Effects of Single and Combined Pesticide Exposure on the Grazer–Aufwuchs–Interaction –: Micro- and Mesocosm Experiments

Die Eutrophierung von Fließgewässern führt besonders im Frühjahr zur Zunahme der Biomasse des benthischen Aufwuchses. Dies kann zur reduzierter Infiltration von Wasser in die Gewässersohle führen und wichtige Funktionen von Fließgewässerökosystems stören. Verschiedene Studien konnten zeigen, dass invertebrate Weidegänger (Grazer) in der Lage sind die Biomasse des Aufwuchses in Fließgewässern effektiv zu reduzieren. Diese wichtige ökologische Funktion, das benthische Grazing, kann dazu beitragen negative Effekte der Eutrophierung in Fließgewässern zu kompensieren. Relevante invertebrate Grazer sind unter anderem die Larven der Eintagsfliege Rhithrogena semicolorata, die hohe Biomassen in Fließgewässern erreichen und eine hohe Spezialisierung auf Aufwuchs zeigen. Die Larven von R. semicolorata sind während ihres einjährigen Lebenszykluses Pestiziden ausgesetzt, welche ihre Entwicklung beeinträchtigen können. Um die Effekte von Pestiziden auf die Entwicklung dieser wichtigen Grazer zu untersuchen wurden drei Expositionsszenarien in Anlehnung an reale Bedingungen im Freiland untersucht. Das erste Szenario untersucht die Effekten einer Exposition mit dem Herbizid Terbutryn auf die Grazer-Aufwuchs-Interaktion, wie sie im Herbst und zeitigen Frühjahr anzutreffen ist. Terbutryn verursachte ab 6 µg/L eine deutliche Reduktion der Aufwuchsquantität, was indirekt das Wachstum und die Fettreserven der Grazer verringerte. Die Grazer zeigten ab 0,6 µg/L eine deutliche erhöhte Drift. Da bei dieser Konzentration noch keine Effekte auf die Aufwuchsbiomasse detektiert wurde, ist dies ein Indiz für verringerte Qualität des Aufwuchses. Insektzide werden verstärkt im Frühjahr ausgebracht und beeinflussen die Entwicklung der Grazer direkt. Das zweite Szenario untersuchte die Effekte des Insektizids lambda-Cyhalotrhin (LCH) auf die Grazer-Aufwuchs-Interaktion. LCH ist stark lipophil und adsorbiert schnell an organisches Material und Aufwüchse in Gewässer, weshalb es besonders für aufwuchsfressende Organismen bioverfügbar ist. Neben den erwarteten letalen Effekten wurden in Bereichen um 1 µg/g OC bereits subletale Effekte auf das Driftverhalten sowie das Wachstum der Grazer gefunden. Die Fettreserven waren ab ca. 0,01 µg/g OC reduziert, was auf verringerte Fraßaktivität in diesen niedrigen Konzentrationsbereichen schließen lässt. Die Analyse des Aufwuchses zeigte einen indirekt Effekt von LCH auf die Aufwuchsentwicklung, der einerseits auf die erhöhte Mortalität der Grazer aber auch auf LCH-induzierte Verhaltensänderungen zurückgeführt werden konnte. Das finale Szenario befasste sich mit den Effekten der kombinierten Exposition von Herbizid und Insektizid, wie sie im Lebenszyklus von R. semicolorata auftreten. Dabei zeigte sich entgegen der Erwartung keine erhöhte Mortalität durch die kombinierte Exposition. Die Ergebnisse weisen jedoch daraufhin, dass die Reduzierung der physiologischen Kondition der Grazer, durch die indirekten Effekte von Terbutryn, in subletalen Konzentrationsbereichen von LCH die Dauer bis zur Wiedererholung verlängert. Außerdem zeigte sich am Ende eine erhöhte Aufwuchsbiomasse, was eine reduzierte Leistung des Grazings durch die kombinierte Exposition indiziert.The eutrophication of running water ecosystems leads especially in spring to an increase of benthic biofilms or aufwuchs, which reduces the infiltration of water into the river bed and, hence, disturb important ecological functions. Different studies already showed that invertebrate grazers of running water ecosystems are able to reduce the aufwuchs biomass effectively. This important ecological function, the benthic grazing, can therefore contribute to compensate the negative effects of eutrophication in lotic ecosystems. Relevant grazers are for instance the larvae of the mayfly Rhithrogena semicolorata, which reach high biomasses in streams and show a high feeding specialisation on aufwuchs. During their on year lasting life cycle the larvae of R. semicolorata are exposed to different pesticides, which can disturb their development. To evaluate the effects of pesticides on the development of this important grazer three exposition scenarios were performed following real exposure patterns from the environment. In the first scenarios the effects of a solely exposure with the herbicide Terbutryn on the grazer-aufwuchs-interaction was investigated, as it occurs preferably during autumn and spring. Terbutryn induced a clear reduction of aufwuchs quantity at 6 µg/L, which indirectly affected the growth and the energy storages of the grazers. Furthermore, the grazers showed an increased drift activity at 0.6 µg/L. Considering that the aufwuchs biomass was not affected at this low concentrations, the increased drift was interpreted as an indirect effect of reduced aufwuchs quantity. Inseciticides are preferably applied during spring and are able to disturb the development of grazers directly. The second scenario investigated therefore the effects of the insecticide lambda-Cyhalothrin (LCH) on the grazer-aufwuchs-interaction. LCH has a high lipophilicity and thus rapidly binds to organic matter or aufwuchs after entering aquatic ecosystems. This behaviour leads to a increased bioavailability of this substance to aufwuchs feeding organisms. Apart from the expected lethal effects, sublethal effects in form of increased drift and reduced grazer growth were found in a range of 1 µg/g OC LCH. Moreover, the energy storages of grazers were reduced at 0,01 µg/g OC, which is an indication of disturbed feeding behaviour at this low concentration. The analysis of the aufwuchs revealed additionally an indirect effect of LCH on the aufwuchs development, which was caused by increased grazer mortality and behavioural changes of the grazers induced by sublethal LCH concentrations. The final scenario aimed on the effects of a combined exposure of herbicide and insecticide, as it occurs during the life cycle of R. semicolorata. A increased mortality of grazers, due to the combinded exporsure, was not found. However, the results indicate that the reduction of the physiological condition by the indirect effect of Terbutryn extends the time until grazers recover from sublethal LCH exposure. Finally, also an increased aufwuchs biomass was found in treatments with combined exposure, indicating a reduced performance of grazers exposed to both substances

Optimizing a Human Papillomavirus Type 16 L1-Based Chimaeric Gene for Expression in Plants

Human papillomaviruses (HPVs) are the causative agents of cervical cancer, the fourth most prevalent cancer in women worldwide. The major capsid protein L1 self-assembles into virus-like particles (VLPs), even in the absence of the minor L2 protein: such VLPs have successfully been used as prophylactic vaccines. There remains a need, however, to develop cheaper vaccines that protect against a wider range of HPV types. The use of all or parts of the L2 minor capsid protein can potentially address this issue, as it has sequence regions conserved across several HPV types, which can elicit a wider spectrum of cross-neutralizing antibodies. Production of HPV VLPs in plants is a viable option to reduce costs; the use of a L1/L2 chimera which has previously elicited a cross-protective immune response is an option to broaden cross-protection. The objective of this study was to investigate the effect of codon optimization and of increasing the G+C content of synthetic L1/L2 genes on protein expression in plants. Additionally, we replaced varying portions of the 5′ region of the L1 gene with the wild type (wt) viral sequence to determine the effect of several negative regulatory elements on expression. We showed that GC-rich genes resulted in a 10-fold increase of mRNA levels and 3-fold higher accumulation of proteins. However, the highest increase of expression was achieved with a high GC-content human codon-optimized gene, which resulted in a 100-fold increase in mRNA levels and 8- to 9-fold increase in protein levels. Changing the 5′ end of the L1 gene back to its wt sequence decreased mRNA and protein expression. Our results suggest that the negative elements in the 5′ end of L1 are inadvertently destroyed by changing the codon usage, which enhances protein expression

Mixed messages from benthic microbial communities exposed to nanoparticulate and ionic silver: 3D structure picks up nano-specific effects, while EPS and traditional endpoints indicate a concentration-dependent impact of silver ions

Silver nanoparticles (AgNP) are currently defined as emerging pollutants in surface water ecosystems. Whether the toxic effects of AgNP towards freshwater organisms are fully explainable by the release of ionic silver (Ag+) has not been conclusively elucidated. Long-term effects to benthic microbial communities (periphyton) that provide essential functions in stream ecosystems are unknown. The effects of exposure of periphyton to 2 and 20 μg/L Ag+ (AgNO3) and AgNP (polyvinylpyrrolidone stabilised) were investigated in artificial indoor streams. The extracellular polymeric substances (EPS) and 3D biofilm structure, biomass, algae species, Ag concentrations in the water phase and bioassociated Ag were analysed. A strong decrease in total Ag was observed within 4 days. Bioassociated Ag was proportional to dissolved Ag indicating a rate limitation by diffusion across the diffusive boundary layer. Two micrograms per liter of AgNO3 or AgNP did not induce significant effects despite detectable bioassociation of Ag. The 20-μg/L AgNO3 affected green algae and diatom communities, biomass and the ratio of polysaccharides to proteins in EPS. The 20-μg/L AgNO3 and AgNP decreased biofilm volume to about 50 %, while the decrease of biomass was lower in 20 μg/L AgNP samples than the 20-μg/L AgNO3 indicating a compaction of the NP-exposed biofilms. Roughness coefficients were lower in 20 μg/L AgNP-treated samples. The more traditional endpoints (biomass and diversity) indicated silver ion concentration-dependent effects, while the newly introduced parameters (3D structure and EPS) indicated both silver ion concentration-dependent effects and effects related to the silver species applied

Simulated 50 % radiation dose reduction in coronary CT angiography using adaptive iterative dose reduction in three-dimensions (AIDR3D)

To compare the image quality of coronary CT angiography (CTA) studies between standard filtered back projection (FBP) and adaptive iterative dose reduction in three-dimensions (AIDR3D) reconstruction using CT noise additional software to simulate reduced radiation exposure. Images from 93 consecutive clinical coronary CTA studies were processed utilizing standard FBP, FBP with 50 % simulated dose reduction (FBP50 %), and AIDR3D with simulated 50 % dose reduction (AIDR50 %). Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were measured within 5 regions-of-interest, and image quality for each reconstruction strategy was assessed by two independent readers using a 4-point scale. Compared to FBP, the SNR measured from the AIDR50 % images was similar or higher (airway: 38.3 ± 12.7 vs. 38.5 ± 14.5, p = 0.81, fat: 5.5 ± 1.9 vs. 5.4 ± 2.0, p = 0.20, muscle: 3.2 ± 1.2 vs. 3.1 ± 1.3, p = 0.38, aorta: 22.6 ± 9.4 vs. 20.2 ± 9.7, p < 0.0001, liver: 2.7 ± 1.0 vs. 2.3 ± 1.1, p < 0.0001), while the SNR of the FBP50 % images were all lower (p values < 0.0001). The CNR measured from AIDR50 % images was also higher than that from the FBP images for the aorta relative to muscle (20.5 ± 9.0 vs. 18.3 ± 9.2, p < 0.0001). The interobserver agreement in the image quality score was excellent (κ = 0.82). The quality score was significantly higher for the AIDR50 % images compared to the FBP images (3.6 ± 0.6 vs. 3.3 ± 0.7, p = 0.004). Simulated radiation dose reduction applied to clinical coronary CTA images suggests that a 50 % reduction in radiation dose can be achieved with adaptive iterative dose reduction software with image quality that is at least comparable to images acquired at standard radiation exposure and reconstructed with filtered back projection

Silac mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function

Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the 13C6-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of β1 integrin, β-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions

Diagnosis of obstructive coronary artery disease using computed tomography angiography in patients with stable chest pain depending on clinical probability and in clinically important subgroups: meta-analysis of individual patient data

OBJECTIVE: To determine whether coronary computed tomography angiography (CTA) should be performed in patients with any clinical probability of coronary artery disease (CAD), and whether the diagnostic performance differs between subgroups of patients. DESIGN: Prospectively designed meta-analysis of individual patient data from prospective diagnostic accuracy studies. DATA SOURCES: Medline, Embase, and Web of Science for published studies. Unpublished studies were identified via direct contact with participating investigators. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Prospective diagnostic accuracy studies that compared coronary CTA with coronary angiography as the reference standard, using at least a 50% diameter reduction as a cutoff value for obstructive CAD. All patients needed to have a clinical indication for coronary angiography due to suspected CAD, and both tests had to be performed in all patients. Results had to be provided using 2×2 or 3×2 cross tabulations for the comparison of CTA with coronary angiography. Primary outcomes were the positive and negative predictive values of CTA as a function of clinical pretest probability of obstructive CAD, analysed by a generalised linear mixed model; calculations were performed including and excluding non-diagnostic CTA results. The no-treat/treat threshold model was used to determine the range of appropriate pretest probabilities for CTA. The threshold model was based on obtained post-test probabilities of less than 15% in case of negative CTA and above 50% in case of positive CTA. Sex, angina pectoris type, age, and number of computed tomography detector rows were used as clinical variables to analyse the diagnostic performance in relevant subgroups. RESULTS: Individual patient data from 5332 patients from 65 prospective diagnostic accuracy studies were retrieved. For a pretest probability range of 7-67%, the treat threshold of more than 50% and the no-treat threshold of less than 15% post-test probability were obtained using CTA. At a pretest probability of 7%, the positive predictive value of CTA was 50.9% (95% confidence interval 43.3% to 57.7%) and the negative predictive value of CTA was 97.8% (96.4% to 98.7%); corresponding values at a pretest probability of 67% were 82.7% (78.3% to 86.2%) and 85.0% (80.2% to 88.9%), respectively. The overall sensitivity of CTA was 95.2% (92.6% to 96.9%) and the specificity was 79.2% (74.9% to 82.9%). CTA using more than 64 detector rows was associated with a higher empirical sensitivity than CTA using up to 64 rows (93.4% v 86.5%, P=0.002) and specificity (84.4% v 72.6%, P<0.001). The area under the receiver-operating-characteristic curve for CTA was 0.897 (0.889 to 0.906), and the diagnostic performance of CTA was slightly lower in women than in with men (area under the curve 0.874 (0.858 to 0.890) v 0.907 (0.897 to 0.916), P<0.001). The diagnostic performance of CTA was slightly lower in patients older than 75 (0.864 (0.834 to 0.894), P=0.018 v all other age groups) and was not significantly influenced by angina pectoris type (typical angina 0.895 (0.873 to 0.917), atypical angina 0.898 (0.884 to 0.913), non-anginal chest pain 0.884 (0.870 to 0.899), other chest discomfort 0.915 (0.897 to 0.934)). CONCLUSIONS: In a no-treat/treat threshold model, the diagnosis of obstructive CAD using coronary CTA in patients with stable chest pain was most accurate when the clinical pretest probability was between 7% and 67%. Performance of CTA was not influenced by the angina pectoris type and was slightly higher in men and lower in older patients. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42012002780

Characterization of Genome-Wide Association-Identified Variants for Atrial Fibrillation in African Americans

Despite a greater burden of risk factors, atrial fibrillation (AF) is less common among African Americans than European-descent populations. Genome-wide association studies (GWAS) for AF in European-descent populations have identified three predominant genomic regions associated with increased risk (1q21, 4q25, and 16q22). The contribution of these loci to AF risk in African American is unknown.We studied 73 African Americans with AF from the Vanderbilt-Meharry AF registry and 71 African American controls, with no history of AF including after cardiac surgery. Tests of association were performed for 148 SNPs across the three regions associated with AF, and 22 SNPs were significantly associated with AF (P<0.05). The SNPs with the strongest associations in African Americans were both different from the index SNPs identified in European-descent populations and independent from the index European-descent population SNPs (r(2)<0.40 in HapMap CEU): 1q21 rs4845396 (odds ratio [OR] 0.30, 95% confidence interval [CI] 0.13-0.67, P = 0.003), 4q25 rs4631108 (OR 3.43, 95% CI 1.59-7.42, P = 0.002), and 16q22 rs16971547 (OR 8.1, 95% CI 1.46-45.4, P = 0.016). Estimates of European ancestry were similar among cases (23.6%) and controls (23.8%). Accordingly, the probability of having two copies of the European derived chromosomes at each region did not differ between cases and controls.Variable European admixture at known AF loci does not explain decreased AF susceptibility in African Americans. These data support the role of 1q21, 4q25, and 16q22 variants in AF risk for African Americans, although the index SNPs differ from those identified in European-descent populations

Diagnosis of obstructive coronary artery disease using computed tomography angiography in patients with stable chest pain depending on clinical probability and in clinically important subgroups: Meta-analysis of individual patient data

Objective To determine whether coronary computed tomography angiography (CTA) should be performed in patients with any clinical probability of coronary artery disease (CAD), and whether the diagnostic performance differs between subgroups of patients. Design Prospectively designed meta-analysis of individual patient data from prospective diagnostic accuracy studies. Data sources Medline, Embase, and Web of Science for published studies. Unpublished studies were identified via direct contact with participating investigators. Eligibility criteria for selecting studies Prospective diagnostic accuracy studies that compared coronary CTA with coronary angiography as the reference standard, using at least a 50% diameter reduction as a cutoff value for obstructive CAD. All patients needed to have a clinical indication for coronary angiography due to suspected CAD, and both tests had to be performed in all patients. Results had to be provided using 2 72 or 3 72 cross tabulations for the comparison of CTA with coronary angiography. Primary outcomes were the positive and negative predictive values of CTA as a function of clinical pretest probability of obstructive CAD, analysed by a generalised linear mixed model; calculations were performed including and excluding non-diagnostic CTA results. The no-treat/treat threshold model was used to determine the range of appropriate pretest probabilities for CTA. The threshold model was based on obtained post-test probabilities of less than 15% in case of negative CTA and above 50% in case of positive CTA. Sex, angina pectoris type, age, and number of computed tomography detector rows were used as clinical variables to analyse the diagnostic performance in relevant subgroups. Results Individual patient data from 5332 patients from 65 prospective diagnostic accuracy studies were retrieved. For a pretest probability range of 7-67%, the treat threshold of more than 50% and the no-treat threshold of less than 15% post-test probability were obtained using CTA. At a pretest probability of 7%, the positive predictive value of CTA was 50.9% (95% confidence interval 43.3% to 57.7%) and the negative predictive value of CTA was 97.8% (96.4% to 98.7%); corresponding values at a pretest probability of 67% were 82.7% (78.3% to 86.2%) and 85.0% (80.2% to 88.9%), respectively. The overall sensitivity of CTA was 95.2% (92.6% to 96.9%) and the specificity was 79.2% (74.9% to 82.9%). CTA using more than 64 detector rows was associated with a higher empirical sensitivity than CTA using up to 64 rows (93.4% v 86.5%, P=0.002) and specificity (84.4% v 72.6%, P&lt;0.001). The area under the receiver-operating-characteristic curve for CTA was 0.897 (0.889 to 0.906), and the diagnostic performance of CTA was slightly lower in women than in with men (area under the curve 0.874 (0.858 to 0.890) v 0.907 (0.897 to 0.916), P&lt;0.001). The diagnostic performance of CTA was slightly lower in patients older than 75 (0.864 (0.834 to 0.894), P=0.018 v all other age groups) and was not significantly influenced by angina pectoris type (typical angina 0.895 (0.873 to 0.917), atypical angina 0.898 (0.884 to 0.913), non-anginal chest pain 0.884 (0.870 to 0.899), other chest discomfort 0.915 (0.897 to 0.934)). Conclusions In a no-treat/treat threshold model, the diagnosis of obstructive CAD using coronary CTA in patients with stable chest pain was most accurate when the clinical pretest probability was between 7% and 67%. Performance of CTA was not influenced by the angina pectoris type and was slightly higher in men and lower in older patients. Systematic review registration PROSPERO CRD42012002780