TY - JOUR A1 - Haueis, Lisa A1 - Stech, Marlitt A1 - Kubick, Stefan T1 - A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies JF - Frontiers in Bioengineering and Biotechnology N2 - 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. KW - cell-free protein synthesis KW - In vitro transcription KW - translation KW - nanobody KW - VHH KW - camelid KW - CHO cell lysate Y1 - 2022 U6 - https://doi.org/10.3389/fbioe.2022.896763 SN - 2296-4185 VL - 10 PB - Frontiers Media CY - Lausanne ER - TY - JOUR A1 - Coudenys, Wim A1 - Warditz, Vladislava T1 - Is translation child's play? BT - Circulation of knowledge in Lomonosov's Kratkij rossijskij letopisec s rodosloviem (1760) and its translations JF - Die Welt der Slaven : internationale Halbjahresschrift für Slavistik N2 - 1765 and 1767 saw the publication of the German, respectively the English translation of Lomonosov's Kratkij rossijskij letopisec s rodosloviem (1760). For the very first time the European reading public could find out how Russians saw their own history. These translations testified to Russia's ascent both as an empire and as a part of European learned society, and were made by youths who wanted to further their own career and were neither professional translators nor historians. In this article, we argue that the translations of Lomonosov's Kratkij rossijskij letopisec should not be studied as an isolated act of cultural transfer but as an episode in a longer history of circulation of knowledge. We demonstrate the complexity of this circulation by reassessing the 'quality' of these translations and positioning them in that longer history of circulation of knowledge by analysing the distribution of historical concepts (Begriffe) in Lomonosov's original and its translations. KW - circulation of knowledge KW - translation KW - historiography KW - 18th century KW - Russia KW - Lomonosov KW - conceptual history Y1 - 2020 U6 - https://doi.org/10.13173/ws.66.1.46 SN - 0043-2520 SN - 2193-5475 VL - 66 IS - 1 SP - 46 EP - 69 PB - Harrassowitz CY - Wiesbaden ER - TY - JOUR A1 - Hermanussen, Michael A1 - Scheffler, Christiane T1 - Evidence of chronic undernutrition in late 19th century German infants of all social classes JF - Human biology and public health N2 - 125 years ago, European infants grew differently from modern infants. We show weight gains of 20 healthy children weighed longitudinally from birth to age 1 year, published by Camerer in 1882. The data illustrate the historically prevalent concepts of infant nutrition practiced by German civil servants, lawyers, merchants, university professors, physicians, foresters and farmers. Breastfeeding by the mother was not truly appreciated in those days; children were often breastfed by wet nurses or received bottled milk. Bottle feeding mainly used diluted cow’s milk with some added carbohydrates, without evidence that appropriate amounts of oil, butter or other fatty components were added. French children from 1914 showed similar weight gain patterns suggesting similar feeding practices. The historical data suggest that energy deficient infant formula was fed regularly in the late 19th and early 20th century Europe, regardless of wealth and social class. The data question current concerns that temporarily feeding energy deficient infant formula may warrant serious anxieties regarding long-term cognitive, social and emotional behavioral development. KW - chronic undernutrition KW - breastfeeding KW - historical growth KW - social class KW - translation Y1 - 2022 U6 - https://doi.org/10.52905/hbph2022.2.42 SN - 2748-9957 VL - 2022 IS - 2 PB - Universitätsverlag Potsdam CY - Potsdam ER - TY - JOUR A1 - Wiemann, Dirk T1 - Layer after Layer BT - aerial roots and routes of translation JF - Thesis Eleven N2 - When the Royal Botanic Gardens at Kew in South London were opened to the general public in the 1840s, they were presented as a ‘world text’: a collection of flora from all over the world, with the spectacular tropical (read: colonial) specimens taking centre stage as indexes of Britain’s imperial supremacy. However, the one exotic plant species that preoccupied the British cultural imagination more than any other remained conspicuously absent from the collection: the banyan tree, whose non-transferability left a significant gap in the ‘text’ of the garden, thereby effectively puncturing the illusion of comprehensive global command that underpins the biopolitical designs of what Richard Grove has aptly dubbed ‘green imperialism’. This article demonstrates how, in the 19th and early 20th centuries, the banyan tree became an object of fascination and admiration for British scientists, painters, writers and photographers precisely because of its obstinate non-availability to colonial control and visual or even conceptual representability. KW - banyan KW - colonial botany KW - historical nature KW - Kew Gardens KW - translation Y1 - 2021 U6 - https://doi.org/10.1177/0725513621990772 SN - 0725-5136 VL - 162 IS - 1 SP - 33 EP - 45 PB - Sage CY - Melbourne ER - TY - THES A1 - Schuster, Maja T1 - High resolution decoding of the tobacco chloroplast translatome and its dynamics during light-intensity acclimation N2 - Chloroplasts are the photosynthetic organelles in plant and algae cells that enable photoautotrophic growth. Due to their prokaryotic origin, modern-day chloroplast genomes harbor 100 to 200 genes. These genes encode for core components of the photosynthetic complexes and the chloroplast gene expression machinery, making most of them essential for the viability of the organism. The regulation of those genes is predominated by translational adjustments. The powerful technique of ribosome profiling was successfully used to generate highly resolved pictures of the translational landscape of Arabidopsis thaliana cytosol, identifying translation of upstream open reading frames and long non-coding transcripts. In addition, differences in plastidial translation and ribosomal pausing sites were addressed with this method. However, a highly resolved picture of the chloroplast translatome is missing. Here, with the use of chloroplast isolation and targeted ribosome affinity purification, I generated highly enriched ribosome profiling datasets of the chloroplasts translatome for Nicotiana tabacum in the dark and light. Chloroplast isolation was found unsuitable for the unbiased analysis of translation in the chloroplast but adequate to identify potential co-translational import. Affinity purification was performed for the small and large ribosomal subunit independently. The enriched datasets mirrored the results obtained from whole-cell ribosome profiling. Enhanced translational activity was detected for psbA in the light. An alternative translation initiation mechanism was not identified by selective enrichment of small ribosomal subunit footprints. In sum, this is the first study that used enrichment strategies to obtain high-depth ribosome profiling datasets of chloroplasts to study ribosome subunit distribution and chloroplast associated translation. Ever-changing light intensities are challenging the photosynthetic capacity of photosynthetic organism. Increased light intensities may lead to over-excitation of photosynthetic reaction centers resulting in damage of the photosystem core subunits. Additional to an expensive repair mechanism for the photosystem II core protein D1, photosynthetic organisms developed various features to reduce or prevent photodamage. In the long-term, photosynthetic complex contents are adjusted for the efficient use of experienced irradiation. However, the contribution of chloroplastic gene expression in the acclimation process remained largely unknown. Here, comparative transcriptome and ribosome profiling was performed for the early time points of high-light acclimation in Nicotiana tabacum chloroplasts in a genome-wide scale. The time- course data revealed stable transcript level and only minor changes in translational activity of specific chloroplast genes during high-light acclimation. Yet, psbA translation was increased by two-fold in the high light from shortly after the shift until the end of the experiment. A stress-inducing shift from low- to high light exhibited increased translation only of psbA. This study indicate that acclimation fails to start in the observed time frame and only short-term responses to reduce photoinhibition were observed. N2 - Chloroplasten sind die photosynthetischen Organellen in Pflanzen- und Algenzellen, die photoautotrophes Wachstum ermöglichen. Aufgrund ihrer prokaryotischen Herkunft besitzen moderne Chloroplasten ein Genom mit 100 bis 200 Gene. Diese kodieren für zentrale Komponenten der Photosynthesekomplexe und des Genexpressionsapparates, was sie für die Lebensfähigkeit des gesamten Organismus essenziell macht. Die leistungsstarke Methode Ribosome Profiling wurde bereits erfolgreich eingesetzt, um hochaufgelöste Bilder der zytosolischen Translationslandschaft von Arabidopsis thaliana zu erstellen, wobei Translation von der Hauptsequenz vorgelagerten, kodierenden Sequenzen und langen, nicht-kodierenden Transkripten identifiziert wurde. Ferner wurden mit dieser Technik Regulationen der Plastidentranslation und spezifische Regionen mit unterschiedlicher Elongationsgeschwindigkeit aufgedeckt. Es fehlen jedoch hochaufgelöste Datensätze des Chloroplasten-Translatoms. Chloroplastenisolation und Affinitätsaufreinigung chloroplastidiärer Ribosomen wurde verwendet, um hochangereicherte Ribosome Profiling-Datensätze des Chloroplastentranslatoms für Nicotiana tabacum im Dunkeln und unter Licht zu erzeugen. Wenngleich sich die Chloroplastenisolation als ungeeignet für eine unverfälschte Analyse der Translation im Chloroplast erwies, ermöglichte sie die Identifizierung von potentiellem co-translationalen Proteinimport. Die entsprechenden Datensätze spiegelten die Ergebnisse des zellulären Ribosome Profilings wider. Für psbA wurde im Licht erhöhte Translationsaktivität festgestellt. Alternative Initiationsmechanismen konnten durch spezifische Anreicherung der kleinen ribosomalen Untereinheit nicht verifiziert werden. Zusammenfassend, dies ist die erste Studie, die mittels Anreicherungsstrategien hochaufgelöste Ribosome Profiling-Datensätze zur Analyse von Ribosomuntereinheitsverteilungen und Chloroplast-assoziierter Translation nutzte. Ständig wechselnde Lichtintensitäten stellen die Photosynthesekapazität von photosynthetischen Organismen auf die Probe. Erhöhte Lichtintensitäten können zu einer Überreizung der photosynthetischen Reaktionszentren führen, was Beschädigungen von zentralen Komplexeinheiten der Photosysteme verursacht. Neben einem aufwändigen Reparaturmechanismus für das Photosystem II-Protein D1 entwickelte der photosynthetische Organismus verschiedene Mechanismen um lichtinduzierte Schäden zu reduzieren oder zu verhindern. Langfristig kommt es zu einer Anreicherung spezifischer Photosynthesekomplexen um eine effiziente Ausnutzung der erhöhten Strahlung zu gewährleisten. Der Beitrag der chloroplastidiäeren Genexpressionsregulation zum Akklimatisierungsprozess ist jedoch weitgehend unbekannt. Hier wurde ein vergleichendes Transkript- und Ribosomen Profiling für die frühen Zeitpunkte der Akklimatisierung unter Starklicht in Tabakchloroplasten in einem genomweiten Maßstab durchgeführt. Die Zeitverlaufsdaten zeigten ein unverändertes Transkriptniveau und nur geringe Änderungen der translationalen Aktivität von chloroplastidiären Genen im Hochlicht im Vergleich zu Kontrollproben. Die psbA-Translation war jedoch unter Hochlicht schon kurz nach Beginn bis zum Ende des Experiments um etwa das Zweifache erhöht. Der stressinduzierende Wechsel von Schwach- zu Hochlicht bewirkte ebenfalls eine auf psbA-beschränkt, erhöhte Translation. Die Ergebnisse zeigen, dass die Akklimatisierung im beobachteten Zeitrahmen nicht begonnen hatte und nur kurzfristige Reaktionen zur Verringerung der Photoinhibition wirksam gewesen sein konnten. KW - translation KW - chloroplast KW - high light KW - ribosome profiling Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-512680 ER - TY - JOUR A1 - Eckstein, Lars A1 - Schwarz, Anja T1 - The making of Tupaia’s map BT - a story of the extent and mastery of Polynesian navigation, competing systems of wayfinding on James Cook’s endeavour, and the invention of an ingenious cartographic system JF - The journal of pacific history N2 - Tupaia’s Map is one of the most famous and enigmatic artefacts to emerge from the early encounters between Europeans and Pacific Islanders. It was drawn by Tupaia, an arioi priest, chiefly advisor and master navigator from Ra‘iātea in the Leeward Society Islands in collaboration with various members of the crew of James Cook’s Endeavour, in two distinct moments of mapmaking and three draft stages between August 1769 and February 1770. To this day, the identity of many islands on the chart, and the logic of their arrangement have posed a riddle to researchers. Drawing in part on archival material hitherto overlooked, in this long essay we propose a new understanding of the chart’s cartographic logic, offer a detailed reconstruction of its genesis, and thus for the first time present a comprehensive reading of Tupaia’s Map. The chart not only underscores the extent and mastery of Polynesian navigation, it is also a remarkable feat of translation between two very different wayfinding systems and their respective representational models. KW - Cartography KW - first contact KW - wayfinding KW - star navigation KW - sea of islands KW - translation KW - Indigenous knowledges and ontologies KW - Tupaia Y1 - 2019 U6 - https://doi.org/10.1080/00223344.2018.1512369 SN - 0022-3344 SN - 1469-9605 VL - 54 IS - 1 SP - 1 EP - 95 PB - Routledge, Taylor & Francis Group CY - London ER - TY - GEN A1 - Bentele, Kajetan A1 - Saffert, Paul A1 - Rauscher, Robert A1 - Ignatova, Zoya A1 - Bluethgen, Nils T1 - Efficient translation initiation dictates codon usage at gene start T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 912 KW - codon usage KW - mRNA structure KW - translation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-441337 SN - 1866-8372 IS - 912 ER - TY - JOUR A1 - Bartholomäus, Alexander A1 - Fedyunin, Ivan A1 - Feist, Peter A1 - Sin, Celine A1 - Zhang, Gong A1 - Valleriani, Angelo A1 - Ignatova, Zoya T1 - Bacteria differently regulate mRNA abundance to specifically respond to various stresses JF - Geology N2 - Environmental stress is detrimental to cell viability and requires an adequate reprogramming of cellular activities to maximize cell survival. We present a global analysis of the response of Escherichia coli to acute heat and osmotic stress. We combine deep sequencing of total mRNA and ribosome-protected fragments to provide a genome-wide map of the stress response at transcriptional and translational levels. For each type of stress, we observe a unique subset of genes that shape the stress-specific response. Upon temperature upshift, mRNAs with reduced folding stability up-and downstream of the start codon, and thus with more accessible initiation regions, are translationally favoured. Conversely, osmotic upshift causes a global reduction of highly translated transcripts with high copy numbers, allowing reallocation of translation resources to not degraded and newly synthesized mRNAs. KW - transcription KW - translation KW - deep sequencing KW - Escherichia coli KW - copy numbers Y1 - 2016 U6 - https://doi.org/10.1098/rsta.2015.0069 SN - 1364-503X SN - 1471-2962 VL - 374 PB - Royal Society CY - London ER - TY - JOUR A1 - Gorochowski, Thomas E. A1 - Aycilar-Kucukgoze, Irem A1 - Bovenberg, Roel A. L. A1 - Roubos, Johannes A. A1 - Ignatova, Zoya T1 - A Minimal Model of Ribosome Allocation Dynamics Captures Trade-offs in Expression between Endogenous and Synthetic Genes JF - ACS synthetic biology N2 - Cells contain a finite set of resources that must be distributed across many processes to ensure survival. Among them, the largest proportion of cellular resources is dedicated to protein translation. Synthetic biology often exploits these resources in executing orthogonal genetic circuits, yet the burden this places on the cell is rarely considered. Here, we develop a minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for the maintenance of cell physiology. Critically, it contains three key variables related to design parameters of the synthetic construct covering transcript abundance, translation initiation rate, and elongation time. We show that model-predicted changes in ribosome allocation closely match experimental shifts in synthetic protein expression rate and cellular growth. Intriguingly, the model is also able to accurately infer transcript levels and translation times after further exposure to additional ambient stress. Our results demonstrate that a simple model of resource allocation faithfully captures the redistribution of protein synthesis resources when faced with the burden of synthetic gene expression and environmental stress. The tractable nature of the model makes it a versatile tool for exploring the guiding principles of efficient heterologous expression and the indirect interactions that can arise between synthetic circuits and their host chassis because of competition for shared translational resources. KW - protein biosynthesis KW - translation KW - synthetic biology KW - systems biology Y1 - 2016 U6 - https://doi.org/10.1021/acssynbio.6b00040 SN - 2161-5063 VL - 5 SP - 710 EP - 720 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Saffert, Paul A1 - Adamla, Frauke A1 - Schieweck, Rico A1 - Atkins, John F. A1 - Ignatova, Zoya T1 - An Expanded CAG Repeat in Huntingtin Causes+1 Frameshifting JF - The journal of biological chemistry N2 - Maintenance of triplet decoding is crucial for the expression of functional protein because deviations either into the -1 or +1 reading frames are often non-functional. We report here that expression of huntingtin (Htt) exon 1 with expanded CAG repeats, implicated in Huntington pathology, undergoes a sporadic +1 frameshift to generate from the CAG repeat a trans-frame AGC repeat-encoded product. This +1 recoding is exclusively detected in pathological Htt variants, i.e. those with expanded repeats with more than 35 consecutive CAG codons. An atypical +1 shift site, UUC C at the 5 end of CAG repeats, which has some resemblance to the influenza A virus shift site, triggers the +1 frameshifting and is enhanced by the increased propensity of the expanded CAG repeats to form a stem-loop structure. The +1 trans-frame-encoded product can directly influence the aggregation of the parental Htt exon 1. KW - aggregation KW - Huntington disease KW - translation KW - translation regulation KW - trinucleotide repeat disease KW - frameshifting KW - seeding Y1 - 2016 U6 - https://doi.org/10.1074/jbc.M116.744326 SN - 0021-9258 SN - 1083-351X VL - 291 SP - 18505 EP - 18513 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER -