Factors affecting SVI in small scale WWTPs.

This paper analyses factors associated with bulking in 195 small scale wastewater treatment plants (WWTPs) in Estonia. Operational data from each plant were collected and analysed statistically. The key factors associated with bulking were infiltration into sewage pipes, the type and purpose of process reactor, operational practices and influent characteristics. Both anaerobic fraction and volumetric fraction of the anaerobic reactor compared to the aerobic reactor resulted in a positive correlation with sludge volume index (SVI) <150 ml/g values. Good operation and maintenance practice as well as an operator's competence play a crucial role in bulking prevention. Using the 30 minute settling test (V30) as the single process control parameter can mislead an operator's judgement in process control strategies and cause effluent violations. Misjudgements in process control decisions can lead to unwanted conditions in small WWTPs (e.g. excessive chemical addition favoured bulking). Use of instrumentation, control and automation helped to keep the process conditions more stable and reduce the probability of bulking. Analyses of variance showed that the factors associated with Microthrix parvicella growth were long solids retention time (SRT), low food-to-microorganism ratio (F/M) and lack of carbon content compared against nitrogen and phosphorus contents.

Treatment of high-strength wastewater by Fe(2+)-activated persulphate and hydrogen peroxide.

Ferrous ion-activated persulphate and hydrogen peroxide were studied for the treatment of real high-strength industrial effluent. The Fenton process demonstrated greater organic load removal, biodegradability improvement and toxicity reduction as well as lower treatment cost than the activated persulphate system. However, the use of an activated persulphate process was more favourable due to the exothermic effect intrinsic to the Fenton reaction, which resulted in a rapid increase in the temperature of the high-strength wastewater along with excessive foam formation. Overall, for the H2O2/Fe(2+) and [Formula: see text] processes, the application of a chemical oxygen demand (COD)/oxidant/Fe(2+) weight ratio of 1/0.4/0.075 resulted in a COD removal of 58 and 50%, a 7-day biochemical oxygen demand/COD ratio increase from 0.14 to 0.3 and 0.23, and an increase in the EC50 (Daphnia magna) by 6.5-fold and 2.9-fold, respectively. The stepwise addition of the oxidant and activator was favourable for the Fenton process and resulted in negligible improvement in the wastewater treatment efficacy in the activated persulphate system.

Achieving nitritation and anammox enrichment in a single moving-bed biofilm reactor treating reject water.

A biofilm with high nitrifying efficiency was converted into a nitritating and thereafter a nitritating-anammox biofilm in a moving-bed biofilm reactor at 26.5 (+/- 0.5) degrees C by means of a combination of intermittent aeration, low dissolved oxygen concentration, low hydraulic retention time, free ammonia and furthermore, also by elevated HCO3- concentration. Nitrite-oxidizing bacteria (NOB) were more effectively suppressed by an enhanced HCO3- concentration range of 1200-2350 mg/L as opposed to free-ammonia-based process control where NOBs recovered from inhibition; the respective total-nitrogen removal rates were 0.3 kg N/(m3 x d) and 0.2 kg N/(m3 x d). The biofilm modification strategies resulted in a shift in bacterial community as the NOB Nitrobacter spp. were replaced with NOB belonging to the genus Nitrospira spp. and were closely related to Candidatus Nitrospira defluvii. A community of anaerobic ammonium-oxidizing microorganisms -uncultured Planctomycetales bacterium clone P4 (closely related to Candidatus Brocadia fulgida)--was developed.

Polycyclic aromatic hydrocarbons (PAHs) in meat products and estimated PAH intake by children and the general population in Estonia.

The concentrations of benzo[a]pyrene and 11 other polycyclic aromatic hydrocarbons (PAHs) were analysed from 322 commercial, cured meat products and 14 home-grilled meat samples as part of the Estonian food safety monitoring programme during 2001-2005. The maximum acceptable concentration of 5 microg kg(-1) for benzo[a]pyrene was exceeded in 3.4% of samples. The highest PAH concentrations were detected in home-grilled pork samples. Using of disposable grilling unit resulted in 1.6 times higher PAH concentrations compared to the traditional wood-burning grill. The average intake of benzo[a]pyrene and sum of 12 PAHs from meat products was estimated for children (age 1-16 years) on the basis of an individual food consumption questionnaire and, for the general population, based on national food consumption data. The highest total PAH concentrations detected were 16 microg kg(-1) in smoked meat and ham, 19 microg kg(-1) in smoked sausage and 6.5 microg kg(-1) in smoked chicken samples. Since smoking and grilling are prevalent meat-cooking methods in Estonia, the impact of meat products is assessed to be significant in overall PAH intake.

Distance determination in human ubiquitin by pulsed double electron-electron resonance and double quantum coherence ESR methods.

Recently, distance measurements by pulsed ESR (electron spin resonance) have been obtained using pulsed DEER (double electron-electron resonance) and DQC (double quantum coherence) in SDSL (site directed spin labeling) proteins. These methods can observe long range dipole interactions (15-80A). We applied these methods to human ubiquitin proteins. The distance between the 20th and the 35th cysteine was estimated in doubly spin labeled human ubiquitin. Pulsed DEER requires two microwave sources. However, a phase cycle is not usually required in this method. On the other hand, DQC-ESR at X-band ( approximately 9GHz) can acquire a large echo signal by using pulses of short duration and high power, but this method has an ESEEM (electron spin echo envelope modulation) problem. We used a commercial pulsed ESR spectrometer and compared these two methods.

Nitrates and nitrites in vegetables and vegetable-based products and their intakes by the Estonian population.

The content of nitrates were determined in 1,349 samples of vegetables and ready-made food in 2003-2004 as a part of the Estonian food safety monitoring programme and the Estonian Science Foundation grant research activities. The results of manufacturers' analyses carried out for internal monitoring were included in the study. The highest mean values of nitrates were detected in dill, spinach, lettuce and beet root. The mean concentrations were 2,936, 2,508, 2,167 and 1,446 mg kg(-1), respectively. The content of nitrites in samples was lower than 5 mg kg(-1). In total, the mean intake of nitrates by the Estonian population was 58 mg day(-1). The mean content of nitrates in vegetable-based infant foods of Estonian origin was 88 mg kg(-1). The average daily intake of nitrates by children in the age group of 4-6 years was 30 mg. The infants' average daily intake of nitrates from consumption of vegetable-based foods was 7.8 mg.

Nitrites, nitrates and N-nitrosoamines in Estonian cured meat products: intake by Estonian children and adolescents.

The contents of nitrate, nitrite and N-nitrosoamines in commercial cured meat products on the Estonian market were determined for 2000-01 and 2003-04 as part of the Estonian food safety monitoring programme and the Estonian Science Foundation grant research activities. The maximum permitted levels of residual nitrites and nitrates were not exceeded in the samples analysed. However, a great variation in the content of nitrate, nitrite and N-nitrosoamines was found for all the products. The concentrations of these compounds in domestic cured meat products showed a decrease from year to year. The mean intake of nitrate, nitrite and N-nitrosoamines by Estonian children (n=346) from cured meat products was calculated on the basis of individual intake data. The mean daily intake of nitrates was 1.7 mg, that of nitrites was 0.83 mg and that of N-nitrosoamines was 0.073 microg. In the 2000-01 study, the calculated nitrite intake exceeded the acceptable daily intake by up to 140% for 1-6-year-old children and up to 105% in 2003-04.

Monocytes, but not macrophages, produce the eosinophil cationic protein.

The eosinophil cationic protein (ECP) is a cytotoxic protein with ribonuclease activity, produced and stored in bone marrow eosinophil myelocytes. Mature circulating eosinophils contain about 10 pg ECP per cell. The aim of this study was to investigate the possibility that monocytes produce and store ECP. By results from flow cytometry and specific protein measurement it is shown that human monocytes contain ECP (monocytes about 10 fg ECP per cell). RT-PCR analysis indicated the presence of mRNA coding for ECP in blood monocytes but not in alveolar macrophages. Furthermore, mRNA coding for ECP and low amounts of the protein were found in three myeloid cell lines representing different stages of monocytic differentiation. Differentiation of U-937 cells to macrophages induced lowered transcription of the ECP gene and reduced protein production. Immunohistochemical staining of lung tissue indicated that lung macrophages do not contain ECP. It is concluded that ECP is produced to a low extent by human monocytes and that the production is shut down during macrophage differentiation. This might indicate an alternative transcriptional regulation of the ECP gene in the monocytic lineage compared to the eosinophil lineage.

Estimation of biodegradation parameters of phenolic compounds on activated sludge by respirometry.

The bio-oxidation of phenol, catechol, resorcinol, m-cresol and 5-methylresorcinol on activated sludge was investigated by oxygen uptake measurements. In addition, the degradation of acetate with the same microbial population was studied. The substrate-dependent oxygen uptake data were analysed on the basis of the Michaelis-Menten kinetics. The extant kinetic parameters, the maximum rates of oxygen consumption and half-saturation constants for the processes with different substrates were determined. The simple respirometric approach also made it possible to determine the short-term oxygen demands of the substrates which formed 23-38% of the theoretical oxygen demand of the studied compounds.

Dynamic model of amperometric biosensors. Characterisation of glucose biosensor output.

An integrated model for the characterisation of the output signal course of oxidase-bound amperometric biosensors is presented and evaluated in the case of glucose biosensors. This model integrates two earlier proposed models, the model of oxygen transducer-based biosensors, allowing the prediction of steady state parameters from the transient response and the dynamic signal lag model, characterising the electrochemical diffusion-limited sensors. The integrated model allows the characterisation of the whole biosensor signal output, originating from the output curve itself with errors less than 3% and no need to determine the system's geometrical, diffusion and partition parameters.

Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression.

Activated platelets can express CD40 ligand (CD40L) and trigger inflammatory response and tissue factor (TF) expression in endothelial cells through interaction with CD40. This pathway is also important for T cell-induced monocyte and endothelial cell procoagulant activity. We have studied the potential role of the CD40-CD40L pathway in platelet-induced TF expression in a monocytic cell line and in whole-blood monocytes. In vitamin D(3)-differentiated U-937 cells, thrombin-stimulated platelets increased TF expression as measured by mRNA quantification, flow cytometry, and procoagulant activity. Maximum antigen expression occurred after 2 hours. Neutralizing anti-P-selectin antibody yielded a 50% suppression of procoagulant activity, whereas antibody to CD40L had no effect. In thrombin receptor activator-stimulated citrated blood, monocytes were up to 77% TF-positive, with peak expression after only 15 minutes. However, no TF mRNA was detectable at that time. Anti-P-selectin antibody reduced TF by 50%, whereas antibody to CD40L gave a 17% reduction. Thus, we conclude that P-selectin exposed on activated platelets induces the expression of TF in both U-937 cells and whole-blood monocytes but by different mechanisms. Platelet CD40L does not display any significant effect on U-937 cells but may be of some importance on whole-blood monocytes. This suggests a possible functional difference between U-937 and monocyte CD40. Another important finding in this study is the rapid appearance of surface TF on monocytes without detectable mRNA formation. This indicates that TF may be stored intracellularly in these cells and can be exposed on the surface independent of de novo protein synthesis.

The role of RAR and RXR activation in retinoid-induced tissue factor suppression.

Excessive expression of tissue factor (TF) is a common finding in leukaemic cells and may contribute to thrombotic complications in patients. Retinoic acid has been shown to induce differentiation and reduce TF expression in acute promyelocytic leukaemia (APL) cells in vitro, and to induce remission in APL patients. Treatment of the APL cell line NB4 with the specific retinoic acid receptor-alpha (RARalpha) agonists Ro4-6055 or TTNPB resulted in down-regulation of TF expression and in induction of differentiation. The activation of RARbeta, RARgamma or retinoid X receptor (RXR) did not suppress the constitutive TF expression in NB4 cells. Moreover, the RARalpha antagonist Ro41-5253 blocked the retinoid-induced down-regulation of TF. In contrast, in the monoblastic U-937 cell line only a partial suppression of TF antigen expression and activity was observed by treatment with the RAR agonist TTNPB or the RXR agonist SR11237 alone. However, the combination of TTNPB and SR11237 resulted in a pronounced down-regulation of TF expression and induction of differentiation in U-937 cells. We show for the first time that the activation of both subunits of the RARalpha-RXR transcriptional complex is needed for TF suppression in U-937 cells, whereas in NB4 cells RARalpha activation alone is sufficient. Thus, distinct molecular mechanisms for TF suppression seem to be operating in leukaemic cell lines of different origin.

Induction of differentiation in U-937 and NB4 cells is associated with inhibition of tissue factor production.

Tissue factor (TF) production is under strict control in mature monocytic cells. However, constitutive expression of TF can be found in myelomonocytic cells and in haematopoietic cells arrested at an early stage of differentiation. In this paper we show that TF expression is down-regulated during the monocyte/granulocyte differentiation process, using the human monoblastic U-937 and the acute promyelocytic leukaemia NB4 cell lines as models. Expression of TF mRNA, protein and procoagulant activity (PCA) was constitutively high in untreated cells. Exposure of U-937 cells to 1alpha,25-dihydroxycholecalciferol (VitD3) and all-trans retinoic acid (ATRA) resulted in down-regulation of TF expression and PCA. In NB4 cells induction by ATRA, but not VitD3, resulted in the down-regulation of TF expression and PCA. Consistent with this, induction of terminal differentiation, as confirmed by the expression of differentiation associated antigens and cell cycle arrest, was inversely correlated to TF expression in U-937 and NB4 cells. Moreover, terminally differentiated U-937 cells retained the capacity to respond to inflammatory mediators, i.e. lipopolysaccharide and interferon-gamma, by a rapid increase in TF expression. In conclusion, we show that not only ATRA but also VitD3 is a potent suppressor of monocytic TF expression and thus might have potential clinical use for the treatment of coagulopathies.

Superoxide electrode based on covalently immobilized cytochrome c: modelling studies.

We have recently described an optimised electrode for the detection of enzymatic and cellular superoxide (O2*-) production based on cytochrome c immobilized covalently at a surface-modified gold electrode and applied this system to the study of free radical production by activated human glioblastoma cells. In this paper we report the development of a mathematical model for the O2*- electrode responding to enzymically produced O2*- which should enable the determination of absolute concentrations of O2*- in biological systems when this electrode is employed for direct, real-time monitoring of free radical release and interactions.

Calibration of glucose biosensors by using pre-steady state kinetic data.

A new method for biosensor calibration and data processing, allowing the prediction of steady state parameters from the analysis of transient response curves (Rinken et al., 1996. Analytical Letters 29, 859), has been evaluated in the case of an oxygen sensor based two-substrate enzyme electrode for glucose determination. The electrochemical glucose biosensor was prepared by covering the surface of oxygen sensor with glucose oxidase (EC 1.1.3.4) immobilized in nylon mesh. This decreased the oxygen flow to the sensor in the presence of glucose and resulted in time-dependent decrease of the biosensor signal. Except the lag period of the response in the beginning of the assay, the oxygen consumption by the immobilized enzyme was described by an exponential function: [formula: see text] The parameter C, which corresponded to the steady-state output of the biosensor, was found to be the most suitable for glucose determination. The non-linear fitting for data of over 1000 independent experiments to the equation above always revealed correlation coefficients greater than 0.97. The calculation of the steady state parameter from the transient phase data makes the analysis fast and precise, especially for sensors with thick membranes, being convenient to use in the case of enzyme electrodes. The theoretical essence of the parameter C also gives valuable information for the optimal design of biosensors.

IL-10 inhibits LPS-induced human monocyte tissue factor expression in whole blood.

Interleukin 4 (IL-4), IL-10 and IL-13 are all known to modulate several proinflammatory functions in human monocytes. They have also previously been shown to down-regulate lipopolysaccharide (LPS)-induced tissue factor (TF) expression in isolated cultured monocytes. In this study we investigated the effect of these three cytokines on the induction of monocytic TF in a whole blood environment at three levels: mRNA quantitation, surface antigen expression and procoagulant activity. We showed that IL-10 attenuated LPS-induced monocyte TF expression and activity in whole blood in a concentration-dependent manner, both when added to the blood prior to LPS and, although to a lesser extent, when added up to 1 h subsequent to LPS challenge. Maximum inhibition occurred at 5 ng/ml of IL-10 when the cytokine was added before LPS. IL-4 and IL-13, however, did not exhibit any inhibitory effect in the whole blood environment, contrary to the reported findings in cell culture experiments. Our results confirm the potential of IL-10 as an anti-inflammatory, TF-preventing drug, whereas the effects of IL-4 and IL-13 on monocytes in whole blood seem more complex, and require further investigation.

Tissue factor expression in human monocytic cell lines.

Tissue factor (TF) is a main initiator of the coagulation protease cascade. Control of the expression of this protein in monocytes is essential, since these cells are the only circulating blood cells responsible for TF expression. In this report we have used two human cell lines, arrested at different stages of monocytic differentiation, to study TF expression. The monoblastic cell line U-937 had a constitutive expression of TF surface protein and low TF mRNA levels detected by immunofluorescence or quantitative reverse transcriptase polymerase chain reaction respectively. The phorbol-12-myristate-13-acetate (PMA) was a potent enhancer of TF expression in U-937. Lipopolysaccharide (LPS) and tumor necrosis factor (TNF) had no effect on TF expression in U-937. The Mono Mac 6 cell line, with phenotypic features similar to that of mature monocytes, expressed lower basal levels of TF mRNA and surface TF antigen. However, in Mono Mac 6 cells TF expression was induced in response to LPS and TNF. These results indicate differences in basal and induced TF expression between U-937 and Mono Mac 6 cell lines.

Inhibition of tissue factor surface expression in human peripheral blood monocytes exposed to cytokines.

Interleukin (IL)-4, IL-10, IL-13 and transforming growth factor beta (TGF-beta) are known to regulate several monocyte functions, including inhibition of the synthesis of different cytokines. Using quantitative RT-PCR and flow cytometry analysis we investigated the effects of these cytokines on bacterial lipopolysaccharide (LPS)-induced tissue factor (TF) expression in human monocytes. The effects of IL-4 and IL-10 on monocyte chemoattractant protein-1 (MCP-1)-and C-reactive protein (CRP)-induced TF expression were also studied. A direct comparison revealed that IL-4, IL-10 and IL-13 all down-regulated LPS-induced TF expression in a concentration-dependent manner without the need for priming. In contrast, TGF-beta required 4 h of priming to inhibit TF expression induced by LPS. IL-10 was the most powerful inhibitor, causing almost complete inhibition at 5 ng/ml. IL-4 and IL-13 exhibited a significantly lower inhibitory capacity even at concentrations of 100 ng/ml. IL-4 and IL-10 showed similar concentration-dependent inhibition of MCP-1- and CRP-induced TF expression. We also showed that the regulatory effect of the interleukins occurred at the mRNA level. In vivo, these inhibitory cytokines may play an important regulatory role in preventing thrombosis. IL-10, in particular, may be a possible candidate as a TF-preventing drug.