@article{ZhangCasertaYarmanetal.2021, author = {Zhang, Xiaorong and Caserta, Giorgio and Yarman, Aysu and Supala, Eszter and Tadjoung Waffo, Armel Franklin and Wollenberger, Ulla and Gyurcsanyi, Robert E. and Zebger, Ingo and Scheller, Frieder W.}, title = {"Out of Pocket" protein binding}, series = {Chemosensors}, volume = {9}, journal = {Chemosensors}, number = {6}, publisher = {MDPI}, address = {Basel}, issn = {2227-9040}, doi = {10.3390/chemosensors9060128}, pages = {13}, year = {2021}, abstract = {The epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.}, language = {en} } @article{SchrapersHartmannKositzkietal.2015, author = {Schrapers, Peer and Hartmann, Tobias and Kositzki, Ramona and Dau, Holger and Reschke, Stefan and Schulzke, Carola and Leimk{\"u}hler, Silke and Haumann, Michael}, title = {'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus}, series = {Inorganic chemistry}, volume = {54}, journal = {Inorganic chemistry}, number = {7}, publisher = {American Chemical Society}, address = {Washington}, issn = {0020-1669}, doi = {10.1021/ic502880y}, pages = {3260 -- 3271}, year = {2015}, abstract = {Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO-) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Moligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.}, language = {en} } @article{UhligMadaboosiSchmidtetal.2012, author = {Uhlig, Katja and Madaboosi, Narayanan and Schmidt, Stephan and J{\"a}ger, Magnus S. and Rose, J{\"u}rgen and Duschl, Claus and Volodkin, Dmitry V.}, title = {3d localization and diffusion of proteins in polyelectrolyte multilayers}, series = {Soft matter}, volume = {8}, journal = {Soft matter}, number = {47}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1744-683X}, doi = {10.1039/c2sm26500a}, pages = {11786 -- 11789}, year = {2012}, abstract = {The interaction of diverse biomaterials with surfaces is more crucial than ever for biomedical applications to ensure efficiency and reproducibility. Very interesting surface materials are micrometer-thick polyelectrolyte multilayers. Not only their surface but also the bulk can be loaded with biomaterials like proteins or DNA for various purposes. Therefore, we established a method to analyze the lateral and vertical distribution of fluorescently labelled proteins of various size and charge in polyelectrolyte films composed of poly(L-lysine) and hyaluronic acid by confocal laser scanning microscopy. This approach enables us to measure the diffusion coefficients of the proteins via fluorescence recovery after photobleaching as a function of their vertical position in the film and facilitates the understanding of molecular interactions in the film with a high resolution in both space and time. As a result, we confirm that protein loading in the film is driven by electrostatic interactions - uncharged dextran molecules of 10 and 500 kDa do not diffuse into the film. Proteins of different sizes (3-11 nm) can diffuse relatively fast (D = 2-4 mm(2) s(-1)) independent of their net charge, indicating complex interpolymer interactions. This approach is a new powerful experimental tool to design the polyelectrolyte multilayers for bio-applications by finding a relationship between intermolecular interactions and mobility and availability of biomolecules to biological samples (e.g. cells) or detection units (e.g. biosensors).}, language = {en} } @article{SchwarzerHuamaniCanoetal.2010, author = {Schwarzer, Christian and Huamani, Fatima Cßceres and Cano, Asunci{\´o}n and La Torre, Mar{\´i}a I. La and Weigend, Maximilian}, title = {400 years for long-distance dispersal and divergence in the northern atacama desert : insights from the Huaynaputina pumice slopes of Moquegua, Peru}, issn = {0140-1963}, doi = {10.1016/j.jaridenv.2010.05.034}, year = {2010}, abstract = {The Huaynaputina eruption (1600 AD, Moquegua, S Peru) in the northern Atacama Desert denuded the Ornate area of all vegetation and deposited deep pumice layers. Data on the flora, climate and soil characteristics of these slopes near Ornate at 1600-2600 m a.s.l. are provided. Fifty-nine angiosperm species established themselves on the pumice slopes in the past ca. 400 years, with the bulk of the small and herbaceous species and several species new records for Peru. Three Ornate sites were sampled in both a dry and a wet year and species numbers differed widely (14 versus 45 spp.). Among areas compared floristic composition is most similar to the Lomas de Tacna, and has less in common with geographically closer Lomas or Sierra formations. Nine species represent highly disjunct populations (200->700 km) from their nearest known living populations in central Peru, Chile, or Argentina/Bolivia and appear to have reached the area via long-distance dispersal. Abiotic conditions may have played an important role in limiting the establishment of species from the neighboring vegetation. Four taxa on the pumice slopes show clear morphological differences to populations elsewhere, two of them may represent neoendemics of the Ornate pumice, indicating rapid morphological divergence. (C) 2010 Elsevier Ltd. All rights reserved.}, language = {en} } @article{GrudenHrenHermanetal.2012, author = {Gruden, Kristina and Hren, Matjaz and Herman, Ana and Blejec, Andrej and Albrecht, Tanja and Selbig, Joachim and Bauer, Christian G. and Schuchardt, Johannes and Or-Guil, Michal and Zupancic, Klemen and Svajger, Urban and Stabuc, Borut and Ihan, Alojz and Kopitar, Andreja Natasa and Ravnikar, Maja and Knezevic, Miomir and Rozman, Primoz and Jeras, Matjaz}, title = {A "Crossomics" study analysing variability of different components in peripheral blood of healthy caucasoid individuals}, series = {PLoS one}, volume = {7}, journal = {PLoS one}, number = {1}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0028761}, pages = {12}, year = {2012}, abstract = {Background: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable. Methodology/Principal Findings: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex "crossomics'' analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4(+)T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20\% of metabolites and less than 10\% of genes, were identified as highly variable in our dataset. Conclusions/Significance: Our study shows that the intra- individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective.}, language = {en} } @article{LeongRaffeinerSpintietal.2022, author = {Leong, Jia Xuan and Raffeiner, Margot and Spinti, Daniela and Langin, Gautier and Franz-Wachtel, Mirita and Guzman, Andrew R. and Kim, Jung-Gun and Pandey, Pooja and Minina, Alyona E. and Macek, Boris and Hafren, Anders and Bozkurt, Tolga O. and Mudgett, Mary Beth and B{\"o}rnke, Frederik and Hofius, Daniel and Uestuen, Suayib}, title = {A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component}, series = {The EMBO journal}, volume = {41}, journal = {The EMBO journal}, number = {13}, publisher = {Wiley}, address = {Hoboken}, issn = {0261-4189}, doi = {10.15252/embj.2021110352}, pages = {17}, year = {2022}, abstract = {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.}, language = {en} } @article{RuzanskiSmirnovaRejzeketal.2013, author = {Ruzanski, Christian and Smirnova, Julia and Rejzek, Martin and Cockburn, Darrell and Pedersen, Henriette L. and Pike, Marilyn and Willats, William G. T. and Svensson, Birte and Steup, Martin and Ebenh{\"o}h, Oliver and Smith, Alison M. and Field, Robert A.}, title = {A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night}, series = {The journal of biological chemistry}, volume = {288}, journal = {The journal of biological chemistry}, number = {40}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M113.497867}, pages = {28581 -- 28598}, year = {2013}, abstract = {Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a glucosyl buffer to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway.}, language = {en} } @article{WolffZhangHaagenDeckeretal.2017, author = {Wolff, Martin and Zhang-Haagen, Bo and Decker, Christina and Barz, Bogdan and Schneider, Mario and Biehl, Ralf and Radulescu, Aurel and Strodel, Birgit and Willbold, Dieter and Nagel-Steger, Luitgard}, title = {A beta 42 pentamers/hexamers are the smallest detectable oligomers in solution}, series = {Scientific reports}, volume = {7}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-017-02370-3}, pages = {13}, year = {2017}, language = {en} } @article{KlauschiesCoutinhoGaedke2018, author = {Klauschies, Toni and Coutinho, Renato Mendes and Gaedke, Ursula}, title = {A beta distribution-based moment closure enhances the reliability of trait-based aggregate models for natural populations and communities}, series = {Ecological modelling : international journal on ecological modelling and engineering and systems ecolog}, volume = {381}, journal = {Ecological modelling : international journal on ecological modelling and engineering and systems ecolog}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0304-3800}, doi = {10.1016/j.ecolmodel.2018.02.001}, pages = {46 -- 77}, year = {2018}, abstract = {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.}, language = {en} } @article{HuangWarsinkeKoroljovaSkorobogatkoetal.1999, author = {Huang, T. and Warsinke, Axel and Koroljova-Skorobogatko, O. V. and Makower, Alexander and Kuwana, T. and Scheller, Frieder W.}, title = {A bienzyme carbon paste electrode for the sensitive detection of NADPH and the measurement of glucose-6- phosphate dehydrogenase}, year = {1999}, language = {en} } @article{GajovicWarsinkeScheller1998, author = {Gajovic, Nenad and Warsinke, Axel and Scheller, Frieder W.}, title = {A bienzyme electrode for L-malate based on a novel and general design}, year = {1998}, language = {en} } @article{EremenkoMakowerBaueretal.1997, author = {Eremenko, A. V. and Makower, Alexander and Bauer, Christian G. and Kurochkin, I. N. and Scheller, Frieder W.}, title = {A bienzyme electrode for tyrosine containing peptides determination}, year = {1997}, language = {en} } @article{LettauWarsinkeKatterleetal.2006, author = {Lettau, Kristian and Warsinke, Axel and Katterle, Martin and Danielsson, Bengt and Scheller, Frieder W.}, title = {A bifunctional molecularly imprinted polymer (MIP): analysis of binding and catalysis by a thermistor}, doi = {10.1002/anie.200601796}, year = {2006}, abstract = {Binding or catalysis? Both can be distinguished with a molecularly imprinted polymer (MIP) by the different patterns of heat generation. The catalytically active sites, like in the corresponding enzyme, generate a steady-state temperature increase. Thus, enzyme-like catalysis and antibody-analogue binding are analyzed simultaneously in a bifunctional MIP for the first time (see scheme).}, language = {en} } @article{ZhangBramskiTutusetal.2019, author = {Zhang, Shuhao and Bramski, Julia and Tutus, Murat and Pietruszka, J{\"o}rg and B{\"o}ker, Alexander and Reinicke, Stefan}, title = {A Biocatalytically Active Membrane Obtained from Immobilization of 2-Deoxy-D-ribose-5-phosphate Aldolase on a Porous Support}, series = {ACS applied materials \& interfaces}, volume = {11}, journal = {ACS applied materials \& interfaces}, number = {37}, publisher = {American Chemical Society}, address = {Washington}, issn = {1944-8244}, doi = {10.1021/acsami.9b12029}, pages = {34441 -- 34453}, year = {2019}, abstract = {Aldol reactions play an important role in organic synthesis, as they belong to the class of highly beneficial C-C-linking reactions. Aldol-type reactions can be efficiently and stereoselectively catalyzed by the enzyme 2-deoxy-D-ribose-5-phosphate aldolase (DERA) to gain key intermediates for pharmaceuticals such as atorvastatin. The immobilization of DERA would open the opportunity for a continuous operation mode which gives access to an efficient, large-scale production of respective organic intermediates. In this contribution, we synthesize and utilize DERA/polymer conjugates for the generation and fixation of a DERA bearing thin film on a polymeric membrane support. The conjugation strongly increases the tolerance of the enzyme toward the industrial relevant substrate acetaldehyde while UV-cross-linkable groups along the conjugated polymer chains provide the opportunity for covalent binding to the support. First, we provide a thorough characterization of the conjugates followed by immobilization tests on representative, nonporous cycloolefinic copolymer supports. Finally, immobilization on the target supports constituted of polyacrylonitrile (PAN) membranes is performed, and the resulting enzymatically active membranes are implemented in a simple membrane module setup for the first assessment of biocatalytic performance in the continuous operation mode using the combination hexanal/acetaldehyde as the substrate.}, language = {en} } @article{BoechatWeithoffKruegeretal.2007, author = {Bo{\"e}chat, Iola G. and Weithoff, Guntram and Kr{\"u}ger, Angela and G{\"u}cker, Bj{\"o}rn and Adrian, Rita}, title = {A biochemical explanation for the success of the mixotrophy in the flagellate Ochromonas sp.}, issn = {0024-3590}, doi = {10.4319/lo.2007.52.4.1624}, year = {2007}, abstract = {We report the influence of different nutritional modes-autotrophy, mixotrophy, and heterotrophy-on the fatty acid and sterol composition of the freshwater flagellate Ochromonas sp. and discuss the ecological significance of our results with respect to the resource competition theory (rct). Polyunsaturated fatty acids (PUFAs) are the most efficient biochemical variable distinguishing between nutritional modes of Ochromonas sp. Decreasing concentrations of PUFAs were observed in the order autotrophs, mixotrophs, heterotrophs. In mixotrophs and heterotrophs, concentrations of saturated fatty acids were higher than those of monounsaturated fatty acids and PUFAs as a result of bacterivory. Stigmasterol was the main sterol in Ochromonas sp., regardless of nutritional mode. Mixotrophs showed higher growth rates than heterotrophs, which could not be explained by rct. Heterotrophs, in turn, exhibited higher growth rates than autotrophs, which were cultured under the same light conditions as mixotrophs. Mixotrophs can synthesize PUFAs, which are important for many physiological functions such as membrane permeability and growth. Thus, mixotrophy facilitated efficient growth as well as the ability to synthesize complex and essential biomolecules. These strong synergetic effects are due to the combination of biochemical benefits of heterotrophic and autotrophic metabolic pathways and cannot be predicted by rct.}, language = {en} } @article{BadalyanNeumannSchaalLeimkuehleretal.2013, author = {Badalyan, Artavazd and Neumann-Schaal, Meina and Leimk{\"u}hler, Silke and Wollenberger, Ursula}, title = {A Biosensor for aromatic aldehydes comprising the mediator dependent PaoABC-Aldehyde oxidoreductase}, series = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis}, volume = {25}, journal = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis}, number = {1}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1040-0397}, doi = {10.1002/elan.201200362}, pages = {101 -- 108}, year = {2013}, abstract = {A novel aldehyde oxidoreductase (PaoABC) from Escherichia coli was utilized for the development of an oxygen insensitive biosensor for benzaldehyde. The enzyme was immobilized in polyvinyl alcohol and currents were measured for aldehyde oxidation with different one and two electron mediators with the highest sensitivity for benzaldehyde in the presence of hexacyanoferrate(III). The benzaldehyde biosensor was optimized with respect to mediator concentration, enzyme loading and pH using potassium hexacyanoferrate(III). The linear measuring range is between 0.5200 mu M benzaldehyde. In correspondence with the substrate selectivity of the enzyme in solution the biosensor revealed a preference for aromatic aldehydes and less effective conversion of aliphatic aldehydes. The biosensor is oxygen independent, which is a particularly attractive feature for application. The biosensor can be applied to detect contaminations with benzaldehyde in solvents such as benzyl alcohol, where traces of benzaldehyde in benzyl alcohol down to 0.0042?\% can be detected.}, language = {en} } @article{BergMohnickeNendel2022, author = {Berg-Mohnicke, Michael and Nendel, Claas}, title = {A case for object capabilities as the foundation of a distributed environmental model and simulation infrastructure}, series = {Environmental modelling \& software with environment data news}, volume = {156}, journal = {Environmental modelling \& software with environment data news}, publisher = {Elsevier}, address = {Oxford}, issn = {1364-8152}, doi = {10.1016/j.envsoft.2022.105471}, pages = {12}, year = {2022}, abstract = {With the advent of increasingly powerful computational architectures, scientists use these possibilities to create simulations of ever-increasing size and complexity. Large-scale simulations of environmental systems require huge amounts of resources. Managing these in an operational way becomes increasingly complex and difficult to handle for individual scientists. State-of-the-art simulation infrastructures usually provide the necessary re-sources in a centralised setup, which often results in an all-or-nothing choice for the user. Here, we outline an alternative approach to handling this complexity, while rendering the use of high-performance hardware and large datasets still possible. It retains a number of desirable properties: (i) a decentralised structure, (ii) easy sharing of resources to promote collaboration and (iii) secure access to everything, including natural delegation of authority across levels and system boundaries. We show that the object capability paradigm will cover these issues, and present the first steps towards developing a simulation infrastructure based on these principles.}, language = {en} } @article{HaueisStechKubick2022, author = {Haueis, Lisa and Stech, Marlitt and Kubick, Stefan}, title = {A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies}, series = {Frontiers in Bioengineering and Biotechnology}, volume = {10}, journal = {Frontiers in Bioengineering and Biotechnology}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {2296-4185}, doi = {10.3389/fbioe.2022.896763}, pages = {11}, year = {2022}, abstract = {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.}, language = {en} } @article{KrebsRakotoarinoroStechetal.2022, author = {Krebs, Simon K. and Rakotoarinoro, Nathanael and Stech, Marlitt and Zemella, Anne and Kubick, Stefan}, title = {A CHO-based cell-free dual fluorescence reporter system for the straightforward assessment of amber suppression and scFv functionality}, series = {Frontiers in Bioengineering and Biotechnology}, volume = {10}, journal = {Frontiers in Bioengineering and Biotechnology}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {2296-4185}, doi = {10.3389/fbioe.2022.873906}, pages = {15}, year = {2022}, abstract = {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).}, language = {en} } @article{CabukUenlue2022, author = {{\c{C}}abuk, Uğur and {\"U}nl{\"u}, Ercan Sel{\c{c}}uk}, title = {A combined de novo assembly approach increases the quality of prokaryotic draft genomes}, series = {Folia microbiologica : international journal for general, environmental and applied microbiology, and immunology}, volume = {67}, journal = {Folia microbiologica : international journal for general, environmental and applied microbiology, and immunology}, publisher = {Springer}, address = {Dordrecht}, issn = {0015-5632}, doi = {10.1007/s12223-022-00980-7}, pages = {801 -- 810}, year = {2022}, abstract = {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.}, language = {en} }