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. Environ Toxicol Chem 2016;35:823-835. # 2015 SETAC

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.

Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40 degrees fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 +/- 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510-560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 approximately 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus.

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.In neuronal and neuroendocrine cells, exocytosis is mediated by the plasma membrane proteins (t-SNAREs) 2 syntaxin and SNAP-25 (synaptosome-associated protein 25 kDa) and the vesicular protein synaptobrevin (v-SNARE) (1, 2). The cytoplasmic regions of these three proteins interact to form a trimeric, four-helical complex, the generation of which drives fusion of the two opposing bilayers (3). This process is regulated by a conserved set of accessory proteins that operate throughout the trafficking pathway. The Sec1p/munc18 (SM) protein family represents one such set of modulators with SM protein mutations characterized by a severe disruption of general secretion or neurotransmitter release (4 -7). The mammalian SM protein munc18-1 was originally isolated as a syntaxin 1-binding protein that binds to the monomeric form of syntaxin 1, rendering the t-SNARE unable to form the SDS-resistant ternary SNARE complex (8, 9). Syntaxin 1 can act as a molecular switch, adopting two structurally distinct forms (10). In the open form, the SNARE helix does not interact with the N-terminal three-helical regulatory domain (termed Habc) and has been shown to not interact with munc18-1 (10). In contrast, the closed form of syntaxin 1, in which the N-terminal Habc domain interacts with the SNARE helix, exhibits a high affinity for munc18-1. Association of syntaxin 1 with its SNARE partners to form the ternary SNARE complex, which prevents syntaxin from adopting the closed conformation, has also been shown to preclude munc18-1 binding (11). However, a recent finding by Zilly et al. (12) using lysed cellular membrane sheets provided evidence that munc18-1 may interact with syntaxin 1 when in the binary SNARE complex (a heterodimer of syntaxin and SNAP-25).The interaction of munc18-1 with its cognate syntaxin is in sharp contrast to the specificity of its yeast homologue Sec1p, which binds its cognate syntaxin, Sso1p, in the ternary SNARE complex and not in the monomeric state (13). This binding specificity has, ...

SummaryWe present a novel, multi-dimensional, time-correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser-scanning microscope operated at a pixel dwell-time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi-exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed-distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non-FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error range. To confirm that this method can be used also to quantify intermolecular FRET, we labelled cultured neurones with the styryl dye FM1-43, quantified the fluorescence lifetime, then quenched its fluorescence using FM4-64, an efficient energy acceptor for FM1-43 emission. These experiments confirmed directly for the first time that FRET occurs between these two chromophores, characterized the lifetimes of these probes, determined the interchromophore distance in the plasma membrane and provided high-resolution two-dimensional images of lifetime distributions in living neurones.

The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).

~u t h o r for correspondence, facsimile: +30 314 2 1122.Abstract. Measurements of time-resolved fluorescence decay, laser-flash-induced absorption changes in the UV and at 820 nm and of the relative fluorescence quantum yield in different preparations (thylakoids, photosystem 11 (PSII) membrane fragments and PSII core complexes) from spinach led to a number of conclusions. (I) Light is transformed into Gibbs energy with trapping times of 250 ps and 130 ps in open reaction centres of PSII membrane fragments and PSII core complexes, respectively. Assuming rapid Boltzmann distribution of excitation energy and taking into account the antenna properties (size and spectral distribution), the molecular rate constant of primary charge separation is estimated to be about (3 ps)-'. (2) The electron transfer from Pheo-to Q, is characterised by a rate constant of (300 p -' . (3) The Q i reoxidation kinetics are significantly retarded in D20 suspensions. These HID isotope effects are interpreted as to reflect hydrogen-bond dependent changes in the protein dynamics that are relevant to electron transfer. (4) In PSII reaction centres closed for photochemical trapping the yield of a primary radical pair with lifetimes exceeding 1 ns is comparatively small (c 30%) at room temperature. Short illumination in the presence of Na2S204 changes the radical pair dynamics. (5) Photoinhibition under aerobic conditions impairs the primary charge separation and leads to formation of quencher(s) of excitation energy.

Advanced time-correlated single-photon counting (TCSPC) devices are able to record several 106 photons per second and deliver an instrument response function down to 25 ps FWHM. Under these conditions the accuracy of fluorescence decay or photon migration times is limited by systematic timing errors rather than by the photon statistics. The experiments described below determined the variation of the instrument response function (IRF) with the count rate and the timing drift for an SPC-140 TCSPC module and a number of commonly used detectors. For count rates from 3⋅10 4 to 4⋅10 6 s-1 a shift of the first moment of the IRF smaller than 2 ps was obtained. The drift over 16 minutes was within ±0.7 ps.