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Wiesner-Reinhold, Melanie ; Barknowitz, Gitte ; Florian, Simone ; Mewis, Inga ; Schumacher, Fabian ; Schreiner, Monika ; Glatt, Hansruedi
We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N-2-(1-MIM)-dG, N-6-(1-MIM)-dA] in six tissues, as well as protein adducts [tau N-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N-2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N-6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.
A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component
(2022)
Leong, Jia Xuan ; Raffeiner, Margot ; Spinti, Daniela ; Langin, Gautier ; Franz-Wachtel, Mirita ; Guzman, Andrew R. ; Kim, Jung-Gun ; Pandey, Pooja ; Minina, Alyona E. ; Macek, Boris ; Hafren, Anders ; Bozkurt, Tolga O. ; Mudgett, Mary Beth ; Börnke, Frederik ; Hofius, Daniel ; Uestuen, Suayib
Beyond its role in cellular homeostasis, autophagy plays anti- and promicrobial roles in host-microbe interactions, both in animals and plants.
One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy.
Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination.
Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown.
Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection.
Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery.
Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense-related autophagy in plant-bacteria interactions.
A balance to death
(2018)
Leaf senescence plays a crucial role in nutrient recovery in late-stage plant development and requires vast transcriptional reprogramming by transcription factors such as ORESARA1 (ORE1). A proteolytic mechanism is now found to control ORE1 degradation, and thus senescence, during nitrogen starvation.
Ecological communities are complex adaptive systems that exhibit remarkable feedbacks between their biomass and trait dynamics. Trait-based aggregate models cope with this complexity by focusing on the temporal development of the community’s aggregate properties such as its total biomass, mean trait and trait variance. They are based on particular assumptions about the shape of the underlying trait distribution, which is commonly assumed to be normal. However, ecologically important traits are usually restricted to a finite range, and empirical trait distributions are often skewed or multimodal. As a result, normal distribution-based aggregate models may fail to adequately represent the biomass and trait dynamics of natural communities. We resolve this mismatch by developing a new moment closure approach assuming the trait values to be beta-distributed. We show that the beta distribution captures important shape properties of both observed and simulated trait distributions, which cannot be captured by a Gaussian. We further demonstrate that a beta distribution-based moment closure can strongly enhance the reliability of trait-based aggregate models. We compare the biomass, mean trait and variance dynamics of a full trait distribution (FD) model to the ones of beta (BA) and normal (NA) distribution-based aggregate models, under different selection regimes. This way, we demonstrate under which general conditions (stabilizing, fluctuating or disruptive selection) different aggregate models are reliable tools. All three models predicted very similar biomass and trait dynamics under stabilizing selection yielding unimodal trait distributions with small standing trait variation. We also obtained an almost perfect match between the results of the FD and BA models under fluctuating selection, promoting skewed trait distributions and ongoing oscillations in the biomass and trait dynamics. In contrast, the NA model showed unrealistic trait dynamics and exhibited different alternative stable states, and thus a high sensitivity to initial conditions under fluctuating selection. Under disruptive selection, both aggregate models failed to reproduce the results of the FD model with the mean trait values remaining within their ecologically feasible ranges in the BA model but not in the NA model. Overall, a beta distribution-based moment closure strongly improved the realism of trait-based aggregate models.
Omidbakhshfard, Mohammad Amin ; Neerakkal, Sujeeth ; Gupta, Saurabh ; Omranian, Nooshin ; Guinan, Kieran J. ; Brotman, Yariv ; Nikoloski, Zoran ; Fernie, Alisdair ; Mueller-Roeber, Bernd ; Gechev, Tsanko S.
Abiotic stresses cause oxidative damage in plants. Here, we demonstrate that foliar application of an extract from the seaweed Ascophyllum nodosum, SuperFifty (SF), largely prevents paraquat (PQ)-induced oxidative stress in Arabidopsis thaliana. While PQ-stressed plants develop necrotic lesions, plants pre-treated with SF (i.e., primed plants) were unaffected by PQ. Transcriptome analysis revealed induction of reactive oxygen species (ROS) marker genes, genes involved in ROS-induced programmed cell death, and autophagy-related genes after PQ treatment. These changes did not occur in PQ-stressed plants primed with SF. In contrast, upregulation of several carbohydrate metabolism genes, growth, and hormone signaling as well as antioxidant-related genes were specific to SF-primed plants. Metabolomic analyses revealed accumulation of the stress-protective metabolite maltose and the tricarboxylic acid cycle intermediates fumarate and malate in SF-primed plants. Lipidome analysis indicated that those lipids associated with oxidative stress-induced cell death and chloroplast degradation, such as triacylglycerols (TAGs), declined upon SF priming. Our study demonstrated that SF confers tolerance to PQ-induced oxidative stress in A. thaliana, an effect achieved by modulating a range of processes at the transcriptomic, metabolic, and lipid levels.
Omidbakhshfard, Mohammad Amin ; Neerakkal, Sujeeth ; Gupta, Saurabh ; Omranian, Nooshin ; Guinan, Kieran J. ; Brotman, Yariv ; Nikoloski, Zoran ; Fernie, Alisdair ; Mueller-Roeber, Bernd ; Gechev, Tsanko S.
Abiotic stresses cause oxidative damage in plants. Here, we demonstrate that foliar application of an extract from the seaweed Ascophyllum nodosum, SuperFifty (SF), largely prevents paraquat (PQ)-induced oxidative stress in Arabidopsis thaliana. While PQ-stressed plants develop necrotic lesions, plants pre-treated with SF (i.e., primed plants) were unaffected by PQ. Transcriptome analysis revealed induction of reactive oxygen species (ROS) marker genes, genes involved in ROS-induced programmed cell death, and autophagy-related genes after PQ treatment. These changes did not occur in PQ-stressed plants primed with SF. In contrast, upregulation of several carbohydrate metabolism genes, growth, and hormone signaling as well as antioxidant-related genes were specific to SF-primed plants. Metabolomic analyses revealed accumulation of the stress-protective metabolite maltose and the tricarboxylic acid cycle intermediates fumarate and malate in SF-primed plants. Lipidome analysis indicated that those lipids associated with oxidative stress-induced cell death and chloroplast degradation, such as triacylglycerols (TAGs), declined upon SF priming. Our study demonstrated that SF confers tolerance to PQ-induced oxidative stress in A. thaliana, an effect achieved by modulating a range of processes at the transcriptomic, metabolic, and lipid levels.
Christakoudi, Sofa ; Tsilidis, Konstantinos K. ; Muller, David C. ; Freisling, Heinz ; Weiderpass, Elisabete ; Overvad, Kim ; Söderberg, Stefan ; Häggström, Christel ; Pischon, Tobias ; Dahm, Christina C. ; Zhang, Jie ; Tjønneland, Anne ; Schulze, Matthias Bernd
Abdominal and general adiposity are independently associated with mortality, but there is no consensus on how best to assess abdominal adiposity. We compared the ability of alternative waist indices to complement body mass index (BMI) when assessing all-cause mortality. We used data from 352,985 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) and Cox proportional hazards models adjusted for other risk factors. During a mean follow-up of 16.1 years, 38,178 participants died. Combining in one model BMI and a strongly correlated waist index altered the association patterns with mortality, to a predominantly negative association for BMI and a stronger positive association for the waist index, while combining BMI with the uncorrelated A Body Shape Index (ABSI) preserved the association patterns. Sex-specific cohort-wide quartiles of waist indices correlated with BMI could not separate high-risk from low-risk individuals within underweight (BMI<18.5 kg/m(2)) or obese (BMI30 kg/m(2)) categories, while the highest quartile of ABSI separated 18-39% of the individuals within each BMI category, which had 22-55% higher risk of death. In conclusion, only a waist index independent of BMI by design, such as ABSI, complements BMI and enables efficient risk stratification, which could facilitate personalisation of screening, treatment and monitoring.
Christakoudi, Sofa ; Tsilidis, Konstantinos K. ; Muller, David C. ; Freisling, Heinz ; Weiderpass, Elisabete ; Overvad, Kim ; Söderberg, Stefan ; Häggström, Christel ; Pischon, Tobias ; Dahm, Christina C. ; Zhang, Jie ; Tjønneland, Anne ; Schulze, Matthias Bernd
Abdominal and general adiposity are independently associated with mortality, but there is no consensus on how best to assess abdominal adiposity. We compared the ability of alternative waist indices to complement body mass index (BMI) when assessing all-cause mortality. We used data from 352,985 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) and Cox proportional hazards models adjusted for other risk factors. During a mean follow-up of 16.1 years, 38,178 participants died. Combining in one model BMI and a strongly correlated waist index altered the association patterns with mortality, to a predominantly negative association for BMI and a stronger positive association for the waist index, while combining BMI with the uncorrelated A Body Shape Index (ABSI) preserved the association patterns. Sex-specific cohort-wide quartiles of waist indices correlated with BMI could not separate high-risk from low-risk individuals within underweight (BMI<18.5 kg/m(2)) or obese (BMI30 kg/m(2)) categories, while the highest quartile of ABSI separated 18-39% of the individuals within each BMI category, which had 22-55% higher risk of death. In conclusion, only a waist index independent of BMI by design, such as ABSI, complements BMI and enables efficient risk stratification, which could facilitate personalisation of screening, treatment and monitoring.
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.
Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation of synthetic proteins with desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment and in vivo imaging. The positioning of the ncAA-responsive codon within the protein's coding sequence is critical in order to maintain protein function, achieve high yields of ncAA-containing protein, and allow effective conjugation. Cell-free ncAA incorporation is of particular interest due to the open nature of cell-free systems and their concurrent ease of manipulation. In this study, we report a straightforward workflow to inquire ncAA positions in regard to incorporation efficiency and protein functionality in a Chinese hamster ovary (CHO) cell-free system. As a model, the well-established orthogonal translation components Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and tRNATyr(CUA) were used to site-specifically incorporate the ncAA p-azido-l-phenylalanine (AzF) in response to UAG codons. A total of seven ncAA sites within an anti-epidermal growth factor receptor (EGFR) single-chain variable fragment (scFv) N-terminally fused to the red fluorescent protein mRFP1 and C-terminally fused to the green fluorescent protein sfGFP were investigated for ncAA incorporation efficiency and impact on antigen binding. The characterized cell-free dual fluorescence reporter system allows screening for ncAA incorporation sites with high incorporation efficiency that maintain protein activity. It is parallelizable, scalable, and easy to operate. We propose that the established CHO-based cell-free dual fluorescence reporter system can be of particular interest for the development of antibody-drug conjugates (ADCs).
Next-generation sequencing methods provide comprehensive data for the analysis of structural and functional analysis of the genome. The draft genomes with low contig number and high N50 value can give insight into the structure of the genome as well as provide information on the annotation of the genome. In this study, we designed a pipeline that can be used to assemble prokaryotic draft genomes with low number of contigs and high N50 value. We aimed to use combination of two de novo assembly tools (SPAdes and IDBA-Hybrid) and evaluate the impact of this approach on the quality metrics of the assemblies. The followed pipeline was tested with the raw sequence data with short reads (< 300) for a total of 10 species from four different genera. To obtain the final draft genomes, we firstly assembled the sequences using SPAdes to find closely related organism using the extracted 16 s rRNA from it. IDBA-Hybrid assembler was used to obtain the second assembly data using the closely related organism genome. SPAdes assembler tool was implemented using the second assembly, produced by IDBA-hybrid as a hint. The results were evaluated using QUAST and BUSCO. The pipeline was successful for the reduction of the contig numbers and increasing the N50 statistical values in the draft genome assemblies while preserving the coverage of the draft genomes.
Mbebi, Alain J. ; Breitler, Jean-Christophe ; Bordeaux, M'elanie ; Sulpice, Ronan ; McHale, Marcus ; Tong, Hao ; Toniutti, Lucile ; Castillo, Jonny Alonso ; Bertrand, Benoit ; Nikoloski, Zoran
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.
A comparative whole-genome approach identifies bacterial traits for marine microbial interactions
(2022)
Luca Zoccarato, Daniel Sher et al. leverage publicly available bacterial genomes from marine and other environments to examine traits underlying microbial interactions.
Their results provide a valuable resource to investigate clusters of functional and linked traits to better understand marine bacteria community assembly and dynamics.
Microbial interactions shape the structure and function of microbial communities with profound consequences for biogeochemical cycles and ecosystem health. Yet, most interaction mechanisms are studied only in model systems and their prevalence is unknown. To systematically explore the functional and interaction potential of sequenced marine bacteria, we developed a trait-based approach, and applied it to 473 complete genomes (248 genera), representing a substantial fraction of marine microbial communities.
We identified genome functional clusters (GFCs) which group bacterial taxa with common ecology and life history. Most GFCs revealed unique combinations of interaction traits, including the production of siderophores (10% of genomes), phytohormones (3-8%) and different B vitamins (57-70%). Specific GFCs, comprising Alpha- and Gammaproteobacteria, displayed more interaction traits than expected by chance, and are thus predicted to preferentially interact synergistically and/or antagonistically with bacteria and phytoplankton. Linked trait clusters (LTCs) identify traits that may have evolved to act together (e.g., secretion systems, nitrogen metabolism regulation and B vitamin transporters), providing testable hypotheses for complex mechanisms of microbial interactions.
Our approach translates multidimensional genomic information into an atlas of marine bacteria and their putative functions, relevant for understanding the fundamental rules that govern community assembly and dynamics.
Noonan, Michael J. ; Tucker, Marlee A. ; Fleming, Christen H. ; Akre, Thomas S. ; Alberts, Susan C. ; Ali, Abdullahi H. ; Altmann, Jeanne ; Antunes, Pamela Castro ; Belant, Jerrold L. ; Beyer, Dean ; Blaum, Niels ; Boehning-Gaese, Katrin ; Cullen Jr, Laury ; de Paula, Rogerio Cunha ; Dekker, Jasja ; Drescher-Lehman, Jonathan ; Farwig, Nina ; Fichtel, Claudia ; Fischer, Christina ; Ford, Adam T. ; Goheen, Jacob R. ; Janssen, Rene ; Jeltsch, Florian ; Kauffman, Matthew ; Kappeler, Peter M. ; Koch, Flavia ; LaPoint, Scott ; Markham, A. Catherine ; Medici, Emilia Patricia ; Morato, Ronaldo G. ; Nathan, Ran ; Oliveira-Santos, Luiz Gustavo R. ; Olson, Kirk A. ; Patterson, Bruce D. ; Paviolo, Agustin ; Ramalho, Emiliano Estero ; Rosner, Sascha ; Schabo, Dana G. ; Selva, Nuria ; Sergiel, Agnieszka ; da Silva, Marina Xavier ; Spiegel, Orr ; Thompson, Peter ; Ullmann, Wiebke ; Zieba, Filip ; Zwijacz-Kozica, Tomasz ; Fagan, William F. ; Mueller, Thomas ; Calabrese, Justin M.
Home range estimation is routine practice in ecological research. While advances in animal tracking technology have increased our capacity to collect data to support home range analysis, these same advances have also resulted in increasingly autocorrelated data. Consequently, the question of which home range estimator to use on modern, highly autocorrelated tracking data remains open. This question is particularly relevant given that most estimators assume independently sampled data. Here, we provide a comprehensive evaluation of the effects of autocorrelation on home range estimation. We base our study on an extensive data set of GPS locations from 369 individuals representing 27 species distributed across five continents. We first assemble a broad array of home range estimators, including Kernel Density Estimation (KDE) with four bandwidth optimizers (Gaussian reference function, autocorrelated‐Gaussian reference function [AKDE], Silverman's rule of thumb, and least squares cross‐validation), Minimum Convex Polygon, and Local Convex Hull methods. Notably, all of these estimators except AKDE assume independent and identically distributed (IID) data. We then employ half‐sample cross‐validation to objectively quantify estimator performance, and the recently introduced effective sample size for home range area estimation ( N̂ area
) to quantify the information content of each data set. We found that AKDE 95% area estimates were larger than conventional IID‐based estimates by a mean factor of 2. The median number of cross‐validated locations included in the hold‐out sets by AKDE 95% (or 50%) estimates was 95.3% (or 50.1%), confirming the larger AKDE ranges were appropriately selective at the specified quantile. Conversely, conventional estimates exhibited negative bias that increased with decreasing N̂ area. To contextualize our empirical results, we performed a detailed simulation study to tease apart how sampling frequency, sampling duration, and the focal animal's movement conspire to affect range estimates. Paralleling our empirical results, the simulation study demonstrated that AKDE was generally more accurate than conventional methods, particularly for small N̂ area. While 72% of the 369 empirical data sets had >1,000 total observations, only 4% had an N̂ area >1,000, where 30% had an N̂ area <30. In this frequently encountered scenario of small N̂ area, AKDE was the only estimator capable of producing an accurate home range estimate on autocorrelated data.
Palkopoulou, Eleftheria ; Lipson, Mark ; Mallick, Swapan ; Nielsen, Svend ; Rohland, Nadin ; Baleka, Sina Isabelle ; Karpinski, Emil ; Ivancevici, Atma M. ; Thu-Hien To ; Kortschak, Daniel ; Raison, Joy M. ; Qu, Zhipeng ; Chin, Tat-Jun ; Alt, Kurt W. ; Claesson, Stefan ; Dalen, Love ; MacPhee, Ross D. E. ; Meller, Harald ; Rocar, Alfred L. ; Ryder, Oliver A. ; Heiman, David ; Young, Sarah ; Breen, Matthew ; Williams, Christina ; Aken, Bronwen L. ; Ruffier, Magali ; Karlsson, Elinor ; Johnson, Jeremy ; Di Palma, Federica ; Alfoldi, Jessica ; Adelsoni, David L. ; Mailund, Thomas ; Munch, Kasper ; Lindblad-Toh, Kerstin ; Hofreiter, Michael ; Poinar, Hendrik ; Reich, David
Chapman, Eric M. ; Lant, Benjamin ; Ohashi, Yota ; Yu, Bin ; Schertzberg, Michael ; Go, Christopher ; Dogra, Deepika ; Koskimaki, Janne ; Girard, Romuald ; Li, Yan ; Fraser, Andrew G. ; Awad, Issam A. ; Abdelilah-Seyfried, Salim ; Gingras, Anne-Claude ; Derry, William Brent
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.
Wolff, Martin ; Gast, Klaus ; Evers, Andreas ; Kurz, Michael ; Pfeiffer-Marek, Stefania ; Schüler, Anja ; Seckler, Robert ; Thalhammer, Anja
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.
Wolff, Martin ; Gast, Klaus ; Evers, Andreas ; Kurz, Michael ; Pfeiffer-Marek, Stefania ; Schüler, Anja ; Seckler, Robert ; Thalhammer, Anja
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.
Buyinza, Daniel ; Derese, Solomon ; Ndakala, Albert ; Heydenreich, Matthias ; Yenesew, Abiy ; Koch, Andreas ; Oriko, Richard
The manuscript describes the phytochemical investigation of the roots, leaves and stem bark of Millettia lasiantha resulting in the isolation of twelve compounds including two new isomeric isoflavones lascoumestan and las-coumaronochromone. The structures of the new compounds were determined using different spectroscopic techniques.
Luetkecosmann, Steffi ; Faupel, Thomas ; Porstmann, Silvia ; Porstmann, Tomas ; Micheel, Burkhard ; Hanack, Katja
Diagnostics of Autoimmune Diseases involve screening of patient samples for containing autoantibodies against various antigens. To ensure quality of diagnostic assays a calibrator is needed in each assay system. Different calibrators as recombinant human monoclonal antibodies as well as chimeric antibodies against the autoantigens of interest are described. A less cost-intensive and also more representative possibility covering different targets on the antigens is the utilization of polyclonal sera from other species. Nevertheless, the detection of human autoantibodies as well as the calibration reagent containing antibodies from other species in one assay constitutes a challenge in terms of assay calibration. We therefore developed a cross-reactive monoclonal antibody which binds human as well as rabbit sera with similar affinities in the nanomolar range. We tested our monoclonal antibody S38CD11B12 successfully in the commercial Serazym (R) Anti-Cardiolipin-beta 2-GPI IgG/IgM assay and could thereby prove the eligibility of S38CD11B12 as detection antibody in autoimmune diagnostic assays using rabbit derived sera as reference material.
A familial congenital heart disease with a possible multigenic origin involving a mutation in BMPR1A
(2019)
Demal, Till Joscha ; Heise, Melina ; Reiz, Benedikt ; Dogra, Deepika ; Braenne, Ingrid ; Reichenspurner, Hermann ; Männer, Jörg ; Aherrahrou, Zouhair ; Schunkert, Heribert ; Erdmann, Jeanette ; Abdelilah-Seyfried, Salim
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.
Xiao, Shangbin ; Liu, Liu ; Wang, Wei ; Lorke, Andreas ; Woodhouse, Jason Nicholas ; Grossart, Hans-Peter
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.
A flexible approach to assess fluorescence decay functions in complex energy transfer systems
(2015)
Background: Time-correlated Forster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions.
Results: Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems.
Conclusion: This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems.
Sharma, Neha ; Ruelens, Philip ; Maggen, Thomas ; Dochy, Niklas ; Torfs, Sanne ; Kaufmann, Kerstin ; Rohde, Antje ; Geuten, Koen
Winter cereals require prolonged cold to transition from vegetative to reproductive development. This process, referred to as vernalization, has been extensively studied in Arabidopsis (Arabidopsis thaliana). In Arabidopsis, a key flowering repressor called FLOWERING LOCUS C (FLC) quantitatively controls the vernalization requirement. By contrast, in cereals, the vernalization response is mainly regulated by the VERNALIZATION genes, VRN1 and VRN2. Here, we characterize ODDSOC2, a recently identified FLC ortholog in monocots, knowing that it belongs to the FLC lineage. By studying its expression in a diverse set of Brachypodium accessions, we find that it is a good predictor of the vernalization requirement. Analyses of transgenics demonstrated that BdODDSOC2 functions as a vernalization-regulated flowering repressor. In most Brachypodium accessions BdODDSOC2 is down-regulated by cold, and in one of the winter accessions in which this down-regulation was evident, BdODDSOC2 responded to cold before BdVRN1. When stably down-regulated, the mechanism is associated with spreading H3K27me3 modifications at the BdODDSOC2 chromatin. Finally, homoeolog-specific gene expression analyses identify TaAGL33 and its splice variant TaAGL22 as the FLC orthologs in wheat (Triticum aestivum) behaving most similar to Brachypodium ODDSOC2. Overall, our study suggests that ODDSOC2 is not only phylogenetically related to FLC in eudicots but also functions as a flowering repressor in the vernalization pathway of Brachypodium and likely other temperate grasses. These insights could prove useful in breeding efforts to refine the vernalization requirement of temperate cereals and adapt varieties to changing climates.
Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin.
Ophiostomatoid fungi are vectored by their bark-beetle associates and colonize different host tree species. To survive and proliferate in the host, they have evolved mechanisms for detoxification and elimination of host defence compounds, efficient nutrient sequestration, and, in pathogenic species, virulence towards plants. Here, we assembled a draft genome of the spruce pathogen Ophiostoma bicolor. For our comparative and phylogenetic analyses, we mined the genomes of closely related species (Ophiostoma piceae, Ophiostoma ulmi, Ophiostoma novo-ulmi, and Grosmannia clavigera). Our aim was to acquire a genomic and evolutionary perspective of gene families important in host colonization. Genome comparisons showed that both the nuclear and mitochondrial genomes in our assembly were largely complete. Our O. bicolor 25.3 Mbp draft genome had 10 018 predicted genes, 6041 proteins with gene ontology (GO) annotation, 269 carbohydrate-active enzymes (CAZymes), 559 peptidases and inhibitors, and 1373 genes likely involved in pathogen-host interactions. Phylogenetic analyses of selected protein families revealed core sets of cytochrome P450 genes, ABC transporters and backbone genes involved in secondary metabolite (SM) biosynthesis (polyketide synthases (PKS) and non-ribosomal synthases), and species-specific gene losses and duplications. Phylogenetic analyses of protein families of interest provided insight into evolutionary adaptations to host biochemistry in ophiostomatoid fungi.
Zhang, Youjun ; Chen, Moxian ; Siemiatkowska, Beata ; Toleco, Mitchell Rey ; Jing, Yue ; Strotmann, Vivien ; Zhang, Jianghua ; Stahl, Yvonne ; Fernie, Alisdair
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.
Zhang, Youjun ; Chen, Moxian ; Siemiatkowska, Beata ; Toleco, Mitchell Rey ; Jing, Yue ; Strotmann, Vivien ; Zhang, Jianghua ; Stahl, Yvonne ; Fernie, Alisdair
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.
Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions: Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources.
Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor.
Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 mu L - 100 mu L). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter.
Conclusions: Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources.
Jantzen, Friederike ; Wozniak, Natalia Joanna ; Kappel, Christian ; Sicard, Adrien ; Lenhard, Michael
Background: The outcrossing rate is a key determinant of the population-genetic structure of species and their long-term evolutionary trajectories. However, determining the outcrossing rate using current methods based on PCRgenotyping individual offspring of focal plants for multiple polymorphic markers is laborious and time-consuming.
Results: We have developed an amplicon-based, high-throughput enabled method for estimating the outcrossing rate and have applied this to an example of scented versus non-scented Capsella (Shepherd’s Purse) genotypes. Our results show that the method is able to robustly capture differences in outcrossing rates. They also highlight potential biases in the estimates resulting from differential haplotype sharing of the focal plants with the pollen-donor population at individual amplicons.
Conclusions: This novel method for estimating outcrossing rates will allow determining this key population-genetic parameter with high-throughput across many genotypes in a population, enabling studies into the genetic determinants of successful pollinator attraction and outcrossing.
Jantzen, Friederike ; Wozniak, Natalia Joanna ; Kappel, Christian ; Sicard, Adrien ; Lenhard, Michael
Background: The outcrossing rate is a key determinant of the population-genetic structure of species and their long-term evolutionary trajectories. However, determining the outcrossing rate using current methods based on PCRgenotyping individual offspring of focal plants for multiple polymorphic markers is laborious and time-consuming.
Results: We have developed an amplicon-based, high-throughput enabled method for estimating the outcrossing rate and have applied this to an example of scented versus non-scented Capsella (Shepherd’s Purse) genotypes. Our results show that the method is able to robustly capture differences in outcrossing rates. They also highlight potential biases in the estimates resulting from differential haplotype sharing of the focal plants with the pollen-donor population at individual amplicons.
Conclusions: This novel method for estimating outcrossing rates will allow determining this key population-genetic parameter with high-throughput across many genotypes in a population, enabling studies into the genetic determinants of successful pollinator attraction and outcrossing.
A macro-tidal freshwater ecosystem recovering from hypereutrophication : the Schelde lease study
(2009)
Cox, Tom ; Maris, Tom ; Soetart, Karline ; Conley, Daniel ; van Damme, Stefan ; Meire, Patrick ; Middelburg, Jack J. ; Vos, Matthijs ; Struyf, Eric
We report a 40 year record of eutrophication and hypoxia on an estuarine ecosystem and its recovery from hypereutrophication. After decades of high inorganic nutrient concentrations and recurring anoxia and hypoxia, we observe a paradoxical increase in chlorophyll-a concentrations with decreasing nutrient inputs. We hypothesise that algal growth was inhibited due to hypereutrophication, either by elevated ammonium concentrations, severe hypoxia or the production of harmful substances in such a reduced environment. We study the dynamics of a simple but realistic mathematical model, incorporating the assumption of algal growth inhibition. It shows a high algal biomass, net oxygen production equilibrium with low ammonia inputs, and a low algal biomass, net oxygen consumption equilibrium with high ammonia inputs. At intermediate ammonia inputs it displays two alternative stable states. Although not intentional, the numerical output of this model corresponds to observations, giving extra support for assumption of algal growth inhibition. Due to potential algal growth inhibition, the recovery of hypereutrophied systems towards a classical eutrophied state, will need reduction of waste loads below certain thresholds and will be accompanied by large fluctuations in oxygen concentrations. We conclude that also flow-through systems, heavily influenced by external forcings which partly mask internal system dynamics, can display multiple stable states.
Karwinkel, Thiemo ; Winklhofer, Michael ; Janner, Lars Erik ; Brust, Vera ; Hüppop, Ommo ; Bairlein, Franz ; Schmaljohann, Heiko
Current evidence suggests that migratory animals extract map information from the geomagnetic field for true navigation. The sensory basis underlying this feat is elusive, but presumably involves magnetic particles.
A common experimental manipulation procedure consists of pre-treating animals with a magnetic pulse, with the aim of re-magnetising particles to alter the internal representation of the external field prior to a navigation task.
Although pulsing provoked deflected bearings in caged songbirds, analogous studies with free-flying songbirds yielded inconsistent results.
Here, we pulsed European robins (Erithacus rubecula) at an offshore stopover site during spring migration and monitored their free-flight behaviour with a regional-scale network of radio-receiving stations.
We found no pulse effect on departure probability, nocturnal departure timing departure direction or consistency of flight direction.
This suggests either no use of the geomagnetic map by our birds, or that magnetic pulses do not affect the sensory system underlying geomagnetic map detection.
A matter of concern
(2021)
Wandt, Viktoria Klara Veronika ; Winkelbeiner, Nicola Lisa ; Bornhorst, Julia ; Witt, Barbara ; Raschke, Stefanie ; Simon, Luise ; Ebert, Franziska ; Kipp, Anna Patricia ; Schwerdtle, Tanja
Neurons are post-mitotic cells in the brain and their integrity is of central importance to avoid neurodegeneration. Yet, the inability of self-replenishment of post-mitotic cells results in the need to withstand challenges from numerous stressors during life. Neurons are exposed to oxidative stress due to high oxygen consumption during metabolic activity in the brain. Accordingly, DNA damage can occur and accumulate, resulting in genome instability. In this context, imbalances in brain trace element homeostasis are a matter of concern, especially regarding iron, copper, manganese, zinc, and selenium. Although trace elements are essential for brain physiology, excess and deficient conditions are considered to impair neuronal maintenance. Besides increasing oxidative stress, DNA damage response and repair of oxidative DNA damage are affected by trace elements. Hence, a balanced trace element homeostasis is of particular importance to safeguard neuronal genome integrity and prevent neuronal loss. This review summarises the current state of knowledge on the impact of deficient, as well as excessive iron, copper, manganese, zinc, and selenium levels on neuronal genome stability
Author summary <br /> The use of orally inhaled drugs for treating lung diseases is appealing since they have the potential for lung selectivity, i.e. high exposure at the site of action -the lung- without excessive side effects. However, the degree of lung selectivity depends on a large number of factors, including physiochemical properties of drug molecules, patient disease state, and inhalation devices. To predict the impact of these factors on drug exposure and thereby to understand the characteristics of an optimal drug for inhalation, we develop a predictive mathematical framework (a "pharmacokinetic model"). In contrast to previous approaches, our model allows combining knowledge from different sources appropriately and its predictions were able to adequately predict different sets of clinical data. Finally, we compare the impact of different factors and find that the most important factors are the size of the inhaled particles, the affinity of the drug to the lung tissue, as well as the rate of drug dissolution in the lung. In contrast to the common belief, the solubility of a drug in the lining fluids is not found to be relevant. These findings are important to understand how inhaled drugs should be designed to achieve best treatment results in patients. <br /> The fate of orally inhaled drugs is determined by pulmonary pharmacokinetic processes such as particle deposition, pulmonary drug dissolution, and mucociliary clearance. Even though each single process has been systematically investigated, a quantitative understanding on the interaction of processes remains limited and therefore identifying optimal drug and formulation characteristics for orally inhaled drugs is still challenging. To investigate this complex interplay, the pulmonary processes can be integrated into mathematical models. However, existing modeling attempts considerably simplify these processes or are not systematically evaluated against (clinical) data. In this work, we developed a mathematical framework based on physiologically-structured population equations to integrate all relevant pulmonary processes mechanistically. A tailored numerical resolution strategy was chosen and the mechanistic model was evaluated systematically against data from different clinical studies. Without adapting the mechanistic model or estimating kinetic parameters based on individual study data, the developed model was able to predict simultaneously (i) lung retention profiles of inhaled insoluble particles, (ii) particle size-dependent pharmacokinetics of inhaled monodisperse particles, (iii) pharmacokinetic differences between inhaled fluticasone propionate and budesonide, as well as (iv) pharmacokinetic differences between healthy volunteers and asthmatic patients. Finally, to identify the most impactful optimization criteria for orally inhaled drugs, the developed mechanistic model was applied to investigate the impact of input parameters on both the pulmonary and systemic exposure. Interestingly, the solubility of the inhaled drug did not have any relevant impact on the local and systemic pharmacokinetics. Instead, the pulmonary dissolution rate, the particle size, the tissue affinity, and the systemic clearance were the most impactful potential optimization parameters. In the future, the developed prediction framework should be considered a powerful tool for identifying optimal drug and formulation characteristics.
A Metabarcoding Analysis of the Mycobiome of Wheat Ears Across a Topographically Heterogeneous Field
(2019)
For the elucidation of the dynamics of signal transduction processes that are induced by cellular interactions, defined events along the signal transduction cascade and subsequent activation steps have to be analyzed and then also correlated with each other. This cannot be achieved by ensemble measurements because averaging biological data ignores the variability in timing and response patterns of individual cells and leads to highly blurred results. Instead, only a multi-parameter analysis at a single-cell level is able to exploit the information that is crucially needed for deducing the signaling pathways involved. The aim of this work was to develop a process line that allows the initiation of cell-cell or cell-particle interactions while at the same time the induced cellular reactions can be analyzed at various stages along the signal transduction cascade and correlated with each other. As this approach requires the gentle management of individually addressable cells, a dielectrophoresis (DEP)-based microfluidic system was employed that provides the manipulation of microscale objects with very high spatiotemporal precision and without the need of contacting the cell membrane. The system offers a high potential for automation and parallelization. This is essential for achieving a high level of robustness and reproducibility, which are key requirements in order to qualify this approach for a biomedical application. As an example process for intercellular communication, T cell activation has been chosen. The activation of the single T cells was triggered by contacting them individually with microbeads that were coated with antibodies directed against specific cell surface proteins, like the T cell receptor-associated kinase CD3 and the costimulatory molecule CD28 (CD; cluster of differentiation). The stimulation of the cells with the functionalized beads led to a rapid rise of their cytosolic Ca2+ concentration which was analyzed by a dual-wavelength ratiometric fluorescence measurement of the Ca2+-sensitive dye Fura-2. After Ca2+ imaging, the cells were isolated individually from the microfluidic system and cultivated further. Cell division and expression of the marker molecule CD69 as a late activation event of great significance were analyzed the following day and correlated with the previously recorded Ca2+ traces for each individual cell. It turned out such that the temporal profile of the Ca2+ traces between both activated and non-activated cells as well as dividing and non-dividing cells differed significantly. This shows that the pattern of Ca2+ signals in T cells can provide early information about a later reaction of the cell. As isolated cells are highly delicate objects, a precondition for these experiments was the successful adaptation of the system to maintain the vitality of single cells during and after manipulation. In this context, the influences of the microfluidic environment as well as the applied electric fields on the vitality of the cells and the cytosolic Ca2+ concentration as crucially important physiological parameters were thoroughly investigated. While a short-term DEP manipulation did not affect the vitality of the cells, they showed irregular Ca2+ transients upon exposure to the DEP field only. The rate and the strength of these Ca2+ signals depended on exposure time, electric field strength and field frequency. By minimizing their occurrence rate, experimental conditions were identified that caused the least interference with the physiology of the cell. The possibility to precisely control the exact time point of stimulus application, to simultaneously analyze short-term reactions and to correlate them with later events of the signal transduction cascade on the level of individual cells makes this approach unique among previously described applications and offers new possibilities to unravel the mechanisms underlying intercellular communication.
An important goal in biotechnology and (bio-) medical research is the isolation of single cells from a heterogeneous cell population. These specialised cells are of great interest for bioproduction, diagnostics, drug development, (cancer) therapy and research. To tackle emerging questions, an ever finer differentiation between target cells and non-target cells is required. This precise differentiation is a challenge for a growing number of available methods.
Since the physiological properties of the cells are closely linked to their morphology, it is beneficial to include their appearance in the sorting decision. For established methods, this represents a non addressable parameter, requiring new methods for the identification and isolation of target cells. Consequently, a variety of new flow-based methods have been developed and presented in recent years utilising 2D imaging data to identify target cells within a sample. As these methods aim for high throughput, the devices developed typically require highly complex fluid handling techniques, making them expensive while offering limited image quality.
In this work, a new continuous flow system for image-based cell sorting was developed that uses dielectrophoresis to precisely handle cells in a microchannel. Dielectrophoretic forces are exerted by inhomogeneous alternating electric fields on polarisable particles (here: cells). In the present system, the electric fields can be switched on and off precisely and quickly by a signal generator. In addition to the resulting simple and effective cell handling, the system is characterised by the outstanding quality of the image data generated and its compatibility with standard microscopes. These aspects result in low complexity, making it both affordable and user-friendly.
With the developed cell sorting system, cells could be sorted reliably and efficiently according to their cytosolic staining as well as morphological properties at different optical magnifications. The achieved purity of the target cell population was up to 95% and about 85% of the sorted cells could be recovered from the system. Good agreement was achieved between the results obtained and theoretical considerations. The achieved throughput of the system was up to 12,000 cells per hour. Cell viability studies indicated a high biocompatibility of the system.
The results presented demonstrate the potential of image-based cell sorting using dielectrophoresis. The outstanding image quality and highly precise yet gentle handling of the cells set the system apart from other technologies. This results in enormous potential for processing valuable and sensitive cell samples.
A model analysis of mechanisms for radial microtubular patterns at root hair initiation sites
(2016)
Krupinski, Pawel ; Bozorg, Behruz ; Larsson, André ; Pietra, Stefano ; Grebe, Markus ; Jönsson, Henrik
Plant cells have two main modes of growth generating anisotropic structures. Diffuse growth where whole cell walls extend in specific directions, guided by anisotropically positioned cellulose fibers, and tip growth, with inhomogeneous addition of new cell wall material at the tip of the structure. Cells are known to regulate these processes via molecular signals and the cytoskeleton. Mechanical stress has been proposed to provide an input to the positioning of the cellulose fibers via cortical microtubules in diffuse growth. In particular, a stress feedback model predicts a circumferential pattern of fibers surrounding apical tissues and growing primordia, guided by the anisotropic curvature in such tissues. In contrast, during the initiation of tip growing root hairs, a star-like radial pattern has recently been observed. Here, we use detailed finite element models to analyze how a change in mechanical properties at the root hair initiation site can lead to star-like stress patterns in order to understand whether a stress-based feedback model can also explain the microtubule patterns seen during root hair initiation. We show that two independent mechanisms, individually or combined, can be sufficient to generate radial patterns. In the first, new material is added locally at the position of the root hair. In the second, increased tension in the initiation area provides a mechanism. Finally, we describe how a molecular model of Rho-of-plant (ROP) GTPases activation driven by auxin can position a patch of activated ROP protein basally along a 2D root epidermal cell plasma membrane, paving the way for models where mechanical and molecular mechanisms cooperate in the initial placement and outgrowth of root hairs.
Winkelbeiner, Nicola Lisa ; Wandt, Viktoria Klara Veronika ; Ebert, Franziska ; Lossow, Kristina ; Bankoglu, Ezgi E. ; Martin, Maximilian ; Mangerich, Aswin ; Stopper, Helga ; Bornhorst, Julia ; Kipp, Anna Patricia ; Schwerdtle, Tanja
Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2’-deoxyguanosine (8-oxodG), 5-hydroxy-2’-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
Winkelbeiner, Nicola Lisa ; Wandt, Viktoria Klara Veronika ; Ebert, Franziska ; Lossow, Kristina ; Bankoglu, Ezgi E. ; Martin, Maximilian ; Mangerich, Aswin ; Stopper, Helga ; Bornhorst, Julia ; Kipp, Anna Patricia ; Schwerdtle, Tanja
Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2’-deoxyguanosine (8-oxodG), 5-hydroxy-2’-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
A feasible approach to construct multilayer films of sulfonated polyanilines – PMSA1 and PABMSA1 – containing different ratios of aniline, 2-methoxyaniline-5-sulfonic acid (MAS) and 3-aminobenzoic acid (AB), with the entrapped redox enzyme pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) on Au and ITO electrode surfaces, is described. The formation of layers has been followed and confirmed by electrochemical impedance spectroscopy (EIS), which demonstrates that the multilayer assembly can be achieved in a progressive and uniform manner. The gold and ITO electrodes subsequently modified with PMSA1:PQQ-GDH and PABMSA1 films are studied by cyclic voltammetry (CV) and UV-Vis spectroscopy which show a significant direct bioelectrocatalytical response to the oxidation of the substrate glucose without any additional mediator. This response correlates linearly with the number of deposited layers. Furthermore, the constructed polymer/enzyme multilayer system exhibits a rather good long-term stability, since the catalytic current response is maintained for more than 60% of the initial value even after two weeks of storage. This verifies that a productive interaction of the enzyme embedded in the film of substituted polyaniline can be used as a basis for the construction of bioelectronic units, which are useful as indicators for processes liberating glucose and allowing optical and electrochemical transduction.
Cheng, Feng ; Dennis, Alice B. ; Osuoha, Josephine Ijeoma ; Canitz, Julia ; Kirschbaum, Frank ; Tiedemann, Ralph
Background
Teleost fishes comprise more than half of the vertebrate species. Within teleosts, most phylogenies consider the split between Osteoglossomorpha and Euteleosteomorpha/Otomorpha as basal, preceded only by the derivation of the most primitive group of teleosts, the Elopomorpha. While Osteoglossomorpha are generally species poor, the taxon contains the African weakly electric fish (Mormyroidei), which have radiated into numerous species. Within the mormyrids, the genus Campylomormyrus is mostly endemic to the Congo Basin. Campylomormyrus serves as a model to understand mechanisms of adaptive radiation and ecological speciation, especially with regard to its highly diverse species-specific electric organ discharges (EOD). Currently, there are few well-annotated genomes available for electric fish in general and mormyrids in particular. Our study aims at producing a high-quality genome assembly and to use this to examine genome evolution in relation to other teleosts. This will facilitate further understanding of the evolution of the osteoglossomorpha fish in general and of electric fish in particular.
Results
A high-quality weakly electric fish (C. compressirostris) genome was produced from a single individual with a genome size of 862 Mb, consisting of 1,497 contigs with an N50 of 1,399 kb and a GC-content of 43.69%. Gene predictions identified 34,492 protein-coding genes, which is a higher number than in the two other available Osteoglossomorpha genomes of Paramormyrops kingsleyae and Scleropages formosus. A Computational Analysis of gene Family Evolution (CAFE5) comparing 33 teleost fish genomes suggests an overall faster gene family turnover rate in Osteoglossomorpha than in Otomorpha and Euteleosteomorpha. Moreover, the ratios of expanded/contracted gene family numbers in Osteoglossomorpha are significantly higher than in the other two taxa, except for species that had undergone an additional genome duplication (Cyprinus carpio and Oncorhynchus mykiss). As potassium channel proteins are hypothesized to play a key role in EOD diversity among species, we put a special focus on them, and manually curated 16 Kv1 genes. We identified a tandem duplication in the KCNA7a gene in the genome of C. compressirostris.
Conclusions
We present the fourth genome of an electric fish and the third well-annotated genome for Osteoglossomorpha, enabling us to compare gene family evolution among major teleost lineages. Osteoglossomorpha appear to exhibit rapid gene family evolution, with more gene family expansions than contractions. The curated Kv1 gene family showed seven gene clusters, which is more than in other analyzed fish genomes outside Osteoglossomorpha. The KCNA7a, encoding for a potassium channel central for EOD production and modulation, is tandemly duplicated which may related to the diverse EOD observed among Campylomormyrus species.
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.
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.
Recently, Kocyan & Wiland-Szymańska (2016) have published a thorough research article on one of the outstanding members of the family Hypoxidaceae on the Seychelles, which resulted in the raise of a new genus (Friedmannia Kocyan & Wiland-Szymańska 2016: 60) to accommodate the former Curculigo seychellensis Bojer ex Baker (1877: 368). However, it has turned out that the name Friedmannia Chantanachat & Bold (1962: 45) already exists in literature for a green alga, which renders the new hypoxid genus illegitimate (Melbourne Code; McNeill et al. 2012). Therefore, we assign a new generic epithet to Curculigo seychellensis.
Van den Wyngaert, Silke ; Seto, Kensuke ; Rojas-Jimenez, Keilor ; Kagami, Maiko ; Grossart, Hans-Peter
Chytrids are a diverse group of ubiquitous true zoosporic fungi. The recent molecular discovery of a large diversity of undescribed chytrids has raised awareness on their important, but so far understudied ecological role in aquatic ecosystems. In the pelagic zone, of both freshwater and marine ecosystems, many chytrid species have been morphologically described as parasites on almost all major groups of phytoplankton. However, the majority of these parasitic chytrids has rarely been isolated and lack DNA sequence data, resulting in a large proportion of "dark taxa" in databases. Here, we report on the isolation and in-depth morphological, molecular and host range characterization of a chytrid infecting the common freshwater desmid Staurastrum sp. We provide first insights on the metabolic activity of the different chytrid development stages by using the vital dye FUN (R)-1 (2-chloro-4-[2,3-dihydro-3-methyl-[benzo-1,3-thiazol-2-yl]-methylidene]-1-phenylquinolinium iodide). Cross infection experiments suggest that this chytrid is an obligate parasite and specific for the genus Staurastrum sp. Phylogenetic analysis, based on ITS1-5.8S-ITS2 and 28S rDNA sequences, placed it in the order Rhizophydiales. Based on the unique zoospore ultrastructure, combined with thallus morphology, and molecular phylogenetic placement, we describe this parasitic chytrid as a new genus and species Staurastromyces oculus, within a new family Staurastromycetaceae. (C) 2017 Elsevier GmbH. All rights reserved.