Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains.

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-ß family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

Transcriptomics and physiological analyses reveal co-ordinated alteration of metabolic pathways in Jatropha curcas drought tolerance.

Jatropha curcas, a multipurpose plant attracting a great deal of attention due to its high oil content and quality for biofuel, is recognized as a drought-tolerant species. However, this drought tolerance is still poorly characterized. This study aims to contribute to uncover the molecular background of this tolerance, using a combined approach of transcriptional profiling and morphophysiological characterization during a period of water-withholding (49 d) followed by rewatering (7 d). Morphophysiological measurements showed that J. curcas plants present different adaptation strategies to withstand moderate and severe drought. Therefore, RNA sequencing was performed for samples collected under moderate and severe stress followed by rewatering, for both roots and leaves. Jatropha curcas transcriptomic analysis revealed shoot- and root-specific adaptations across all investigated conditions, except under severe stress, when the dramatic transcriptomic reorganization at the root and shoot level surpassed organ specificity. These changes in gene expression were clearly shown by the down-regulation of genes involved in growth and water uptake, and up-regulation of genes related to osmotic adjustments and cellular homeostasis. However, organ-specific gene variations were also detected, such as strong up-regulation of abscisic acid synthesis in roots under moderate stress and of chlorophyll metabolism in leaves under severe stress. Functional validation further corroborated the differential expression of genes coding for enzymes involved in chlorophyll metabolism, which correlates with the metabolite content of this pathway.

The transcriptomic profile of Pseudozyma aphidis during production of mannosylerythritol lipids.

The basidiomycetous fungus Pseudozyma aphidis is able to convert vegetable oils to abundant amounts of the biosurfactant mannosylerythritol lipid (MEL) with a unique product pattern of MEL-A, MEL-B, MEL-C, and MEL-D. To investigate the metabolism of MEL production, we analyzed the transcriptome of P. aphidis DSM 70725 under MEL-inducing and non-inducing conditions using deep sequencing. Following manual curation of the previously described in silico gene models based on RNA-Seq data, we were able to generate an experimentally verified gene annotation containing 6347 genes. Using this database, our expression analysis revealed that only four of the five cluster genes required for MEL synthesis were clearly induced by the presence of soybean oil. The acetyltransferase encoding gene PaGMAT1 was expressed on a much lower level, which may explain the secretion of MEL with different degrees of acetylation in P. aphidis. In parallel to MEL synthesis, microscopic observations showed morphological changes accompanied by expression of genes responsible for cell development, indicative of a coregulation between MEL synthesis and cell morphology. In addition a set of transcription factors was identified which may be responsible for regulation of MEL synthesis and cell development. The upregulation of genes required for nitrogen metabolism and other assimilation processes indicate additional metabolic pathways required under the MEL-inducing conditions used. We also searched for a conserved gene cluster for cellobiose lipids (CL) but only found seven genes with limited homology distributed over the genome. However, we detected characteristic TLC spots in fermentations using P. aphidis DSM 70725, indicative of CL secretion.

Sediment characteristics mediate mixture effect of metconazole and thiacloprid on the activity behavior of the amphipod Hyalella azteca.

Pesticide mixtures occur frequently in freshwaters. Here, pesticides can persist over long periods and alter aquatic communities and ecosystems by causing chronic indirect effects. Particularly effects on activity behavior of organisms can be considered as starting points of cascading effects as they provide the basis for further sublethal responses such as reproduction or feeding. Therefore, the impact of two pesticides in combination, the fungicide metconazole and the insecticide thiacloprid, was evaluated on the immobilization and activity behavior of Hyalella azteca with varying sediment conditions. The results showed a change from additive effects to synergism in the mobility tests for sediment with higher contents of total carbon but not for the activity behavior tests using a Multispecies Freshwater Biomonitoring system. However, sediments with high carbon, nitrogen and phosphorous contents led to comparable activity behavior of H. azteca to control conditions after three days of contaminant exposure which was not the case in all other treatments. The autoregressive integrated moving average (ARIMA) forecast approach used showed that this activity behavior remained constant after recovery to pre-exposure levels at least for a time period of 16 h. This study showed that mobility and activity of H. azteca are largely affected by the exposure to pesticides, which is mediated by the structure of the sediment. However, further studies are needed that test activity behavior impairments in environments where the individuals are in direct contact with the sediment that may buffer the pesticide exposure from the water column.

Species and condition specific adaptation of the transcriptional landscapes in Candida albicans and Candida dubliniensis.

BACKGROUND: Although Candida albicans and Candida dubliniensis are most closely related, both species behave significantly different with respect to morphogenesis and virulence. In order to gain further insight into the divergent routes for morphogenetic adaptation in both species, we investigated qualitative along with quantitative differences in the transcriptomes of both organisms by cDNA deep sequencing. RESULTS: Following genome-associated assembly of sequence reads we were able to generate experimentally verified databases containing 6016 and 5972 genes for C. albicans and C. dubliniensis, respectively. About 95% of the transcriptionally active regions (TARs) contain open reading frames while the remaining TARs most likely represent non-coding RNAs. Comparison of our annotations with publically available gene models for C. albicans and C. dubliniensis confirmed approximately 95% of already predicted genes, but also revealed so far unknown novel TARs in both species. Qualitative cross-species analysis of these databases revealed in addition to 5802 orthologs also 399 and 49 species-specific protein coding genes for C. albicans and C. dubliniensis, respectively. Furthermore, quantitative transcriptional profiling using RNA-Seq revealed significant differences in the expression of orthologs across both species. We defined a core subset of 84 hyphal-specific genes required for both species, as well as a set of 42 genes that seem to be specifically induced during hyphal morphogenesis in C. albicans. CONCLUSIONS: Species-specific adaptation in C. albicans and C. dubliniensis is governed by individual genetic repertoires but also by altered regulation of conserved orthologs on the transcriptional level.

Reprogramming the infection mechanism of a filamentous phage.

When they infect Escherichia coli cells, the filamentous phages IF1 and fd first interact with a pilus and then target TolA as their common receptor. They use the domains N2 and N1 of their gene-3-proteins (G3P) for these interactions but differ in the mechanism of infection. In G3P of phage IF1, N1 and N2 are independent modules that are permanently binding-active. G3P of phage fd is usually in a closed state in which N1 and N2 are tightly associated. The TolA binding site is thus inaccessible and the phage incompetent for infection. Partial unfolding and prolyl isomerization must occur to abolish the domain interactions and expose the TolA binding site. This complex mechanism of phage fd could be changed to the simple infection mechanism of phage IF1 by reprogramming its G3P following physicochemical rules of protein stability. The redesigned phage fd was robust and as infectious as wild-type phage fd.

Estimating the recreational carrying capacity of a lowland river section.

Recreational boating represents a major human use of inland waters in many regions. However, boating tourism may affect the ecological integrity of surface waters in multiple ways. In particular, surface waves produced by boating may disturb freshwater invertebrates, such as interrupting the filtration activity of benthic mussels. As mussels may significantly contribute to self-purification, disturbance may have crucial impacts on water quality, and thus on water tourism. In this paper we calculate the carrying capacity of a river section for sustainable boating tourism based on the preservation of water quality. This approach is complemented by spatial and social approaches for carrying capacity estimates. The ecological carrying capacity significantly decreases with lower water levels during summer. Hence, the analysis of variables that influence the river's carrying capacity allows the formation of recommendations for management measures that integrate social, touristic and ecological aspects.

Multiple pesticides in lentic small water bodies: Exposure, ecotoxicological risk, and contamination origin.

Lentic small water bodies (LSWB) are a highly valuable landscape element with important ecosystem services and benefits for humans and the environment. However, data about their pesticide contamination dynamic and the associated ecotoxicological effects are scarce. To overcome these knowledge gaps, five LSWBs located in agricultural fields in Northern Germany were studied during the spring pesticide application period (April to July 2018) and the concentrations of 94 pesticides were measured in weekly intervals. The goals of this study were to observe the trends of pesticide contamination during the application period, assess the ecotoxicity of the contamination, and assign the findings to temporal and spatial origins. Samples contained pesticide concentrations between 0.12 and 4.83 µg L-1 as sums. High detection frequencies (81% of samples) and concentrations (max 1.2 µg L-1) were observed for metazachlor transformation products. Contamination from multiple pesticides was detected with up to 25 compounds per sample and a maximum of 37 compounds per LSWB during the entire sampling period. High toxicities for algae and macrophytes were recorded using toxic units (TU) of -0.2 to -3.5. TUs for invertebrates were generally lower than for algae/macrophytes (-2.7 to -5.2) but were also recorded at levels with ecological impacts. Pesticide detections were separated into four categories to assign them to different temporal and spatial origins. Pesticides from the spring (5-11%) and the previous autumn (0-36%) application periods were detected in the LSWB. Some pesticides could be related to the application of the previous crop on the same field (0-39%), but most of the compounds (44-85%) were not related to the crop management in the last two years on the respective LSWB fields. The relevance of different input pathways is still unknown. Particularly, the effect of long-distance transport needs to be clarified to protect aquatic biota in LSWBs.

A novel tool for objective assessment of femorotibial rotation: a cadaver study.

Traumatic rupture of the anterior cruciate ligament (ACL) of the knee is one of the most frequent orthopaedic sports' injuries. However, the best operative reconstruction technique is still the focus of current discussions among experts. While single-bundle reconstruction primarily addresses anterior-posterior instability, the anatomical double-bundle reconstruction aims to stabilise anterior-posterior as well as rotational instability. So far no definite evidence to favour the one or the other technique exists due to the lack of an objective method for quantifying rotational knee stability. In this context several authors have recently reported on devices for the analysis of femorotibial rotation. However, most of these tools are still in the developmental stage. Therefore, the aim of this study was (1) to develop a new instrument for assessing rotational knee stability independent from the surrounding soft tissue with an adequate method of analysis and (2) to establish the possible field of application of this device in a human cadaver study. The so-called torsiometer evaluated was designed to assess internal and external knee joint rotation objectively in different flexion angles. Measurements were performed implying internal and external rotation at 90°, 30° and 0° knee flexion with and without intact ACL, respectively. Each measurement revealed valid and reproducible values. The restraint in ACL-absent knees was clearly lower and the course of rotation explicitly higher than in knee joints with intact ACL.

Influences of pesticides, nutrients, and local environmental variables on phytoplankton communities in lentic small water bodies in a German lowland agricultural area.

Agrochemicals such as pesticides and nutrients are concurrent chemical stressors in freshwater aquatic ecosystems surrounded by agricultural areas. Lentic small water bodies (LSWB) are ecologically significant habitats especially for maintaining biodiversity but highly understudied. Phytoplankton are ideal indicator species for stress responses. Functional features of the phytoplankton are important in revealing the processes that determine the structure of the communities. In this study, we investigated the effects of pesticides, nutrients, and local environmental variables on the species composition and functional features of phytoplankton communities in LSWB. We studied pesticide toxicity of ninety-four pesticides, three nutrients (NH4-N, NO3-N and PO4-P) and local environment variables (precipitation, water level change, temperature, dissolved oxygen concentration, electrical conductivity, pH) in five LSWB over twelve weeks during the spring pesticide application period. We explored respective changes in species composition of phytoplankton community and functional features. Redundancy analysis and variance partitioning analysis were applied to correlate phytoplankton community compositions with the pesticide toxicity (as maximum toxicity in toxic units), nutrients and local environment variables. We used multiple linear regression models to identify the main environmental variables driving the functional features of phytoplankton communities. Pesticide toxicity, nutrients and local environmental variables significantly (p < 0.001) contributed to shaping phytoplankton community composition individually. Local environment variables showed the highest pure contribution for driving phytoplankton composition (12%), followed by nutrients (8%) and pesticide toxicity (2%). Functional features (represented by functional diversity and functional redundancy) of the phytoplankton community were significantly affected by pesticide toxicity and nutrients concentrations. The functional richness and functional evenness were negatively affected by PO4-P concentrations. Pesticide toxicity was positively correlated with functional redundancy indices. Our findings emphasized the relative importance of concurrent multiple stressors (e.g., pesticides and nutrients) on phytoplankton community structure, directing potential effects on metacommunity structures in aquatic ecosystems subjected to agricultural runoff.

Data on three-year monitoring of benthic macroinvertebrates in ditches of the orchard region of Altes Land, Germany.

The data presented in this article are related to the research article 'Chemical and biological monitoring of the load of plant protection products and of zoocoenoses in ditches of the orchard region Altes Land' (Süß et al., 2006) [1], which is only available in German. The benthic macro invertebrate data presented here were acquired from four ditches (three ditches were located in apple orchards, and one ditch was located in a grassland region) between 2001 and 2003 (Süß & Lorenz, 2020) [2]. This article describes the methods used to record the benthic macro invertebrate species. The field data set is publicly available at the OpenAgrar repository under https://doi.org/10.5073/20201029-170047[2]. It is related to two field data sets, in which pesticide monitoring data (Lorenz et al., 2018) [3] and zooplankton monitoring data (Lorenz & Mueller, 2019) [4] from the same ditches and time period have been presented.

Terrestrial adult stages of freshwater insects are sensitive to insecticides.

Terrestrial adult stages of freshwater insects may be exposed to pesticides by wind drift, over-spray, contact or feeding. However, studies addressing insecticide effects on freshwater invertebrates focus primarily on the impact of pesticides reaching the streams and potentially harming the aquatic juvenile stages. This is also reflected in the current risk assessment procedures, which do not include testing of adult freshwater insects. In order to assess the potential impact of insecticides on adult stages of freshwater insects, we exposed six common species to the insecticides Karate (lambda-cyhalothrin) and Confidor (imidacloprid). Dose-response relations were established, and LD50 estimates were compared to those of the honey bee, Apis mellifera L. (Hymenoptera: Apidae), which is the standard terrestrial test insect when pesticides are evaluated prior to commercial release. Generally, the tested species were more sensitive to the studied insecticides than the honey bee. In order to examine whether the sensitivity of adult stages of freshwater insects corresponds with the sensitivity of the juvenile stages of the same species, the ranking of the two life stages with respect to the toxicity of Karate was compared, revealing some correspondence, but also some dissimilarities. Our results strongly indicate that terrestrial adult stages of aquatic insects are not adequately protected by current risk assessment procedures.

Rabbits transgenic for human IgG genes recapitulating rabbit B-cell biology to generate human antibodies of high specificity and affinity.

Transgenic animals incorporating human antibody genes are extremely attractive for drug development because they obviate subsequent antibody humanization procedures required for therapeutic translation. Transgenic platforms have previously been established using mice, but also more recently rats, chickens, and cows and are now in abundant use for drug development. However, rabbit-based antibody generation, with a strong track record for specificity and affinity, is able to include gene conversion mediated sequence diversification, thereby enhancing binder maturation and improving the variance/selection of output antibodies in a different way than in rodents. Since it additionally frequently permits good binder generation against antigens that are only weakly immunogenic in other organisms, it is a highly interesting species for therapeutic antibody generation. We report here on the generation, utilization, and analysis of the first transgenic rabbit strain for human antibody production. Through the knockout of endogenous IgM genes and the introduction of human immunoglobulin sequences, this rabbit strain has been engineered to generate a highly diverse human IgG antibody repertoire. We further incorporated human CD79a/b and Bcl2 (B-cell lymphoma 2) genes, which enhance B-cell receptor expression and B-cell survival. Following immunization against the angiogenic factor BMP9 (Bone Morphogenetic Proteins 9), we were able to isolate a set of exquisitely affine and specific neutralizing antibodies from these rabbits. Sequence analysis of these binders revealed that both somatic hypermutation and gene conversion are fully operational in this strain, without compromising the very high degree of humanness. This powerful new transgenic strategy will allow further expansion of the use of endogenous immune mechanisms in drug development.

Data on three-year zooplankton monitoring in ditches of the apple orchard region of Altes Land, Germany.

The data presented in this article are related to the research article 'Chemical and biological monitoring of the load of plant protection products and of zoocoenoses in ditches of the orchard region Altes Land' (Süß et al., 2006), which is only available in the German language. The zooplankton data presented here were acquired from four ditches (three ditches were located in apple orchards, and one ditch was located in a grassland region) between 2001 and 2003 (Lorenz & Müller, 2018). This article describes the methods used to determine zooplankton species in the samples. The field data set is publicly available at the OpenAgrar repository under (Lorenz & Müller, 2018). It is related to the field data set of Lorenz et al. (2018) where pesticide monitoring data from the same ditches and time period were presented.

Data on three-year pesticide monitoring in ditches of the apple orchard region of Altes Land, Germany.

The data presented in this article are related to the research article 'Chemical and biological monitoring of the load of plant protection products and of zoocoenoses in ditches of the orchard region Altes Land' (Süß et al., 2006) [1], which is only available in the German language. The pesticide data presented here were acquired from four ditches (three ditches were located in apple orchards, and one ditch was located in a grassland region) between 2001 and 2003 (Lorenz et al., 2018) [2]. Two different monitoring strategies were applied: event-driven sampling after pesticide applications and weekly integrated sampling using automatic water samplers. A total of 70 active substances were monitored while farmers applied 25 active substances. This article describes the study sites and the analytical methods used to quantify the pesticides in the water samples. The field data set is publicly available at the OpenAgrar repository under https://doi.org/10.5073/20180213-144359 (Lorenz et al., 2018) [2].

Two-year field data on neonicotinoid concentrations in guttation drops of seed treated maize (Zea mays).

We present neonicotinoid concentrations in guttation drops of commonly used maize (Zea mays) cultivars, germinated from seeds coated with active substances (a.s.): i) imidacloprid (IMD), ii) clothianidin (CTN) and iii) thiamethoxam (THM) over two growing seasons. In one variant clothianidin was applied as seed granule. The trial took place at the experimental fields of the Julius Kühn-Institut in Berlin in 2010 and 2011. Data from 2010 are related to a presentation of "Pesticides in guttation droplets following seed treatment - field studies" (Schenke et al., 2011) [1] presented at the SETAC North America conference and only some figures were used in the "Scientific opinion on the science behind the development of a risk assessement of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees)" (EFSA, 2012) [2]. Only parts of the data from 2011 was presented in relation to the "Exposure of Coccinellidae to guttation droplets on maize seedlings with seed or granule treatment of neonicotinoids" (Schenke and Heimbach, 2014) [3]. The article describes the study sites, the variants of treated maize seeds, sample collection and the analytical methods used to quantify the neonicotinoids and relevant metabolites of IMD (5-OH-IMD and IMD-olefine) and of THM (CTN) in guttation drop samples. The complete field data set is publicly available at the OpenAgrar repository under https://doi.org/10.5073/20180907-142020 (Schenke et al., 2018) [4].

Effects of ship-induced waves on aquatic ecosystems.

Most larger water bodies worldwide are used for navigation, and the intensity of commercial and recreational navigation is expected to further increase. Navigation profoundly affects aquatic ecosystems. To facilitate navigation, rivers are trained and developed, and the direct effects of navigation include chemical and biological impacts (e.g., inputs of toxic substances and dispersal of non-native species, respectively). Furthermore, propagating ships create hydrodynamic alterations, often simply summarized as waves. Although ship-induced waves are recognized as influential stressors, knowledge on their effects is poorly synthesized. We present here a review on the effects of ship-induced waves on the structure, function and services of aquatic ecosystems based on more than 200 peer reviewed publications and technical reports. Ship-induced waves act at multiple organizational levels and different spatial and temporal scales. All the abiotic and biotic components of aquatic ecosystems are affected, from the sediment and nutrient budget to the planktonic, benthic and fish communities. We highlight how the effects of ship-induced waves cascade through ecosystems and how different effects interact and feed back into the ecosystem finally leading to altered ecosystem services and human health effects. Based on this synthesis of wave effects, we discuss strategies for mitigation. This may help to develop scientifically based and target-oriented management plans for navigational waters that optimize abiotic and biotic integrity and their ecosystem services and uses.

GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies.

The determination of the binding strength of immunoglobulins (IgGs) to targets can be influenced by avidity when the targets are soluble di- or multimeric proteins, or associated to cell surfaces, including surfaces introduced from heterogeneous assays. However, for the understanding of the contribution of a second drug-to-target binding site in molecular design, or for ranking of monovalent binders during lead identification, affinity-based assessment of the binding strength is required. Typically, monovalent binders like antigen-binding fragments (Fabs) are generated by proteolytic cleavage with papain, which often results in a combination of under- and over-digestion, and requires specific optimization and chromatographic purification of the desired Fabs. Alternatively, the Fabs are produced by recombinant approaches. Here, we report a lean approach for the functional assessment of human IgG1s during lead identification based on an in-solution digestion with the GingisKHAN™ protease, generating a homogenous pool of intact Fabs and Fcs and enabling direct assaying of the Fab in the digestion mixture. The digest with GingisKHAN™ is highly specific and quantitative, does not require much optimization, and the protease does not interfere with methods typically applied for lead identification, such as surface plasmon resonance or cell-based assays. GingisKHAN™ is highly suited to differentiate between affinity and avidity driven binding of human IgG1 monoclonal and bispecific antibodies during lead identification.