Performance of full scale constructed wetlands in removing antibiotics and antibiotic resistance genes.

Additional treatment of wastewater, such as constructed wetlands (CWs), is a possible solution to reduce the discharge of antibiotics and antibiotic resistance genes (ARGs) from households and industry to the environment. This study aims to investigate the occurrence and removal of antibiotics and ARGs by two full scale CWs operated at different hydraulic retention times (HRT), namely 1 day and 3 days. Both CWs were receiving the same wastewater treatment plant (WWTP) effluent. Temporally and spatially distributed sampling of water and sediment was conducted for one year and samples were analyzed for antibiotics and ARGs by using LC-MS/MS and qPCR. Results showed that both CWs removed antibiotics significantly with a comparable overall removal of 28%-100%, depending on the type of antibiotics. However, some of the antibiotics showed higher concentration after the CW treatment. Five antibiotics (tiamulin, tylosin, oxytetracycline, sulfamethoxazole and trimethoprim) were the most abundant (>1500 ng/l on average) in winter. Meanwhile, ermB was the most abundant (average of 5.0 log) in winter compared to summer (average of 3.5 log). Other ARGs did not show a significant increase or decrease between winter and summer. ARGs were removed from the wastewater by 0.8 to 1.5 log. The HRT did not influence the removal of either the antibiotics or the ARGs. A strong correlation was found between sul genes and intI1. The results also revealed a positive and a negative relationship from sampling point 1 to sampling point 5: a positive relation between abundance of antibiotics, ARGs, and of NO3-N, NH4-N, TP, COD and a negative relation between antibiotics, ARGs and temperature. This relationship showed the effect between antibiotics and ARGs concentrations with physicochemical parameters and nutrients. The ability of CWs to reduce the input of micropollutants into the environment makes CWs a potential post treatment to WWTP.

Fate of antibiotics and antibiotic resistance genes during conventional and additional treatment technologies in wastewater treatment plants.

Information on the removal of antibiotics and ARGs in full-scale WWTPs (with or without additional treatment technology) is limited. However, it is important to understand the efficiency of full-scale treatment technologies in removing antibiotics and ARGs under a variety of conditions relevant for practice to reduce their environmental spreading. Therefore, this study was performed to evaluate the removal of antibiotics and ARGs in a conventional wastewater treatment plant (WWTP A) and two full-scale combined with additional treatment technologies. WWTP B, a conventional activated sludge treatment followed by an activated carbon filtration step (1-STEP® filter) as a final treatment step. WWTP C, a treatment plant using aerobic granular sludge (NEREDA®) as an alternative to activated sludge treatment. Water and sludge were collected and analysed for 52 antibiotics from four target antibiotic groups (macrolides, sulfonamides, quinolones, tetracyclines) and four target ARGs (ermB, sul 1, sul 2 and tetW) and integrase gene class 1 (intI1). Despite the high removal percentages (79-88%) of the total load of antibiotics in all WWTPs, some antibiotics were detected in the various effluents. Additional treatment technology (WWTP C) showed antibiotics removal up to 99% (tetracyclines). For ARGs, WWTP C reduced 2.3 log followed by WWTP A with 2.0 log, and WWTP B with 1.3 log. This shows that full-scale WWTP with an additional treatment technology are promising solutions for reducing emissions of antibiotics and ARGs from wastewater treatment plants. However, total removal of the antibiotics and ARGS cannot be achieved for all types of antibiotics and ARGs. In addition, the ARGs were more abundant in the sludge compared to the wastewater effluent suggesting that sludge is an important reservoir representing a source for later ARG emissions upon reuse, i.e. as fertilizer in agriculture or as resource for bioplastics or bioflocculants. These aspects require further research.

Towards robust and versatile single nanoparticle fiducial markers for correlative light and electron microscopy.

Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye-labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can (partially) withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold-core rhodamine B-labelled silica particles, 15 nm CdSe/CdS/ZnS core-shell-shell quantum dots (QDs) and 230 nm Y2 O3 :Eu3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold-core rhodamine B-labelled silica NPs and QDs are quenched after a single exposure to 60 ke-  nm-2 with an energy of 120 keV, while Y2 O3 :Eu3+ NPs are robust and still show luminescence after five doses of 60 ke- nm-2 . In addition, the luminescence intensity of Y2 O3 :Eu3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y2 O3 :Eu3+ NPs are promising as robust fiducial marker in CLEM. LAY DESCRIPTION: Luminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye-labelled silica particles, quantum dots and lanthanide-doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye-labelled silica NPs and QDs are quenched after a single exposure to 60 ke- nm-2 with an energy of 120 keV, while lanthanide-doped inorganic NPs are robust and still show luminescence after five doses of 60 ke- nm-2 . In addition, the luminescence intensity of lanthanide-doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide-doped NPs are promising as robust fiducial marker in CLEM.

Spatial Overlap of Grey Seals and Fisheries in Irish Waters, Some New Insights Using Telemetry Technology and VMS.

Seals and humans often target the same food resource, leading to competition. This is of mounting concern with fish stocks in global decline. Grey seals were tracked from southeast Ireland, an area of mixed demersal and pelagic fisheries, and overlap with fisheries on the Celtic Shelf and Irish Sea was assessed. Overall, there was low overlap between the tagged seals and fisheries. However, when we separate active (e.g. trawls) and passive gear (e.g. nets, lines) fisheries, a different picture emerged. Overlap with active fisheries was no different from that expected under a random distribution, but overlap with passive fisheries was significantly higher. This suggests that grey seals may be targeting the same areas as passive fisheries and/or specifically targeting passive gear. There was variation in foraging areas between individual seals suggesting habitat partitioning to reduce intra-specific competition or potential individual specialisation in foraging behaviour. Our findings support other recent assertions that seal/fisheries interactions in Irish waters are an issue in inshore passive fisheries, most likely at the operational and individual level. This suggests that seal population management measures would be unjustifiable, and mitigation is best focused on minimizing interactions at nets.

Snapshot coherence-gated direct wavefront sensing for multi-photon microscopy.

Deep imaging in turbid media such as biological tissue is challenging due to scattering and optical aberrations. Adaptive optics has the potential to compensate the tissue aberrations. We present a wavefront sensing scheme for multi-photon scanning microscopes using the pulsed, near-infrared light reflected back from the sample utilising coherence gating and a confocal pinhole to isolate the light from a layer of interest. By interfering the back-reflected light with a tilted reference beam, we create a fringe pattern with a known spatial carrier frequency in an image of the back-aperture plane of the microscope objective. The wavefront aberrations distort this fringe pattern and thereby imprint themselves at the carrier frequency, which allows us to separate the aberrations in the Fourier domain from low spatial frequency noise. A Fourier analysis of the modulated fringes combined with a virtual Shack-Hartmann sensor for smoothing yields a modal representation of the wavefront suitable for correction. We show results with this method correcting both DM-induced and sample-induced aberrations in rat tail collagen fibres as well as a Hoechst-stained MCF-7 spheroid of cancer cells.

Switching CdSe quantum dot luminescence with a-Si:H.

Dynamical control of the luminescence of quantum dots is highly important for technology in the field of telecommunication, displays, and photovoltaics. In this work we use an a-Si:H solar cell structure in which CdSe quantum dots are sandwiched. By applying a positive potential over the device, charge carriers generated in the quantum dots are transported to the a-Si:H layer and transformed into electrical energy, changing the luminescence intensity with a switching time lower than 60 ms. This is a promising new step towards using quantum dots in optical switching devices.

Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells.

A modified phasor approach for analyzing time-gated fluorescence lifetime images.

Fluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, time-correlated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens. Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (∼10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells. We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data.

High sensitivity and specificity of the C6-peptide ELISA on cerebrospinal fluid in Lyme neuroborreliosis patients.

Lyme neuroborreliosis (LNB) is a serious but treatable disease. The diagnosis of LNB poses a challenge to clinicians, and improved tests are needed. The C6-peptide ELISA is frequently used on serum but not on cerebrospinal fluid (CSF). Data on the sensitivity of the C6-peptide ELISA in CSF in patients suffering from LNB have been conflicting. Serum-CSF pairs from 59 LNB patients, 36 Lyme non-neuroborreliosis cases, 69 infectious meningitis/encephalitis controls and 74 neurological controls were tested in a C6-peptide ELISA. With the optimal cut-off of 1.1, the sensitivity of the C6-peptide ELISA for LNB patients in CSF was 95%, and the specificity was 83% in the Lyme non-neuroborreliosis patients, 96% in the infectious controls, and 97% in the neurological controls. These results suggest that the C6-peptide ELISA has a high sensitivity and good specificity for the diagnosis of LNB patients in CSF. The C6-peptide ELISA can be used on CSF in a clinical setting to screen for LNB.

Axillary recurrences after negative sentinel lymph node biopsy under local anaesthesia for breast cancer: a follow-up study after 5 years.

INTRODUCTION: Sentinel lymph node biopsy (SLNB) is accepted as a standard surgical staging procedure for determining the tumour status of the regional lymph nodes. Until September 2000 we performed SLNB in general anaesthesia. Since 1999, after validation of the SLNB concept, axillary dissection was omitted in SLN-negative patients. This study presents our data after SLNB under local anaesthesia after a follow-up of at least 5 years. MATERIALS AND METHODS: Between September 2000 and May 2003, 356 SLNBs were performed under local anaesthesia without sedation in patients with proven breast cancer (T4-tumours and small in situ carcinomas excluded) and without clinically or ultrasound guided cytological evidence of axillary node involvement. Lymphatic mapping and SLN identification were performed through the combination of blue dye and 99m Tc-nanocolloid. All positive SLNs were followed by an axillary dissection up to level three. SLN-negative patients were followed without axillary clearance. RESULTS: In 353/356 SLNBs at least one sentinel node was found. 254/353 SLNs were tumour free. After a median follow-up of 73 months loco-regional and distant events were encountered in 10/353 SLNBs. Four patients (SLN-negative) showed tumour localization in the residual breast or chest wall (1.1%). Three patients (SLN-negative) presented with supraclavicular metastases (0.8%). In three patients (one SLN-negative and two SLN-positive followed by ALND) an axillary recurrence was encountered (0.8%). CONCLUSION: This survey confirms the safety of the SLNB under local anaesthesia in selecting patients for axillary lymph node dissection in breast cancer.

Implementation of diffractive optical element in four-wave mixing scheme for ex situ characterization of hydride vapor phase epitaxy-grown GaN layers.

A holographic beam splitter has been integrated into a picosecond four-wave mixing (FWM) scheme. This modification significantly simplified the procedure of dynamic grating recording, thus making the FWM technique an easy-to-use tool for the holographic characterization of wide band gap materials. The novel FWM scheme was applied for characterization of hydride vapor phase epitaxy-grown undoped GaN layers of different thickness. It allowed the determination of carrier lifetime, diffusion coefficient, and carrier diffusion length by optical means, as well as the study of carrier recombination peculiarities with respect to dislocation and excess carrier density.

Quantitative analysis of Tenecteplase in rat plasma samples using LC-MS/MS as an alternative for ELISA.

An LC-MS/MS method has been developed for the quantitative determination of a protein drug (Tenecteplase; M(W) 58,777 Da) in rat plasma. The protein was digested with trypsin without prior clean-up of the plasma sample, without the use of a label nor internal standard. A limited validation was performed to assess the linearity, the sensitivity and the specificity of the method. In addition, the developed method was applied to the quantitative analysis of Tenecteplase in rat plasma samples originating from a single-dose study in rats.

In vivo nonlinear spectral imaging in mouse skin.

We report on two-photon autofluorescence and second harmonic spectral imaging of live mouse tissues. The use of a high sensitivity detector and ultraviolet optics allowed us to record razor-sharp deep-tissue spectral images of weak autofluorescence and short-wavelength second harmonic generation by mouse skin. Real-color image representation combined with depth-resolved spectral analysis enabled us to identify tissue structures. The results show that linking nonlinear deep-tissue imaging microscopy with autofluorescence spectroscopy has the potential to provide important information for the diagnosis of skin tissues.

Short-wavelength two-photon excitation fluorescence microscopy of tryptophan with a photonic crystal fiber based light source.

We report on a novel and simple light source for short-wavelength two-photon excitation fluorescence microscopy based on the visible nonsolitonic radiation from a photonic crystal fiber. We demonstrate tunability of the light source by varying the wavelength and intensity of the Ti:Sapphire excitation light source. The visible nonsolitonic radiation is used as an excitation light source for two-photon fluorescence microscopy of tryptophan powder.

All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique that is increasingly being used in the life sciences during the past decades. However, a broader application of FLIM requires more cost-effective and user-friendly solutions. We demonstrate the use of a simple CCD/CMOS lock-in imager for fluorescence lifetime detection. The SwissRanger SR-2 time-of-flight detector, originally developed for 3D vision, embeds all the functionalities required for FLIM in a compact system. The further development of this technology and its combination with light-emitting-and laser diodes could drive a wider spreading of thuse of FLIM including high-throughput applications.

Fast fluorescence lifetime imaging of calcium in living cells.

A fast fluorescence lifetime imaging (FLIM) system is developed that can acquire images at a rate of hundreds of frames per second. The FLIM system is based on a wide-field microscope equipped with a time-gated intensified CCD detector and a pulsed laser. The time-gated detector acquires the signals from two time gates simultaneously and is therefore insensitive to movements of the specimen and photo-bleaching. The system is well suited for quantitative biological FLIM experiments and its performance is evaluated in calcium imaging experiments on beating neonatal rat myocytes. Several calcium sensitive dyes are characterized and tested for their suitability for fast FLIM experiments: Oregon Green Bapta-1 (OGB1), Oregon Green Bapta-2 (OGB2), and Oregon Green Bapta-5N (OGB5N). Overall the sensitivity range of these dyes is shifted to low calcium concentrations when used as lifetime dyes. OGB1 and OGB2 behave very similarly and can be used for FLIM-based calcium imaging in the range 1 to approximately 500 nM and OGB5N can be used up to 3 microM. The fast FLIM experiments on the myocytes could be carried out at a 100-Hz frame rate. During the beating of the myocytes a lifetime change of about 20% is observed. From the lifetime images a rest calcium level of about 65 nM is found.

Ambulatory sentinel node biopsy under local anaesthesia for patients with early breast cancer.

AIMS: Sentinel lymph node biopsy (SLNB) may permit reliable identification of patients with axillary node involvement. The aim of this study was to report our experience with this procedure under local anaesthesia. METHODS: One hundred and sixty-two patients underwent a sentinel node procedure under local anaesthesia without sedation. The SLN was identified by (99m)Tc-nano-colloid and patent blue. Immediate histopathologic examination and immunohistochemistry was performed. Patients with positive SLNs proceded to axillary dissection under general anaesthesia. RESULTS: In all 162 patients the SLN ('s) were found using blue dye and gamma-probe. The SLN was positive in 55/162 patients (34%). Five of these were detected using immunohistochemistry only. CONCLUSIONS: A 100% detection rate of sentinel nodes in early breast cancer harvested under local anaesthesia was achieved without serious morbidity. This allows the surgeon to select preoperatively the treatment given to the patient.

Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution.

In this paper a detailed discussion is presented of the factors that affect the fluorescence lifetime imaging performance of a scanning microscope equipped with a single photon counting based, two- to eight-channel, time-gated detection system. In particular we discuss the sensitivity, lifetime resolution, acquisition speed, and the shortest lifetimes that can be measured. Detection systems equipped with four to eight time-gates are significantly more sensitive than the two time-gate system. Only minor sensitivity differences were found between systems with four or more time-gates. Experiments confirm that the lifetime resolution is dominated by photon statistics. The time response of the detector determines the shortest lifetimes that can be resolved; about 25 ps for fast MCP-PMTs and 300-400 ps for other detectors. The maximum count rate of fast MCP-PMTs, however, is 10-100 times lower than that of fast PMTs. Therefore, the acquisition speed with MCP-PMT based systems is limited. With a fast PMT operated close to its maximum count rate we were able to record a fluorescence lifetime image of a beating myocyte in less than one second.

Partial amino acid sequence of purified von Willebrand factor-cleaving protease.

von Willebrand factor-cleaving protease (vWF-cp) is responsible for the continuous degradation of plasma vWF multimers released from endothelial cells. It is deficient in patients with thrombotic thrombocytopenic purpura, who show unusually large vWF multimers in plasma. Purified vWF-cp may be useful for replacement in these patients, who are now treated by plasma therapy. In this study, vWF-cp was purified from normal human plasma by affinity chromatography on the IgG fraction from a patient with autoantibodies to vWF-cp and by a series of further chromatographic procedures, including affinity chromatography on Protein G, Ig-TheraSorb, lentil lectin, and heparin. Four single-chain protein bands, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, showed M(r) of 150, 140, 130, and 110 kd and were found to share the same N-terminal amino acid sequence, suggesting that they were derived from the same polypeptide chain that had been partially degraded at the carboxy-terminal end. A hydrophobic sequence (Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly) of the first 15 residues was established. The protease migrates in gel filtration as a high-molecular-weight complex with clusterin, a 70-kd protein with chaperonelike activity. vWF-cp bound to clusterin is dissociated by the use of concentrated chaotropic salts. vWF-cp in normal human plasma or serum is not associated with clusterin, suggesting that the observed complex is due to vWF-cp denaturation during the purification procedure. Activity of vWF-cp is unusually stable during incubation at 37 degrees C; its in vitro half-life in citrated human plasma, heparin plasma, or serum is longer than 1 week. There was even a temporary increase in protease activity during the first 3 days of incubation.

Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy.

Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.