Publications by authors named "Stephen J Clark"

23 Publications

  • Page 1 of 1

Increased transcriptome variation and localised DNA methylation changes in oocytes from aged mice revealed by parallel single-cell analysis.

Aging Cell 2020 12 17;19(12):e13278. Epub 2020 Nov 17.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Advancing maternal age causes a progressive reduction in fertility. The decline in developmental competence of the oocyte with age is likely to be a consequence of multiple contributory factors. Loss of epigenetic quality of the oocyte could impair early developmental events or programme adverse outcomes in offspring that manifest only later in life. Here, we undertake joint profiling of the transcriptome and DNA methylome of individual oocytes from reproductively young and old mice undergoing natural ovulation. We find reduced complexity as well as increased variance in the transcriptome of oocytes from aged females. This transcriptome heterogeneity is reflected in the identification of discrete sub-populations. Oocytes with a transcriptome characteristic of immature chromatin configuration (NSN) clustered into two groups: one with reduced developmental competence, as indicated by lower expression of maternal effect genes, and one with a young-like transcriptome. Oocytes from older females had on average reduced CpG methylation, but the characteristic bimodal methylation landscape of the oocyte was preserved. Germline differentially methylated regions of imprinted genes were appropriately methylated irrespective of age. For the majority of differentially expressed transcripts, the absence of correlated methylation changes suggests a post-transcriptional basis for most age-related effects on the transcriptome. However, we did find differences in gene body methylation at which there were corresponding changes in gene expression, indicating age-related effects on transcription that translate into methylation differences. Interestingly, oocytes varied in expression and methylation of these genes, which could contribute to variable competence of oocytes or penetrance of maternal age-related phenotypes in offspring.
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http://dx.doi.org/10.1111/acel.13278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744954PMC
December 2020

Multi-omics profiling of mouse gastrulation at single-cell resolution.

Nature 2019 12 11;576(7787):487-491. Epub 2019 Dec 11.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
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http://dx.doi.org/10.1038/s41586-019-1825-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924995PMC
December 2019

Successful Multidisciplinary Management of an Endodontic-Periodontal Lesion Associated With a Palato-Radicular Groove: A Case Report.

Clin Adv Periodontics 2020 06 31;10(2):88-93. Epub 2019 Oct 31.

Department of Diagnosis and Oral Health, University of Louisville, Louisville, KY.

Introduction: This case report describes the collaborative management of an extensive combined endodontic-periodontal lesion related to a long palato-radicular groove (PRG) on a maxillary lateral incisor. Cases with similar severity have been reported minimally in the endodontic journals but even less in the periodontal journals. This case report illustrates the result of multidisciplinary treatment of the combined lesions associated with PRG.

Case Presentation: A 63-year-old patient presented with a periapical radiolucency on tooth #10. After evaluation, the patient was diagnosed with an endodontic-periodontal lesion associated with PRG. After being informed of a guarded prognosis, the patient consented to a surgical procedure in an effort to retain the tooth. Management of the case involved a combination of endodontic therapy, odontoplasty under dental operating microscopy to attempt to eliminate the root anomaly, and periodontal regenerative procedures with allografts and a resorbable barrier membrane. Clinical examination and the cone-beam computed tomography scan at a 2-year postoperative visit revealed a substantial reduction in probing depth and significant bone fill of the defect.

Conclusions: In the past, a long PRG in combination with a periapical lesion often resulted in extraction of the tooth. With accurate assessment of the etiology of the defect, patient education, and a multidisciplinary approach, teeth with a PRG may be retained with a stable outcome for years.
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http://dx.doi.org/10.1002/cap.10079DOI Listing
June 2020

Combined single-cell profiling of expression and DNA methylation reveals splicing regulation and heterogeneity.

Genome Biol 2019 02 11;20(1):30. Epub 2019 Feb 11.

European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, Cambridge, UK.

Background: Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic features, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remains poorly understood.

Results: We applied parallel DNA methylation and transcriptome sequencing to differentiating human induced pluripotent stem cells to characterize splicing variation (exon skipping) and its determinants. Our results show that variation in single-cell splicing can be accurately predicted based on local sequence composition and genomic features. We observe moderate but consistent contributions from local DNA methylation profiles to splicing variation across cells. A combined model that is built based on genomic features as well as DNA methylation information accurately predicts different splicing modes of individual cassette exons. These categories include the conventional inclusion and exclusion patterns, but also more subtle modes of cell-to-cell variation in splicing. Finally, we identified and characterized associations between DNA methylation and splicing changes during cell differentiation.

Conclusions: Our study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated link between DNA methylation variation and splicing.
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http://dx.doi.org/10.1186/s13059-019-1644-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371455PMC
February 2019

Genome-Scale Oscillations in DNA Methylation during Exit from Pluripotency.

Cell Syst 2018 07;7(1):63-76.e12

Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK; Epigenetics Programme, Babraham Institute, Cambridge, UK; Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK; Centre for Trophoblast Research, University of Cambridge, Cambridge, UK. Electronic address:

Pluripotency is accompanied by the erasure of parental epigenetic memory, with naïve pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, co-expression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modeling, we show that this variability is associated with coherent, genome-scale oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.
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http://dx.doi.org/10.1016/j.cels.2018.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066359PMC
July 2018

scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells.

Nat Commun 2018 02 22;9(1):781. Epub 2018 Feb 22.

Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT, UK.

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.
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http://dx.doi.org/10.1038/s41467-018-03149-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823944PMC
February 2018

Histopathological tumour viability after neoadjuvant chemotherapy influences survival in resected pancreatic cancer: analysis of early outcome data.

ANZ J Surg 2018 Mar 20;88(3):E167-E172. Epub 2017 Mar 20.

Department of Gastrointestinal Surgery, Royal North Shore Hospital, Sydney, New South Wales, Australia.

Background: Neoadjuvant therapy is increasingly recognized as an effective strategy prior to pancreatoduodenectomy. We investigate the role of neoadjuvant chemotherapy (NAC) followed by surgery and the predictive role of viable residual tumour cells histopathologically on outcomes.

Methods: The study population comprised of 195 consecutive patients with pancreatic adenocarcinoma who were treated with either NAC or a surgery-first (SF) strategy. Histopathological viable tumour cells were examined in the NAC patients and clinicopathological factors were correlated with overall survival.

Results: Forty-two patients (22%) were treated with NAC and 153 patients (78%) underwent SF. NAC was associated with higher estimated blood loss during surgery (928 mL versus 615 mL; P = 0.004), fewer (<15) excised lymph nodes (37% versus 17%; P = 0.015) and lower rates of lymphovascular invasion (65% versus 45%; P = 0.044) when compared with SF. Two-year survival of patients undergoing NAC was 63% and 51% in patients undergoing SF (P = 0.048). The 2-year survival of patients who had >65% residual tumour cells was 45% and 90% in patients who had <65% residual tumour cells (P = 0.022). Favourable responders (<65% viable tumour cells) were observed to have shorter operation time (<420 min) (55% versus 13%; P = 0.038), trend towards negative lymph node status (38% versus 10%; P = 0.067) and greater lymph node harvest in node positive patients (≥4 positive lymph nodes) (77% versus 37%; P = 0.045).

Conclusion: The improved survival of patients undergoing NAC indicates effective management of micrometastatic disease and is an effective option requiring further investigation. Histopathological viable tumour cells after NAC was a surrogate marker for survival.
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http://dx.doi.org/10.1111/ans.13897DOI Listing
March 2018

Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

Nat Protoc 2017 03 9;12(3):534-547. Epub 2017 Feb 9.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.
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http://dx.doi.org/10.1038/nprot.2016.187DOI Listing
March 2017

Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.

Genome Biol 2016 Apr 18;17:72. Epub 2016 Apr 18.

Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT, UK.

Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.
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http://dx.doi.org/10.1186/s13059-016-0944-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834828PMC
April 2016

Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity.

Nat Methods 2016 Mar 11;13(3):229-232. Epub 2016 Jan 11.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing that allows for the discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.
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http://dx.doi.org/10.1038/nmeth.3728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770512PMC
March 2016

Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis.

Mol Cell 2015 Nov 5;60(4):611-25. Epub 2015 Nov 5.

Medical Research Council Clinical Sciences Centre (MRC CSC), Faculty of Medicine, Imperial College London, London W12 0NN, UK. Electronic address:

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.
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http://dx.doi.org/10.1016/j.molcel.2015.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672152PMC
November 2015

Genome-wide bisulfite sequencing in zygotes identifies demethylation targets and maps the contribution of TET3 oxidation.

Cell Rep 2014 Dec 12;9(6):1990-2000. Epub 2014 Dec 12.

Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK; Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK. Electronic address:

Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome. We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development. Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup.
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http://dx.doi.org/10.1016/j.celrep.2014.11.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542306PMC
December 2014

Summary of: Regenerative endodontics.

Authors:
Stephen J Clark

Br Dent J 2014 Mar;216(6):356-7

Professor of Endodontics Department of Oral Health and Rehabilitation University of Louisville Louisville, KY.

Background: Significant advances in our understanding of the biological processes involved in tooth development and repair at the cellular and molecular levels have underpinned the newly emerging area of regenerative endodontics. Development of treatment protocols based on exploiting the natural wound healing properties of the dental pulp and applying tissue engineering principles has allowed reporting of case series showing preservation of tissue vitality and apexogenesis.

Aim: To review current case series reporting regenerative endodontics.

Results: Current treatment approaches tend to stimulate more reparative than regenerative responses in respect of the new tissue generated, which often does not closely resemble the physiological structure of dentine-pulp. However, despite these biological limitations, such techniques appear to offer significant promise for improved treatment outcomes.

Conclusions: Improved biological outcomes will likely emerge from the many experimental studies being reported and will further contribute to improvements in clinical treatment protocols.
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http://dx.doi.org/10.1038/sj.bdj.2014.225DOI Listing
March 2014

Association of sirtuin 1 (SIRT1) gene SNPs and transcript expression levels with severe obesity.

Obesity (Silver Spring) 2012 Jan 14;20(1):178-85. Epub 2011 Jul 14.

Department of Genomics of Common Diseases, School of Public Health, Imperial College London, Hammersmith Hospital, London, UK.

Recent studies have reported associations of sirtuin 1 (SIRT1) single nucleotide polymorphisms (SNPs) to both obesity and BMI. This study was designed to investigate association between SIRT1 SNPs, SIRT1 gene expression and obesity. Case-control analyses were performed using 1,533 obese subjects (896 adults, BMI >40 kg/m(2) and 637 children, BMI >97th percentile for age and sex) and 1,237 nonobese controls, all French Caucasians. Two SNPs (in high linkage disequilibrium (LD), r(2) = 0.96) were significantly associated with adult obesity, rs33957861 (P value = 0.003, odds ratio (OR) = 0.75, confidence interval (CI) = 0.61-0.92) and rs11599176 (P value: 0.006, OR = 0.74, CI = 0.61-0.90). Expression of SIRT1 mRNA was measured in BMI-discordant siblings from 154 Swedish families. Transcript expression was significantly correlated to BMI in the lean siblings (r(2) = 0.13, P value = 3.36 × 10(-7)) and lower SIRT1 expression was associated with obesity (P value = 1.56 × 10(-35)). There was also an association between four SNPs (rs11599176, rs12413112, rs33957861, and rs35689145) and BMI (P values: 4 × 10(-4), 6 × 10(-4), 4 × 10(-4), and 2 × 10(-3)) with the rare allele associated with a lower BMI. However, no SNP was associated with SIRT1 transcript expression level. In summary, both SNPs and SIRT1 gene expression are associated with severe obesity.
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http://dx.doi.org/10.1038/oby.2011.200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760128PMC
January 2012

Prevalence of carotid and pulp calcifications: a correlation using digital panoramic radiographs.

Int J Comput Assist Radiol Surg 2009 Mar 15;4(2):169-73. Epub 2008 Nov 15.

Department of Periodontics, Endodontics and Dental Hygiene, School of Dentistry, University of Louisville, 501 South Preston Street, Room LL35, Louisville, KY 40292, USA.

Objective: To compare the prevalence of pulp calcification with that of carotid calcification using digital panoramic dental radiographs.

Study Design: Digital panoramic radiographs of patients at a dental oncology clinic were included if (1) the carotid artery bifurcation region was visible bilaterally and (2) the patient had non-restored or minimally restored molars and/or canines. An endodontist evaluated the images for pulpal calcifications in the selected teeth. An oral and maxillofacial radiologist independently evaluated the same images for calcifications in the carotid bifurcation region. Odds-ratio and Pearson chi(2) were used for data analysis. Presence of pulpal calcification was also evaluated as a screening test for the presence of carotid calcification.

Results: A total of 247 panoramic radiographs were evaluated. 32% (n = 80) had pulpal calcifications and 25% (n = 61) had carotid calcifications with 12% (n = 29) having both carotid and pulp calcifications. A significantly higher prevalence of both pulp and carotid calcification was found in subjects older than age 60 years compared to younger age groups. Accuracy of pulpal calcification in screening for carotid calcification was 66.4%.

Conclusions: Both pulp and carotid calcifications were more prevalent in older individuals. The presence of pulp calcification was not a strong predictor for the presence of carotid calcification.
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http://dx.doi.org/10.1007/s11548-008-0277-7DOI Listing
March 2009

Correlation between carotid area calcifications and periodontitis: a retrospective study of digital panoramic radiographic findings in pretreatment cancer patients.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007 Mar 4;103(3):359-66. Epub 2006 Dec 4.

School of Dentistry, The University of Louisville, Louisville, KY 40292, USA.

Objectives: The objective of this study was to examine the prevalence of carotid area calcifications retrospectively detected on digital panoramic radiographs of pretreatment cancer subjects, and to correlate the finding of such calcifications with radiographic evidence of periodontal bone loss in the same subjects.

Study Design: Digital panoramic radiographs of 201 subjects were evaluated for calcifications projected in the carotid artery bifurcation area as well as for alveolar bone loss as a result of periodontal disease. Inclusion criteria were unobscured carotid artery bifurcation regions bilaterally and sufficient index teeth present with a definable cemento-enamel junction and alveolar crest. Radiographs were independently observed for carotid area calcifications and for periodontal status. Image enhancements permitted for detection of calcifications projected in the carotid area included window/level, inverse, and emboss. Periodontal measurements were made on index teeth using proprietary imaging software and a mouse-driven measurement algorithm. A 3-factor analysis of variance was performed with 3 between-subjects comparisons. Percentage of bone loss was the dependent variable. Independent variables were age, subject sex, and the presence or absence of carotid area calcifications.

Results: Differences measured in percentage of bone loss between sexes were not statistically significant. While bone loss did increase with age, comparison of the mean bone loss of each age category revealed no statistical significance. There was a highly significant correlation between carotid artery area calcifications visible on panoramic radiographs and percent alveolar bone loss. Radiographs showing unilateral and bilateral calcifications had a mean percent bone loss of 24.2% +/- 12.6% and 25.7% +/- 13.0% respectively, compared to those with no calcification at 10.4% +/- 9.9%.

Conclusions: Nearly 1 in 4 subjects in this study evidenced calcifications projected in the carotid bifurcation region. The finding of such calcifications was significantly related to the calculated percentage of alveolar bone loss.
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http://dx.doi.org/10.1016/j.tripleo.2006.08.016DOI Listing
March 2007

Effects of imaging system and exposure on accurate detection of the second mesio-buccal canal in maxillary molar teeth.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006 Dec 10;102(6):796-802. Epub 2006 Aug 10.

University of California San Francisco School of Dentistry, San Francisco, CA, USA.

Objectives: The objectives of this study were to determine observer ability to detect the second mesio-buccal canal (MB2) in maxillary molars using analog film, CMOS, and photostimulable phosphor x-ray detectors across a wide range of radiation exposures and to determine the optimal exposures for these modalities accurately detecting presence of the MB2.

Research Design: Using 6 experimental models with 2 maxillary molars in each, images were generated with the 3 detector modalities at nominal x-ray beam angulations of 0 degrees and 10 degrees across a range of radiation exposures. Observers independently assessed presence of the MB2 in images presented in random order. Horizontal root sections were then evaluated to determine true MB2 presence.

Results: For single images at optimal exposures, the correct number of mesiobuccal canals could be identified 55% of the time for CMOS (RVG 6000), 44% of the time for analog x-ray film (InSight), and 39% of the time for photostimulable phosphor (DenOptix). Statistically significant differences were observed between the imaging modalities (chi2 = 23.4, crucial value = 5.99, P < or = .05). RVG 6000 CMOS outperformed both DenOptix photostimulable phosphor (z score = -5.5) and InSight analog direct exposure x-ray film (z score = 4). Exposure affected the rate of accuracy for MB2 detection across the exposure range tested for RVG 6000 and for film, but not to any appreciable degree for DenOptix.

Conclusions: The CMOS detector (RVG-6000) performed best for evaluating presence of the MB2. This was the only modality to exceed 50% reliability with optimal exposure when single images were considered.
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http://dx.doi.org/10.1016/j.tripleo.2006.02.009DOI Listing
December 2006

Gay priests and other bogeymen.

Authors:
Stephen J Clark

J Homosex 2006 ;51(4):1-13

Department of Psychology, Keene State College, Keene, NH 03435-3400, USA.

The sexual abuse of boys by priests was at the center of the 2002 scandal in the Roman Catholic Church. This scandal has resurrected the misconception that a link exists between having a homosexual orientation and being at increased risk for being a pedophile or child molester. This paper reviews and debunks the arguments in support of this misconception. Central to these arguments is what might be called the "proportionality argument": that the ratio of homosexuals to heterosexuals among child molesters is higher than the ratio of homosexuals to heterosexuals in the general population. The flaws in the proportionality argument and several other misconceptions are discussed.
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http://dx.doi.org/10.1300/J082v51n04_01DOI Listing
January 2007

Perceived quality of radiographic images after rapid processing of D- and F-speed direct-exposure intraoral x-ray films.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003 Oct;96(4):486-91

School of Dentistry, The University of Louisville, KY 40292, USA.

Objective: We sought to compare the densitometric properties and perceived image quality of InSight (F-speed) and Ultra-Speed (D-speed) film radiographs processed with rapid chemistry. The effects of density, contrast, and film speed on perceived image quality were also studied.

Study Design: Images were made of a human cadaver phantom with exposures to achieve background densities approximating 1.5, 2.0, and 3.0. Films were processed in a radiographic darkroom by using Insta-Neg and Insta-Fix rapid chemistry as the manufacturer had recommended. Five endodontic residents independently analyzed images of varying density, speed, and contrast that were presented in a randomized manner. They were required to evaluate the perceived image quality of 5 specifically designated areas on the film, using a labeled photograph as a guide. These areas included root canal obturation, periodontal ligament space, dentinoenamel junction, and crestal bone height. In addition, they were also asked to assess the overall perceived image quality. Statistical analysis consisted of ordinal regression and 2-factor analysis of variance.

Results: No statistically significant differences were proved between F- and D-speed radiographs within the same density group. Higher density and higher contrast resulted in a statistically significant positive impact (P <.01) on the ranking for all 5 subjective determinations. Observers preferred the films exposed to a background density of 3.0 over those of a lower density (P <.01).

Conclusions: InSight (F-speed film) can be used with rapid chemistry to ensure less radiation exposure to patients than is necessary with D-speed film. The observers participating in this study preferred radiographs from the 3.0 background density group to those from the 2.0 and 1.5 density groups.
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http://dx.doi.org/10.1016/s1079-2104(03)00062-3DOI Listing
October 2003

Endodontic measurement accuracy and perceived radiograph quality: effects of film speed and density.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003 Oct;96(4):441-8

School of Dentistry, The University of Louisville, Ky, USA.

Objectives: This study sought to determine the effects of direct exposure x-ray film speed and background density on observer assessment of endodontic working lengths and on perceived radiographic image quality.

Study Design: A human cadaver maxilla section with surrounding soft tissues was used for the study. The canal length to the radiographic apex was determined on 4 canals in maxillary posterior teeth by using Trophy RVG images and adjusting the position of a No. 15 file in each canal until the file tip coincided with the radiographic apex in images made at 3 different vertical angulations. The files were measured with a micrometer from the file stop to the file tip to obtain the length to the radiographic apex. Then No. 10 files were placed in the 4 canals at varying lengths short of this previously determined length, and 5 observers assessed the distance from the file tip to the radiographic apex on radiographs made with Kodak D-, E-, and F-speed and Flow D- and E-speed direct exposure x-ray films that were exposed to produce background densities of 1.5, 2.0, and 3.0. Subjective appraisal of radiographic quality was also assessed.

Results: Analysis of variance and Tukey honestly significantly different post-hoc analysis results concerning measurement errors made with each film type revealed significantly less error for Kodak Ektaspeed Plus (E-speed) intraoral x-ray film than for Kodak InSight (F-speed) and Flow E; however, no difference was detected among Kodak Ektaspeed Plus (E-speed), Kodak Ultra-Speed (D-speed), and Flow D. Films with a background optical density of 3.0 received 98% favorable ratings; radiographs with a background optical density of 2.0 received 77% favorable ratings; and those with background optical density of 1.5 received only 18% favorable ratings at the 95% confidence level. Flow D film received the most favorable ratings, but there was no statistically significant difference among other film types at the 95% confidence level.

Conclusions: Underexposed radiographs are perceived as inferior to slightly overexposed radiographs for endodontic file length assessment regardless of the film speed used. Current Flow and Kodak E-speed and F-speed radiographs appear to be as accurate as other accepted radiographs used in determining endodontic working lengths. Image background density should be kept constant when making comparisons among x-ray films.
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http://dx.doi.org/10.1016/s1079-2104(03)00035-0DOI Listing
October 2003

Tuned-aperture computed tomography versus parallax analog and digital radiographic images in detecting second mesiobuccal canals in maxillary first molars.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003 Aug;96(2):223-8

Endodontics, The University of Louisville, KY 40292, USA.

Objective: We sought to evaluate the detectability of second mesiobuccal (MB2) canals in the MB root of maxillary first molar teeth by comparing (1) parallax with pairs of conventional direct-exposure film intraoral radiographs (both D-speed and F-speed), (2) parallax with pairs of charge-coupled device-based digital images acquired through the use of Trophy RVG-ui, and (3) charge-coupled device-based images acquired through the use of a Trophy RVG-ui sensor and tomosynthetically reconstructed by TACT (tuned-aperture computed tomography) Workbench Software.

Study Design: Maxillary first molars were mounted in simulated bone. Pairs of images were generated by conventional D-speed and F-speed radiography and digital radiography with a charge-coupled device-based sensor, the RVG-ui. Sequences of images were also acquired for TACT reconstruction by using the digital sensor. Observers viewed sets of images to determine the number of canals present within the MB root of each tooth. Roots were horizontally cross-sectioned and viewed under an operating microscope to determine the actual number of canals present.

Results: The frequency of detection of MB2 canals was remarkably similar across techniques: 39.2% to 39.6% with parallax for both types of film and for RVG-ui images, and 37.9% with TACT. No statistically significant difference was found in the detectability of MB2 canals between the modalities tested. TACT had higher correlation coefficients than the other 3 modalities with respect to intrarater and interrater reliabilities.

Conclusions: (1) There was a less than 40% chance of locating MB2 canals in the MB root of maxillary first molar teeth by using parallax with pairs of digital or analog radiographs. (2) TACT did not significantly affect the rate of detection of MB2 canals.
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http://dx.doi.org/10.1016/s1079-2104(03)00061-1DOI Listing
August 2003

A comparison of D-, E-, and F-speed conventional intraoral radiographic films in endodontic measurement.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002 Mar;93(3):337-40

School of Dentistry, The University of Louisville, Kentucky 40292, USA.

Objectives: We sought to compare observer measurement error and subjective ratings for International Standards Organization D-, E-, and F-speed direct exposure dental x-ray films used to determine endodontic working lengths.

Methods: Radiographs were exposed to achieve a standard background density of approximately 2.0. Three human maxillary posterior teeth for which No. 10 K-files had been placed at various lengths within 4 canals were studied in a cadaver section. A total of 30 radiographs were used (10 from each film type). Five licensed dentists excluding all authors viewed the radiographs independently and in random sequence. Distances from the file tips to the radiographic apices were measured and were compared with known lengths for error determination. The observing dentists also subjectively rated the images as desirable or undesirable. Statistical methods included a 3-factor ANOVA with Tukey honestly significant difference post hoc analysis to compare objective measurements and chi-square with respect to subjective ratings.

Results: No significant differences were attributable to film speed grouping or observers (P > .05); however, there was a specimen effect in that a significant difference was found in measurement accuracy among the 4 canals (P < .05). Differences in subjective ratings for the 3 film types were not statistically significant (P > .05).

Conclusion: All 3 film types were similar in objective and subjective ratings. This being the case, the faster film is preferred to minimize the radiation dose to the patient.
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http://dx.doi.org/10.1067/moe.2002.122336DOI Listing
March 2002

A comparison of pain levels during pulpectomy, extractions, and restorative procedures.

J Endod 2002 Feb;28(2):108-10

University of Louisville School of Dentistry, KY, USA.

Most previous studies on pain in endodontics have focused on pain that occurs after root canal therapy. Very few studies have compared pain during the root canal procedure with pain occurring during other dental procedures. In the present study, 250 patients were queried following dental procedures regarding their pain levels prior to treatment and their pain levels during the treatment procedure. Of the total number of patients, 150 had a pulpectomy, 50 patients had a single extraction, and 50 patients had a single restoration. These patients reported significantly more pain during extractions than during root canal therapy. Ninety-two percent of patients undergoing root canal therapy reported that pain during the procedure was less than or much less than anticipated. Eighty-three percent of the patients undergoing root canal therapy experienced less pain during the treatment procedure than they experienced prior to the treatment.
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http://dx.doi.org/10.1097/00004770-200202000-00015DOI Listing
February 2002