Mouthwash Samples Research Articles

Evaluation of concentration and purity of DNA extracted from mouthwash, saliva and buccal cytobrush samples

IntroductionNon-invasive sampling has been highlighted as an alternative sampling for molecular analysis of diseases. Various methods of sampling and DNA isolation have been introduced. This study includes collection of mouthwash, saliva and buccal swab/cytobrush sampling, and extraction using conventional methods. ObjectiveTo assess the DNA quantity and quality by evaluating the concentration and purity of DNA isolated from mouthwash, saliva and buccal cytobrush using in-house DNA extraction methods. MethodsMouthwash, saliva and buccal cytobrush samples were collected from paediatric patients attending monthly blood transfusions in University Malaya Medical Centre. The samples were extracted using alkaline lysis method and purified with phenol:chloroform. The DNA purity and concentration was measured by spectrophotometry. Mean and standard error of mean were calculated and analysed for significance in difference. Results & DiscussionThe DNA with highest purity (1.743 ± 0.031) was obtained from mouthwash samples while DNA with highest concentration (0.796 ± 0.095 μg/μL) was obtained from saliva. Buccal cytobrush samples produced DNA with high purity comparable to mouthwash samples, however, at a lower concentration (0.162 ± 0.041 μg/μL). ConclusionHigh quality DNA can be obtained from mouthwash, saliva and buccal cytobrush samples. Saliva samples may serve as the best alternative sampling for extraction of genomic DNA for its high concentration and acceptable purity.

Interleukin-7 receptor gene polymorphism at +1237 locus and its effect on susceptibility to opportunistic infections among HIV and AIDS patients in Limpopo Province, South Africa.

Over the past decade, an increasing number of studies have demonstrated correlations between host genetics and susceptibility to diseases. However, few studies have investigated the effects of host genetics on the occurrence of opportunistic infections among human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) patients. In the present study, the frequency of the interleukin (IL)-7Rα+1237 A/G single nucleotide polymorphisms was determined in relation to opportunistic infection occurrence among HIV and AIDS patients in the Vhembe District. Demographic, clinical, and socioeconomic status data were collected from patients using a structured questionnaire. Genomic DNA was extracted from mouthwash samples using the QIAmp Blood Mini Kit. Genotyping of the IL-7Rα+1237 gene was conducted using a sequence-specific polymerase chain reaction method. We found that the IL-7Rα+1237 genotype distribution in our study population differed from those in European populations with a predominance of the A/G genotype. Individuals carrying the A/G genotype primarily suffered from chest pain (χ(2) = 5.016, P = 0.025), while individuals carrying the G/G genotype were protected from chest pain but had a higher prevalence of sexually transmitted disease (23 vs 16.9%); however, the difference was not statistically significant (P = 0.435). Individuals carrying the A/A genotype were more susceptible to diarrhea (32 vs 13.6%) (P = 0.034). Our data will support gene therapy and may be used to modify the course of diseases among HIV patients as well as the general population. Further studies using larger populations are needed to confirm these hypotheses.

Comparative evaluation of the antiplaque effectiveness of green tea catechin mouthwash with chlorhexidine gluconate

Aim:The aim of this study is to compare the antiplaque efficacy of green tea catechin mouthwash with chlorhexidine gluconate mouthwash.Materials and Methods:A single blinded cross-over study was conducted among 30 participants in the age group of 18-25 years. The mouthwash samples for the study were previously labeled assigning the letters: A (0.25% of green tea catechin mouthwash) and B (0.12% of chlorhexidine mouthwash). The study subjects were randomly divided into two groups of 15 each and the study was divided into two phases. In phase I, the mouthwash A was given to one group and other group was given mouthwash B. After a 15 day washout period, in phase II, both groups were given other mouthwash. At the end of each phase of 1 week, plaque score was recorded by using Turesky modification of the Quigley-Hein plaque index.Results:The plaque scores were compared and the difference between the green tea catechin and chlorhexidine mouthwash was determined by t-test. The difference between plaque scores were not statistically significant (P > 0.05). The results showed that both the groups that is green tea catechin mouthwash (0.25%) and chlorhexidine mouthwash (0.12%) have comparable results in plaque reduction.Conclusion:This study supports the effectiveness of green tea catechin mouthwash as an antiplaque agent. It should be explored as a cost-effective, long-term antiplaque rinse with prophylactic benefits.

Electrochemical derivatization‐capillary electrophoresis‐contactless conductivity detection: A versatile strategy for simultaneous determination of cationic, anionic, and neutral analytes

The simultaneous determination of cationic, anionic, and neutral analytes in a real sample was demonstrated by coupling electrochemical (EC) derivatization with counter-EOF CE-C(4) D. An EC flow cell was used to oxidize alcohols from an antiseptic mouthwash sample into carboxylic acids at a platinum electrode in acid medium. The carboxylates formed in the derivatization process and other sample ingredients, such as benzoate, saccharinate, and sodium ions, were separated in counter-flow mode and detected in one run in Tris-HCl buffer, pH 8.6. Fewer than 5 min were needed to complete each analysis with the automated flow system comprising solenoid pumps for the management of solutions. Insights into the electrochemistry of benzoic acid, present in the sample matrix, were also gained by EC-CE-C(4) D; more specifically, by applying potentials higher than 1.47 V to the platinum electrode, some formiate and minute amounts of salicylate were detected.

Determination of antibiotics in oral hygiene products by high performance liquid chromatography-tandem mass spectrometry

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) method was developed for simultaneous determination of 13 antibiotics in oral hygiene products, including five tetracyclines, three macrolides, two quinolones, one beta-lactam and two lincosamides. The sample was extracted with 0.1% (volume percentage, same hereinafter) formic acid-acetonitrile (95:5, v/v), then centrifuged, filtered and diluted. The target compounds were separated on a C18 column (150 mm x 2.1 mm, 5 microm) with a gradient elution of 0. 1% formic acid and acetonitrile as the mobile phases, and detected by tandem mass spectrometry in positive electrospray ionization and multiple reaction monitoring (MRM) mode. The quantification of 13 antibiotics was performed by the external standard method. The calibration curves showed good linearity in the range of 5.0-50.0 microg/L with detection limits of 10.0 mg/kg. The recoveries of antibiotics in mouthwash and toothpaste samples at the three spiked levels of 10, 20 and 100 mg/kg were in the range of 80.1%-115% with the relative standard deviations in the range of 0.94%-8.69%. This method is accurate, reliable, simple, and suitable for the analysis of antibiotics in oral hygiene products.

Analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in human exfoliated oral mucosa cells by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Quantitation of DNA adducts could provide critical information on the relationship between exposure to tobacco smoke and cancer risk in smokers. In this study, we developed a robust and sensitive liquid chromatography-tandem mass spectrometry method for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in human oral cells, a noninvasive source of DNA for biomarker studies. Isolated DNA undergoes acid hydrolysis, after which samples are purified by solid-phase extraction and analyzed by LC-ESI-MS/MS. The developed method was applied to the analysis of samples obtained via collection with a commercial mouthwash from 30 smokers and 15 nonsmokers. In smokers, the levels of HPB-releasing DNA adducts averaged 12.0 pmol HPB/mg DNA (detected in 20 out of 28 samples with quantifiable DNA yield), and in nonsmokers, the levels of adducts averaged 0.23 pmol/mg DNA (detected in 3 out of 15 samples). For the 30 smoking subjects, matching buccal brushings were also analyzed, and HPB-releasing DNA adducts were detected in 24 out of 27 samples with quantifiable DNA yield, averaging 44.7 pmol HPB/mg DNA. The levels of adducts in buccal brushings correlated with those in mouthwash samples of smokers (R = 0.73, p < 0.0001). Potentially, the method can be applied in studies of individual susceptibility to tobacco-induced cancers in humans.

What happens if people start drinking mouthwash as surrogate alcohol? A quantitative risk assessment

Mouthwash ingestion has been observed in settings of restricted availability to alcoholic beverages such as in hospitals, prisons or military establishments. The literature offers limited evidence that ingredients of mouthwash may have health effects above the effects of ethanol. This study provides a quantitative risk assessment based on analysis of 30 mouthwash samples. All investigated brands contained alcohol, most of them menthol (93%), eucalyptol (90%), benzoic acid (87%), methyl salicylate (67%), and thymol (30%). For low risk drinking scenarios with average levels, only ethanol will exceed acceptable daily intakes (ADI). In worst case scenarios for alcohol dependent consumers ingesting 100g ethanol per day in the form of mouthwash, methyl salicylate will also exceeded the ADI by a factor of 17. The margin of exposure (MOE) for methyl salicylate, benzoates and thymol was below 100 for average scenarios, and below 10 for worst case scenarios, but ethanol is still the most toxic ingredient with MOE below 1. The occasional or even chronic ingestion of mouthwash may not cause health effects except the effects of ethanol alone. Only in extreme exposures, ingredients such as thymol or methyl salicylate could exacerbate the effects of ethanol, especially by contributing to metabolic acidosis.

The Adult Cystic Fibrosis Airway Microbiota Is Stable over Time and Infection Type, and Highly Resilient to Antibiotic Treatment of Exacerbations

Cystic fibrosis (CF) is characterized by defective mucociliary clearance and chronic airway infection by a complex microbiota. Infection, persistent inflammation and periodic episodes of acute pulmonary exacerbation contribute to an irreversible decline in CF lung function. While the factors leading to acute exacerbations are poorly understood, antibiotic treatment can temporarily resolve pulmonary symptoms and partially restore lung function. Previous studies indicated that exacerbations may be associated with changes in microbial densities and the acquisition of new microbial species. Given the complexity of the CF microbiota, we applied massively parallel pyrosequencing to identify changes in airway microbial community structure in 23 adult CF patients during acute pulmonary exacerbation, after antibiotic treatment and during periods of stable disease. Over 350,000 sequences were generated, representing nearly 170 distinct microbial taxa. Approximately 60% of sequences obtained were from the recognized CF pathogens Pseudomonas and Burkholderia, which were detected in largely non-overlapping patient subsets. In contrast, other taxa including Prevotella, Streptococcus, Rothia and Veillonella were abundant in nearly all patient samples. Although antibiotic treatment was associated with a small decrease in species richness, there was minimal change in overall microbial community structure. Furthermore, microbial community composition was highly similar in patients during an exacerbation and when clinically stable, suggesting that exacerbations may represent intrapulmonary spread of infection rather than a change in microbial community composition. Mouthwash samples, obtained from a subset of patients, showed a nearly identical distribution of taxa as expectorated sputum, indicating that aspiration may contribute to colonization of the lower airways. Finally, we observed a strong correlation between low species richness and poor lung function. Taken together, these results indicate that the adult CF lung microbiome is largely stable through periods of exacerbation and antibiotic treatment and that short-term compositional changes in the airway microbiota do not account for CF pulmonary exacerbations.

Rapid molecular detection of pulmonary tuberculosis in HIV-infected patients in Santiago, Chile

Santiago, Chile, has a mean annual tuberculosis (TB) rate of 13 per 100 000 population; however, TB incidence in human immunodeficiency virus (HIV) infected individuals is at least 20 times higher. To assess the accuracy of rapid molecular testing for pulmonary TB (PTB) detection in routine care in HIV-infected patients. Cross-sectional study, conducted prospectively in five hospitals between March 2010 and June 2011. HIV-positive subjects with suspected PTB provided sputum or mouth wash samples that were directly processed for acid-fast smear, mycobacterial cultures and Xpert® MTB/RIF. Positive test results were reported on the same day. We enrolled 166 subjects into the study; 50.6% provided two sputum samples, 33.1% only one sputum sample and 16.3% a mouth wash sample. The prevalence of TB was 8.1% (13/160). Diagnostic sensitivity increased from 66.7% (95%CI 39.1-86.2) for acid-fast smear to 91.7% (95%CI 64.6-98.5) for Xpert MTB/RIF, with comparable specificity at 98.6% (146/148, 95%CI 95.2-99.6) and 99.3% (147/148, 95%CI 96.3-99.9). Xpert MTB/RIF allowed early detection of rifampicin resistance in 16.6% of cases, with rapid adjustment to multidrug-resistant treatment. Xpert MTB/RIF provided earlier TB diagnosis in 25% more cases than acid-fast smear alone. Its implementation should be considered for TB diagnosis in HIV-positive patients even outside TB-endemic areas.

Potentiometric Study of Coated Graphite Electrode and Polymeric Membrane Electrode for the Determination of Sm3+ Ion

AbstractPreliminary studies on the two Schiff bases N2‐((3H‐indol‐3‐yl)methylene)‐6‐phenyl‐1,3,5‐triazine‐2,4‐diamine (L1) and N2,N4‐bis((3H‐indol‐3‐yl)methylene)‐6‐phenyl‐1,3,5‐triazine‐2,4‐diamine (L2) have shown that they can act as Sm3+ selective electrodes. The performances of a polymeric membrane electrode and a coated graphite electrode based on L2 were compared and the CGE proved to be better, as it shows a low detection limit of 1.8×10−8 mol L−1, a Nernstian slope of 19.6±0.4 mV decade−1 of activity with a response time of 11 s in the pH range of 3.0–9.0. The CGE was used to determine Sm3+ in medicinal plants and soil samples. It was also used to determine fluoride ions in mouthwash samples and in toothpaste.

Evaluation of the Effect of Green Tea Extract on Mouth Bacterial Activity in the Presence of Propylene Glycol

Compounds present in green tea have proved to inhibit the growth and activity of bacteria associated with infections. To assess the effects of green tea leaves extract in presence of propylene glycol on the aerobic mouth bacteria load. Saliva of 25 volunteer girl students aging 20-25 years were selected and evaluated by a mouthwash sample containing 1% tannin, as the most effective antibacterial complex in green tea. Comparative studies were also conducted between green tea mouthwashes containing 1% tannin and a similar sample with 10% propylene glycol added during extraction. This comparison was applied for a chlorhexidine 0.2% sample as a chemical mouthwash brand, too. There was a meaningful difference between the green tea mouthwashes containing 10% propylene glycol and the simple green tea extract (P < 0.05). Significant difference was also seen between the herbal and chemical mouthwashes (P < 0.05). The extract 1% tannin containing 10% propylene glycol reduced the aerobic mouth bacterial load of the student salvia about 64 percent. The pH monotonousness in different days and temperatures approved the stability of tannin in liquid water medium. Using green tea extract as a herbal mouthwash is safe and harmless specially for children and pregnant women. This result led us to suppose that green tea may prevent plaque formation on teeth, coming over halitosis due to mouth infection, too. These effects need to be approved in an in vivo trial as a second study.

Evaluation of the Effect of Green Tea Extract on Mouth Bacterial Activity in the Presence of Propylene Glycol

Background: Compounds present in green tea have proved to inhibit the growth and activity of bacteria associated with infections. Objectives: To assess the effects of green tea leaves extract in presence of propylene glycol on the aerobic mouth bacteria load. Materials and Methods: Saliva of 25 volunteer girl students aging 20-25 years were selected and evaluated by a mouthwash sample containing 1% tannin, as the most effective antibacterial complex in green tea. Comparative studies were also conducted between green tea mouthwashes containing 1% tannin and a similar sample with 10% propylene glycol added during extraction. This comparison was applied for a chlorhexidine 0.2% sample as a chemical mouthwash brand, too. Results: There was a meaningful difference between the green tea mouthwashes containing 10% propylene glycol and the simple green tea extract (P < 0.05). Significant difference was also seen between the herbal and chemical mouthwashes (P < 0.05). The extract 1% tannin containing 10% propylene glycol reduced the aerobic mouth bacterial load of the student salvia about 64 percent. The pH monotonousness in different days and temperatures approved the stability of tannin in liquid water medium. Conclusions: Using green tea extract as a herbal mouthwash is safe and harmless specially for children and pregnant women. This result led us to suppose that green tea may prevent plaque formation on teeth, coming over halitosis due to mouth infection, too. These effects need to be approved in an in vivo trial as a second study.

Theoretical and Experimental Investigation of a Fast Eu-PVC Sensor and Its Interaction With 4-Methylphenylisothiocyanate

A europium selective PVC membrane sensor based on 4-methylphenylisothiocyanate (4-MePIC) as a suitable neutral ionophore is introduced. The semiempirical PM6/SPARKLE calculations were performed to investigate the interaction between the 4-MePIC ligand, Eu(III) and interfering cations. The obtained results were used to predict the selectivity of the proposed sensor. The standard enthalpy of reaction (ΔHr°) has been used to predict the selectivity of the 4-MePIC ligand to a particular cation, except when the complex formation was controlled by the kinetic parameters. The computational data have been shown that the 4-MePIC ligand could be used as a suitable neutral ionophore in fabrication of a Eu(III) ion selective electrode. The nephelauxetic parameter (β) indicates a significant covalent character in the Eu(III)- ionophore complex. The proposed sensor has a very fast response time with a near Nernstian response of 19.7 ± 0.3 mV decade-1 for Eu(III) over a concentration range from 1 × 10-6 to 1 × 10-2 M with a detection limit of 1 × 10-6 M. It was applied for Eu(III) determination in various synthetic and mouthwash samples with satisfactory results.

Kinetics of DNA Adduct Formation in the Oral Cavity after Drinking Alcohol

Alcohol consumption is one of the top 10 risks for the worldwide burden of disease and an established cause of head and neck cancer, as well as cancer at other sites. Acetaldehyde, the major metabolite of ethanol, reacts with DNA to produce adducts, which are critical in the carcinogenic process and can serve as biomarkers of exposure and, possibly, of disease risk. Acetaldehyde associated with alcohol consumption is considered "carcinogenic to humans." We have previously developed the technology to quantify acetaldehyde-DNA adducts in human tissues, but there are no studies in the literature defining the formation and removal of acetaldehyde-DNA adducts in people who consumed alcohol. We investigated levels of N(2)-ethylidene-dGuo, the major DNA adduct of acetaldehyde, in DNA from human oral cells at several time points after consumption of increasing alcohol doses. Ten healthy nonsmokers were dosed once a week for three weeks. Mouthwash samples were collected before and at several time points after the dose. N(2)-Ethylidene-dGuo was measured as its NaBH(3)CN reduction product N(2)-ethyl-dGuo by liquid chromatography-electrospray-tandem mass spectrometry. N(2)-ethylidene-dGuo levels increased as much as 100-fold from baseline within 4 hours after each dose for all subjects and in a dose-responsive manner (P = 0.001). These results show an effect of alcohol on oral cell DNA adduct formation, strongly supporting the key role of acetaldehyde in head and neck cancer caused by alcohol drinking. Our results provide some of the first conclusive evidence linking exposure to a lifestyle carcinogen and kinetics of DNA adduct formation in humans.

Experimental and Semiempirical Investigation of Interaction Between Fast Sm Membrane Sensor and 1, 3-Di(Thiophene Imino) Benzoic Acid

This work describes, for the first time, the theoretical modeling of structure and interaction between Sm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> ion and 1, 3-di(thiophene imino)benzoic acid (DTBA) as a chelating reagent in the membrane and solution which were performed using the PM6/SPARKLE semiempirical method in the MOPAC program. Computational modeling results can be predicted behavior of Sm-DTBA interactions which was used as an ionophore for construction of samarium-selective potentiometric membrane sensor. The best performance was obtained with a membrane composition of 31% (w/w) PVC, 62% tributylphosphate (TBP), 4.8% DTBA and 3.2% sodium tetraphenyl borate (NaTBP). This sensor has a Nernstian slope of 19.6 mV decade for Sm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> ions in the range of 1.0 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-1</sup> - 5.0 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">6</sup> M and the detection limit was 3.1 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-6</sup> M. The response time of the sensor is very fast (≤ 5 s) and the potentiometric response is independent of the pH of the solution in the pH range of 4.5-8.5. This sensor shows good selectivity for Sm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> ion with respect to alkali, alkaline earth and heavy metal cations. The proposed sensor has been used for indirect determination of some drugs such as homatropine hydrobromide and hyoscine N-butylbromide in their pharmaceutical formulations and fluoride ions in mouthwash samples.

Abstract 4639: Effects of defined amounts of alcohol consumption on levels of N2-ethylidene-dGuo in DNA from human buccal cells and peripheral blood nucleated cells

Abstract Acetaldehyde, the primary metabolite of ethanol, is implicated in the carcinogenic effect of alcohol. This oxidized metabolite of ethanol reacts with DNA bases forming adducts, which play a key role in chemically induced carcinogenesis. N2-Ethylidenedeoxyguanosine (N2-Eti-dGuo) is the major adduct formed by this reaction. Several studies have shown an association between alcohol drinking and levels of this DNA adduct, suggesting its potential use as biomarker for the study of alcohol related carcinogenesis. However, there are no reports on the kinetics of formation and repair of N2-Eti-dGuo after alcohol consumption. Formation and lifetime of DNA adducts in humans is not always clear and can vary according to tissue or cell type. Therefore we investigated levels of N2-Eti-dGuo in DNA from peripheral blood monocytes, granulocytes, and buccal cells at several time points after consumption of different doses of alcohol. We recruited ten non-smokers, who regularly consume alcohol in moderation but with no history of alcohol abuse. Subjects participated to three drinking sessions targeting increasing blood alcohol levels: 0.03%, 0.05%, and 0.07%. Participants refrained from drinking for the duration of the study except for the administered alcohol dose. Mouthwash and blood samples were taken 1 h before the dose and 2, 4, 6, 24 h and 2, 5 and 7 days after. Blood samples were immediately processed to isolate granulocytes and monocytes using a Ficoll gradient. Efficiency of the cell separation was confirmed by flow-cytometry. DNA was extracted from these cells and from the buccal cells collected with the mouthwash. N2-Eti-dGuo was measured by LC-ESI-MS/MS. Preliminary results from the samples from the highest dose tested showed an average ten-fold increase of levels of N2-Eti-dGuo in buccal cell DNA 2 and 4 h after exposure, the levels of the adduct decreased after 24 h but showed a new two-fold increase, between 2 and 5 days from exposure. Levels of N2-Eti-dGuo in monocytes and granulocytes DNA did not increase within 6 h from the dose but only after 24 h from exposure. The results obtained from the granulocytes’ DNA showed an average three-fold increase after 24 h reaching a peak 2 or 5 days after the dose. The results obtained from the monocytes followed a similar trend but showed an average two-fold increase 24 h or 2 days after the dose. These results indicate a clear effect of alcohol on buccal cell DNA within 6 h after exposure and an unexpected increase of N2-Eti-dGuo beginning 24 h after alcohol consumption in monocytes and granulocytes. These unexpected results underline the importance of selecting the right surrogate tissue and time-point for the analysis of effects of alcohol on N2-Eti-dGuo and thus for further investigation of the use of this DNA adduct as a biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2011-4639

Experimental and PM6/SPARKLE Semiempirical Study of Interaction between 4‐Methoxyphenylcyanamide and Gadolinium(III) as a Fast Polymeric Membrane Sensor

AbstractIn this study, a highly sensitive PVC–membrane sensor based on 4‐methoxyphenylcyanamide (4‐MeOpcyd) as a neutral carrier was prepared and the specific interaction between Gd3+ ions and ionophore at the surface of selective polymeric membrane was calculated using the PM6/SPARKLE semiempirical method. The results of the Mopac2009 calculations show selective complexation of Gd3+ by 4‐MeOpcyd ligand that correlate quite well with experimental data. This membrane sensor has a linear response in the range of 1.0×10−6–1.0×10−2 M Gd3+ with detection limit of 6.2×10−7 M. This electrode was applied in potentiometric titration for determination of gadolinium and also for fluoride ions in the mouthwash samples with satisfactory results.

Maternal de novo triple mosaicism for two single OCRL nucleotide substitutions (c.1736A&gt;T, c.1736A&gt;G) in a Lowe syndrome family

Since the identification of the Lowe's oculocerebrorenal syndrome gene, more than 100 distinct OCRL mutations have been observed. Germline mosaicism has rarely been detected in Lowe families; however, the presence of mosaic mutations, in particular triple mosaicism, may often remain undiagnosed. In the course of OCRL analysis in a Polish family, the index case showed a hemizygous nucleotide transition (c.1736A>G, p.His507Arg). Gene analysis in the patient's mother not only provided evidence that she is a carrier of the mutant allele transmitted to her son but also showed an additional c.1736A>T (p.His507Leu) transversion affecting the same base position. DNA from a mouthwash sample from the mother showed a similar fluorescence intensity pattern at the affected nucleotide. These data, together with the findings that maternal grandparents solely showed wildtype sequence, implied a de novo mosaicism in the mother. Triple X syndrome was ruled out by karyotype analysis and a partial or complete gene duplication could be excluded. Allele-specific amplification confirmed the results of three alleles being present in the mother. The amount of wildtype allele detected in qPCR implied the presence of cells solely harboring c.1736A and single-cell PCR experiments confirmed the presence of non-mutant cells in the mother's blood. These data suggest that the mutations observed are the result of two de novo events in early embryogenesis of the mother. To the best of our knowledge, this is the first observation of triple mosaicism at a single nucleotide.

GSTM1 null and NAT2 slow acetylation genotypes, smoking intensity and bladder cancer risk: results from the New England bladder cancer study and NAT2 meta-analysis

Associations between bladder cancer risk and NAT2 and GSTM1 polymorphisms have emerged as some of the most consistent findings in the genetic epidemiology of common metabolic polymorphisms and cancer, but their interaction with tobacco use, intensity and duration remain unclear. In a New England population-based case-control study of urothelial carcinoma, we collected mouthwash samples from 1088 of 1171 cases (92.9%) and 1282 of 1418 controls (91.2%) for genotype analysis of GSTM1, GSTT1 and NAT2 polymorphisms. Odds ratios and 95% confidence intervals of bladder cancer among New England Bladder Cancer Study subjects with one or two inactive GSTM1 alleles (i.e. the 'null' genotype) were 1.26 (0.85-1.88) and 1.54 (1.05-2.25), respectively (P-trend = 0.008), compared with those with two active copies. GSTT1 inactive alleles were not associated with risk. NAT2 slow acetylation status was not associated with risk among never (1.04; 0.71-1.51), former (0.95; 0.75-1.20) or current smokers (1.33; 0.91-1.95); however, a relationship emerged when smoking intensity was evaluated. Among slow acetylators who ever smoked at least 40 cigarettes/day, risk was elevated among ever (1.82; 1.14-2.91, P-interaction = 0.07) and current heavy smokers (3.16; 1.22-8.19, P-interaction = 0.03) compared with rapid acetylators in each category; but was not observed at lower intensities. In contrast, the effect of GSTM1-null genotype was not greater among smokers, regardless of intensity. Meta-analysis of the NAT2 associations with bladder cancer showed a highly significant relationship. Findings from this large USA population-based study provided evidence that the NAT2 slow acetylation genotype interacts with tobacco smoking as a function of exposure intensity.

Pediatric Epstein‐Barr Virus Carriers With or Without Tonsillar Enlargement May Substantially Contribute to Spreading of the Virus

Human-to-human transmission of the persistent infection establishing Epstein-Barr virus (EBV) occurs via saliva. Tonsils act as important portal of entry and exit of EBV. The contagiousness of pediatric EBV carriers and the role played by tonsillar enlargement (TE) are not known. We compared EBV shedding in mouthwash samples from pediatric EBV carriers with or without TE to that in mouthwash samples from pediatric patients with infectious mononucleosis (IM), the symptomatic form of primary infection if delayed after the age of 5 years. EBV DNA was quantified by polymerase chain reaction, and contagiousness was assessed using the cord lymphocyte transformation assay. EBV carriers with TE shed EBV DNA at an almost similar frequency (although in lower amounts) as pediatric patients with acute IM but more frequently (P <.001) and in higher amounts (P = .038) than EBV carriers without TE. EBV DNA levels in mouthwash samples from EBV carriers with TE mirrored levels in tonsils and gradually declined after tonsillectomy. Almost half of the mouthwash samples from pediatric EBV carriers contained infectious EBV. Pediatric EBV carriers--in particular, those with TE-may considerably contribute to the spreading of EBV in industrialized countries.