Feature-based molecular networking for identification of organic micropollutants including metabolites by non-target analysis applied to riverbank filtration.

Due to growing concern about organic micropollutants and their transformation products (TP) in surface and drinking water, reliable identification of unknowns is required. Here, we demonstrate how non-target liquid chromatography (LC)-high-resolution tandem mass spectrometry (MS/MS) and the feature-based molecular networking (FBMN) workflow provide insight into water samples from four riverbank filtration sites with different redox conditions. First, FBMN prioritized and connected drinking water relevant and seasonally dependent compounds based on a modification-aware MS/MS cosine similarity. Within the resulting molecular networks, forty-three compounds were annotated. Here, carbamazepine, sartans, and their respective TP were investigated exemplarily. With chromatographic information and spectral similarity, four additional TP (dealkylated valsartan, dealkylated irbesartan, two oxygenated irbesartan isomers) and olmesartan were identified and partly verified with an authentic standard. In this study, sartans and TP were investigated and grouped regarding their removal behavior under different redox conditions and seasons for the first time. Antihypertensives were grouped into compounds being well removed during riverbank filtration, those primarily removed under anoxic conditions, and rather persistent compounds. Observed seasonal variations were mainly limited to varying river water concentrations. FBMN is a powerful tool for identifying previously unknown or unexpected compounds and their TP in water samples by non-target analysis.

Seasonal performance assessment of four riverbank filtration sites by combined non-target and effect-directed analysis.

Targeting the most relevant organic micropollutants (OMP) in routine analysis appears difficult due to formation of transformation products of unknown concentration or toxicity. Performance assessment of water purification processes is still based upon limited target data. Therefore, we broadened the assessment of the removal efficiencies with combined non-target and effect-directed analysis at four riverbank filtration (RBF) sites in Germany. To assess micropollutant elimination, constancy and formation during different seasons, considering local redox conditions, travel distances and total component number in the river, non-target analysis features were grouped into categories. Furthermore, RBF sites were investigated with four endpoints (baseline toxicity, acetylcholinesterase inhibition, antibiotic effects and estrogenic effects) for thin-layer chromatography - effect-directed analysis for the first time. Results showed elimination or reduction of many features and effects, but also constancy and formation of varying proportions. Fall river samples showed precipitation-caused dilution in both tests. Spring samples showed increased effects only in acetylcholinesterase inhibition and estrogenic effects, probably due to phytoestrogens or algae bloom during vegetation period. Sites were ranked considering the total number of features, group proportions, seasonal variations and intensity and number of effects in abstraction wells. Oxic conditions and low initial component numbers in the river (Ruhr sites) resulted in less effects and fewer formations. Longer travel distances were important for a more efficient reduction of effects and features. Combination of non-target and effect-directed analysis proved to be valuable for a more comprehensive assessment of process performance beyond target analysis as also unknown OMP are observed with both methods.

Impact of seasonality, redox conditions, travel distances and initial concentrations on micropollutant removal during riverbank filtration at four sites.

Riverbank filtration (RBF) is a reliable water purification technique that has proven to be suitable for the removal of organic micropollutants. Its removal efficiency and dependency on a variety of factors such as redox conditions, temperatures, geology, travel times, level of initial micropollutant concentrations and seasonality were investigated during three seasonal sampling campaigns. Two anoxic (silty sand, Ems river) and two oxic (gravel, Ruhr river) RBF sites in Germany with different travel distances (42-633 m) were studied. Micropollutant concentrations were examined using a large-volume direct injection liquid chromatography method coupled to high-resolution mass spectrometry. Seasonal differences in micropollutant concentrations in the rivers were observed for chlorotolurone, diclofenac, terbuthylazine, mecoprop-P, MCPA (2-methyl-4-chlorophenoxyacetic acid) and propyphenazone. Redox dependencies in RBF were only found for sulfamethoxazole, propyphenazone, terbuthylazine and carbamazepine. Data for oxazepam, tramadol, N-desmethyl-tramadol, tilidin-desmethyl, carbamazepine and carbendazim indicate a required minimum travel distance of e.g. 100-200 m for the complete removal. Notably, travel time did not seem to be a substantial factor for their removal. High conductivity aquifers are also well suited for micropollutant removal. Seasonal initial concentration level variations showed no impact on the resulting abstraction well concentrations. Although the calculated removal efficiencies varied, they proved to be improper for seasonal raw water quality comparison. Knowledge of micropollutant behavior in riverbank filtration was broadened and RBF proved to be well suited for effective micropollutant reduction throughout the year, yet for a complete removal long travel distances or further technical purification steps are required.

Pharmaceuticals in the environment--Global occurrences and perspectives.

Pharmaceuticals are known to occur widely in the environment of industrialized countries. In developing countries, more monitoring results have recently become available, but a concise picture of measured environmental concentrations (MECs) is still elusive. Through a comprehensive literature review of 1016 original publications and 150 review articles, the authors collected MECs for human and veterinary pharmaceutical substances reported worldwide in surface water, groundwater, tap/drinking water, manure, soil, and other environmental matrices in a comprehensive database. Due to the heterogeneity of the data sources, a simplified data quality assessment was conducted. The database reveals that pharmaceuticals or their transformation products have been detected in the environment of 71 countries covering all continents. These countries were then grouped into the 5 regions recognized by the United Nations (UN). In total, 631 different pharmaceutical substances were found at MECs above the detection limit of the respective analytical methods employed, revealing distinct regional patterns. Sixteen substances were detected in each of the 5 UN regions. For example, the anti-inflammatory drug diclofenac has been detected in environmental matrices in 50 countries, and concentrations found in several locations exceeded predicted no-effect concentrations. Urban wastewater seems to be the dominant emission pathway for pharmaceuticals globally, although emissions from industrial production, hospitals, agriculture, and aquaculture are important locally. The authors conclude that pharmaceuticals are a global challenge calling for multistakeholder approaches to prevent, reduce, and manage their entry into and presence in the environment, such as those being discussed under the Strategic Approach to International Chemicals Management, a UN Environment Program.

Yangtze Three Gorges Reservoir, China: A holistic assessment of organic pollution, mutagenic effects of sediments and genotoxic impacts on fish.

Besides obvious benefits, the Three Gorges Dam's construction resulted in new pollution scenarios with the potentials to threaten the Three Gorges Reservoir (TGR) ecosystem. In order to record organic contamination, to find links to ecotoxicological impacts and to serve as reference for ensuing monitoring, several sites in the TGR area were screened applying the triad approach with additional lines-of-evidence as a holistic assessment method. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011 and 2012 to determine organic pollution levels, mutagenic potentials and genotoxic impacts. Two regional hot-spots near the cities of Chongqing and Kaixian were identified and further investigated in 2013. Only polycyclic aromatic hydrocarbons (PAHs) could be detected in sediments in 2011 (165-1653ng/g), emphasizing their roles as key pollutants of the area. Their ubiquity was confirmed at Chongqing (150-433ng/g) and Kaixian (127-590ng/g) in 2013. Concentrations were comparable to other major Chinese and German rivers. However, the immense sediment influx suggested a deposition of 216-636kgPAH/day (0.2-0.6mgPAH/(m(2)·day)), indicating an ecotoxicological risk. PAH source analysis highlighted primary impacts of combustion sources on the more industrialized upper TGR section, whereas petrogenic sources dominated the mid-low section. Furthermore, sediment extracts from several sites exhibited significant activities of frameshift promutagens in the Ames fluctuation assay. Additionally, significant genotoxic impairments in erythrocytes of P. vachellii were detected (Chongqing/Kaixian), demonstrating the relevance of genotoxicity as an important mode of action in the TGR's fish. PAHs, their derivatives and non-target compounds are considered as main causative agents.

Selection of organic process and source indicator substances for the anthropogenically influenced water cycle.

An increasing number of organic micropollutants (OMP) is detected in anthropogenically influenced water cycles. Source control and effective natural and technical barriers are essential to maintain a high quality of drinking water resources under these circumstances. Based on the literature and our own research this study proposes a limited number of OMP that can serve as indicator substances for the major sources of OMP, such as wastewater treatment plants, agriculture and surface runoff. Furthermore functional indicators are proposed that allow assessment of the proper function of natural and technical barriers in the aquatic environment, namely conventional municipal wastewater treatment, advanced treatment (ozonation, activated carbon), bank filtration and soil aquifer treatment as well as self-purification in surface water. These indicator substances include the artificial sweetener acesulfame, the anti-inflammatory drug ibuprofen, the anticonvulsant carbamazepine, the corrosion inhibitor benzotriazole and the herbicide mecoprop among others. The chemical indicator substances are intended to support comparisons between watersheds and technical and natural processes independent of specific water cycles and to reduce efforts and costs of chemical analyses without losing essential information.

Occurrence and distribution of organic trace substances in waters from the Three Gorges Reservoir, China.

This study deals with the evaluation of water quality of the Three Gorges Reservoir (TGR) in order to assess its suitability as a raw water source for drinking water production. Therefore, water samples from (1) surface water, (2) tap water, and (3) wastewater treatment plant effluents were taken randomly by 2011-2012 in the area of the TGR and were analyzed for seven different organic contaminant groups (207 substances in total), applying nine different analytical methods. In the three sampled water sources, typical contaminant patterns were found, i.e., pesticides and polycyclic aromatic hydrocarbons (PAH) in surface water with concentrations of 0.020-3.5 µg/L and 0.004-0.12 µg/L, disinfection by-products in tap water with concentrations of 0.050-79 µg/L, and pharmaceuticals in wastewater treatment plant effluents with concentrations of 0.020-0.76 µg/L, respectively. The most frequently detected organic compounds in surface water (45 positives out of 57 samples) were the pyridine pesticides clopyralid and picloram. The concentrations might indicate that they are used on a regular basis and in conjunction in the area of the TGR. Three- and four-ring PAH were ubiquitously distributed, while the poorly soluble five- and six-ring members, perfluorinated compounds, polychlorinated biphenyls, and polybrominated diphenyl ethers, were below the detection limit. In general, the detected concentrations in TGR are in the same range or even lower compared to surface waters in western industrialized countries, although contaminant loads can still be high due to a high discharge. With the exception of the two pesticides, clopyralid and picloram, concentrations of the investigated organic pollutants in TGR meet the limits of the Chinese Standards for Drinking Water Quality GB 5749 (Ministry of Health of China and Standardization Administration of China 2006) and the European Union (EU) Council Directive 98/83/EC on the quality of water intended for human consumption (The Council of the European Union 1998), or rather, the EU Directive on environmental quality standards in the field of water policy (The European Parliament and The Council of the European Union 2008). Therefore, the suggested use of surface water from TGR for drinking water purposes is a valid option. Current treatment methods, however, do not seem to be efficient since organic pollutants were detected in significant concentrations in purified tap water.

Multi-dimensional fluorescence lifetime and FRET measurements.

When and where proteins associate with each other in living cells are key questions in many biological research projects. One way to address these questions is to measure the extent of Förster resonance energy transfer (FRET) between proteins that have been labeled with appropriate donor and acceptor fluorophores. When both proteins interact, donor and acceptor fluorophores are brought into close vicinity so that the donor can transmit a part of its excitation energy to the acceptor. As a result, both the intensity and the lifetime of the donor fluorescence decrease, whereas the intensity of the acceptor emission increases. This offers different approaches to determine FRET efficiency: One is to detect changes in the intensity of donor and acceptor emission, the other is to measure changes in the lifetime of the donor molecule. One important advantage of the fluorescence lifetime approach is that it allows to distinguish between free and associated donor molecules. However, like intensity measurements it lacks an intrinsic control ensuring that changes in the measured parameters are only due to FRET and not other quenching processes. Here, we show how this limitation can be overcome by spectrally resolved fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting approach. Both approaches allow biologists to record both donor and acceptor fluorescence emitted by the sample in a single measurement.

Multispectral fluorescence lifetime imaging by TCSPC.

We present a fluorescence lifetime imaging technique with simultaneous spectral and temporal resolution. The technique is fully compatible with the commonly used multiphoton microscopes and nondescanned (direct) detection. An image of the back-aperture of the microscope lens is projected on the input of a fiber bundle. The input of the fiber bundle is circular, and the output is flattened to match the input slit of a spectrograph. The spectrum at the output of the spectrograph is projected on a 16-anode PMT module. For each detected photon, the encoding logics of the PMT module deliver a timing pulse and the number of the PMT channel in which the photon was detected. The photons are accumulated by a multidimensional time-correlated single photon counting (TCSPC) process. The recording process builds up a four-dimensional photon distribution over the times of the photons in the excitation pulse period, the wavelengths of the photons, and the coordinates of the scan area. The method delivers a near-ideal counting efficiency and is capable of resolving double-exponential decay functions. We demonstrate the performance of the technique for autofluorescence imaging of tissue.

Time-correlated single photon counting FLIM: some considerations for physiologists.

Recent developments in cellular imaging now permit the minimally invasive study of protein interactions in living cells. These advances are of enormous interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Up until recently, all protein interactions had to be determined in vitro using biochemical approaches. This biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with spatial resolution. More recent developments in TCSPC imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. Here, we discuss some findings we have made along the way which may be useful for physiologists to consider.

Functionally and spatially distinct modes of munc18-syntaxin 1 interaction.

Eukaryotic membrane trafficking is a conserved process under tight temporal and spatial regulation in which the fusion of membranes is driven by the formation of the ternary SNARE complex. Syntaxin 1a, a core component of the exocytic SNARE complex in neurons and neuroendocrine cells, is regulated directly by munc18-1, its cognate Sec1p/munc18 (SM) protein. SM proteins show remarkable structural conservation throughout evolution, indicating a common binding mechanism and function. However, SM proteins possess disparate binding mechanisms and regulatory effects with munc18-1, the major brain isoform, classed as atypical in both its binding specificity and its mode. We now show that munc18-1 interacts with syntaxin 1a through two mechanistically distinct modes of binding, both in vitro and in living cells, in contrast to current models. Furthermore, these functionally divergent interactions occur at distinct cellular locations. These findings provide a molecular explanation for the multiple, spatially distinct roles of munc18-1.

[Cotyla quid? On the early history of late medieval medical volume calculations]. / Cotyla quid? Zur Vorgeschichte des spätmittelalterlichen Medizinalhohlmasses.

As can be made evident chiefly by their comparative numerical examination, the Egyptian pyramids (the step pyramids being excluded for the present purpose) have been, from the beginning up to the Egyptian fashion in early Imperial Rome, designed and built with the additional intention of physically manifesting a volume of pi x 10k x (average value) 0.96824 cm3, where k is either a positive integer or zero, and where pi is a short product, following very restrictive formation rules which to some extent are traceable in the papyrus Rhind, of prime numbers. Conceptually (but not really as to the Hin at least) this establishes the capacity units 1 [2]Heqat = 9682.4 cm3 and 1 Hin = 484.12 cm3 already for the Old Kingdom. It is shown further that the Attic Medimnos as introduced in the course of finishing Solon's reforms is identical with the Egyptian volume system's standard unification: pisigma = 2 x 3 x 5 x 7 x 11 x 23, and k = 0, so that 1 Medimnos = about 51443 cm3. Accordingly and by means of some adjacent considerations a Kotyle / Cotyla of 269 cm3 +/- 1 cm3 is established for the Hellenistic, early Arabic, and Medieval Latin medicine.

Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging.

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.