PMP2 regulates myelin thickening and ATP production during remyelination.

It is well established that axonal Neuregulin 1 type 3 (NRG1t3) regulates developmental myelin formation as well as EGR2-dependent gene activation and lipid synthesis. However, in peripheral neuropathy disease context, elevated axonal NRG1t3 improves remyelination and myelin sheath thickness without increasing Egr2 expression or activity, and without affecting the transcriptional activity of canonical myelination genes. Surprisingly, Pmp2, encoding for a myelin fatty acid binding protein, is the only gene whose expression increases in Schwann cells following overexpression of axonal NRG1t3. Here, we demonstrate PMP2 expression is directly regulated by NRG1t3 active form, following proteolytic cleavage. Then, using a transgenic mouse model overexpressing axonal NRG1t3 (NRG1t3OE) and knocked out for PMP2, we demonstrate that PMP2 is required for NRG1t3-mediated remyelination. We demonstrate that the sustained expression of Pmp2 in NRG1t3OE mice enhances the fatty acid uptake in sciatic nerve fibers and the mitochondrial ATP production in Schwann cells. In sum, our findings demonstrate that PMP2 is a direct downstream mediator of NRG1t3 and that the modulation of PMP2 downstream NRG1t3 activation has distinct effects on Schwann cell function during developmental myelination and remyelination.

Accuracy assessment of inverse distance weighting interpolation of groundwater nitrate concentrations in Bavaria (Germany).

For the designation of nitrate vulnerable zones under the EU Nitrate Directive, some German federal states use inverse distance weighting (IDW) as interpolation method. Our study quantifies the accuracy of IDW with respect to the designation of areas with a groundwater nitrate concentration above the threshold of 50 mg NO3/l using a dataset of 5790 groundwater monitoring sites in Bavaria. The results show that the absolute differences of nitrate concentrations between the monitoring sites are only weakly correlated within a range of no more than 0.4 km. The IDW cross-validated nitrate concentration of measurement sites shows a mean absolute error of 7.0 mg NO3/l and the number of measurement sites above 50 mg NO3/l is 44% too low by interpolation for all sites as a whole. The corresponding values for interpolation separately for the 18 hydrogeological regions in Bavaria are 7.1 mg NO3/l and 38%. The sensitivity and the accuracy of nitrate concentration maps due to the variation of IDW parameters and the position of sampling points are analysed by Monte Carlo IDW interpolations using a Random Forest modelled map as reference spatial distribution. Compared to this reference map, the area with a concentration above 50 mg NO3/l in groundwater is estimated by IDW to be 46% too low for the best IDW parametrization. Overall, IDW interpolation systematically underrates the occurrence of higher range nitrate concentrations. In view of these underestimations, IDW does not appear to be a suitable regionalization method for the designation of nitrate vulnerable zones, neither when applied for a federal state as a whole nor when interpolated separately for hydrogeological regions.

Large scale prediction of groundwater nitrate concentrations from spatial data using machine learning.

Reducing nitrogen inputs, in particular nitrate, to groundwater is becoming increasingly important to fulfil requirements of the European Water Framework Directive. When developing management plans for mitigation measures at larger scales, complex hydro-biogeochemical models reach their limits due to data availability and spatial discretization. To circumvent this problem, the spatial distribution of nitrate concentration in groundwater is estimated using a parsimonious GIS-based statistical approach. Point nitrate concentrations and spatial environmental data as predictors are used to train statistical models. In order to compile the spatial predictors with the respective monitoring sites, different designs of contributing areas (buffer zones) and their effects on the performance of different statistical models are investigated. Multiple Linear Regression (MLR), Classification and Regression Trees (CART), Random Forest (RF) and Boosted Regression Trees (BRT) are compared in terms of the predictive performance of each model according to various objective functions. We determine the most influential spatial predictors used in the respective models. After training the models with a subset of the data, we then predict the spatial nitrate distribution in groundwater for the entire federal state of Hesse, Germany on a 1 × 1 km grid by only the spatial environmental data. The Random Forest model outperforms the other models (R2 = 0.54), relying on hydrogeological units, the percentage of arable land and the nitrogen balance as the three most influencing predictors based on a 1000 m circular contributing area. The use of exclusively spatial available predictors is a big step forward in the prediction of nitrate in groundwater on regional scale.

Metabolic constraints of swelling-activated glutamate release in astrocytes and their implication for ischemic tissue damage.

Volume-regulated anion channel (VRAC) is a glutamate-permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes - the cell type which shows prominent swelling in stroke - to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post-ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin-luciferase ATP assays and a Seahorse analyzer. Swelling-activated glutamate release was quantified with the radiotracer D-[3 H]aspartate. The specific contribution of VRAC to swelling-activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine-rich repeat-containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT-PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2-deoxy-D-glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP-depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty-four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.

Stress-induced modulation of volume-regulated anions channels in human alveolar carcinoma cells.

Shift in the cellular homeostasis of the organic osmolyte taurine has been associated with dysregulation of the volume-regulated anion channel (VRAC) complex, which comprises leucine-rich repeat-containing family 8 members (LRRC8A-E). Using SDS-PAGE, western blotting, qRT-PCR, and tracer technique ([3 H]taurine) we demonstrate that reactive oxygen species (ROS) and the cell growth-associated kinases Akt/mTOR, play a role in the regulation of VRAC in human alveolar cancer (A549) cells. LRRC8A is indispensable for VRAC activity and long-term exposure to hypoosmotic challenges and/or ROS impairs VRAC activity, not through reduction in total LRRC8A expression or LRRC8A availability in the plasma membrane, but through oxidation/inactivation of kinases/phosphatases that control VRAC activity once it has been instigated. Pursuing Akt signaling via the serine/threonine kinase mTOR, using mTORC1 inhibition (rapamycin) and mTORC2 obstruction (Rictor knockdown), we demonstrate that interference with the PI3K-mTORC2-Akt signaling-axes obstructs stress-induced taurine release. Furthermore, we show that an increased LRRC8A expression, following exposure to cisplatin, ROS, phosphatase/lipoxygenase inhibitors, and antagonist of CysLT1-receptors, correlates an increased activation of the proapoptotic transcription factor p53. It is suggested that an increase in LRRC8A protein expression could be taken as an indicator for cell stress and limitation in VRAC activity.

Downregulation of Leucine-Rich Repeat-Containing 8A Limits Proliferation and Increases Sensitivity of Glioblastoma to Temozolomide and Carmustine.

BACKGROUND: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Ubiquitously expressed volume-regulated anion channels (VRAC) are thought to play a role in cell proliferation, migration, and apoptosis. VRAC are heteromeric channel complexes assembled from proteins belonging to the leucine-rich repeat-containing 8A (LRRC8A through E), among which LRRC8A plays an indispensable role. In the present work, we used an RNAi approach to test potential significance of VRAC and LRRC8A in GBM survival and sensitivity to chemotherapeutic agents. METHODS: Primary GBM cells were derived from a human surgical tissue sample. LRRC8A expression was determined with quantitative RT-PCR and downregulated using siRNA. The effects of LRRC8A knockdown on GBM cell viability, proliferation, and sensitivity to chemotherapeutic agents were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and Coulter counter assays. Cell cycle progression was further explored using fluorescence-activated cell sorting analysis of propidium iodide-stained cells. RESULTS: Temozolomide (TMZ), carmustine, and cisplatin reduced GBM cell survival with the IC50 values of ~1,250, 320, and 30 µM, respectively. Two of three tested gene-specific siRNA constructs, siLRRC8A_3 and siLRRC8A_6, downregulated LRRC8A expression by >80% and significantly reduced GBM cell numbers. The most potent siLRRC8A_3 itself reduced viable cell numbers by ≥50%, and significantly increased toxicity of the sub-IC50 concentrations of TMZ (570 µM) and carmustine (167 µM). In contrast, the effects of siLRRC8A_3 and cisplatin (32 µM) were not additive, most likely because cisplatin uptake is VRAC-dependent. The results obtained in primary GBM cells were qualitatively recapitulated in U251 human GBM cell line. CONCLUSION: Downregulation of LRRC8A expression reduces GBM cell proliferation and increases sensitivity to the clinically used TMZ and carmustine. These findings indicate that VRAC represents a potential target for the treatment of GBM, alone or in combination with the current standard-of-care.

Pesticide exposure assessment for surface waters in the EU. Part 2: Determination of statistically based run-off and drainage scenarios for Germany.

BACKGROUND: In order to assess surface water exposure to active substances of plant protection products (PPPs) in the European Union (EU), the FOCUS (FOrum for the Co-ordination of pesticide fate models and their USe) surface water workgroup introduced four run-off and six drainage scenarios for Step 3 of the tiered FOCUSsw approach. These scenarios may not necessarily represent realistic worst-case situations for the different Member States of the EU. Hence, the suitability of the scenarios for risk assessment in the national authorisation procedures is not known. RESULTS: Using Germany as an example, the paper illustrates how national soil-climate scenarios can be developed to model entries of active substances into surface waters from run-off and erosion (using the model PRZM) and from drainage (using the model MACRO). In the authorisation procedure for PPPs on Member State level, such soil-climate scenarios can be used to determine exposure endpoints with a defined overall percentile. CONCLUSION: The approach allows the development of national specific soil-climate scenarios and to calculate percentile-based exposure endpoints. The scenarios have been integrated into a software tool analogous to FOCUS-SWASH which can be used in the future to assess surface water exposure in authorisation procedures of PPPs in Germany. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Pesticide exposure assessment for surface waters in the EU. Part 1: Some comments on the current procedure.

In 2001, the European Commission introduced a risk assessment project known as FOCUS (FOrum for the Coordination of pesticide fate models and their USe) for the surface water risk assessment of active substances in the European Union. Even for the national authorisation of plant protection products (PPPs), the vast majority of EU member states still refer to the four runoff and six drainage scenarios selected by the FOCUS Surface Water Workgroup. However, our study, as well as the European Food Safety Authority (EFSA), has stated the need for various improvements. Current developments in pesticide exposure assessment mainly relate to two processes. Firstly, predicted environmental concentrations (PECs) of pesticides are calculated by introducing model input variables such as weather conditions, soil properties and substance fate parameters that have a probabilistic nature. Secondly, spatially distributed PECs for soil-climate scenarios are derived on the basis of an analysis of geodata. Such approaches facilitate the calculation of a spatiotemporal cumulative distribution function (CDF) of PECs for a given area of interest and are subsequently used to determine an exposure concentration endpoint as a given percentile of the CDF. For national PPP authorisation, we propose that, in the future, exposure endpoints should be determined from the overall known statistical PEC population for an area of interest, and derived for soil and climate conditions specific to the particular member state. © 2016 Society of Chemical Industry.

HydroCrowd: a citizen science snapshot to assess the spatial control of nitrogen solutes in surface waters.

We organized a crowdsourcing experiment in the form of a snapshot sampling campaign to assess the spatial distribution of nitrogen solutes, namely, nitrate, ammonium and dissolved organic nitrogen (DON), in German surface waters. In particular, we investigated (i) whether crowdsourcing is a reasonable sampling method in hydrology and (ii) what the effects of population density, soil humus content and arable land were on actual nitrogen solute concentrations and surface water quality. The statistical analyses revealed a significant correlation between nitrate and arable land (0.46), as well as soil humus content (0.37) but a weak correlation with population density (0.12). DON correlations were weak but significant with humus content (0.14) and arable land (0.13). The mean contribution of DON to total dissolved nitrogen was 22%. Samples were classified as water quality class II or above, following the European Water Framework Directive for nitrate and ammonium (53% and 82%, respectively). Crowdsourcing turned out to be a useful method to assess the spatial distribution of stream solutes, as considerable amounts of samples were collected with comparatively little effort.

Relevance of nonfunctional linear polyacrylic acid for the biodegradation of superabsorbent polymer in soils.

Biodegradability is a desired characteristic for synthetic soil amendments. Cross-linked polyacrylic acid (PAA) is a synthetic superabsorbent used to increase the water availability for plant growth in soils. About 4% within products of cross-linked PAA remains as linear polyacrylic acid (PAAlinear). PAAlinear has no superabsorbent function but may contribute to the apparent biodegradation of the overall product. This is the first study that shows specifically the biodegradation of PAAlinear in agricultural soil. Two (13)C-labeled PAAlinear of the average molecular weights of 530, 400, and 219,500 g mol(-1) were incubated in soil. Mineralization of PAAlinear was measured directly as the (13)CO2 efflux from incubation vessels using an automatic system, which is based on (13)C-sensitive wavelength-scanned cavity ring-down spectroscopy. After 149 days, the PAAlinear with the larger average molecular weight and chain length showed about half of the degradation (0.91% of the initial weight) of the smaller PAAlinear (1.85%). The difference in biodegradation was confirmed by the δ(13)C signature of the microbial biomass (δ(13)Cmic), which was significantly enriched in the samples with short PAAlinear (-13‰ against reference Vienna Pee Dee Belemnite,VPDB) as compared to those with long PAAlinear (-16‰ VPDB). In agreement with other polymer studies, the results suggest that the biodegradation of PAAlinear in soil is determined by the average molecular weight and occurs mainly at terminal sites. Most importantly, the study outlines that the size of PAA that escapes cross-linking can have a significant impact on the overall biodegradability of a PAA-based superabsorbent.

Influence of murine mesenchymal stem cells on proliferation, phenotype, vitality, and cytotoxicity of murine cytokine-induced killer cells in coculture.

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell-cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.

Biodegradability of a polyacrylate superabsorbent in agricultural soil.

Superabsorbent polymers (SAP) are used, inter alia, as soil amendment to increase the water holding capacity of soils. Biodegradability of soil conditioners has become a desired key characteristic to protect soil and groundwater resources. The present study characterized the biodegradability of one acrylate based SAP in four agricultural soils and at three temperatures. Mineralisation was measured as the (13)CO2 efflux from (13)C-labelled SAP in soil incubations. The SAP was either single-labelled in the carboxyl C-atom or triple-labelled including additionally the two C-atoms interlinked in the SAP backbone. The dual labelling allowed estimating the degradation of the polyacrylate main chain. The (13)CO2 efflux from samples was measured using an automated system including wavelength-scanned cavity ring-down spectroscopy. Based on single-labelled SAP, the mean degradation after 24 weeks varied between 0.45% in loamy sand and 0.82% in loam. However, the differences between degradation rates in different soils were not significant due to a large intra-replicate variability. Similarly, mean degradation did not differ significantly between effective temperature regimes of 20° and 30 °C after 12 weeks. Results from the triple-labelled SAP were lower as compared to their single-labelled variant. Detailed results suggest that the polyacrylate main chain degraded in the soils, if at all, at rates of 0.12-0.24 % per 6 months.

Trend of herbicide loads in the river Rhine and its tributaries.

Many policies have been established and actions have been taken to reduce pesticide pollution of surface waters. However, the effectiveness of these initiatives was rarely tested on an empirical basis. This study suggests that evidence on the policy effects can be evaluated by analyzing the temporal changes in the loads rather than the concentrations of active ingredients in rivers. It is shown that the long-term change of pesticide emissions into surface waters can be tested statistically by the number of upward versus downward trends in river load. To evaluate the situation in Germany, 57 concentration time series of 14 herbicide substances at 7 river monitoring stations with a minimum of 24 analyses per year were assembled and annual substance river loads were calculated. The longest time course was 17 years (1990-2006). The significance of trends of data rows was analyzed by an univariate Mann-Kendall test that evaluates 27 (47.4%) of the 57 time series as statistically significant downward trended. It took a period of 10 years and longer before the high annual atrazine and simazine loads measured in the years 1990-1991 had been diminished to a drastically lowered level after ban of the herbicides. Data are available on the yearly consumption of 8 substances used in German agriculture. A total of 36 time series for this subset were tested with a partial Mann-Kendall test with the consumption as covariance factor, which reduces the number of significant trends in river load noticeably. Based on this test, only 7 (19.4% of 36) declining time series remain. As a result, the intended effect of measures to reduce surface water contamination by the use of pesticides seems to be only partially successful, however, the database to justify this statement is small. For the water monitoring strategies in Germany, it is recommend to enhance the sampling frequency at river stations. A minimum of a semimonthly sampling interval would facilitate the calculation of valuable annual river loads and would therefore allow a pronounced validation of the long-term change of pesticide emission into rivers.

Mitigation strategies to reduce pesticide inputs into ground- and surface water and their effectiveness; a review.

In this paper, the current knowledge on mitigation strategies to reduce pesticide inputs into surface water and groundwater, and their effectiveness when applied in practice is reviewed. Apart from their effectiveness in reducing pesticide inputs into ground- and surface water, the mitigation measures identified in the literature are evaluated with respect to their practicability. Those measures considered both effective and feasible are recommended for implementing at the farm and catchment scale. Finally, recommendations for modelling are provided using the identified reduction efficiencies. Roughly 180 publications directly dealing with or being somehow related to mitigation of pesticide inputs into water bodies were examined. The effectiveness of grassed buffer strips located at the lower edges of fields has been demonstrated. However, this effectiveness is very variable, and the variability cannot be explained by strip width alone. Riparian buffer strips are most probably much less effective than edge-of-field buffer strips in reducing pesticide runoff and erosion inputs into surface waters. Constructed wetlands are promising tools for mitigating pesticide inputs via runoff/erosion and drift into surface waters, but their effectiveness still has to be demonstrated for weakly and moderately sorbing compounds. Subsurface drains are an effective mitigation measure for pesticide runoff losses from slowly permeable soils with frequent waterlogging. For the pathways drainage and leaching, the only feasible mitigation measures are application rate reduction, product substitution and shift of the application date. There are many possible effective measures of spray drift reduction. While sufficient knowledge exists for suggesting default values for the efficiency of single drift mitigation measures, little information exists on the effect of the drift reduction efficiency of combinations of measures. More research on possible interactions between different drift mitigation measures and the resulting overall drift reduction efficiency is therefore indicated. Point-source inputs can be mitigated against by increasing awareness of the farmers with regard to pesticide handling and application, and encouraging them to implement loss-reducing measures of "best management practice". In catchments dominated by diffuse inputs at least in some years, mitigation of point-source inputs alone may not be sufficient to reduce pesticide loads/concentrations in water bodies to an acceptable level.

Point- and nonpoint-source pesticide contamination in the Zwester Ohm catchment, Germany.

Reducing pesticide loads in surface waters implies identifying the pathways responsible for the pollution. The current study documents the pesticide contamination of the river Zwester Ohm, a 4917-ha catchment in Germany with 41% of the land used for crop production. Discharges and concentrations of 19 pesticides were measured continuously at three locations for 15 mo. The load detected at the outlet of the catchment amounted to 9048 g a.i. The losses represent 0.22% of the pesticides applied by the farmers. The contamination showed a seasonal pattern following the pesticide application times. The wastewater treatment plant system (WWTPS) in the catchment (two wastewater treatment plants [WWTP], 14 combined sewer overflows (CSO), four CSO tanks) emits during dry weather periods purified sewage and during storm events sewage mixed with stormwater runoff into the river. The contribution by the WWTPS to the pesticide load was defined as point-source pollution (PSP). The load was dominated by PSP with at least 77% of the total pollution. No significant interdependencies between intrinsic properties of the pesticides, hydrometeorological factors, and the loads occurring in the stream could be found. Therefore, it is not possible to predict PSP for other catchments based on the results from this study. Whereas 65% of the total load entered the river via the WWTP, a portion of 12% was attributed to the CSO. The study points out that the influence of CSO on PSP should be taken into account in future catchment studies in areas with comparable agricultural structure.