Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster.

The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

The influence of vitamin C on systemic markers of endothelial and inflammatory cell activation in smokers and non-smokers.

OBJECTIVE AND DESIGN: To determine the influence of vitamin C supplementation (500 mg, bd, 14 days) on the circulating concentrations of soluble ICAM-1 (a marker of endothelial activation), neopterin (a marker of monocyte activation), and neutrophil elastase (a marker of neutrophil activation) in smokers and non-smokers in a randomised, double-blind, placebo-controlled trial in a hospital setting. SUBJECTS: Twenty smokers (serum cotinine > or = 20 ng ml(-1)) and 20 age- and gender-matched non-smokers (serum cotinine < or = 13.7 ng ml(-1)). RESULTS: At baseline, there was a significant elevation in the concentration of sICAM-1 in smokers (median 247, IQR 199 to 357 ng ml(-1)) compared to non-smokers (median 207, IQR 189 to 227 ng ml(-1); p = 0.014). Vitamin C supplementation did not influence the circulating concentrations of ICAM-1 or neopterin, or leukocyte elastase activity, in smokers, non-smokers, or in the total population. CONCLUSIONS: Markers of monocyte and neutrophil activation were not influenced by smoking status in this study population. However, sICAM-1 concentrations were significantly elevated in tobacco smokers, reflecting tobacco-induced vascular activation that is unaffected by Vitamin C supplementation.

A prospective study to investigate the relationship between periodontal disease and adverse pregnancy outcome.

OBJECTIVE: This study aimed to investigate a relationship between maternal periodontal disease and preterm birth, low birth weight and late miscarriage. DESIGN: Prospective study in a single centre. SETTING: Guy's and St Thomas' Hospital Trust, London, UK between August 1998 and July 2001. SUBJECTS AND METHODS: Pregnant women were recruited on attending an ultrasound scan at approximately 12 weeks of pregnancy. Subjects completed a questionnaire and underwent periodontal examination. MAIN OUTCOME MEASURES: Plaque and bleeding scores, pocket probing depth and loss of attachment. Pregnancy outcome data was collected retrospectively, including gestational age and birth weight at delivery. RESULTS: Data were collected for 3,738 subjects. Regression analysis indicated that there were no significant relationships between the severity of periodontal disease and either preterm birth (PTB) or low birth weight (LBW). In contrast, there did appear to be a correlation between poorer periodontal health and those that experienced a late miscarriage. CONCLUSIONS: There was no association between either preterm birth or low birth weight and periodontal disease in this population. There is evidence of a correlation between markers of poorer periodontal health and late miscarriage.

Effect of treatment of chronic periodontitis on levels of serum markers of acute-phase inflammatory and vascular responses.

AIMS: Recent epidemiological work suggests an association between periodontal disease severity and cardiovascular disease risk. This study aimed to ascertain if circulating levels of cardiovascular and systemic inflammatory markers could be modified following treatment of periodontal disease. METHOD: Adult subjects were recruited from those awaiting periodontal treatment and randomised to either immediate (test, n=24) or delayed treatment (control, n=15). Demographic and clinical data were collected and venous blood was taken before and either 6 weeks after completion of treatment or after an equivalent 3-month control period. Periodontal examination included probing depth, loss of attachment, plaque scores and bleeding scores. Blood was analysed to determine serum and plasma fibrinogen, C-reactive protein, sialic acid, tumour necrosis factor-alpha and interleukin -6 and -1beta. Effects of treatment were assessed by paired tests and analysis of variance by treatment group with baseline covariates. RESULTS: Treatment improved plaque and bleeding scores and reduced probing depths (p<0.002). However, there were no statistically significant changes in levels of any of the systemic markers. CONCLUSION: Improvement in periodontal health did not influence the levels of vascular markers.

Effect of nicotine replacement and quitting smoking on circulating adhesion molecule profiles (sICAM-1, sCD44v5, sCD44v6).

BACKGROUND: Soluble ICAM-1 (sICAM-1; sCD54), sCD44v5 and sCD44v6 are circulating adhesion molecules, with immunomodulatory potential, that have been frequently attributed diagnostic, prognostic and aetiological significance in a number of inflammatory and malignant diseases. We have previously shown that systemic concentrations of these molecules are increased significantly in tobacco smokers, but reduce to within normal levels at 12 months following successful quitting. MATERIALS AND METHODS: We have been able to extend these observations by measuring levels before and 4, 8, 22 and 52 weeks after smoking cessation in subjects receiving high-dose nicotine replacement therapy (25 mg of nicotine; n = 34) or placebo patches (n = 34) for 26 weeks. Smoking cessation was confirmed by regular measurement of expired-air CO levels and by plasma cotinine analysis. RESULTS: Plasma sICAM-1, sCD44v5 and sCD44v6 concentrations all declined rapidly within 4 weeks of smoking cessation (P < 0.001 for all declines). Additionally, no differences were observed between those using nicotine replacement and those who were not for sICAM-1, sCD44v5, or sCD44v6. CONCLUSIONS: The recovery in smoking-associated adhesion molecule profiles represents an almost immediate beneficial effect of smoking cessation. Nicotine replacement therapy is an effective aid to quitting and does not affect these recoveries. The elevated levels of these important risk factors in smokers (sICAM-1, sCD44v5 and sCD44v6) are linked to noxious element(s) in tobacco smoke other than nicotine or nicotine metabolites.

Analysis of circulating insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 (IGFBP-3) in tobacco smokers and non-smokers.

BACKGROUND: IGF-1 and the major serum IGF-1 binding protein, IGFBP-3, are under extensive investigation as potential prognostic markers of specific malignancies and vascular diseases. However, there is conflicting evidence that tobacco smoking may influence systemic concentrations of IGF-1 and IGFBP-3. SUBJECTS AND METHODS: Serum concentrations of IGF-1 and IGFBP-3 were measured in 20 smokers and 20 non-smokers, matched for age and gender. Serum concentrations of cotinine, the major metabolite of nicotine, and ICAM-1, known to exhibit a dose-dependent relationship with cotinine, were also assayed. RESULTS: There was no difference between the systemic concentrations of IGF-1 or IGFBP-3 found in smokers and non-smokers (IGF-1: mean [s.d]; 104 29 vs 101 24 ng ml-1, respectively; and IGFBP-3: 2562 [522] vs 2447 [570] ng ml-1, respectively). Similarly, there was no correlation between serum cotinine and IGF-1 or IGFBP-3 concentrations in smokers. Soluble ICAM-1 concentrations were significantly increased in smokers, compared to non-smokers (mean [s.d]; 258 [60] vs 194 [50] ng ml-1, respectively; p = 0.002). CONCLUSION: There was no relationship noted between tobacco smoking and either IGF-1 or IGFBP-3. These data suggest that smoking would not appear to be a major confounder of the reported clinical associations between IGF-1, IGFBP-3, or IGF-1/IGFBP-3 ratios and specific disease entities.

Comparison of the erosive potential of gastric juice and a carbonated drink in vitro.

The aim of this study was to compare the erosive effect of gastric juice and a carbonated drink on enamel and dentine by measuring release of calcium from 30 hemisectioned teeth in vitro. In addition, the titrable acidity (mL of 0.05 M sodium hydroxide required to neutralize) and pH of the fluids was estimated. The mean pH of the seven gastric acid samples was 2.92 (range 1.2-6.78) and mean titratable acidity 0.68 mL (range 0.03-1.64). Both the pH and the titratable acidity of the gastric juice varied between patients all of whom suffered from symptoms of reflux disease. The carbonated drink had a pH of 2.45 and a titratable acidity of 0.29 mL. The median amount of calcium released by the gastric acids from enamel was 69.6 microg L-1 (interquartile range 5.4-144) and 62.4 microg L-1 (2.2-125.3) from dentine. The carbonated drink released 18.7 microg L-1 (13.4-23.4) and 18.6 microg L-1 (11.9-35.3), respectively. The differences in calcium release by gastric juice and the carbonated drink were statistically significant for both enamel (P < 0.005) and dentine (P < 0.01). It is concluded that gastric juice has a greater potential, per unit time, for erosion than a carbonated drink.

Elevated systemic concentrations of soluble ICAM-1 (sICAM) are not reflected in the gingival crevicular fluid of smokers with periodontitis.

Raised serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) in smokers could have immunomodulatory effects in periodontitis. The aim of this study was to compare serum and gingival crevicular fluid (GCF) concentrations of sICAM-1 in smokers and non-smokers with periodontal disease. sICAM-1 in serum and GCF collected from age- and gender-matched smokers (n = 14) and non-smokers (n = 14) with periodontitis were measured by ELISA. Mean serum sICAM-1 concentrations were significantly elevated in smokers (331 ng mL(-1)), compared with non-smokers (238 ng mL(-1), p = 0.008). However, the concentration of sICAM-1 in the GCF was significantly lower in the smokers (83 ng mL(-1)), compared with non-smokers (212 ng mL(-1), p = 0.013). The difference between concentrations of sICAM-1 in GCF and serum was significant only in smokers (p < 0.001). Since GCF is a serum-derived tissue exudate, these results suggest that, in smokers, circulating sICAM-1 molecules are affected either in their passage from the periodontal microvasculature or within the periodontal tissues.

Experiences and perceptions of vocational training reported by the 1999 cohort of vocational dental practitioners and their trainers in England and Wales.

OBJECTIVE: To assess the self-reported confidence of vocational dental practitioners (VDPs) in clinical procedures together with vocational trainers' perceptions of the VDPs confidence in the same procedures, immediately after qualification and towards the end of the vocational training year. DESIGN: A questionnaire-based cohort study. SETTING: A general practice study carried out in 1999. SUBJECTS: Vocational Dental Practitioners and vocational trainers in England and Wales. METHOD: VDPs and trainers were asked on a single occasion to grade the clinical confidence of the VDP at the beginning and near the end of vocational training as high, satisfactory or low. RESULTS: Questionnaires were sent to 531 VDPs and 555 trainers; 82 per cent of VDPs and trainers responded. Approximately half the VDPs were male and 57 per cent were white, whilst 89 per cent of trainers were male and 81 per cent white. A large proportion of both VDPs and trainers reported low confidence in orthodontics, molar endodontics and surgical extractions at the start of the training year. Towards the end of training, both groups reported improved confidence levels in most clinical procedures. However, a higher proportion of trainers reported low confidence than their VDPs in most clinical procedures at both time points (p<0.001). VDPs appeared to gain most from experience and training in administration/management and interpersonal skills. CONCLUSION: Vocational training appears to satisfy its aim to enhance clinical and administrative confidence.

Periodontal health of London women during early pregnancy.

OBJECTIVES: A descriptive cross-sectional study to determine the severity of periodontal disease in early pregnancy and its relation to demographic variables in a South East London population. METHODS: 2,027 pregnant women attending Guy's Hospital for an ultrasound scan at 10 to 14 weeks gestation were assessed. Data were collected via questionnaire and periodontal examination, including plaque and bleeding scores, pocket probing depth and loss of attachment. RESULTS: Mean age was 29.8 years (sd 5.5). Of these, 61.8% were white, 28.5% black, and 9.7% of other ethnic group. A total of 15% reported smoking during pregnancy. The mean number of teeth present was 28 (sd 2) per subject, mean percentage of sites with plaque present was 60.5% (sd 22.6), mean pocket depth was 2.0 mm (sd 0.4), mean loss of attachment was 0.4 mm (sd 0.3), and the mean percentage of sites bleeding on probing was 20.2% (sd 16.3). Linear regression demonstrated that probing depth was related to age, ethnicity, socioeconomic status and plaque score but not to smoking whereas loss of attachment demonstrated relationships with age, smoking status and plaque score but not ethnicity or socioeconomic status. CONCLUSION: There was a relatively high proportion of subjects with deep periodontal pockets in this pregnant population compared to the Adult Dental Health Survey 1998 but with similar levels of loss of attachment and percentage of sites with plaque present. Several demographic factors were associated with the level of periodontal disease in this population.

Plasma concentrations of reputed tumor-associated soluble CD44 isoforms (v5 and v6) in smokers are dose related and decline on smoking cessation.

There is some evidence to suggest that smoking may affect circulating levels of CD44 (sCD44) molecules. Therefore, we investigated the effect of smoking on the circulating level of sCD44 by comparing the change in total sCD44, sCD44v5, and sCD44v6 concentrations over 1 year in a group of people who quit smoking (n = 30) and a control group of people who continued to smoke (n = 30). Smoking status and compliance were monitored by analysis of plasma cotinine and expired CO levels and also by self-reported tobacco use. We show a dose-dependent relationship between smoke intake and baseline plasma concentrations of reputed tumor-associated CD44 variant isoforms (sCD44v5 and sCD44v6) in smokers (n = 60). There was a significant decline in the level of both sCD44v5 and sCD44v6 in quitters as compared with continuing smokers [-13.2 (95% confidence interval, -7.6 to -18.8; P < 0.001) and -62.2 ng/ml (95% confidence interval, -33.9 to -90.6; P < 0.001), respectively], but not in the total sCD44 concentration. These results show that the increased concentrations of sCD44v5 and sCD44v6 in smokers are dose related and reversible and suggest that the attributed diagnostic specificity and prognostic value of sCD44 molecules in malignant and inflammatory disease may be affected by smoking status.

Dramatic decline in circulating intercellular adhesion molecule-1 concentration on quitting tobacco smoking.

The concentration of soluble ICAM-1 (sICAM-1) is significantly elevated in smokers, but it is unclear if smoking is the direct cause of elevated sICAM-1 levels, if the relationship between smoking and sICAM-1 level is dose-dependent, and if smoking cessation may lead to a decline in sICAM-1. We sought to clarify the relationship between smoking and sICAM-1 in a group of smokers who quit smoking for 1 year (n = 30) and a control group who continued to smoke (n = 30). A dose-dependent relationship between plasma sICAM-1 concentration and daily cigarette consumption (P = 0.02), plasma cotinine level (P = 0.02), and expired CO level (P = 0.007) was observed at baseline (n = 60). The mean change in sICAM-1 concentration after 52 weeks was greater for quitters than for continuing smokers (mean difference = -71.1 ng/ml, P < 0.001). The influence of smoking on sICAM-1 needs to be carefully considered in clinical trials. Soluble ICAM-1 remains bioactive and may contribute to pathogenic processes; therefore, reduction in the concentration of circulating ICAM-1 molecules may directly contribute to the health benefits associated with smoking cessation.

The acute influence of tobacco smoking on adhesion molecule expression on monocytes and neutrophils and on circulating adhesion molecule levels in vivo.

Soluble adhesion molecules have been reported as risk markers of a wide range of human diseases and specific adhesion molecules may play a direct role in pathological processes. Serum soluble intercellular adhesion molecule-1 (sICAM-1) is known to be significantly elevated in smokers compared to non-smokers. We examined the acute effects of smoking a standard 2R1 research cigarette on the serum concentration of sICAM-1 and other circulating adhesion molecules (sP-selectin, sE-selectin, sL-selectin, sVCAM-1 and sPECAM-1) in heavy smokers (serum cotinine >/= 100 ng/ml), light smokers (serum cotinine

Serum concentration of total soluble CD44 is elevated in smokers.

Soluble CD44 isoforms have been reported as markers of specific malignancies and inflammatory diseases. However, recent reports suggest tobacco smoking may lead to an elevation in the circulating concentration of specific CD44 variants. We, therefore, investigated the effect of smoking status on circulating levels of total sCD44. Total soluble CD44 was measured by enzyme-linked immunosorbent assay in the serum of two age- and gender-matched groups consisting of smokers (n = 19) and non-smokers (n = 20). Smoking status was confirmed by analysis of serum cotinine. The concentration of total sCD44 was found to be significantly elevated in smokers compared with non-smokers (p = 0.025). The observation that total sCD44 concentration is raised in smokers may have relevance to the aetiology of smoking-associated diseases. The effect of smoking on sCD44 concentrations should be considered when assessing the role of sCD44 as a marker of inflammatory disease, cancer, or other disease processes.

Perceived sources of occupational stress in general dental practitioners.

AIM: To evaluate levels of occupational stress in general dental practitioners (GDPs) and compare them with those reported by Cooper et al. (1987). DESIGN/SETTING: Postal questionnaire in England and Wales. SUBJECTS: A sample of 1007 GDPs was chosen from the Dental Register. MAIN OUTCOME MEASURES: 30 separate stressors were scored on a 5-point scale. Summated scores within time-, job-, income-, staff- and patient-related categories were submitted to analysis of variance by sex, type of practice, years since qualification and geographic location. RESULTS: 823 questionnaires were returned (82%) of which 667 were from practising GDPs (63% male, 26% female, 1% undetermined) working full- (87%) or part- (13%) time in NHS (61%), private (8%), mixed (26%), or community (5%) practice. In general, problems associated with time management were the most stressful. The highest ranked individual stressors were 'running behind schedule' (percentage responding with scores 4 or 5 = 61.9, 95% confidence intervals = 58.2-65.6), followed by 'coping with difficult or uncooperative patients' (58.2, 54.5-61.9) and the 'working constraints set by the NHS' (57.5, 53.8-61.2). Differences in levels of reported stress (P < 0.05) were observed for at least one group category of stressor between practitioners with different types of practice, between males and females, by geographical area (north against south) and by the length of time since qualification. CONCLUSIONS: The results indicate that GDPs now rank factors related to time management as major job stressors, with an apparently dramatic elevation of 'NHS working constraints' to a top-ranking stressor since 1986.

The prevalence of tooth wear in a cluster sample of adolescent schoolchildren and its relationship with potential explanatory factors.

OBJECTIVE: To assess the prevalence of tooth wear in adolescents and its relationship with diet, saliva and gastro-oesophageal reflux. DESIGN: Single centre cluster sample epidemiological study. SETTING: A school in London in the summer of 1996. SUBJECTS: 11-14-year-old schoolchildren. MAIN OUTCOME MEASURES: The Smith and Knight tooth wear index (TWI), salivary factors, diet and symptoms of gastro-oesophageal reflux were recorded for all subjects. RESULTS: Results were obtained from 210 subjects. One subject refused to provide a saliva sample and 11 subjects provided insufficient saliva for analysis of buffering power (n = 198). 57% (95% confidence intervals 50.3-63.7%) of subjects had tooth wear on more than ten teeth and a median 12% (interquartile range 6-18%, 95% confidence intervals 8-14%) of surfaces were affected. However, dentine involvement was rare. The median intake of carbonated drinks was 2 cans (interquartile range 1-3) a day. However, there was no correlation with TWI (r = -0.09, P = 0.19). There was no relationship between tooth wear index (TWI) and salivary flow rate (r = -0.02, P = 0.78) or buffering capacity (r = -0.02, P = 0.76). A trend was observed for those with a reported history of regurgitation (n = 27) to have a higher maxillary TWI (median 8, interquartile range 2-13) compared with those who did not (5, 2-9, P = 0.06). CONCLUSIONS: Tooth wear is common in adolescents and the relationship with dietary acid, salivary buffering and symptoms of gastro-oesophageal reflux is complex and requires further investigation.

Experience of undergraduates from three London dental schools and trainers from the south east of England on interviews for vocational training in 1996.

OBJECTIVE: To assess the current perceptions, motivations and expectations of trainers and vocational dental practitioners (VDPs) of vocational training (VT) interviews. DESIGN: Questionnaire based study. SETTING: General practice study carried out in 1996. SUBJECTS: Undergraduates from three London dental schools and vocational trainers from south-east England. MAIN OUTCOME MEASURES: Perceptions, motivation and experiences were assessed by a questionnaire. RESULTS: 111 questionnaires were posted to trainers with 72 (65%) returned. 108 (70%) were returned from the dental schools after 155 were posted. Graduates employed in hospital training posts, armed forces and community dentistry were eliminated from the results. Most trainers (74%) had previous experience of VT and 89% of trainers thought that a CV was important for their choice of VDPs. However, only 11% of trainers sought references before interviewing. 34% of VDPs preferred jobs close to their dental school but 75% considered location to be vital to their choice. 67% of VDPs made 10 or more applications and 57% visited 10 or more practices for interviews. 78% of VDPs considered that either genderism or racism was likely to or possibly influenced the trainers choice. However, the converse was not true, 62% of trainers considered it unlikely that the VDPs were influenced by their gender or race. CONCLUSIONS: Trainers and VDPs have different perceptions concerning the protocol of interviews for VT.

Elastase in gingival crevicular fluid from smokers and non-smokers with chronic inflammatory periodontal disease.

OBJECTIVE: To compare elastase concentrations in gingival crevicular fluid (GCF) from individual sites of smokers and non-smokers. MATERIALS AND METHODS: Twelve pairs of smokers and non-smokers with untreated, moderate to advanced chronic inflammatory periodontal disease were matched for gender, age, ethnicity and the clinical and radiographic extent of disease. Durapore filter strip samples were collected over 30 s from two mesiopalatal sites on upper left posterior teeth. Samples were analysed for: 1) polymorphonuclear neutrophil leucocyte (PMNL) cell counts; 2) PMNL elastase-alpha 1-antitrypsin complex in the GCF supernatant by ELISA; and 3) functional elastase, free or bound to alpha 2-macroglobulin, estimated from activity against N-tert-butoxycarbonyl-alanyl-prolyl-norvalyl-p-chlorothiobenzyl ester in supernatant and lysates of GCF PMNLs. RESULTS: There were no differences in disease parameters between groups except that bleeding on probing was less extensive in smokers (P<0.001). Cell counts and elastase content of crevicular PMNLs showed no differences between groups. Lower concentrations of elastase were found in GCF supernatants from smokers than non-smokers. This difference was observed for functional elastase (mean [s.d.] = 30.21 [17.60] against 73.77 [75.26] ng microliter(-1), p<0.05) and elastase complexed with alpha 1-antitrypsin (8.97 [6.54] ng microliter(-1) against 25.71 [22.07] ng microliter(-1), p<0.001). CONCLUSIONS: Smokers have lower elastase concentrations in GCF than non-smokers. Further investigation is required to elucidate the underlying cause and its relationship with periodontal disease.

Relationship between calcium and inorganic phosphorus concentrations of both resting and stimulated saliva and dental plaque in children and young adults.

Calcium and inorganic phosphorus concentrations of both resting and paraffin-wax stimulated whole saliva and dental plaque were estimated in 39 young adults and 98 children aged 12-14 yr. Plaque was collected from the adults after 48 h without tooth-brushing and consumption of a standardized diet. Plaque was collected from the children without any dietary or oral hygiene restrictions. The results from the study with children provided consistent evidence for a statistically significant relationship between salivary and plaque concentrations of both calcium and inorganic phosphorus. However, multiple regression indicated that only the concentrations in the stimulated secretion were directly associated with concentrations in plaque. The apparent relationship between concentrations in resting saliva and plaque was due to correlation between resting and stimulated saliva. These relationships were less apparent in the young adults, in whom the controlled conditions resulted in a narrower range of mineral concentrations in dental plaque.