A Survey on Vaccination and Disease Occurrence in Municipal and Non-Profit Animal Shelters in Portugal.

Few studies are available describing animal shelters in Portugal. The aim was to characterize prophylactic measures and disease occurrence in shelters with a questionnaire. The response rates of 67 shelters (42 municipal shelters, 25 associations) were compared by the Fisher's exact test. More veterinarians answered for municipal shelters (98%) than for associations (40%; p < 0.001). Over 80% of the respondents indicated using individual medical records and routine prophylaxis. Excessive length of stay for dogs was reported by 54% of associations and 33% of municipal shelters. Management tools should be promoted to improve the situation. Puppy vaccinations were similar and a final vaccination at 16 weeks was indicated by >33% of shelters. Annual revaccination of dogs was reported more frequently by associations (88%) than municipal shelters (55%; p = 0.02). The three most reported diseases were parvovirus and mange in dogs, upper respiratory disease and panleukopenia in cats, and dermatophytosis in both species. Similar response rates for diagnostic options were obtained by both shelter types, except for distemper. Testing for feline retroviruses was indicated by most shelters (>69%), but only a few (<24%) confirmed positive test results. Clinical diagnoses should be complemented by testing. Additional information on disease occurrence should be obtained by objective monitoring.

Releases of micropollutants from building surface materials into rainwater and snowmelt induced runoff.

Building surface materials, exposed to wash-off by rainwater or snowmelt, are recognised as one of the significant urban diffuse pollution sources contributing to the impairment of stormwater quality. The pollution conveyed by roof runoff originates from two potential sources, migration of surface material constituents, or wash-off of pollutants deposited on the surface by atmospheric deposition. This study investigated the releases of metals and several groups of contaminants of emerging concern: alkylphenols, alkylphenol ethoxylates, and phthalates, from commercially available materials, which are commonly used on buildings and structure surfaces in the urban environment. The materials tested included the following: metal sheets of stainless steel, copper, zinc, galvanised steel, corten steel, corrugated and coated steel, coated zinc; and bitumen-based roofing felt and shingles, as well as polyvinyl chloride (PVC) from two manufacturers. The stainless steel was considered a control material serving to estimate pollutant contributions deposited on the pilot panels from the surrounding environment. Moreover, this study presents novel data on roof snowmelt induced runoff quality, not reported in the previous literature. The experimental setup consisted of 2-m2 rectangular panels mounted in triplicates of each material and placed in an open-air setting on the campus of Luleå University of Technology, Sweden. Runoff leaving the gently sloping material panels was collected during 11 rain and three snowmelt driven runoff events occurring over a five-year period. The results showed that, in general, the micropollutant concentrations and loads were lower in snowmelt than rain induced runoff, and no decreasing trend was detected in the releases of phthalates or metals during the study period. Moreover, on a yearly basis, copper sheets were estimated to release 0.6 g/m2 Cu to runoff, zinc and galvanised sheets 1.3 and 0.7 g/m2 Zn, respectively, and, PVC sheets were estimated to release up to 78 mg/m2 of diisononyl phthalate (DINP).

G-quadruplex ligands as potent regulators of lysosomes.

Guanine-quadruplex structures (G4) are unusual nucleic acid conformations formed by guanine-rich DNA and RNA sequences and known to control gene expression mechanisms, from transcription to protein synthesis. So far, a number of molecules that recognize G4 have been developed for potential therapeutic applications in human pathologies, including cancer and infectious diseases. These molecules are called G4 ligands. When the biological effects of G4 ligands are studied, the analysis is often limited to nucleic acid targets. However, recent evidence indicates that G4 ligands may target other cellular components and compartments such as lysosomes and mitochondria. Here, we summarize our current knowledge of the regulation of lysosome by G4 ligands, underlying their potential functional impact on lysosome biology and autophagic flux, as well as on the transcriptional regulation of lysosomal genes. We outline the consequences of these effects on cell fate decisions and we systematically analyzed G4-prone sequences within the promoter of 435 lysosome-related genes. Finally, we propose some hypotheses about the mechanisms involved in the regulation of lysosomes by G4 ligands.

Synthetic stormwater for laboratory testing of filter materials.

Synthetic stormwater was tested to determine the ageing effects on dissolved metal concentrations and used in a column experiment to determine efficiency of four different filter materials (milkweed, bark, peat, polypropylene) in removing total and dissolved metals. Synthetic stormwater was created by adding metal salts, oil and collected stormwater sediment to tap water. Two ageing experiments were performed to determine the change of synthetic stormwater quality over time. One experiment lasted for 11 days and another focused on rapid concentration changes one day after preparation. The one-day ageing experiment showed rapid decrease in dissolved concentration of certain metals, specifically Cu. To consider this change, correction coefficients for each metal were developed and used to estimate the average dissolved metal concentration in the synthetic stormwater during the experiment to determine filter treatment efficiency. During the 11-day experiment on metal concentrations, no noticeable quality changes were observed for at least six days after the preparation of synthetic stormwater. Furthermore, a column experiment was run with duplicate filter columns. Inflow and outflow samples were analysed for total and dissolved metals, turbidity, particle size distribution, and pH. High removal of total metal concentrations was noticed in all tested filter media (58-94%). Dissolved metal concentration removal varied among different filter media. In general, columns with bark and peat media were able to treat dissolved metals better than polypropylene and milkweed. The level of treatment of dissolved metals between the different filter media columns were bark > peat > milkweed > polypropylene.

On the flexibility of the cellular amination network in E coli.

Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine sources. We found that several amino acids complemented the auxotrophy either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids ß-alanine, alanine, glycine, and serine as new amine sources and identified d-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.

Nitrogen is an essential part of many of the cell's building blocks, including amino acids and nucleotides, which form proteins and DNA respectively. Therefore, nitrogen has to be available to cells so that they can survive and grow. In nature, some microorganisms convert the gaseous form of nitrogen into ammonium, which then acts as the nitrogen source of most organisms, including bacteria, plants and animals. Once cells take up ammonium, it is 'fixed' by becoming part of an amino acid called glutamate, which has a so-called 'amine group' that contains a nitrogen. Glutamate then becomes the central source for passing these amines on to other molecules, distributing nitrogen throughout the cell. This coupling between ammonium fixation and glutamate production evolved over millions of years and occurs in all organisms. However, the complete metabolic network that underlies the distribution of amines remains poorly understood despite decades of research. Furthermore, it is not clear whether ammonium can be fixed in a way that is independent of glutamate. To answer these questions, Schulz-Mirbach et al. used genetic engineering to create a strain of the bacterium E. coli that was unable to make glutamate. These mutant cells could only grow in the presence of certain amino acids, which acted as alternative amine sources. Schulz-Mirbach et al. found that enzymes called transaminases, and one called AspC in particular, were required for the cells to be able to produce glutamate using the amine groups from other amino acids. Notably, Schulz-Mirbach et al. showed that AspC, which had previously been shown to use an amino acid called aspartate as a source of amine groups, is indispensable if the cell is to use the amine groups from other amino acids ­ including histidine, tyrosine, phenylalanine, tryptophan, methionine, isoleucine and leucine. Schulz-Mirbach et al. also discovered that if they engineered the E. coli cells to produce transaminases from other species, the repertoire of molecules that the cells could use as the source of amines to generate glutamate increased. In a final set of experiments, Schulz-Mirbach et al. were able to engineer the cells to fix ammonium by producing aspartate and leucine, thus entirely bypassing the deleted routes of glutamate synthesis. These data suggest that fixing ammonium and distributing nitrogen in E. coli can be very flexible. The results from these experiments may shed light on how cells adapt when there is not a lot of ammonium available. Moreover, this study could advance efforts at metabolic engineering, for example, to create molecules through new pathways or to boost the production of amino acids needed for industrial purposes.

Evaluation of the Introduction of a Colorectal Bundle in Left Sided Colorectal Resections (EvaCol): Study Protocol of a Multicentre, Observational Trial.

Purpose: Overall complication and leak rates in colorectal surgery showed only minor improvements over the last years and remain still high. While the introduction of the WHO Safer Surgery Checklist has shown a reduction of overall operative mortality and morbidity in general surgery, only minor attempts have been made to improve outcomes by standardizing perioperative processes in colorectal surgery. Nevertheless, a number of singular interventions have been found reducing postoperative complications in colorectal surgery. The aim of the present study is to combine nine of these measures to a catalogue called colorectal bundle (CB). This will help to standardize pre-, intra-, and post-operative processes and therefore eventually reduce complication rates after colorectal surgery. Methods: The study will be performed among nine contributing hospitals in the extended north-western part of Switzerland. In the 6-month lasting control period the patients will be treated according to the local standard of each contributing hospital. After a short implementation phase all patients will be treated according to the CB for another 6 months. Afterwards complication rates before and after the implementation of the CB will be compared. Discussion: The overall complication rate in colorectal surgery is still high. The fact that only little progress has been made in recent years underlines the relevance of the current project. It has been shown for other areas of surgery that standardization is an effective measure of reducing postoperative complication rates. We hypothesize that the combination of effective, individual components into the CB can reduce the complication rate. Trial registration: Registered in ClinicalTrials.gov on 11/03/2020; NCT04550156. Highlights: Purpose: Overall complications in colorectal surgery remain still highStandardizing can reduce overall operative mortality and morbidityOnly minor attempts have been made to standardize perioperative processes in colorectal surgerySingular interventions have been found reducing postoperative complicationsThe aim is to combine nine of these measures to a colorectal bundle (CB)The CB will help to reduce complication rates after colorectal surgery Methods: The observational study will be performed among nine hospitals in SwitzerlandSix month the patients will be treated according to the local standardsAfterwards patients will be treated according to the CB for another six monthsComplication rates before and after the implementation of the CB will be compared Discussion: Only little progress has been made to reduce complication rate in colorectal surgeryStandardization is an effective measure of reducing complication ratesThe combination of effective, individual components into the CB can reduce the complication rate.

Exploiting urban roadside snowbanks as passive samplers of organic micropollutants and metals generated by traffic.

Stormwater and snowmelt runoff is known to contribute to the deterioration of quality of urban surface waters. Vehicular traffic is recognised as a major source of a wide range of pollutants to urban runoff, including conventional pollutants, such as suspended solids and metals, and those referred to as 'contaminants of emerging concern'. The aim of this study was to investigate the contribution of selected metal(loid)s (Cd, Cr, Cu, Ni, Pb, Pd, Sb, W, Zn), polycyclic aromatic hydrocarbons (PAHs), nonylphenols, octylphenols and -ethoxylates, phthalates and bisphenol A (BPA) from vehicular traffic by sampling urban roadside snow at eight sites, with varying traffic intensities, and one control site without direct impacts of traffic. Our results confirmed that vehicles and traffic-related activities were the sources of octylphenols, BPA and phthalates as well as the metal(loid)s Sb and W, infrequently reported in previous studies. Among metal(loid)s, Cu, Zn and W occurred in the highest concentrations (up to 1.2 mg/L Cu, 2.4 mg/L Zn and 1.9 mg/L W), while PAHs and phthalates occurred in the highest concentrations among the trace organic pollutants (up to 540 µg/L phthalate diisononyl phthalate). Among the phthalates, di-(2-ethylhexyl)phthalate had the highest frequency of detection (43% of the roadside samples). While BPA and octylphenols had relatively high frequencies of detection (50% for BPA and 81% for octylphenols), they were present in comparatively low concentrations (up to 0.2 µg/L BPA and 1.1 µg/L octylphenols). The control site displayed generally low concentrations of the pollutants studied, indicating that atmospheric deposition was not a significant source of the pollutants found in the roadside snow. Several of the pollutants in the roadside snow exceeded the applicable surface water and stormwater effluent guideline values. Thus, the transport of these pollutants with runoff posed risk of causing adverse effects in the receiving surface waters.

Influence of processing and storage on the iodine content of meat and fish products using iodized salt.

Table salt fortified with KIO3 is commonly used to prevent iodine deficiency disorders. However, there is a lack of reliable data about the stability of KIO3 during food processing. In this study several meat and fish products were prepared with iodized salt and the iodine stability was determined through the whole production process. Applied processes included heating, fermenting, freezing, hot smoking, ripening by enzymes and storing. In all products an increase in iodine content was observed after addition of iodized salt. The iodine content remained constant during most of the applied processes. The only iodine loss was observed in ham after heating and can be explained by loss of iodine containing brine. During subsequent storage no iodine loss was observed in any of the products. The use of KIO3 fortified salt in the investigated products might therefore be beneficial for the iodine supply.

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA.

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

Phenotypic antimicrobial resistance (AMR) of avian pathogenic Escherichia coli (APEC) from broiler breeder flocks between 2009 and 2018.

Colibacillosis is one of the most important diseases in poultry production. The use of antimicrobials remains a therapeutic cornerstone for avian pathogenic E. coli (APEC), thereby contributing to the development of antimicrobial resistance (AMR). The aim of this study was to characterize AMR in broiler breeder flocks reared under commercial conditions. Data covering 10 years, from 2009 to 2018, were used to evaluate the phenotypic AMR of 264 APEC isolates obtained from 158 broiler breeder flocks of a large company in Portugal. The APEC isolates were tested against eleven antimicrobials by the Kirby-Bauer disc diffusion test. The annual proportion of AMR was calculated by dividing the number of APEC isolates with phenotypic resistance by the total number of APEC isolated that year. Similarly, the overall AMR of the whole period was calculated. The relationship of antimicrobial resistance with time (years) was investigated with a generalized linear model using logistic regression. The overall AMR of the 10-year period was: amoxicillin 78%, ampicillin 73.5%, tetracycline 63.3%, doxycycline 56.4%, apramycin 34.5%, neomycin 68.2%, enrofloxacin 32.6%, flumequine 39.4%, co-trimoxazole 47.7%, florfenicol 46.6% and lincospectin 66.3%. Over time, a significant decrease in AMR was observed for amoxicillin and ampicillin, neomycin, flumequine, co-trimoxazole, florfenicol and lincospectin. Multidrug resistance (MDR) decreased from 100% in 2009 to 48% in 2018. Only seven (2.7%) APEC strains were fully susceptible to all tested antimicrobials. The decrease over time of AMR in APEC likely reflects the efficacy of manifold improvements in broiler breeder production systems. A further reduction in AMR is still desirable. RESEARCH HIGHLIGHTSDecreasing trend of antimicrobial resistance in avian pathogenic E. coli over time.Over 50% of isolates still resistant to amoxicillin, tetracycline and doxycycline.Multidrug resistance decreased from 100% in 2009 to 48% in 2018.Further reduction of antimicrobial resistance in broiler breeders desirable.

Sporadic nonfunctional pancreatic neuroendocrine tumors: Risk of lymph node metastases and aggressiveness according to tumor size: A multicenter international study.

BACKGROUND: Although the correlation between tumor size and aggressiveness is clearly established in sporadic nonfunctional pancreatic neuroendocrine tumors, the management of tumors ≤2 cm remains debated. In recent guidelines, the cut-off size to operate ranged from 1 to 2 cm. The aim of this retrospective study was to report the rate of lymph nodes metastases in resected sporadic nonfunctional pancreatic neuroendocrine tumors, according to tumor size and, second, to identify risk factors of lymph node metastases and disease-free survival. METHODS: Resected sporadic nonfunctional pancreatic neuroendocrine tumors from 9 international expert centers were included (1999-2017). Functional pancreatic neuroendocrine tumors, genetic syndromes, and R2 resection were excluded. Aggressiveness was defined as microvascular invasion, perineural invasion, lymph node metastases, G3 grading, distant metastases, and/or recurrence. RESULTS: Overall, 495 resected sporadic nonfunctional pancreatic neuroendocrine tumors were included. For tumors up to 5 cm, the risk of lymph node metastases was increased by 1.73 for every 1 cm increase in size (odds ratio = 1.73; 95% confidence interval = 1.46-2.03). Tumor size >2 cm (P < .001), perineural invasion (P = .002), microvascular invasion (P < .001), and distant metastases (P = .008) were independently associated with lymph node metastases. Tumor size >2 cm (P = .003), R1 status (P = .004), lymph node metastases (P < .001), and World Health Organization grade 3 (P = .002) were independently associated with disease-free survival. Aggressiveness rate was 13.1% in tumors ≤1 cm and 29% in tumors between 1.1 and 2 cm. CONCLUSION: In resected sporadic nonfunctional pancreatic neuroendocrine tumors, the risk of lymph node metastases is correlated to tumor size. Considering that sporadic nonfunctional pancreatic neuroendocrine tumors between 1.1 and 2 cm had a higher risk of lymph node metastases and recurrence compared to tumors ≤1 cm, the decision to perform surgery in this subgroup of patients should be individualized in surgically fit patients.

Determination of the Transport Efficiency in spICP-MS Analysis Using Conventional Sample Introduction Systems: An Interlaboratory Comparison Study.

In single particle inductively coupled plasma mass spectrometry (spICP-MS), the transport efficiency is fundamental for the correct determination of both particle number concentration and size. In the present study, transport efficiency was systematically determined on three different days with six carefully characterised gold nanoparticle (AuNP) suspensions and in seven European and US expert laboratories using different ICP-MS instruments and spICP-MS software. Both particle size-(TES)-and particle frequency-(TEF)-methods were applied. The resulting transport efficiencies did not deviate much under ideal conditions. The TEF method however systematically resulted in lower transport efficiencies. The extent of this difference (0-300% rel. difference) depended largely on the choice and storage conditions of the nanoparticle suspensions used for the determination. The TES method is recommended when the principal measurement objective is particle size. If the main aim of the measurement is the determination of the particle number concentration, the TEF approach could be preferred as it might better account for particle losses in the sample introduction system.

Early assessment of circulating tumor DNA after curative-intent resection predicts tumor recurrence in early-stage and locally advanced non-small-cell lung cancer.

Circulating tumor DNA (ctDNA) has demonstrated great potential as a noninvasive biomarker to assess minimal residual disease (MRD) and profile tumor genotypes in patients with non-small-cell lung cancer (NSCLC). However, little is known about its dynamics during and after tumor resection, or its potential for predicting clinical outcomes. Here, we applied a targeted-capture high-throughput sequencing approach to profile ctDNA at various disease milestones and assessed its predictive value in patients with early-stage and locally advanced NSCLC. We prospectively enrolled 33 consecutive patients with stage IA to IIIB NSCLC undergoing curative-intent tumor resection (median follow-up: 26.2 months). From 21 patients, we serially collected 96 plasma samples before surgery, during surgery, 1-2 weeks postsurgery, and during follow-up. Deep next-generation sequencing using unique molecular identifiers was performed to identify and quantify tumor-specific mutations in ctDNA. Twelve patients (57%) had detectable mutations in ctDNA before tumor resection. Both ctDNA detection rates and ctDNA concentrations were significantly higher in plasma obtained during surgery compared with presurgical specimens (57% versus 19% ctDNA detection rate, and 12.47 versus 6.64 ng·mL-1 , respectively). Four patients (19%) remained ctDNA-positive at 1-2 weeks after surgery, with all of them (100%) experiencing disease progression at later time points. In contrast, only 4 out of 12 ctDNA-negative patients (33%) after surgery experienced relapse during follow-up. Positive ctDNA in early postoperative plasma samples was associated with shorter progression-free survival (P = 0.013) and overall survival (P = 0.004). Our findings suggest that, in early-stage and locally advanced NSCLC, intraoperative plasma sampling results in high ctDNA detection rates and that ctDNA positivity early after resection identifies patients at risk for relapse.

Open aneurysm repair in patients with concomitant abdominal aortic aneurysm and aorto-iliac occlusive disease is associated with a high mortality and surgical complication rate.

AIM OF THE STUDY: To evaluate whether the outcome after open aneurysm repair combined with aorto-femoral bypass in patients with concomitant abdominal aortic aneurysm (AAA) and aorto-iliac occlusive disease (AIOD) is inferior to open aneurysm repair for isolated AAA or aorto-femoral bypass for isolated AIOD. METHODS: We performed a retrospective analysis of 30-day mortality, 1-year mortality and surgical complications of consecutive patients undergoing elective aneurysm repair, aorto-femoral bypass or a combination of these at two vascular surgery departments from 2003 to 2013. Potential risk factors were investigated by multivariable analysis. RESULTS: Overall, 511 patients underwent open repair for isolated AAA, 104 aorto-femoral bypass for isolated AIOD and 46 open AAA repair combined with aorto-femoral bypass for concomitant AAA and AIOD. Surgical complications occurred in 17% of AAA, 23% of AIOD and 37% of combined patients (odds ratio [OR] combined vs AAA 2.76, 95% confidence interval [CI] 1.43-5.34; p = 0.003). Colon ischaemia occurred in 3.7% of AAA, 2.9% of AIOD and 13% of combined patients (incicidence rate ratio [IRR] combined vs AAA 3.27, 95% CI 1.37-7.81; p = 0.01). The 30-day mortality was 3.1% in AAA, 4.8% in AIOD, and 11% in combined patients (IRR combined vs AAA 3.17, 95% CI 1.26-7.96; p = 0.01). One-year mortality was 5.7% in AAA, 5.8% in AIOD and 15% in combined patients (IRR combined vs AAA 2.50, 95% CI 1.17-5.35; p = 0.02). CONCLUSIONS: Combined AAA repair and aorto-femoral bypass has a significantly higher 30-day mortality and postoperative complication rate than isolated AAA repair. Patients with concomitant AAA and AIOD thus represent a high-risk population, which should be considered when deciding on the indication for AAA treatment.

Impact of COVID-19 Restrictions in Portugal: A Questionnaire to Municipal and Animal Association Shelters.

The COVID-19 pandemic has an indirect impact on the health and welfare of animals. The aim of this work was to investigate the effect of COVID-19 on Municipal and Association animal shelters. A questionnaire was sent to 97 Municipal shelters and 65 Associations. Questions focused on public funding, management and animal welfare during COVID-19 restrictions. The response rate was 43.3% (42/97) for Municipal shelters and 38.5% (25/65) for Associations. Municipal shelters (67%) received over 80% of public funding, whereas 68% of the Associations received less than 50%. During the COVID-19 restrictions, financial difficulties were observed by 52% of Associations and 5% of Municipal shelters, and a lack of volunteers was observed by 56% of Associations and 17% of Municipal shelters. Operational difficulties were indicated by 43% of Associations and 12% of Municipal shelters, and a lack of instructions was observed by 31% of Municipal shelters and 4% of Associations. No significant differences were obtained on abandonment, adoption, clinical support, exercise and interaction. Decreased feed supply was reported by 40% of Associations and 5% of Municipal shelters. During the COVID-19 restrictions, Municipal shelters were more affected by the lack of instructions, and Associations were more affected by external factors such as a decrease in feed supply, volunteers and funding. Preparedness and contingency plans seem essential to face unpredicted crises.

Comparison of three explorative methods for identifying building surface materials contributing pollutants to stormwater.

Runoff from building and structure surfaces may contribute to the pollution of urban stormwater and, thereby, to the degradation of the receiving water quality. Various micropollutants have been found in surface runoff from buildings in the urban environment, including metals and organic micropollutants. Effective methods for identification of such pollutants and their sources are the prerequisites for the development of control measures. In this paper, three different methods for the identification of building surface materials acting as sources of metals (Cd, Cr, Cu, Ni, Pb and Zn), nonylphenols and phthalates are presented: (i) screening of the material composition, (ii) laboratory leaching experiments with synthetic rainwater, and (iii) open-air pilot testing of material panels exposed to actual rainfall and runoff. These three methods cover a wide span of experimental aspects, including, e.g., size of material samples, resource demands, and control of influential factors. Nine materials commonly used on building and structure surfaces in the urban environment were tested: metal sheets of zinc, copper, galvanised steel, coated corrugated steel and stainless steel; and, four different roofing membranes of bitumen as well as polyvinyl chloride (PVC). The experimental results indicated that all three methods were meritorious in providing some information contributing to the identification of pollutant sources. The screening of material composition for targeted pollutants is relatively quick and inexpensive, but may fail to identify minor sources of pollutants, or may identify the substances present in the material, but not released in contact with water. Laboratory leaching was generally effective in identifying sources of substances present in surface runoff, but was unsuitable for estimating the magnitude of actual concentrations in building runoff. Open-air pilot studies of material samples (exposed area = 2 m2) were thought to provide the results corresponding well to concentrations in runoff from actual building surfaces, but required relatively large financial and labour resources. Thus, the choice of the method for pollutant identification should be based on study objectives, and some benefits may be achieved using more than one method in an integrated manner; e.g., composition screening and lab or open-air leaching of targeted materials.

Tumor microenvironmental cytokines bound to cancer exosomes determine uptake by cytokine receptor-expressing cells and biodistribution.

Metastatic spread of a cancer to secondary sites is a coordinated, non-random process. Cancer cell-secreted vesicles, especially exosomes, have recently been implicated in the guidance of metastatic dissemination, with specific surface composition determining some aspects of organ-specific localization. Nevertheless, whether the tumor microenvironment influences exosome biodistribution has yet to be investigated. Here, we show that microenvironmental cytokines, particularly CCL2, decorate cancer exosomes via binding to surface glycosaminoglycan side chains of proteoglycans, causing exosome accumulation in specific cell subsets and organs. Exosome retention results in changes in the immune landscape within these organs, coupled with a higher metastatic burden. Strikingly, CCL2-decorated exosomes are directed to a subset of cells that express the CCL2 receptor CCR2, demonstrating that exosome-bound cytokines are a crucial determinant of exosome-cell interactions. In addition to the finding that cytokine-conjugated exosomes are detected in the blood of cancer patients, we discovered that healthy subjects derived exosomes are also associated with cytokines. Although displaying a different profile from exosomes isolated from cancer patients, it further indicates that specific combinations of cytokines bound to exosomes could likewise affect other physiological and disease settings.

Pesticides are the dominant stressors for vulnerable insects in lowland streams.

Despite elaborate regulation of agricultural pesticides, their occurrence in non-target areas has been linked to adverse ecological effects on insects in several field investigations. Their quantitative role in contributing to the biodiversity crisis is, however, still not known. In a large-scale study across 101 sites of small lowland streams in Central Europe, Germany we revealed that 83% of agricultural streams did not meet the pesticide-related ecological targets. For the first time we identified that agricultural nonpoint-source pesticide pollution was the major driver in reducing vulnerable insect populations in aquatic invertebrate communities, exceeding the relevance of other anthropogenic stressors such as poor hydro-morphological structure and nutrients. We identified that the current authorisation of pesticides, which aims to prevent unacceptable adverse effects, underestimates the actual ecological risk as (i) measured pesticide concentrations exceeded current regulatory acceptable concentrations in 81% of the agricultural streams investigated, (ii) for several pesticides the inertia of the authorisation process impedes the incorporation of new scientific knowledge and (iii) existing thresholds of invertebrate toxicity drivers are not protective by a factor of 5.3 to 40. To provide adequate environmental quality objectives, the authorisation process needs to include monitoring-derived information on pesticide effects at the ecosystem level. Here, we derive such thresholds that ensure a protection of the invertebrate stream community.

AUJESZKY'S DISEASE IN HUNTED WILD BOAR (SUS SCROFA) IN THE IBERIAN PENINSULA.

Aujeszky's disease (AD, pseudorabies) eradication programs in domestic pigs are implemented in several European countries where AD virus (ADV) circulates in local wild boar (Sus scrofa), making studies on ADV infection dynamics in wild boar increasingly relevant. The objective of our study was to characterize ADV dynamics in wild boar at a site in central Portugal and compare this site to three enzootic sites in central Spain. A total of 235 wild boar were sampled during the hunting season 2014-15. We collected serum, tissues (oropharyngeal tonsils and trigeminal and sacral ganglia), and swabs (oral, nasal, and genital) and analyzed these samples to detect ADV antibodies (enzyme-linked immunosorbent assay) and DNA (PCR). An overall seroprevalence of 42.6% was found (range 12.7-57.7%), being highest in adults (54.1%; 72/133). Overall, 2.8% (3/108) oral, 6.4% (7/109) nasal, and 12.8% (12/94) genital swabs were PCR positive. We found 20.4% (20/98) of the wild boar had at least one positive swab and were considered shedders. We found ADV in tissues of five animals; of 111 tonsils, three (2.7%) were PCR positive. Trigeminal (2/48; 4%) and sacral (2/53; 4%) ganglia collected in central Portugal, pertaining to three animals, were positive for ADV DNA. Logistic regression models showed that seroprevalence was influenced by site and age, whereas ADV shedding was influenced by site. Our study describes patterns of ADV infection in wild boar in Portugal and shows that wild boar also pose a risk, albeit lower than that in central Spain, for the eradication of AD from extensively managed domestic pigs in Portugal.

Particle size analysis of pristine food-grade titanium dioxide and E 171 in confectionery products: Interlaboratory testing of a single-particle inductively coupled plasma mass spectrometry screening method and confirmation with transmission electron microscopy.

Titanium dioxide is a white colourant authorised as food additive E 171 in the EU, where it is used in a range of alimentary products. As these materials may contain a fraction of particulates with sizes below 100 nm and current EU regulation requires specific labelling of food ingredient to indicate the presence of engineered nanomaterials there is now a need for standardised and validated methods to appropriately size and quantify (nano)particles in food matrices. A single-particle inductively coupled plasma mass spectrometry (spICP-MS) screening method for the determination of the size distribution and concentration of titanium dioxide particles in sugar-coated confectionery and pristine food-grade titanium dioxide was developed. Special emphasis was placed on the sample preparation procedure, crucial to reproducibly disperse the particles before analysis. The transferability of this method was tested in an interlaboratory comparison study among seven experienced European food control and food research laboratories equipped with various ICP-MS instruments and using different software packages. The assessed measurands included the particle mean diameter, the most frequent diameter, the percentage of particles (in number) with a diameter below 100 nm, the particles' number concentration and a number of cumulative particle size distribution parameters (D0, D10, D50, D99.5, D99.8 and D100). The evaluated method's performance characteristics were, the within-laboratory precision, expressed as the relative repeatability standard deviation (RSDr), and the between-laboratory precision, expressed as the relative reproducibility standard deviation (RSDR). Transmission electron microscopy (TEM) was used as a confirmatory technique and served as the basis for bias estimation. The optimisation of the sample preparation step showed that when this protocol was applied to the relatively simple sample food matrices used in this study, bath sonication turned out to be sufficient to reach the highest, achievable degree of dispersed constituent particles. For the pristine material, probe sonication was required. Repeatability and reproducibility were below 10% and 25% respectively for most measurands except for the lower (D0) and the upper (D100) bound of the particle size distribution and the particle number concentration. The broader distribution of the lower and the upper bounds could be attributed to instrument-specific settings/setups (e.g. the timing parameters, the transport efficiency, type of mass-spectrometer) and software-specific data treatment algorithms. Differences in the upper bound were identified as being due to the non-harmonised application of the upper counting limit. Reporting D99.5 or D99.8 instead of the effectively largest particle diameter (D100) excluded isolated large particles and considerably improved the reproducibility. The particle number-concentration was found to be influenced by small differences in the sample preparation procedure. The comparison of these results with those obtained using electron microscopy showed that the mean and median particle diameter was, in all cases, higher when using spICP-MS. The main reason for this was the higher size detection limit for spICP-MS plus the fact that some of the analysed particles remained agglomerated/aggregated after sonication. Single particle ICP-MS is a powerful screening technique, which in many cases provides sufficient evidence to confirm the need to label a food product as containing (engineered) titanium dioxide nanomaterial according to the current EU regulatory requirements. The overall positive outcome of the method performance evaluation and the current lack of alternative standardised procedures, would indicate this method as being a promising candidate for a full validation study.