was improved with ABT-239 (0.1-1.0 mg/kg), a 10- to 150-fold gain in potency, with similar efficacy, over previous antagonists such as thioperamide, ciproxifan, A-304121 [(4-(3-(4-((2R)-2-aminopropanoyl)-1-piperazinyl)propoxy)phenyl)(cyclopropyl) methanone], A-317920 [N-((1R)-2-(4-(3-(4-(cyclopropylcarbonyl) phenoxy)propyl)-1-piperazinyl)-1-methyl-2-oxoethyl)-2-furamide], and A-349821 [(4'-(3-((R,R)2,5-dimethyl-pyrrolidin-1-yl)-propoxy)-biphenyl-4-yl)-morpholin-4-yl-methanone]. Efficacy in this model was maintained for 3 to 6 h and following repeated dosing with ABT-239. Social memory was also improved in adult (0.01-0.3 mg/kg) and aged (0.3-1.0 mg/kg) rats. In schizophrenia models, ABT-239 improved gating deficits in DBA/2 mice using prepulse inhibition of startle (1.0-3.0 mg/kg) and N40 (1.0-10.0 mg/kg). Furthermore, ABT-239 (1.0 mg/kg) attenuated methamphetamine-induced hyperactivity in mice. In freely moving rat microdialysis studies, ABT-239 enhanced acetylcholine release (0.1-3.0 mg/kg) in adult rat frontal cortex and hippocampus and enhanced dopamine release in frontal cortex (3.0 mg/kg), but not striatum. In summary, broad efficacy was observed with ABT-239 across animal models such that potential clinical efficacy may extend beyond disorders such as ADHD to include Alzheimer's disease and schizophrenia.

Mechanism of action of A-85380 in an animal model of depression.

A role for neuronal nicotinic receptor (NNR) activation in animal models of depression has been established. In order to determine the mechanism by which NNR ligands exert their antidepressant effects, experiments using different NNR receptor antagonists in both the mouse and the rat forced swim test (RFST) were performed. In the mouse forced swim test (MFST), A-85380 (0.62 micromol/kg = 0.14 mg/kg, i.p.), an NNR agonist, increased swim distance when administered 15 min prior to test. This effect was blocked by pre-treatment with mecamylamine (1.5 micromol/kg = 0.3 mg/kg, i.p.), suggesting that an NNR mechanism is involved. Further, chlorisondamine at a non-central nervous system (CNS) penetrating dose (1.6 micromol/kg = 1 mg/kg, i.p.) did not antagonize A-85380 in this model, thus implicating central rather than peripheral nicotinic receptors. Dihydro-beta-erythroidine (DHbetaE, 0.3 micromol/kg = 0.1 mg/kg, i.p.) pre-treatment also blocked this effect, indicating that the alpha4beta2 receptor subtype may be involved in A-85380-induced antidepressant effects. Finally, methiothepin (0.33 micromol/kg = 0.14 mg/kg, i.p.) pre-treatment antagonized this effect, suggesting serotonergic involvement. In the rat modified forced swim test, sub-acute administration of A-85380 (0.62 micromol/kg, i.p.) increased swimming behavior and decreased immobility. Climbing behavior was unaffected. In contrast, desipramine treatment (33 micromol/kg = 10 mg/kg, i.p.) resulted in an increase in climbing behavior with no effect on swimming. This behavioral profile has been shown to be more typical of serotonergic rather than noradrenergic antidepressants, suggesting that A-85380 exerts its effects via NNR activation of serotonergic systems.

ABT-089: pharmacological properties of a neuronal nicotinic acetylcholine receptor agonist for the potential treatment of cognitive disorders.

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine dihydrochloride salt] is a selective neuronal nicotinic receptor (NNR) modulator with cognitive enhancing properties in animal models of cognitive functioning. Amongst NNR subtypes, ABT-089 shows selectivity for the cytisine binding site on the alpha4beta2 receptor subtype as compared to the alpha-bungarotoxin (alpha-BgT) binding sites on the alpha7 and alpha1beta1deltagamma receptor subtypes. In functional in vitro electrophysiological and cation flux assays, ABT-089 displays differential activity including agonism, partial agonism and antagonism depending upon the NNR subtype and assay. ABT-089 is as potent and efficacious as (-)-nicotine at evoking acetylcholine (ACh) release from hippocampal synaptosomes. Furthermore, ABT-089 is neuroprotective against excitotoxic glutamate insults, with even greater potency seen after chronic treatment. Similarly, ABT-089 is effective in models of cognitive functioning, including enhancement of baseline functioning as well as improvement of impaired cognitive functioning seen following septal lesioning and natural aging. In neuroprotective assays the compound is most potent by chronic administration. In stark contrast to the positive effects in the cognitive models, ABT-089 shows little propensity to induce adverse effects such as ataxia, hypothermia, seizures, cardiovascular or gastrointestinal side effects. Together these data suggest that ABT-089 is a NNR modulator with the potential for treating cognitive disorders with markedly limited adverse cardiovascular and gastrointestinal side effects.

The effects of Le Fort I osteotomies on velopharyngeal and speech functions in cleft patients.

PURPOSE: Orthognathic surgery alters or even worsens symptoms of velopharyngeal insufficiency in cleft patients. The goal of this study was to evaluate how advancing the maxilla would affect the speech and articulation disorders of these patients. PATIENTS AND METHODS: This was a retrospective study in which we compiled and evaluated the speech scores of 54 cleft lip and palate patients who underwent maxillary advancement between 1981 and 2001. Although 34 individuals underwent an isolated Le Fort I advancement, 20 patients had a combined Le Fort I advancement/mandibular setback operation. The following variables were recorded from both preoperative and postoperative speech evaluations: presence of a pharyngeal flap at the time of surgery, oronasal fistulas, nasality, 7 different articulation errors, velopharyngeal function assessment, and overall speech score. Preoperative and postoperative changes in the data were analyzed using the McNemar test and paired t test. RESULTS: A decrease in competent velopharyngeal function mechanisms was noted postoperatively (42% to 18%), increased borderline incompetence (9% to 22%), and complete velopharyngeal insufficiency (13% to 20%). Speech scores deteriorated significantly (P <.05), whereas articulation defects insignificantly (P =.146) improved after surgery (84% to 73%), with those related to the anterior dentition (P =.064) showing the greatest change (64% to 47%). The frequency of hyponasality decreased after surgery. The number of cases of mild to moderate hypernasality increased. CONCLUSION: This study confirms previous findings that patients with clefts of the lip and palate or palate alone are predisposed to velopharyngeal function alteration after maxillary advancement, particularly with borderline function preoperatively. However, the results show that surgical correction of skeletal relationships and occlusion may translate into improvements in certain aspects of speech disorders.

The effects of mechanical strain on synovial fibroblasts.

PURPOSE: Arthritic diseases of the temporomandibular joint, such as rheumatoid arthritis and osteoarthritis, suggest that inflammatory mediators and metalloproteinases may play a role in their pathogenesis. Recent clinical evidence from physical therapy and other modalities has shown a significant decrease in temporomandibular joint symptoms in patients with early disease. This project examines the effect of mechanical strain on synovial fibroblasts' production of inflammatory mediators including prostaglandin E(2) (PGE(2)) and proteinases. MATERIALS AND METHODS: An established synovial fibroblast cell line (HIG-82) was grown to confluency in modified Eagle's medium supplemented with 10% fetal calf serum. The monolayer of fibroblasts was then subjected to mechanical strain using the Flexercell Strain Unit (Flexcell International Corporation, McKeesport, PA) at 3 cycles per minute, with 10 seconds' elongation of up to 24% and 10 seconds of relaxation. Levels of PGE(2) were determined by radioimmunoassay using commercially available product and measured in nanograms per milliliter of supernatant. Proteinases collagenase, gelatinase, and stromelysin were measured by H(3) radioactive labeling of acidic anhydride to the specific substrate. Enzymatic proteolysis of the radiolabeled substrate was then measured in supernate as units per milliliter. Statistical analysis of all results was performed using Student's t test in triplicate. RESULTS: PGE(2) levels of mechanically activated cells was 18.1 +/- 13.4 ng/mL, with control levels being 58.0 +/- 9.2 ng/mL. This is a statistically significant decrease, between strained and unstrained cells with P <.05. In control cells, proteinase activity that degrades collagen, gelatin, or casein was 4.27 +/- 1.5, 4.62 +/- 0.11, or 0.11 +/- 0.01 U/mL, respectively. Levels for mechanically strained cells were 3.99 +/- 1.90, 4.02 +/- 0.90, and 0.12 +/- 0.01 U/mL, respectively. These results show that there is a significant decrease in PGE(2) levels of synovial fibroblasts undergoing mechanical strain. Proteinases examined show no difference in levels between mechanically activated fibroblasts and their controls. CONCLUSION: This decrease in PGE(2) production in synovial fibroblasts could help elucidate the mechanism by which physical therapy, and in particular continuous passive motion, may decrease inflammatory mediators of the temporomandibular joint.

Recovery after third molar surgery: clinical and health-related quality of life outcomes.

PURPOSE: The study goal was to assess both clinical and health-related quality of life (HRQOL) outcomes after third molar surgery. METHODS: Patients who were having 4 third molars removed were enrolled in a prospective clinical trial. Baseline data were recorded that included demographics, the patient's and surgeon's assessment of third molar conditions, and details of the surgical procedure. After surgery, clinical data were collected that detailed healing and any treatment that was rendered. Each patient was given an HRQOL instrument to complete on each postsurgery day for 14 days; the instrument was designed to assess a patient's perception of recovery in 4 main categories: pain, lifestyle, oral function, and other symptoms related to the procedure. RESULTS: Recovery data were available for 630 of 740 enrolled patients. The median age of the 630 patients was 21 years, and the median operation time was 30 minutes. Recovery for most HRQOL measures occurred within 5 days after surgery. However, recovery from pain to the criterion of "little or none" was delayed relative to other HRQOL measures. Twenty-two percent of patients were treated for delayed healing after surgery. CONCLUSIONS: Having both clinical and HRQOL data on recovery after third molar surgery could assist the surgeon when informing prospective patients about what to expect after surgery to remove third molars.