Sociodemographic trends in a UK temporomandibular joint disorder clinic.

Introduction Temporomandibular disorders (TMD) are a common source of facial pain, resulting from an interaction of biopsychosocial factors. However, social risk factors related to TMD have been researched very little, particularly in patients attending a tertiary care service.Aims To review sociodemographic trends among patients referred to a UK tertiary TMD clinic for specialist management. To provide an insight into the sociodemographic risk factors associated with TMD in patients referred for specialist input and to discuss the surrounding literature.Methods Retrospective review of notes of patients referred to a UK tertiary TMD clinic. Trends in areas of deprivation from which patients came from were quantified into deciles using the English Multiple Indices of Deprivation.Results The mean age of patients was 41.1 years, with women being overrepresented compared to men. Most patients were referred by their general dental practitioner, although a variety of secondary care specialties also referred to this service. A disproportionate number of patients came from the highest decile of deprivation, a trend seen throughout the entire sample and in patients seen by dental professionals specifically. There was no apparent association between this and longer symptom duration.Conclusion Women and those from areas with the highest levels of deprivation were overrepresented in this tertiary clinic setting. Dentists should maintain an awareness of the risk factors for TMD development as well as those which may complicate its management in cases requiring specialist input.

Audit of flumazenil use in special care and oral surgery sedation services.

Background The National Patient Safety Agency (2008) has advised against routine reliance on flumazenil for reversal of sedation and encourages regular audit to help identify issues with excessive dosing of midazolam. This multi-centre audit of flumazenil use across both community-based special care and dental hospital oral surgery specialist services was conducted to compare practice against that reported from other UK sedation services.Methods A six-year retrospective audit was conducted using controlled drug records and patient case notes.Results Both services used flumazenil at very infrequent levels and far below the agreed standard. The dose of flumazenil and justification for its use was recorded in all records. There was variability in the nature of the justifications between the two services, which likely relates to the differing patient groups seen by the specialties. The majority of cases related to supporting the patient's escort for their journey home; however, this was not always pre-planned.Conclusions There was a low level of flumazenil use over an extended period of time, supporting the concept of a culture of safe sedation provision in both services. The audit highlighted variation in record-keeping and need for improved communication with patients about escort requirements.

Clinical presentations on a facial pain clinic.

Introduction The complex nature of facial pain conditions creates a diagnostic challenge which may necessitate specialist referral.Aim To identify the case mix presenting to a specialist tertiary care facial pain clinic.Methods A retrospective review of 112 patient records was undertaken. Trends in provisional diagnoses from referrers and the correlation to diagnoses made following specialist consultation were reviewed.Results The most common provisional diagnoses recorded in referral letters were painful temporomandibular disorders, trigeminal neuralgia and persistent idiopathic facial pain (PIFP). Over a quarter of referrals did not include a provisional diagnosis. Following assessment, only one case was not given a definitive diagnosis and no patients were diagnosed with PIFP. A causative factor was identified in all the initially queried PIFP cases, and painful post-traumatic trigeminal neuropathic pain was found in multiple patients.Conclusions Painful post-traumatic trigeminal neuropathic pain should be considered if pain onset coincides with dental treatment or other traumatic events. PIFP is a rare facial pain diagnosis and may be over-diagnosed by dental and medical practitioners. It is important to systematically exclude other causes before reaching this diagnosis. This will facilitate effective treatment, manage patient expectations and potentially reduce unnecessary referrals.

Analytical performance of a formalin-fixed paraffin-embedded tissue-based 634-probe prognostic assay for predicting outcome of patients with stage II colon cancer.

A formalin-fixed paraffin-embedded tissue-based prognostic assay to assess the risk for recurrence in stage II colon cancer has recently been clinically validated. This study describes the analytical performance and quality control measures of the assay. The reportable range was determined to be [-1.129, 1.414] in risk score units. The accuracy was evaluated with a split sample comparison within the production lab and between the production lab and a reference lab. The concordance between the replicates within the production lab was 79% (95% confidence interval, 64%-91%). There was no evidence of bias, and the concordance was 78% (95% confidence interval, 61%-90%) between the labs. The lab-to-lab concordance was further evaluated by simulating risk scores from the full reportable range. The simulation suggested a higher concordance. The sensitivity study demonstrated that the percentage of tumor tissue did not impact the risk score and that RNA concentration of 9.5 ng/µL was a conservative determination of the analyte lower limit of quantification. From the precision study, the repeatability and reproducibility estimates were 0.1267 and 0.0548 in risk score units, respectively. Furthermore, multifaceted quality control measures were implemented, such as proper tissue processing steps, high-risk and low-risk controls, nontemplate control, and a gene expression-based classifier to evaluate the cDNA amplification kit, a key reagent in the assay. In conclusion, this study demonstrates the strong analytical performance of the assay and further supports its use as an objective standardized prognostic test for stage II colon cancer.

Implications for powering biomarker discovery studies.

This study examined variations in gene expression between FFPE blocks within tumors of individual patients. Microarray data were used to measure tumor heterogeneity within and between patients and disease states. Data were used to determine the number of samples needed to power biomarker discovery studies. Bias and variation in gene expression were assessed at the intrapatient and interpatient levels and between adenocarcinoma and squamous samples. A mixed-model analysis of variance was fitted to gene expression data and model signatures to assess the statistical significance of observed variations within and between samples and disease states. Sample size analysis, adjusted for sample heterogeneity, was used to determine the number of samples required to support biomarker discovery studies. Variation in gene expression was observed between blocks taken from a single patient. However, this variation was considerably less than differences between histological characteristics. This degree of block-to-block variation still permits biomarker discovery using either macrodissected tumors or whole FFPE sections, provided that intratumor heterogeneity is taken into account. Failure to consider intratumor heterogeneity may result in underpowered biomarker studies that may result in either the generation of longer gene signatures or the inability to identify a viable biomarker. Moreover, the results of this study indicate that a single biopsy sample is suitable for applying a biomarker in non-small-cell lung cancer.

The role of hippocampal subregions in memory for stimulus associations.

The hippocampus is hypothesised to be critical for episodic memory in humans and episodic-like memory in animals. Human data regarding the roles of the various subregional networks within the hippocampus is difficult to obtain. In this article we examine the current rodent literature on episodic-like memory and associative recognition and review the roles of the hippocampal subregions in these behavioural tasks. We focus on the large amount of recent data reporting roles for CA3 and CA1 in allocentric spatial and temporal associative memory respectively. Our own recent data are then presented detailing critical roles for CA3 and CA1 in an associative recognition task which does not require allocentric spatial or temporal processing. These data support more generic roles for CA3 and CA1 in episodic-like memory, based on anatomical and theoretical literature on hippocampal function. We also present a novel analysis of our data in which we suggest that the encoding of object, place and context information is unaffected by lesions of the hippocampus and therefore infer that it may be the storage or retrieval phase of this associative memory which is critically dependent on hippocampal function. In conclusion however, more specific anatomically and temporally controlled methods are needed to fully define the role of hippocampal subregions in episodic-like memory.

Functional characterization of GABAergic pallidopallidal and striatopallidal synapses in the rat globus pallidus in vitro.

As a central integrator of basal ganglia function, the external segment of the globus pallidus (GP) plays a critical role in the control of voluntary movement. Driven by intrinsic mechanisms and excitatory glutamatergic inputs from the subthalamic nucleus, GP neurons receive GABAergic inhibitory input from the striatum (Str-GP) and from local collaterals of neighbouring pallidal neurons (GP-GP). Here we provide electrophysiological evidence for functional differences between these two inhibitory inputs. The basic synaptic characteristics of GP-GP and Str-GP GABAergic synapses were studied using whole-cell recordings with paired-pulse and train stimulation protocols and variance-mean (VM) analysis. We found (i) IPSC kinetics are consistent with local collaterals innervating the soma and proximal dendrites of GP neurons whereas striatal inputs innervate more distal regions. (ii) Compared to GP-GP synapses Str-GP synapses have a greater paired-pulse ratio, indicative of a lower probability of release. This was confirmed using VM analysis. (iii) In response to 20 and 50 Hz train stimulation, GP-GP synapses are weakly facilitatory in 1 mM external calcium and depressant in 2.4 mM calcium. This is in contrast to Str-GP synapses which display facilitation under both conditions. This is the first quantitative study comparing the properties of GP-GP and Str-GP synapses. The results are consistent with the differential location of these inhibitory synapses and subtle differences in their release probability which underpin stable GP-GP responses and robust short-term facilitation of Str-GP responses. These fundamental differences may provide the physiological basis for functional specialization.

The utility of MAS5 expression summary and detection call algorithms.

BACKGROUND: Used alone, the MAS5.0 algorithm for generating expression summaries has been criticized for high False Positive rates resulting from exaggerated variance at low intensities. RESULTS: Here we show, with replicated cell line data, that, when used alongside detection calls, MAS5 can be both selective and sensitive. A set of differentially expressed transcripts were identified that were found to be changing by MAS5, but unchanging by RMA and GCRMA. Subsequent analysis by real time PCR confirmed these changes. In addition, with the Latin square datasets often used to assess expression summary algorithms, filtered MAS5.0 was found to have performance approaching that of its peers. CONCLUSION: When used alongside detection calls, MAS5 is a sensitive and selective algorithm for identifying differentially expressed genes.

Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells.

The proteome is determined by rates of transcription, translation, and protein turnover. Definition of stem cell populations therefore requires a stem cell proteome signature. However, the limit to the number of primary cells available has restricted extensive proteomic analysis. We present a mass spectrometric method using an isobaric covalent modification of peptides for relative quantification (iTRAQ), which was employed to compare the proteomes of approximately 1 million long-term reconstituting hematopoietic stem cells (Lin(-)Sca(+)Kit(+); LSK(+)) and non-long-term reconstituting progenitor cells (Lin(-)Sca(+)Kit(-); LSK(-)), respectively. Extensive 2-dimensional liquid chromatography (LC) peptide separation prior to mass spectrometry (MS) enabled enhanced proteome coverage with relative quantification of 948 proteins. Of the 145 changes in the proteome, 54% were not seen in the transcriptome. Hypoxia-related changes in proteins controlling metabolism and oxidative protection were observed, indicating that LSK(+) cells are adapted for anaerobic environments. This approach can define proteomic changes in primary samples, thereby characterizing the molecular signature of stem cells and their progeny.

Simpleaffy: a BioConductor package for Affymetrix Quality Control and data analysis.

UNLABELLED: Quality Control is a fundamental aspect of successful microarray data analysis. Simpleaffy is a BioConductor package that provides access to a variety of QC metrics for assessing the quality of RNA samples and of the intermediate stages of sample preparation and hybridization. Simpleaffy also offers fast implementations of popular algorithms for generating expression summaries and detection calls. AVAILABILITY: Simpleaffy can be downloaded from http://www.bioconductor.org. SUPPLEMENTARY INFORMATION: Additional information can be found on the supplementary website located at http://bioinformatics.picr.man.ac.uk.

Improved prediction for N-termini of alpha-helices using empirical information.

The prediction of the secondary structure of proteins from their amino acid sequences remains a key component of many approaches to the protein folding problem. The most abundant form of regular secondary structure in proteins is the alpha-helix, in which specific residue preferences exist at the N-terminal locations. Propensities derived from these observed amino acid frequencies in the Protein Data Bank (PDB) database correlate well with experimental free energies measured for residues at different N-terminal positions in alanine-based peptides. We report a novel method to exploit this data to improve protein secondary structure prediction through identification of the correct N-terminal sequences in alpha-helices, based on existing popular methods for secondary structure prediction. With this algorithm, the number of correctly predicted alpha-helix start positions was improved from 30% to 38%, while the overall prediction accuracy (Q3) remained the same, using cross-validated testing. Although the algorithm was developed and tested on multiple sequence alignment-based secondary structure predictions, it was also able to improve the predictions of start locations by methods that use single sequences to make their predictions. Furthermore, the residue frequencies at N-terminal positions of the improved predictions better reflect those seen at the N-terminal positions of alpha-helices in proteins. This has implications for areas such as comparative modeling, where a more accurate prediction of the N-terminal regions of alpha-helices should benefit attempts to model adjacent loop regions. The algorithm is available as a Web tool, located at http://rocky.bms.umist.ac.uk/elephant.

Amplification protocols introduce systematic but reproducible errors into gene expression studies.

The desire to perform microarray experiments with small starting amounts of RNA has led to the development of a variety of protocols for preparing and amplifying mRNA. This has consequences not only for the standardization of experimental design, but also for reproducibility and comparability between experiments. Here we investigate the differences between the Affymetrix standard and small sample protocols and address the data analysis issues that arise when comparing samples and experiments that have been processed in different ways. We show that data generated on the same platform using different protocols are not directly comparable. Further, protocols introduce systematic biases that can be largely accounted for by using the correct data analysis techniques.

Extracellular peptidases of imaginal discs of Drosophila melanogaster.

The imaginal discs of Drosophila melanogaster give rise to the adult epidermis during metamorphosis. During this developmental period several peptidase genes are expressed in disc cells, but there is a paucity of biochemical information regarding substrate specificity. We have used peptides and peptidyl 7-amino-4-methylcoumarin (AMC) substrates to detect several peptidases either positioned on the surface of wing discs or secreted by the imaginal cells. Using [Leu(5)]enkephalin as a substrate, a captopril sensitive dipeptidyl carboxypeptidase (angiotensin I-converting enzyme) and an amastatin-sensitive aminopeptidase were detected as prominent activities associated with intact discs. The formation of [Leu(5)]enkephalin-derived Phe was attributed to the concerted action of the D. melanogaster angiotensin I-converting enzyme (Ance) and a dipeptidase. The disc Ance also showed endopeptidic activity towards locust tachykinin-1 (LomTK-I) by cleaving the Gly-Val peptide bond, but this enzyme was not the sole endopeptidase activity associated with discs. Complete inhibition of the endopeptidic hydrolysis of the LomTK-1 by a disc homogenate required a combination of captopril and the neprilysin inhibitor, phosphoramidon, providing biochemical evidence for a neprilysin-like peptidase, in addition to Ance, in imaginal discs of D. melanogaster. Peptidyl AMC substrates for furin, prohormone convertase and tryptase provided evidence for trypsin-like serine endopeptidases in addition to the metalloendopeptidases. We conclude that imaginal discs are endowed with a variety of peptidases from different families that together are capable of hydrolyzing a broad range of peptides and proteins. Some of these peptidases might be responsible for the metabolic activation/inactivation of signaling peptides, as well as being involved in the production of dipeptides and free amino acids required for protein synthesis and osmotic balance during adult morphogenesis.

A critical assessment of the secondary structure alpha-helices and their termini in proteins.

Secondary structure prediction from amino acid sequence is a key component of protein structure prediction, with current accuracy at approximately 75%. We analysed two state-of-the-art secondary structure prediction methods, PHD and JPRED, comparing predictions with secondary structure assigned by the algorithms DSSP and STRIDE. The specific focus of our study was alpha-helix N-termini, as empirical free energy scales are available for residue preferences at N-terminal positions. Although these prediction methods perform well in general at predicting the alpha-helical locations and length distributions in proteins, they perform less well at predicting the correct helical termini. For example, although most predicted alpha-helices overlap a real alpha-helix (with relatively few completely missed or extra predicted helices), only one-third of JPRED and PHD predictions correctly identify the N-terminus. Analysis of neighbouring N-terminal sequences to predicted helical N-termini shows that the correct N-terminus is often within one or two residues. More importantly, the true N-terminal motif is, on average, more favourable as judged by our experimentally measured free energies. This suggests a simple, but powerful, strategy to improve secondary structure prediction using empirically derived energies to adjust the predicted output to a more favourable N-terminal sequence.