Gold Open-Access
Refine
Year of publication
Document Type
- Article (355)
- Part of Periodical (7)
- Review (6)
- Other (3)
- Postprint (2)
Is part of the Bibliography
- yes (373)
Keywords
- Dictyostelium (8)
- centrosome (6)
- climate change (6)
- Arabidopsis thaliana (5)
- metabarcoding (5)
- Brachionus calyciflorus (4)
- DNA methylation (4)
- Myodes glareolus (4)
- animal personality (4)
- conservation (4)
Institute
- Institut für Biochemie und Biologie (373) (remove)
A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies
(2022)
Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds of recombinant proteins ranging from membrane proteins to soluble proteins, enzymes and even toxins. Also within the antibody field the cell-free technology has gained considerable attention with respect to the clinical research pipeline including antibody discovery and production. Besides the classical full-length monoclonal antibodies (mAbs), so-called "nanobodies" (Nbs) have come into focus. A Nb is the smallest naturally-derived functional antibody fragment known and represents the variable domain (VHH, similar to 15 kDa) of a camelid heavy-chain-only antibody (HCAb). Based on their nanoscale and their special structure, Nbs display striking advantages concerning their production, but also their characteristics as binders, such as high stability, diversity, improved tissue penetration and reaching of cavity-like epitopes. The classical way to produce Nbs depends on the use of living cells as production host. Though cell-based production is well-established, it is still time-consuming, laborious and hardly amenable for high-throughput applications. Here, we present for the first time to our knowledge the synthesis of functional Nbs in a standardized mammalian cell-free system based on Chinese hamster ovary (CHO) cell lysates. Cell-free reactions were shown to be time-efficient and easy-to-handle allowing for the "on demand" synthesis of Nbs. Taken together, we complement available methods and demonstrate a promising new system for Nb selection and validation.
Genomic prediction has revolutionized crop breeding despite remaining issues of transferability of models to unseen environmental conditions and environments. Usage of endophenotypes rather than genomic markers leads to the possibility of building phenomic prediction models that can account, in part, for this challenge. Here, we compare and contrast genomic prediction and phenomic prediction models for 3 growth-related traits, namely, leaf count, tree height, and trunk diameter, from 2 coffee 3-way hybrid populations exposed to a series of treatment-inducing environmental conditions. The models are based on 7 different statistical methods built with genomic markers and ChlF data used as predictors. This comparative analysis demonstrates that the best-performing phenomic prediction models show higher predictability than the best genomic prediction models for the considered traits and environments in the vast majority of comparisons within 3-way hybrid populations. In addition, we show that phenomic prediction models are transferrable between conditions but to a lower extent between populations and we conclude that chlorophyll a fluorescence data can serve as alternative predictors in statistical models of coffee hybrid performance. Future directions will explore their combination with other endophenotypes to further improve the prediction of growth-related traits for crops.
Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1(-/-) zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.
Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix–helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.
A familial congenital heart disease with a possible multigenic origin involving a mutation in BMPR1A
(2019)
The genetics of many congenital heart diseases (CHDs) can only unsatisfactorily be explained by known chromosomal or Mendelian syndromes. Here, we present sequencing data of a family with a potentially multigenic origin of CHD. Twelve of nineteen family members carry a familial mutation [NM_004329.2:c.1328 G > A (p.R443H)] which encodes a predicted deleterious variant of BMPR1A. This mutation co-segregates with a linkage region on chromosome 1 that associates with the emergence of severe CHDs including Ebstein’s anomaly, atrioventricular septal defect, and others. We show that the continuous overexpression of the zebrafish homologous mutation bmpr1aap.R438H within endocardium causes a reduced AV valve area, a downregulation of Wnt/ß-catenin signalling at the AV canal, and growth of additional tissue mass in adult zebrafish hearts. This finding opens the possibility of testing genetic interactions between BMPR1A and other candidate genes within linkage region 1 which may provide a first step towards unravelling more complex genetic patterns in cardiovascular disease aetiology.
Biogenic greenhouse gas emissions, e.g., of methane (CH4) and carbon dioxide (CO2) from inland waters, contribute substantially to global warming. In aquatic systems, dissolved greenhouse gases are highly heterogeneous in both space and time. To better understand the biological and physical processes that affect sources and sinks of both CH4 and CO2, their dissolved concentrations need to be measured with high spatial and temporal resolution. To achieve this goal, we developed the Fast-Response Automated Gas Equilibrator (FaRAGE) for real-time in situ measurement of dissolved CH4 and CO2 concentrations at the water surface and in the water column. FaRAGE can achieve an exceptionally short response time (t(95%) = 12 s when including the response time of the gas analyzer) while retaining an equilibration ratio of 62.6% and a measurement accuracy of 0.5% for CH4. A similar performance was observed for dissolved CO2 (t(95%) = 10 s, equilibration ratio 67.1 %). An equilibration ratio as high as 91.8% can be reached at the cost of a slightly increased response time (16 s). The FaRAGE is capable of continuously measuring dissolved CO2 and CH4 concentrations in the nM-to-submM (10(-9)-10(-3) mol L-1) range with a detection limit of subnM (10(-10) mol L-1), when coupling with a cavity ring-down greenhouse gas analyzer (Picarro GasScouter). FaRAGE allows for the possibility of mapping dissolved concentration in a "quasi" three-dimensional manner in lakes and provides an inexpensive alternative to other commercial gas equilibrators. It is simple to operate and suitable for continuous monitoring with a strong tolerance for suspended particles. While the FaRAGE is developed for inland waters, it can be also applied to ocean waters by tuning the gas-water mixing ratio. The FaRAGE is easily adapted to suit other gas analyzers expanding the range of potential applications, including nitrous oxide and isotopic composition of the gases.
Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.
A Metabarcoding Analysis of the Mycobiome of Wheat Ears Across a Topographically Heterogeneous Field
(2019)
Anthropogenic changes in climate, land use, and disturbance regimes, as well as introductions of non-native species can lead to the transformation of many ecosystems. The resulting novel ecosystems are usually characterized by species assemblages that have not occurred previously in a given area. Quantifying the ecological novelty of communities (i.e., biotic novelty) would enhance the understanding of environmental change. However, quantification remains challenging since current novelty metrics, such as the number and/or proportion of non-native species in a community, fall short of considering both functional and evolutionary aspects of biotic novelty. Here, we propose the Biotic Novelty Index (BNI), an intuitive and flexible multidimensional measure that combines (a) functional differences between native and non-native introduced species with (b) temporal dynamics of species introductions. We show that the BNI is an additive partition of Rao's quadratic entropy, capturing the novel interaction component of the community's functional diversity. Simulations show that the index varies predictably with the relative amount of functional novelty added by recently arrived species, and they illustrate the need to provide an additional standardized version of the index. We present a detailed R code and two applications of the BNI by (a) measuring changes of biotic novelty of dry grassland plant communities along an urbanization gradient in a metropolitan region and (b) determining the biotic novelty of plant species assemblages at a national scale. The results illustrate the applicability of the index across scales and its flexibility in the use of data of different quality. Both case studies revealed strong connections between biotic novelty and increasing urbanization, a measure of abiotic novelty. We conclude that the BNI framework may help building a basis for better understanding the ecological and evolutionary consequences of global change.
Metagenomic sequencing has revolutionised our knowledge of virus diversity, with new virus sequences being reported faster than ever before. However, virus discovery from metagenomic sequencing usually depends on detectable homology: without a sufficiently close relative, so-called ‘dark’ virus sequences remain unrecognisable. An alternative approach is to use virus-identification methods that do not depend on detecting homology, such as virus recognition by host antiviral immunity. For example, virus-derived small RNAs have previously been used to propose ‘dark’ virus sequences associated with the Drosophilidae (Diptera). Here, we combine published Drosophila data with a comprehensive search of transcriptomic sequences and selected meta-transcriptomic datasets to identify a completely new lineage of segmented positive-sense single-stranded RNA viruses that we provisionally refer to as the Quenyaviruses. Each of the five segments contains a single open reading frame, with most encoding proteins showing no detectable similarity to characterised viruses, and one sharing a small number of residues with the RNA-dependent RNA polymerases of single- and double-stranded RNA viruses. Using these sequences, we identify close relatives in approximately 20 arthropods, including insects, crustaceans, spiders, and a myriapod. Using a more conserved sequence from the putative polymerase, we further identify relatives in meta-transcriptomic datasets from gut, gill, and lung tissues of vertebrates, reflecting infections of vertebrates or of their associated parasites. Our data illustrate the utility of small RNAs to detect viruses with limited sequence conservation, and provide robust evidence for a new deeply divergent and phylogenetically distinct RNA virus lineage.