Retronasal testing of olfactory function: an investigation and comparison in seven countries.

Flavor perception is to a large extent determined by olfaction, and persons who lost their sense of smell consequently complain about strongly reduced enjoyment of food. The retronasal olfactory function is especially important for flavor appreciation. The aim of this study was to compare retronasal function across different cultures and to develop a test that is applicable across cultures. Identification of 39 retronasal applied odor probes was tested in a total of 518 participants of seven countries; 292 of them were healthy, and 226 exhibited a smell disorder. A retest was performed with 224 of the healthy participants. Furthermore, all participants were tested for orthonasal threshold, identification, and discrimination ability. Significant cultural differences in identification ability were found in 92% of the probes. The 20 probes that could be identified above chance in healthy participants of all countries and that could differentiate between patients and controls were selected for the final retronasal test. This test was well able to differentiate between controls and patients in different countries and showed a good coherence with the orthonasal test (r = 0.80) and a good retest-reliability (r = 0.76). Furthermore, it is age-independent. The strong cultural differences observed in retronasal identification underline the necessity to develop a culturally independent instrument. This retronasal test is easy to apply and can be used across different countries for diagnostics and clinical research.

The role of microorganisms in a full-scale sequencing batch reactor under low aeration and different cycle times.

This study describes the role of microorganisms in a full-scale step-feed sequencing batch reactor (SBR) system for urban wastewater treatment. Chemical profiles for three different cycle times were measured under low aeration conditions with a high carbon-to-nitrogen ratio. The applied organic load was above 1.0 g chemical oxygen demand (COD)/L x d. The removal efficiencies were higher than 81%, 93%, and 76% for soluble COD, N-NH4+, and total Kjeldahl nitrogen, respectively. The ratio of volatile suspended solids (VSS) to total suspended solids was 78%, and the food-to-microorganism ratio was an average of 1.41 g COD/g VSS x d. The active biomass was comprised of 87.8% heterotrophic and 12.2% autotrophic organisms. Nitrifying organisms were found with a low amount of ammonia-oxidizing bacteria (5%) and a much higher amount of nitrite-oxidizing bacteria. Polyphosphate-accumulating organisms (PAOs) were found at high amounts (25%) compared to glycogen-accumulating organisms, even in a system with a high carbon to phosphorus ratio. The activity of denitrifying PAOs was 72%.

Genotoxic effects of exposure to radiofrequency electromagnetic fields (RF-EMF) in cultured mammalian cells are not independently reproducible.

Conflicting results have been published regarding the induction of genotoxic effects by exposure to radiofrequency electromagnetic fields (RF-EMF). Using the comet assay, the micronucleus test and the chromosome aberration test with human fibroblasts (ES1 cells), the EU-funded "REFLEX" project (Risk Evaluation of Potential Environmental Hazards From Low Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods) reported clearly positive effects for various exposure conditions. Because of the ongoing discussion on the biological significance of the effects observed, it was the aim of the present study to independently repeat the results using the same cells, the same equipment and the same exposure conditions. We therefore exposed ES1 cells to RF-EMF (1800 MHz; SAR 2 W/kg, continuous wave with intermittent exposure) for different time periods and then performed the alkaline (pH>13) comet assay and the micronucleus test (MNT). For both tests, clearly negative results were obtained in independently repeated experiments. We also performed these experiments with V79 cells, a sensitive Chinese hamster cell line that is frequently used in genotoxicity testing, and also did not measure any genotoxic effect in the comet assay and the MNT. Appropriate measures of quality control were considered to exclude variations in the test performance, failure of the RF-EMF exposure or an evaluation bias. The reasons for the difference between the results reported by the REFLEX project and our experiments remain unclear.

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test.

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.

Enhancement of genotoxic effects in the comet assay with human blood samples by aphidicolin.

The comet assay (single cell gel electrophoresis) has become increasingly used in human biomonitoring. In its standard version at pH > 13, DNA lesions such as DNA double-strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. Besides DNA damage, strand break formation during excision repair can also increase DNA migration. Inhibitors of DNA repair have been shown to enhance the DNA effects of mutagens and the use of repair inhibitors has been proposed for human biomonitoring studies to increase the sensitivity of the comet assay. To further evaluate the usefulness of such an approach we performed an experimental study with human blood and tested the enhancing effect of aphidicolin (APC) on DNA effects induced by different mutagens. Our results clearly show that APC enhances the genotoxic effects of benzo[a]pyrene diolepoxide (BPDE), bischloroethylnitrosurea (BCNU) and methyl methanesulfonate (MMS), but has no significant effect on gamma radiation-induced DNA effects. The enhancing effect is seen in unstimulated and PHA-stimulated blood, indicating repair activity under both conditions but the effect is stronger in stimulated blood. Our results indicate that APC can be used to increase the sensitivity of the comet assay towards a broad spectrum of induced primary DNA lesions and support the usefulness of this approach. However, for human biomonitoring, a sensitive protocol still has to be established.

Sensitivity of the FPG protein towards alkylation damage in the comet assay.

The comet assay (single cell gel electrophoresis) is widely used for the evaluation of DNA-damaging effects in genotoxicity testing and population monitoring. In its standard version at pH >13, DNA double strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. At reduced pH (12.5-12.1) the expression of ALS as SSB can be eliminated and the effect of SSB only can be identified. Specific endonucleases have been used to characterize specific classes of DNA damage. The formamido pyrimidine glycosylase (FPG) protein has been used to assess oxidative DNA base damage because it detects 8-OH guanine and other oxidatively damaged purines. Here, we show that the FPG protein also detects alkylation damage with high sensitivity in the comet assay. Human whole blood, isolated lymphocytes and V79 cells were treated with alkylating agents and post-incubated with FPG. FPG strongly enhanced MMS- and EMS-induced DNA damage but had no significant effect on ENU-induced DNA damage, indicating that the amount of N-7 guanine alkylation is responsible for the observed effect. Reducing the pH during alkali unwinding and electrophoresis to 12.5 to avoid the contribution of ALS to the comet assay effects, strongly decreased the sensitivity of the comet assay with and without FPG treatment and prevented DNA migration. We conclude that enhanced DNA effects in the comet assay by FPG after exposure to genotoxins with unknown mode of action should not directly be regarded as evidence for the presence of oxidative damage. Furthermore, reducing the pH leads to a considerable loss in sensitivity and should not be used in biomonitoring and other applications which require a sensitive protocol.

Investigations on the effect of cigarette smoking in the comet assay.

The comet assay (single-cell gel electrophoresis, SCG) is widely accepted as an in vitro and in vivo genotoxicity test. Because of its demonstrated ability to detect various kinds of DNA damage and its ease of application, the technique is being increasingly used in human biomonitoring. However, the assessment of small genotoxic effects as typically obtained in biomonitoring may be limited by the different sources of assay variability and the lack of an optimal protocol with high sensitivity. To better characterize the suitability of the comet assay for biomonitoring, we are performing a comprehensive investigation on blood samples from smokers and non-smokers. Because tobacco smoke is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds, smokers should be a suitable study group with relevant mutagen exposure. Here, we report our results for the first sample of 20 healthy male smokers and 20 healthy male non-smokers. Baseline and benzo[a]pyrene diolepoxide (BPDE)-induced effects were analysed by two investigators using two image analysis systems. The study was repeated within 4 months. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline and BPDE-induced DNA damage was comparatively analysed. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. None of these approaches revealed significant differences between smokers and non-smokers. Although more data is needed for a final conclusion, this study indicates some limitations of the comet assay with regard to the detection of DNA damage induced by environmental mutagens in peripheral blood cells.

Full-scale sequencing batch reactor (SBR) for domestic wastewater: performance and diversity of microbial communities.

This work describes the performance and microbial diversity in a sequencing batch reactor of a decentralized full-scale system for urban wastewater treatment under limited aeration. The removal efficiency was: 83% for soluble chemical oxygen demand (SCOD), 60% for N-NH4(+), 70% for total suspended solids (TSS) and 80% for volatile suspended solids (VSS). The biomass concentration had a maximum value around 8.7gVSSL(-1) for organic load rate of 0.6gCODL(-1)d(-1). The food/microorganism ratios showed average of 0.2gCOD/gVSSd. The sludge bacterial flocs were formed an irregular arrangement with organisms attached such as Euglypha sp. and pedunculate ciliates. It was observed the presence of Bacteria domains including Nitrosomonas spp., Nitrobacter spp., Nitrospira and C. "Accumulibacter" cluster. The DPAO activity was 70%. Denaturing gradient gel electrophoresis showed changes in ribotype number over biological treatment time among the groups observed being some are linked to nutrient removal. The reactor showed viability to treat domestic wastewater.

Femtosecond lentotomy: generating gliding planes inside the crystalline lens to regain accommodation ability.

Based on Helmholtz Theory for accommodation the increasing sclerosis of lens nucleus and cortex is the main cause for the developments of presbyopia. Existing therapies, however, do not reverse the stiffness of the crystalline lens and thus do not regain real accommodation ability. A new approach to restore the flexibility of the lens could be realized by photodisruption using ultrafast laser pulses. This process, known as fs-lentotomy, was used to create micro-incisions which act as gliding planes inside the crystalline lens without opening the eye globe.

The effect of smoking on DNA effects in the comet assay: a meta-analysis.

The comet assay (alkaline single-cell gel electrophoresis, SCG or SCGE) is frequently used in biomonitoring to detect genotoxic effects in humans exposed at the workplace or in their environment. Because of its ready accessibility, blood is most frequently used in such studies. Many studies investigated cigarette smoking either as a genotoxic exposure itself or as a potential confounding factor in occupational studies. However, although smoking is considered to be a relevant exposure towards various genotoxins, conflicting results have been reported in the comet assay studies. The actual reasons for this discrepancy are not known. To further evaluate evidence for smoking-related DNA effects in the comet assay, we now used a meta-analysis approach based on a literature search. We identified 38 studies from 37 publications which were suited for a formal meta-analysis based on the standardized mean difference (SMD) between the study groups. The evaluation of these 38 studies indicated higher levels of DNA damage in smokers than in non-smokers [under a random effects model, SMD = 0.55, 95% confidence interval = (0.16-0.93)]. Subdividing these studies into studies investigating the effect of smoking as a genotoxic exposure (Type A studies, n = 12) and studies investigating smoking as a potential confounder in occupational studies (Type B, n = 26) indicated a significant difference only in Type A studies but not in Type B studies. Furthermore, studies using image analysis or image length measurements (n = 23) only indicated a tendency for a genotoxic effect of smoking, whereas studies using an arbitrary score (n = 15) found a significantly higher level of DNA damage in smokers.

Genetic polymorphisms and the effect of cigarette smoking in the comet assay.

A potential genotoxic effect of cigarette smoking has repeatedly been investigated with the comet assay (alkaline single cell gel electrophoresis) and conflicting results have been reported. Besides differences in the methodology and the study design used, genetic differences between the subjects investigated might contribute to the variability of test results. Considering genetic polymorphisms of genes involved in metabolism or DNA repair has led to a better discrimination of smoking-related genotoxic effects in some cases but also led to discrepant results. We therefore evaluated our baseline comet assay effects obtained for nonsmokers and smokers in relation to selected genetic polymorphisms. Our study group comprised 52 nonsmokers and 51 smokers who were strictly selected to exclude potential confounding factors. We chose polymorphisms in the genes GSTM1 and CYP1A1 (Ile462Val) because they take part in the metabolism of genotoxins contained in tobacco smoke. In a subgroup of 32 nonsmokers and 31 smokers we also studied polymorphisms in XPD (Lys751Gln), XRCC1 (Arg399Gln) and XRCC3 (Thr241Val) because they are part of DNA repair pathways involved in the repair of tobacco-related DNA damage. Freshly collected peripheral whole blood samples were tested in the alkaline (pH > 13) comet assay. In all experiments a reference standard (untreated V79 cells) was included to correct for assay variability. An independent second evaluation was carried out for all experiments. None of these approaches revealed a significant difference between nonsmokers and smokers.

The potential of denitrification for the stabilization of activated sludge processes affected by low alkalinity problems

In this study, the problems provoked by nitrification of wastewater with low alkalinity were analyzed in a pilot sequencing batch activated sludge reactor (SBR). Decrease in pH resulted in disappearence of protozoa. De-flocculation of the activated sludge floc started below pH 6.5, resulting in enhanced effluent turbidity and loss of bacteria. Nitrification efficiency was affected below pH 6.2. The denitrification activity was not sufficient to keep up the pH, due to a low C/N ratio of the wastewater. Based on alkalinity and ammonia concentration of the wastewater and the necessary denitrification rate to prevent operational problems, was developed a prognostic diagram. The applicability of this diagram was tested for the SBR with excellent results. The diagram could be applied to optimize the operation of wastewater treatment plants affected by problems with low alkalinity wastewater.

Os problemas provocados pela nitrificação no esgoto com baixa alcalinidade foram analisados num reator piloto do tipo lodos ativados seqüencial por batelada (RSB), alimentado por esgoto urbano. A diminuição do pH se mostrou em três níveis: com pH de 6,8 - 6,0 os protozoários, responsáveis para a filtração da fase liquida, desaparecerem; os flocos de lodos ativados começaram a se destruir abaixo pH 6,5 resultando em elevação da turbidez no efluente final e abaixo de pH 6,2-6,0 a nitrificação foi afetada. A influência da desnitrificação para manter o pH foi analisada. Devido a baixa relação C:N no esgoto pré-tratado, a desnitrificação não se mostrou suficiente para manter o pH estável. Este trabalho apresenta o cálculo da alcalinidade que considera a influência da nitrificação e desnitrificação, de acordo com os resultados obtidos no RSB. Baseado nesse cálculo, foi desenvolvida uma recomendação na forma gráfica para usar em ETE´s afetadas por baixa alcalinidade.