Behavior of Glyphosate in Wastewater Treatment Plants.

The herbicide glyphosate is frequently detected in surface waters and its occurrence is linked to agricultural as well as urban uses. Elevated concentrations downstream of wastewater treatment plants (WWTPs) suggest that municipal wastewater is an important source of glyphosate in surface waters. We therefore conducted a study at a typical municipal WWTP in Switzerland to characterize the seasonality of glyphosate occurrence, the removal efficiency, and the processes involved in glyphosate removal. Glyphosate was present in raw (mechanically treated) wastewater during the whole study period (April to November). A lab incubation experiment with activated sludge indicated negligible degradation of glyphosate. Lack of degradation combined with strong adsorption lead to substantial enrichment of the compound in the sludge. Due to this enrichment and the long residence time of activated sludge (several days, compared to hours for wastewater itself), concentrations in treated wastewater show comparatively little variation, whereas concentrations in raw wastewater may fluctuate considerably. Overall removal efficiencies were in the range of 71-96%. This behavior could be described qualitatively using a numerical model that included input of glyphosate via raw wastewater, adsorption to activated sludge, and export via treated wastewater and excess sludge, but excluded degradation processes.

Detection of koala retrovirus subgroup B (KoRV-B) in animals housed at European zoos.

Many koalas carry an endogenous retrovirus, KoRV-A, in their genome. Recently, a second retrovirus, KoRV-B, was detected in koalas in Japanese and U.S. zoos. However, this virus is not endogenous, differs in the receptor binding site of the surface envelope protein, and uses a receptor different from that of KoRV-A. We describe here a KoRV-B found in koalas at zoos in Germany and Belgium that differs slightly from that found in the Los Angeles zoo.

A new Western blot assay for the detection of porcine cytomegalovirus (PCMV).

Porcine cytomegalovirus (PCMV) may be harmful for human recipients if xenotransplantation using pig cell, tissue or organ will be performed transmitting the virus from donor pigs to human recipients. PCMV is widespread in pigs and closely related to human pathogenic herpesviruses, however there are no data concerning infection of humans. In contrast, recently it had been shown that transplantation of organs from pigs infected with PCMV into non-human primate recipients resulted in a significant reduction of the survival time compared with the transplantation of organs from uninfected pigs. To prevent transmission of PCMV in future pig to human xenotransplantations, sensitive and specific detection methods should be used. Here a new Western blot assay using recombinant proteins corresponding to two domains of the glycoprotein gB of PCMV is described. With this assay, the presence of PCMV-specific antibodies in different pig breeds was analysed. Antibodies were detected in a high percentage of animals, in one breed up to 85%.

Immunological methods for the detection of porcine lymphotropic herpesviruses (PLHV).

Porcine lymphotropic herpesviruses (PLHV-1, -2, and -3) are widespread in pigs and closely related to the human pathogenic gammaherpesviruses Epstein-Barr virus (human herpesvirus 4, HHV-4) and Kaposi sarcoma herpesvirus (HHV-8). In minipigs, PLHV-1 causes a porcine post-transplantation lymphoproliferative disorder (PTLD) after experimental transplantations. Porcine PTLD comes with clinical symptoms similar to those of human PTLD, a serious complication of solid organ and allogeneic bone marrow transplantation linked to HHV-4. Since PLHVs may be transmitted from donor pigs to the human recipient of xenotransplants (pig cells, tissues or organs), sensitive and specific methods should be developed to detect and eliminate PLHVs. Here we describe an ELISA and a Western blot assay using recombinant glycoprotein B of PLHV-1. Using both assays, the presence of specific antibodies in different pig breeds as well as in German slaughterhouse workers was analysed. Antibodies were detected in some animals, but not in human subjects.

Environmental behavior of the chiral herbicide haloxyfop. 2. Unchanged enantiomer composition in blackgrass (Alopecurus myosuroides) and garden cress (Lepidium sativum).

Haloxyfop-methyl is a chiral herbicide against grasses in dicotyledonous crops. In plants and soil, haloxyfop-methyl is rapidly hydrolyzed to haloxyfop-acid, whose R-enantiomer carries the actual herbicidal activity. In soil, S-haloxyfop-acid is converted within less than 1 day and almost completely into R-haloxyfop-acid. In this study, we investigated the possible interconversion of the enantiomers of haloxyfop-methyl and haloxyfop-acid in blackgrass and garden cress. Racemic or enantiopure haloxyfop-methyl was applied to the leaves of plants grown in agar. The metabolism was followed during 4 days using enantioselective GC-MS. In contrast to soils, no interconversion was observed in plants, and metabolism was nonenantioselective. These findings are consistent with the fact that after pre-emergence application to soil and uptake by roots, the observed herbicidal effect is basically independent of the enantiomer composition of the applied substance, whereas after postemergence application, the efficacy clearly is different for the two enantiomers.

Lack of antiviral antibody response in koalas infected with koala retroviruses (KoRV).

Many wild koalas are infected with the koala retrovirus, KoRV, some of which suffer from lymphoma and chlamydial disease. Three subgroups, KoRV-A, KoRV-B and KoRV-J, have so far been described. It is well known that other closely related gammaretroviruses can induce tumours and severe immunodeficiencies in their respective hosts and a possible role for KoRV infection in lymphoma and chlamydial disease in koalas has been suggested. In many wild koalas, KoRV-A has become endogenised, i.e., it is integrated in the germ-line and is passed on with normal cellular genes. In this study, sera from koalas in European zoos and from wild animals in Australia were screened for antibodies against KoRV-A. These naturally infected animals all carry endogenous KoRV-A and two zoo animals are also infected with KoRV-B. The antibody response is generally an important diagnostic tool for detecting retrovirus infections. However, when Western blot analyses were performed using purified virus or recombinant proteins corresponding to KoRV-A, none of the koalas tested positive for specific antibodies, suggesting a state of tolerance. These results have implications for koala vaccination, as they suggest that therapeutic immunisation of animals carrying and expressing endogenous KoRV-A will not be successful. However, it remains unclear whether these animals can be immunised against KoRV-B and immunisation of uninfected koalas could still be worthwhile.

Tolerability of the SQ-standardised grass sublingual immunotherapy tablet in patients treated with concomitant allergy immunotherapy: a non-interventional observational study.

BACKGROUND: The majority of allergic patients are poly-sensitized. For causal treatment by allergy immunotherapy (AIT) a single or few allergen products containing the clinically most relevant allergens are applied, but few data on tolerability of multiple application of AIT is available. The aim of our study was to investigate safety and tolerability in patients who started treatment by sublingual immunotherapy (SLIT) with the standardised SQ(®) grass SLIT-tablet and were treated with concomitant AIT products. METHODS: In a non-interventional, open-label, observational study in Germany treatment of patients with the SQ(®) grass SLIT-tablet and concomitant AIT (SCIT or SLIT) was documented between January 2012 and January 2014. Patients were followed at visits at first administration of the SQ(®) grass SLIT-tablet and after 1-3 months of treatment. Tolerability of the treatment with the SQ(®) grass SLIT-tablet and concomitant AIT were assessed by the physician and administration of AIT and adverse events (AEs) were recorded by the patients in diaries. AEs and adverse drug reactions (ADRs) were coded by using the Medical Dictionary for Regulatory Activities. RESULTS: In total, 181 patients were documented by 48 allergists and 160 patients treated with a concomitant AIT (SCIT 130, SLIT 30). AEs were reported in 58 (36.3 %) patients with concomitant AIT, and AEs considered related with the SQ(®) grass SLIT-tablet in 49 (30.6 %) and with concomitant AIT in 18 (11.3 %) patients. Treatment was discontinued due to ADRs in 12 (7.5 %) patients and severity of ADRs was assessed mild or moderate in 29 (18.1 %), and severe in 20 (12.5 %) patients. Most common reactions were localised at the application site of the SQ(®) grass SLIT-tablet as oral pruritus, throat irritation, oedema mouth and paraesthesia oral; no serious ADRs were reported. Overall tolerability of the SQ(®) grass SLIT-tablet if given with concomitant AIT was assessed as "good" or "very good" by 91.0 % of patients and 91.6 % of physicians. CONCLUSIONS: In comparison to data from previous studies no increase in frequency of AEs or change in the tolerability profile was observed when SLIT with the SQ(®) grass SLIT-tablet was administered with concomitant SCIT or SLIT.

Unspecific abdominal symptoms and pneumobilia: a rare case of gastrointestinal obstruction.

The case of a 77-year-old woman with symptoms of gastric outlet obstruction is presented. Transabdominal ultrasonography findings were suspicious of Bouveret's syndrome. Upper endoscopy confirmed this diagnosis. Bouveret's syndrome is a rare complication of gallstone disease caused by a bilioenteric fistula leading to gastric outlet obstruction by a gallstone and should be suspected in any patient who presents with pneumobilia without recent endoscopic retrograde cholangiopancreatography or biliary surgery.

Lack of antibody response in pigs immunized with the transmembrane envelope protein of porcine endogenous retroviruses.

Recently, we immunized different mammalian species (goats, mice, rats, rabbits, guinea pigs and hamsters) with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV). In all cases, neutralizing immune sera were induced, which recognized epitopes in the fusion peptide proximal region and the membrane proximal external region of p15E. In order to analyse whether pigs are also able to produce such antibodies, and whether such antibodies can be used to study the involvement of the TM protein in placental development (as was shown for endogenous retroviruses of other species), German landrace pigs were immunized with PERV p15E. No binding and neutralizing antibodies were produced as shown in three Western blot analyses and in a neutralization assay, indicating that pigs are tolerant to their endogenous retroviruses, at least for the ectodomain of the TM protein.

Immunization with hybrid proteins containing the membrane proximal external region of HIV-1.

The transmembrane envelope (TM) protein gp41 of HIV-1 is an attractive target when designing a vaccine to induce neutralizing antibodies. A few broadly neutralizing antibodies (2F5, 4E10, and 10E8) that target conserved epitopes in the membrane proximal external region (MPER) of gp41 have been isolated from infected individuals. However, attempts to induce such antibodies by immunizations with gp41 and Env derivatives containing the MPER were successful only to some extent. In contrast, immunizations with the ectodomain of the TM protein p15E of different gamma retroviruses resulted in the induction of neutralizing antibodies. These sera recognized epitopes located in the MPER and in the fusion peptide proximal region (FPPR) of p15E. Based on these results, both regions of p15E were substituted with the corresponding sequences derived from gp41 of HIV-1. Thus, four different hybrid antigens were produced. One of the inserted sequences contained the epitopes of 2F5 and 4E10 in the MPER; the other corresponded to the FPPR. Vaccination of rats, guinea pigs, and a goat induced binding antibodies directed against the FPPR of gp41 and the 2F5 epitope (ELDKWA) located in the MPER. Despite the exact recognition of the 2F5 epitope, no or very weak neutralization of HIV-1NL4-3 by the immune sera was demonstrated. Nonetheless, using the strategy of hybrid proteins, antibodies targeting the desired epitope were successfully induced.

Estimating economic thresholds for site-specific weed control using manual weed counts and sensor technology: an example based on three winter wheat trials.

BACKGROUND: Precision experimental design uses the natural heterogeneity of agricultural fields and combines sensor technology with linear mixed models to estimate the effect of weeds, soil properties and herbicide on yield. These estimates can be used to derive economic thresholds. Three field trials are presented using the precision experimental design in winter wheat. Weed densities were determined by manual sampling and bi-spectral cameras, yield and soil properties were mapped. RESULTS: Galium aparine, other broad-leaved weeds and Alopecurus myosuroides reduced yield by 17.5, 1.2 and 12.4 kg ha(-1) plant(-1) m(2) in one trial. The determined thresholds for site-specific weed control with independently applied herbicides were 4, 48 and 12 plants m(-2), respectively. Spring drought reduced yield effects of weeds considerably in one trial, since water became yield limiting. A negative herbicide effect on the crop was negligible, except in one trial, in which the herbicide mixture tended to reduce yield by 0.6 t ha(-1). Bi-spectral cameras for weed counting were of limited use and still need improvement. Nevertheless, large weed patches were correctly identified. CONCLUSION: The current paper presents a new approach to conducting field trials and deriving decision rules for weed control in farmers' fields.

Development and testing of a decision making based method to adjust automatically the harrowing intensity.

Harrowing is often used to reduce weed competition, generally using a constant intensity across a whole field. The efficacy of weed harrowing in wheat and barley can be optimized, if site-specific conditions of soil, weed infestation and crop growth stage are taken into account. This study aimed to develop and test an algorithm to automatically adjust the harrowing intensity by varying the tine angle and number of passes. The field variability of crop leaf cover, weed density and soil density was acquired with geo-referenced sensors to investigate the harrowing selectivity and crop recovery. Crop leaf cover and weed density were assessed using bispectral cameras through differential images analysis. The draught force of the soil opposite to the direction of travel was measured with electronic load cell sensor connected to a rigid tine mounted in front of the harrow. Optimal harrowing intensity levels were derived in previously implemented experiments, based on the weed control efficacy and yield gain. The assessments of crop leaf cover, weed density and soil density were combined via rules with the aforementioned optimal intensities, in a linguistic fuzzy inference system (LFIS). The system was evaluated in two field experiments that compared constant intensities with variable intensities inferred by the system. A higher weed density reduction could be achieved when the harrowing intensity was not kept constant along the cultivated plot. Varying the intensity tended to reduce the crop leaf cover, though slightly improving crop yield. A real-time intensity adjustment with this system is achievable, if the cameras are attached in the front and at the rear or sides of the harrow.

Saccharin and other artificial sweeteners in soils: estimated inputs from agriculture and households, degradation, and leaching to groundwater.

Artificial sweeteners are consumed in substantial quantities as sugar substitutes and were previously shown to be ubiquitously present in the aquatic environment. The sweetener saccharin is also registered as additive in piglet feed. Saccharin fed to piglets was largely excreted and, consequently, found in liquid manure at concentrations up to 12 mg/L, where it was stable during 2 months of storage. Saccharin may thus end up in soils in considerable quantities with manure. Furthermore, other studies showed that saccharin is a soil metabolite of certain sulfonylurea herbicides. Sweeteners may also get into soils via irrigation with wastewater-polluted surface water, fertilization with sewage sludge (1-43 µg/L), or through leaky sewers. In soil incubation experiments, cyclamate, saccharin, acesulfame, and sucralose were degraded with half-lives of 0.4-6 d, 3-12 d, 3-49 d, and 8-124 d, respectively. The relative importance of entry pathways to soils was compared and degradation and leaching to groundwater were evaluated with computer simulations. The data suggest that detection of saccharin in groundwater (observed concentrations, up to 0.26 µg/L) is most likely due to application of manure. However, elevated concentrations of acesulfame in groundwater (up to 5 µg/L) may result primarily from infiltration of wastewater-polluted surface water through stream beds.

Effects of expectation and caffeine on arousal, well-being, and reaction time.

The objective of this study is to determine the impact of expectation associated with placebo and caffeine ingestion. We used a three-armed, randomized, double-blind design. Two three-armed experiments varying instruction (true, false, control) investigated the role of expectations of changes in arousal (blood pressure, heart rate), subjective well-being, and reaction time (RT). In Experiment 1 (N = 45), decaffeinated coffee was administered, and expectations were produced in one group by making them believe they had ingested caffeinated coffee. In Experiment 2 (N = 45), caffeinated orange juice was given in both experimental groups, but only one was informed about the true content. In Experiment 1, a significant effect for subjective alertness was found in the placebo treatment compared to the control group. However, for RT and well-being no significant effects were found. In Experiment 2, no significant expectancy effects were found. Caffeine produced large effects for blood pressure in both treatments compared to the control group, but the effects were larger for the false information group. For subjective well-being (alertness, calmness), considerable but nonsignificant changes were found for correctly informed participants, indicating possible additivity of pharmacologic effect and expectations. The results tentatively indicate that placebo and expectancy effects primarily show through introspection.

Thrombin inhibitors identified by computer-assisted multiparameter design.

Here, we present a series of thrombin inhibitors that were generated by using powerful computer-assisted multiparameter optimization process. The process was organized in design cycles, starting with a set of randomly chosen molecules. Each cycle combined combinatorial synthesis, multiparameter characterization of compounds in a variety of bioassays, and algorithmic processing of the data to devise a set of compounds to be synthesized in the next cycle. The identified lead compounds exhibited thrombin inhibitory constants in the lower nanomolar range. They are by far the most selective synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore, these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic stability, low serum protein binding, good solubility, high anticoagulant activity, and a slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray crystallographic analysis of a thrombin-inhibitor complex revealed a binding mode with a neutral moiety in the S1 pocket of thrombin.

Seasonality of energetic functioning and production of reactive oxygen species by lugworm (Arenicola marina) mitochondria exposed to acute temperature changes.

The influence of seasonal and acute temperature changes on mitochondrial functions were studied in isolated mitochondria of the eurythermal lugworm Arenicola marina (Polychaeta), with special emphasis on the interdependence of membrane potential and radical production. Acclimatisation of lugworms to pre-spawning/summer conditions is associated with rising mitochondrial substrate oxidation rates, higher proton leakage rates, elevated membrane potentials, and increased production of reactive oxygen species (ROS) in isolated mitochondria, compared with mitochondria from winter animals. However, a high ROS production was compensated for by higher activities of the antioxidant enzymes catalase and superoxide dismutase, as well as lower mitochondrial densities in summer compared with winter animals. In summer animals, a higher sensitivity of the proton leakage rate to changes of membrane potential will confer better flexibility for metabolic regulation (mild uncoupling) in response to temperature change. These seasonal alterations in mitochondrial functions suggest modifications of energy metabolism in eurythermal and euryoxic organisms on intertidal mudflats during summer. In winter, low and less changeable temperatures in intertidal sedimentary environments permit higher respiratory efficiency at low aerobic metabolic rates and lower membrane potentials in A. marina mitochondria.

Directed evolution towards protease-resistant hirudin variants.

Hirudin, a thrombin-specific inhibitor, is efficiently digested and inactivated by proteases with pepsin- and chymotrypsin-like specificity. Using a combination of phage display selection and high-throughput screening methods, several variants of recombinant hirudin were generated. Only very few variants comprising amino acid substitutions in the amino-terminal domain (residues 1-5) and in the carboxyl-terminal tail (residues 49, 50, and/or 56, 57, 62-64) were identified that showed thrombin inhibition activities similar to those of the wild-type polypeptide. Analysis of protease susceptibility, however, revealed that mutations, which conferred protease resistance, simultaneously diminish thrombin inhibition activity. This is particularly apparent for substitutions in the region of residues 56-64, which forms a large number of electrostatic and hydrophobic interactions with thrombin in the crystal structure of the complex. Unlike wild-type hirudin, the variant comprising Pro(50)- ...-His(56)-Asp(57)- ...-Pro(62)-Pro(63)-His(64) is completely resistant to pepsin and chymotrypsin cleavage; however, this is at the expense of thrombin inhibition activity where there is a 100-fold increase in the IC50 value. The frequent replacement of wild-type amino acids by proline at major protease cleavage sites indicates that at least pepsin- and chymotrypsin-like enzymes may exhibit a (conformational) specificity concerning the P1 and P2 positions. On the basis of these results, proline substitutions appear to be a general strategy to design polypeptides that are not susceptible to digestion by a broader range of different proteases.