Epidemiology of morbidity and mortality in US and Canadian recreational scuba diving.

OBJECTIVES: This study investigates morbidity and mortality suffered by divers in the USA and Canada. STUDY DESIGN: Prospectively recruited probability-weighted sample for estimating the national burden of injury and a weighted retrospective survey for estimating exposure. METHODS: The National Electronic Surveillance System and Canadian Hospitals Injury Reporting and Prevention Program (CHIRPP) were searched for scuba diving injuries. The Divers Alert Network diving fatality database was searched for deaths, and Sports and Fitness Industry Association estimates for diving were obtained from annual surveys. RESULTS: In the USA, there were an estimated 1394 emergency department (ED) presentations annually for scuba-related injuries. The majority (80%) were treated and/or released. There were an estimated 306 million dives made by the US residents 2006-2015 and concurrently 563 recreational diving deaths, a fatality rate of 0.18 per 105 dives and 1.8 per 105 diver-years. There were 658 diving deaths in the US 2006-2015 and 13,943 ED presentations for scuba injuries, giving a ratio of 47 diving deaths in the USA for every 1000 ED presentations. There were 98 cases of scuba-related injuries identified in the CHIRPP data. The prevalence of scuba-related injuries for patients aged 3-17 years was 1.5 per 105 cases, and the prevalence of scuba-related injuries to patients 18-62 years was 16.5 per 105 cases. DISCUSSION: In Canada and the USA, only one out of every 10,000 ED presentations is due to a scuba-related injury. That there are 47 deaths for every 1000 ED presentations for scuba injuries speaks to the relatively unforgiving environment in which scuba diving takes place. For 1.8 deaths per million recreational dives, mortality in scuba diving is nonetheless relatively low.

IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen.

BACKGROUND: Allergen-specific immunotherapy (AIT) with birch pollen generates Bet v 1-specific immunoglobulin (Ig)G4 which blocks IgE-mediated hypersensitivity mechanisms. Whether IgG4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG4 antibodies are developed is under debate. OBJECTIVE: We sought to analyze the epitope specificities of IgE and IgG4 antibodies from sera of patients who received AIT. METHODS: 15 sera of patients (13/15 received AIT) with Bet v 1a-specific IgE and IgG4 were analyzed. The structural arrangements of recombinant (r)Bet v 1a and rBet v 1a_11x , modified in five potential epitopes, were analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. IgE binding to Bet v 1 was assessed by ELISA and mediator release assays. Competitive binding of monoclonal antibodies specific for Bet v 1a and serum IgE/IgG4 to rBet v 1a and serum antibody binding to a non-allergenic Bet v 1-type model protein presenting an individual epitope for IgE was analyzed in ELISA and western blot. RESULTS: rBet v 1a_11x had a Bet v 1a - similar secondary and tertiary structure. Monomeric dispersion of rBet v 1a_11x was concentration and buffer-dependent. Up to 1500-fold increase in the EC50 for IgE-mediated mediator release induced by rBet v 1a_11x was determined. The reduction of IgE and IgG4 binding to rBet v 1a_11x was comparable in 67% (10/15) of sera. Bet v 1a-specific monoclonal antibodies inhibited binding of serum IgE and IgG4 to 66.1% and 64.9%, respectively. Serum IgE and IgG4 bound specifically to an individual epitope presented by our model protein in 33% (5/15) of sera. CONCLUSION AND CLINICAL RELEVANCE: Patients receiving AIT develop Bet v 1a-specific IgG4 which competes with IgE for partly identical or largely overlapping epitopes. The similarities of epitopes for IgE and IgG4 might stimulate the development of epitope-specific diagnostics and therapeutics.

The conformational IgE epitope profile of soya bean allergen Gly m 4.

BACKGROUND: Birch pollen-related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown. OBJECTIVE: To identify the IgE epitope profile of Gly m 4 in subjects with birch pollen-related soya allergy utilizing an epitope library presented by Gly m 4-type model proteins. METHODS: Sera from patients with (n = 26) and without (n = 19) allergy to soya as determined by oral provocation tests were studied. Specific IgE (Bet v 1/Gly m 4) was determined by ImmunoCAP. A library of 59 non-allergenic Gly m 4-type model proteins harbouring individual and multiple putative epitopes for IgE was tested in IgE binding assays. Primary, secondary and tertiary protein structures were assessed by mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy. RESULTS: All subjects were sensitized to Gly m 4 and Bet v 1. Allergen-specific serum IgE levels ranged from 0.94 to > 100 kUA /L. The avidities of serum IgE were 5.06 ng (allergic) and 1.8 ng (tolerant) as determined by EC50 for IgE binding to Gly m 4. 96% (46/48) of the protein variants bound IgE. Model proteins had Gly m 4-type conformation and individual IgE binding clustered in six major surface areas. Gly m 4-specific IgE binding could be inhibited to up to 80% by model proteins harbouring individual IgE binding sites in an epitope-wise equimolar fashion. Receiver operating curve analysis revealed an area under fitted curve of up to 0.88 for model proteins and 0.66 for Gly m 4. CONCLUSION AND CLINICAL RELEVANCE: Serum levels and avidity of Gly m 4-specific IgE do not correlate with clinical reactivity to soya. Six IgE-binding areas, represented by 23 amino acids, account for more than 80% of total IgE binding capacity of Gly m 4. Model proteins may be used for epitope-resolved diagnosis to differentiate birch-soya allergy from clinical tolerance.

Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen.

BACKGROUND: IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP-SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross-reactivity of BP-SIT-induced Bet v 1-specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and IgE-blocking activity of Bet v 1-specific IgG4 antibodies emerging during conventional BP-SIT and whether IgG4-epitopes overlapped with IgE epitopes. METHODS: IgE and IgG4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen-allergic patients before and during BP-SIT. Inhibition of IgE binding was studied by IgE-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of IgE-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by IgE and IgG4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3D structure of the allergens using a computer-based algorithm. RESULTS: BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies. In parallel, allergen-specific IgE levels decreased significantly. Sera containing food allergen-reactive IgG4 antibodies inhibited IgE binding, basophil activation, and IgE-mediated food allergen-induced T-cell proliferation. Predicted IgE and IgG4 epitopes on all allergens showed high overlap. CONCLUSION: Our results indicate that BP-SIT may induce Bet v 1-specific IgG4 antibodies that cross-react with related food allergens and inhibit IgE binding by epitope competition.

Ultracold electron bunch generation via plasma photocathode emission and acceleration in a beam-driven plasma blowout.

Beam-driven plasma wakefield acceleration using low-ionization-threshold gas such as Li is combined with laser-controlled electron injection via ionization of high-ionization-threshold gas such as He. The He electrons are released with low transverse momentum in the focus of the copropagating, nonrelativistic-intensity laser pulse directly inside the accelerating or focusing phase of the Li blowout. This concept paves the way for the generation of sub-µm-size, ultralow-emittance, highly tunable electron bunches, thus enabling a flexible new class of an advanced free electron laser capable high-field accelerator.

3D printing of resilient biogels for omnidirectional and exteroceptive soft actuators.

Soft robotics greatly benefits from nature as a source of inspiration, introducing innate means of safe interaction between robotic appliances and living organisms. In contrast, the materials involved are often nonbiodegradable or stem from nonrenewable resources, contributing to an ever-growing environmental footprint. Furthermore, conventional manufacturing methods, such as mold casting, are not suitable for replicating or imitating the complexity of nature's creations. Consequently, the inclusion of sustainability concepts alongside the development of new fabrication procedures is required. We report a customized 3D-printing process based on fused deposition modeling, printing a fully biodegradable gelatin-based hydrogel (biogel) ink into dimensionally stable, complex objects. This process enables fast and cost-effective prototyping of resilient, soft robotic applications from gels that stretch to six times their original length, as well as an accessible recycling procedure with zero waste. We present printed pneumatic actuators performing omnidirectional movement at fast response times (less than a second), featuring integrated 3D-printed stretchable waveguides, capable of both proprio- and exteroception. These soft devices are endowed with dynamic real-time control capable of automated search-and-wipe routines to detect and remove obstacles. They can be reprinted several times or disposed of hazard-free at the end of their lifetime, potentially unlocking a sustainable future for soft robotics.

Hepatitis C virus replication in mice with chimeric human livers.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Species-specific binding of CD4 to the beta 2 domain of major histocompatibility complex class II molecules.

Exon-shuffled constructs between mouse (IA beta b) and human (DR3 beta) class II beta chains were made to study the interaction sites between CD4 and major histocompatibility complex (MHC) class II molecules, and to determine whether a species barrier is involved. The overall structure and the peptide binding groove appeared to be unaffected by the exon shuffling procedure as determined by monoclonal antibody and peptide binding assays, respectively. While purified CD4+ BALB/c T cells responded strongly in a mixed leukocyte reaction to transfectants expressing the whole IA molecule, the response to IA molecules containing a DR beta 2 domain was substantially reduced. In addition, the presence of an IA beta 2 domain in DR failed to restore the weak xenoreactivity to the whole DR molecule. Similar observations were made with murine HEL-specific, IA alpha k beta b-restricted T cell hybridomas which responded significantly stronger to the whole compared with the exon-shuffled IA molecules. The involvement of CD4 in these differential responses was confirmed by the observation that CD4 loss variants responded to both molecules comparably, and transfection of CD4 into these cells restored the parental phenotype. In contrast, CD4 loss variants transfected with human CD4 responded equally to both the whole and the exon-shuffled molecules. Taken together, these data imply the existence of a partial species barrier, and suggest that CD4 interacts with the beta 2 domain of MHC class II molecules, probably in addition to other contact sites. Models for the interaction of CD4 with MHC class II molecules are presented.

Global CO2 emissions from dry inland waters share common drivers across ecosystems.

Many inland waters exhibit complete or partial desiccation, or have vanished due to global change, exposing sediments to the atmosphere. Yet, data on carbon dioxide (CO2) emissions from these sediments are too scarce to upscale emissions for global estimates or to understand their fundamental drivers. Here, we present the results of a global survey covering 196 dry inland waters across diverse ecosystem types and climate zones. We show that their CO2 emissions share fundamental drivers and constitute a substantial fraction of the carbon cycled by inland waters. CO2 emissions were consistent across ecosystem types and climate zones, with local characteristics explaining much of the variability. Accounting for such emissions increases global estimates of carbon emissions from inland waters by 6% (~0.12 Pg C y-1). Our results indicate that emissions from dry inland waters represent a significant and likely increasing component of the inland waters carbon cycle.

Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns.

A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.

Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland.

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-order fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1.5 M KCl) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of Mr 55,000, 48,000, and 40,000 was noted. On two-dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (Mr 48,000, and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the Mr 55,000 protein which focused at pH values higher than 6.4. The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins. Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell. The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue-specific patterns of their protein subunits.

Neoadjuvant radiotherapy followed by surgery compared with surgery alone in the treatment of retroperitoneal sarcoma: a population-based comparison.

Introduction: Retroperitoneal sarcoma (rps) encompasses a heterogeneous group of malignancies with a high recurrence rate after resection. Neoadjuvant radiotherapy (nrt) is often used in the hope of sterilizing margins and decreasing local recurrence after excision. We set out to compare local recurrence-free survival (lrfs) and overall survival (os) in patients treated with or without nrt before resection. Methods: Patients diagnosed with rps from February 1990 to October 2014 were identified in the Alberta Cancer Registry. Patients with complete gross resection of rps and no distant disease were included. Patient, tumour, treatment, and outcomes data were abstracted in a primary chart review. Baseline characteristics were compared using the Wilcoxon nonparametric test for continuous data and the Fisher exact test for dichotomous and categorical data. Survival was analyzed using Kaplan-Meier curves with log-rank test. Cox regression was performed to control for age, sex, tumour size, tumour grade, date of diagnosis, multivisceral resection, and intraoperative rupture. Results: Resection alone was performed in 62 patients, and resection after nrt, in 40. Use of nrt was associated with multivisceral resection and negative microscopic margins. On univariate analysis, nrt was associated with superior median lrfs (89.3 months vs. 28.4 months, p = 0.04) and os (119.4 months vs. 75.9 months, p = 0.04). On multivariate analysis, nrt, younger age, and lower tumour grade predicted improved lrfs and os; sex, tumour size, date of diagnosis, multivisceral resection, and tumour rupture did not. Conclusions: In this population-based study, nrt was associated with superior lrfs and os on both univariate and multivariate analysis. When feasible, nrt should be considered until a randomized controlled trial is completed.

Factors associated with negative margins of lumpectomy specimen: potential use in selecting patients for intraoperative radiotherapy.

BACKGROUND: Lumpectomy followed by radiation is standard treatment for early breast cancer. Recently, the use of partial breast intraoperative radiation (IORT) has been developed, and patients selected for IORT should not have positive margins. This study's purpose was to identify factors predicting negative margins after lumpectomy. METHODS: Patient age, preoperative investigations, surgery, final pathology, and margin status were examined using a prospective database between 1999 and 2005. Univariate and multivariate logistic regression analysis were performed to identify patient and tumor factors predicting an increased rate of negative margins. The results were used to generate a patient selection algorithm. RESULTS: The rate of positive margins at first resection was 17% in 730 lumpectomies (708 patients). Multivariate analysis revealed that older age (P = .0006), smaller tumor size (P < .0025), type of surgery (OR = 3.4 for ultrasound vs mammogram-guided wire localization, P = .003), and having a core needle biopsy (CNB) with preoperative cancer diagnosis (P < .0001) were predictive for having a negative margin. Patients older than age 50 with a preoperative CNB showing invasive cancer less that 3 cm that can be localized under ultrasound had a negative margin rate of 98% (n = 178). These patients would be ideal for consideration of IORT. CONCLUSIONS: Negative margin rates after lumpectomy are predicted by age, tumor size, preoperative investigations, and localization technique. These variables can be used to select patients for IORT with a 2.2% chance of positive margins.

Combined treatment of colorectal tumours with agonistic TRAIL receptor antibodies HGS-ETR1 and HGS-ETR2 and radiotherapy: enhanced effects in vitro and dose-dependent growth delay in vivo.

We and others have demonstrated already that TRAIL (TNF-related apoptosis-inducing ligand) is a very promising candidate for molecular targeted anticancer therapy, especially when combined with ionizing radiation or other DNA-damaging agents. Agonist monoclonal antibodies that activate and are specific for the death signaling TRAIL receptors are an alternative method to stimulate the programmed cell death pathway. Phase 1 clinical trials have subsequently been conducted and shown a very good tolerability of these antibodies. In order to assess the efficacy of TRAIL receptor stimulation to induce cell death by this alternate method, we studied the combination of the agonistic-TRAIL receptor antibodies HGS-ETR1 and HGS-ETR2 with radiation in vitro and in vivo. Induction of apoptosis after combined treatment with TRAIL receptor antibodies HGS-ETR1 and/or HGS-ETR2 (0.01, 0.1, 1.0 mg/ml) and irradiation with 2, 5 or 10 Gy was determined by fluorescence microscopy and Western blot analysis of caspase-8 and PARP. The colorectal tumour cell lines Colo 205, HCT 116 and HCT-15 were used for in vitro experiments. Growth delay experiments were performed with combined treatment with fractionated irradiation (days 1-5 and 3 Gy single dose/day) and the receptor antibodies (intraperitonially, three different concentrations, application on days 1, 4 and 8) on Colo 205 xenograft-bearing NMRI (nu/nu) nude mice. HGS-ETR1 and HGS-ETR2 induced apoptotic cell death in a dose-dependent fashion and significantly increased cell death in combination with irradiation in vitro when compared to either irradiation or antibody treatment alone. The efficacy of the combined treatment seems to be at least partially Bax-dependent. Similar to the results from cell culture experiments, in vivo experiments demonstrated a dose-dependent delay in tumour growth after combined treatment. In vivo, in the Colo205 xenograft model, HGS-ETR2 revealed a higher activity than HGS-ETR1. This is the first study to demonstrate significant efficacy of combined treatment with the monoclonal agonistic TRAIL receptor antibodies HGS-ETR1 and HGS-ETR2 and ionising radiation in in vitro and in vivo models. We postulate that HGS-ETR1 and HGS-ETR2 will be very promising new agents in the field of molecular targeted multi-modality anticancer therapy.

Ultra low dose interleukin-2 therapy promotes a type 1 cytokine profile in vivo in patients with AIDS and AIDS-associated malignancies.

This study was undertaken to determine if prolonged daily subcutaneous administration of ultra low dose IL-2 could influence the constitutive endogenous production of a type 1 (IFN-gamma) cytokine in patients with AIDS or AIDS-associated malignancies. Using a quantitative reverse transcription PCR assay, we demonstrate that daily administration of one type 1 cytokine, IL-2, for 3 mo increases significantly the constitutive endogenous gene expression of another type 1 cytokine, IFN-gamma, in vivo. The predominant source of IFN-gamma appears to be IL-2-expanded natural killer cells and CD8(+) T cells. Moreover, PBMC obtained from these patients during IL-2 therapy showed normalization of a profound deficit in IFN-gamma protein production after stimulation with extracts from infectious agents in vitro. Our data suggest that prolonged exogenous administration of a type 1 cytokine in a nontoxic fashion to patients with AIDS and AIDS-associated malignancies can enhance significantly the endogenous type 1 cytokine profile in vivo. Consequently, ultra low dose IL-2 therapy has the potential to improve the immunodeficient hosts' immune response to infectious pathogens that require IFN-gamma for clearance.

Attenuation of pharmaceuticals and their transformation products in a wastewater treatment plant and its receiving river ecosystem.

Pharmaceuticals are designed to improve human and animal health, but may also be a threat to freshwater ecosystems, particularly after receiving urban or wastewater treatment plant (WWTP) effluents. Knowledge on the fate and attenuation of pharmaceuticals in engineered and natural ecosystems is rather fragmented, and comparable methods are needed to facilitate the comprehension of those processes amongst systems. In this study the dynamics of 8 pharmaceuticals (acetaminophen, sulfapyridine, sulfamethoxazole, carbamazepine, venlafaxine, ibuprofen, diclofenac, diazepam) and 11 of their transformation products were investigated in a WWTP and the associated receiving river ecosystem. During 3 days, concentrations of these compounds were quantified at the influents, effluents, and wastage of the WWTP, and at different distances downstream the effluent at the river. Attenuation (net balance between removal and release from and to the water column) was estimated in both engineered and natural systems using a comparable model-based approach by considering different uncertainty sources (e.g. chemical analysis, sampling, and flow measurements). Results showed that pharmaceuticals load reduction was higher in the WWTP, but attenuation efficiencies (as half-life times) were higher in the river. In particular, the load of only 5 out of the 19 pharmaceuticals was reduced by more than 90% at the WWTP, while the rest were only partially or non-attenuated (or released) and discharged into the receiving river. At the river, only the load of ibuprofen was reduced by more than 50% (out of the 6 parent compounds present in the river), while partial and non-attenuation (or release) was observed for some of their transformation products. Linkages in the routing of some pharmaceuticals (venlafaxine, carbamazepine, ibuprofen and diclofenac) and their corresponding transformation products were also identified at both WWTP and river. Finally, the followed procedure showed that dynamic attenuation in the coupled WWTP-river system could be successfully predicted with simple first order attenuation kinetics for most modeled compounds.

Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum.

The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum, was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in beta-dodecylmaltoside and purified by streptactin affinity chromatography. Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution. Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane. Reconstitution with E.coli polar lipids produced 2D crystals with unit cell parameters of 182A x 154A, gamma=90 degrees exhibiting p22(1)2(1) symmetry. Electron cryo-microscopy yielded a projection map at 7.5A. The unit cell contains four non-crystallographic trimers of BetP. Within each monomer, ten to 12 density peaks characteristic of transmembrane alpha-helices surround low-density regions that define potential transport pathways. Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation. The projection map of BetP shows no clear resemblance to other secondary transporters of known structure.