TY - JOUR A1 - Chang, Dan A1 - Knapp, Michael A1 - Enk, Jacob A1 - Lippold, Sebastian A1 - Kircher, Martin A1 - Lister, Adrian M. A1 - MacPhee, Ross D. E. A1 - Widga, Christopher A1 - Czechowski, Paul A1 - Sommer, Robert A1 - Hodges, Emily A1 - Stümpel, Nikolaus A1 - Barnes, Ian A1 - Dalén, Love A1 - Derevianko, Anatoly A1 - Germonpré, Mietje A1 - Hillebrand-Voiculescu, Alexandra A1 - Constantin, Silviu A1 - Kuznetsova, Tatyana A1 - Mol, Dick A1 - Rathgeber, Thomas A1 - Rosendahl, Wilfried A1 - Tikhonov, Alexey N. A1 - Willerslev, Eske A1 - Hannon, Greg A1 - Lalueza i Fox, Carles A1 - Joger, Ulrich A1 - Poinar, Hendrik N. A1 - Hofreiter, Michael A1 - Shapiro, Beth T1 - The evolutionary and phylogeographic history of woolly mammoths BT - a comprehensive mitogenomic analysis JF - Scientific reports N2 - Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0–2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths. Y1 - 2017 U6 - https://doi.org/10.1038/srep44585 SN - 2045-2322 VL - 7 PB - Nature Publishing Group CY - London ER - TY - JOUR A1 - Czernitzki, Anna-Franziska A1 - Pospisil, Christina A1 - Musalek, Martin A1 - Mumm, Rebekka A1 - Scheffler, Christiane T1 - Analysis of longitudinal data of height z-scores in kindergarten children BT - a pilot study JF - Journal of biological and clinical anthropology : Anthropologischer Anzeiger ; Mitteilungsorgan der Gesellschaft für Anthropologie N2 - Changes in body height throughout extended historic periods are very complex and dynamic processes. Thispilot study aimed to investigate the pattern of longitudinal height z-scores changes in children before and after entering kindergarten. In summer 2016, we measured height and weight of 32 children from 4 groups of two kindergartens aged 3–6 years. All ages were centered according to the age of entry into the kindergarten. For each child we determined mean z-scores for height before and after entering the kindergarten, and assessed the variances for each kindergarten group. Twenty-two children targeted in height z-scores towards average height of their respective kindergarten group, 10 children did not. Due to the small numbers, the convergence in height variance however, remained insignificant (chi-squared independence test, p = 0.127). Additional studies with larger sample sizes are needed to confirm this pilot study. KW - Height z-score KW - kindergarten children KW - secular trend KW - strategic growth adjustment KW - social signal Y1 - 2017 U6 - https://doi.org/10.1127/anthranz/2017/0708 SN - 0003-5548 VL - 74 IS - 2 SP - 109 EP - 112 PB - Schweizerbart science publishers CY - Stuttgart ER - TY - THES A1 - de Abreu e Lima, Francisco Anastacio T1 - Experimental validation of hybrid performance predictive models in Zea mays L. Y1 - 2017 ER - TY - THES A1 - de Souza, Leonardo Perez T1 - Functional characterization of biosynthesis and regulation of plant secondary metabolism Y1 - 2017 ER - TY - THES A1 - Diez Cocero, Mercedes T1 - Analysis of Rubisco – carbonic anhydrase fusions in tobacco as an approach to reduce photorespiration N2 - Rubisco catalyses the first step of CO2 assimilation into plant biomass. Despite its crucial role, it is notorious for its low catalytic rate and its tendency to fix O2 instead of CO2, giving rise to a toxic product that needs to be recycled in a process known as photorespiration. Since almost all our food supply relies on Rubisco, even small improvements in its specificity for CO2 could lead to an improvement of photosynthesis and ultimately, crop yield. In this work, we attempted to improve photosynthesis by decreasing photorespiration with an artificial CCM based on a fusion between Rubisco and a carbonic anhydrase (CA). A preliminary set of plants contained fusions between one of two CAs, bCA1 and CAH3, and the N- or C-terminus of RbcL connected by a small flexible linker of 5 amino acids. Subsequently, further fusion proteins were created between RbcL C-terminus and bCA1/CAH3 with linkers of 14, 23, 32, and 41 amino acids. The transplastomic tobacco plants carrying fusions with bCA1 were able to grow autotrophically even with the shortest linkers, albeit at a low rate, and accumulated very low levels of the fusion protein. On the other hand, plants carrying fusions with CAH3 were autotrophic only with the longer linkers. The longest linker permitted nearly wild-type like growth of the plants carrying fusions with CAH3 and increased the levels of fusion protein, but also of smaller degradation products. The fusion of catalytically inactive CAs to RbcL did not cause a different phenotype from the fusions with catalytically active CAs, suggesting that the selected CAs were not active in the fusion with RbcL or their activity did not have an effect on CO2 assimilation. However, fusions to RbcL did not abolish RbcL catalytic activity, as shown by the autotrophic growth, gas exchange and in vitro activity measurements. Furthermore, Rubisco carboxylation rate and specificity for CO2 was not altered in some of the fusion proteins, suggesting that despite the defect in RbcL folding or assembly caused by the fusions, the addition of 60-150 amino acids to RbcL does not affect its catalytic properties. On the contrary, most growth defects of the plants carrying RbcL-CA fusions are related to their reduced Rubisco content, likely caused by impaired RbcL folding or assembly. Finally, we found that fusions with RbcL C-terminus were better tolerated than with the N-terminus, and increasing the length of the linker relieved the growth impairment imposed by the fusion to RbcL. Together, the results of this work constitute considerable relevant findings for future Rubisco engineering. N2 - Rubisco katalysiert den ersten Schritt der CO2-Assimilierung. Trotz seiner bedeutenden Rolle, zeichnet sich Rubisco durch eine niedrige katalytische Geschwindigkeit aus. Außerdem, entsteht bei der Bindung von O2 anstatt CO2 ein toxisches Zwischenprodukt, welches in einem Prozess, genannt Photorespiration, aufbereitet wird. Da fast die gesamte Nahrungsmittelversorgung auf der Aktivität von Rubisco basiert, könnten schon kleine Verbesserungen in der Spezifität für CO2 zu einem großen Effekt in der Photosysntheserate und letztendlich größeren Ernteerträgen führen. In dieser Arbeit wurde versucht die Effizienz der Photosynthese zu verbessern, indem ein künstlicher CO2 konzentrierender Mechanismus aus einer Fusion von RbcL und einer Carboanhydrase (CA) gebildet wird. Als Vorversuch wurden je bCA1 und CAH3 an Rubiscos C- beziehungsweise N-Terminus mittels eines kleinen, flexiblen Linkers aus 5 Aminosäuren fusioniert. Anschließend wurden weitere Fusionsproteine zwischen dem C-Terminus von RbcL und bCA1/CAH3 mittels Linkern von 14, 23, 32 und 41 Aminosäuren Länge in Chloroplasten von Tabak eingebracht. Die entstandenen transplastomischen Pflanzen mit bCA1-Fusionen waren trotz ihres sehr langsamen Wachstums dazu fähig schon bei kurzen Linkern autotroph zu wachsen und geringe Mengen an Fusionsproteinen zu akkumulieren. Pflanzen mit CAH3 Fusionsproteinen hingegen waren nur mit längeren Linkern autotroph, zeigten aber dafür ähnliche Wachstumsraten zum Wildtyp bei Nutzung des längsten Linkers. Außerdem enthielten diese Pflanzen größere Mengen an Fusionsproteinen aber auch eine erhöhte Anreicherung von kleineren Abbauprodukten. Bei den in dieser Arbeit gewählten CA als Fusionsprotein mit RbcL konnte im Vergleich mit katalytisch inaktiven Varianten kein Effekt auf die CO2-Assimilierung gefunden werden. Wie das autotroph Wachstum sowie die Gaswechsel- und in-vitro-Aktivitätsmessungen zeigen, haben die Fusionen allerdings nicht die katalytische Aktivität von Rubisco blockiert. Ebenso verhielt sich die Carboxylierungsrate von Rubisco und deren Spezifität für CO2 unverändert. Dies weist darauf hin, dass trotz Rubiscos Faltungs- oder Assemblierungsdefekten das Anfügen von 60-150 Aminosäure an den C-Terminus von RbcL nicht die katalytische Leistung des Enzyms beeinträchtigt. Im Gegenteil, die Wachstumsdefekte waren durch die geringe Menge an Rubisco begründet, vermutlich verursacht durch Defekte in der Faltung oder Assemblierung von RbcL. Schlussendlich konnten wichtige Erkenntnisse für zukünftige gentechnische Veränderungen von Rubisco gemacht werden: Fusionen mit dem C-Terminus von RbcL wurden besser toleriert als mit dem N-Terminus und längere Linker verringerten die von der Fusion ausgelösten Wachstumsdefekte. KW - Rubisco KW - fusion Y1 - 2017 ER - TY - THES A1 - Diez Cocero, Mercedes T1 - Analysis of Rubisco - carbonic anhydrase fusions in tobacco as an approach to reduce photorespiration Y1 - 2017 ER - TY - THES A1 - Dippong, Martin T1 - Direkte und indirekte Hapten-selektive Immunfluoreszenzmarkierung von Hybridomzellen zur Generierung monoklonaler Antikörper T1 - Direct and indirect hapten-specific immunofluorescence labeling of hybridoma cells for the generation of monoclonal antibodies N2 - Die Hybridomtechnik zur Produktion von monoklonalen Antikörpern ermöglichte einen großen Schritt in der Entwicklung von Immunoassays für die biochemische Forschung und klinische Diagnostik. Auch die Produktion von Antikörpern gegen niedermolekulare Analyten, Haptene, typische Targets in der Lebensmittel- und Umweltanalytik, erlangte in den letzten Jahren eine immer größere Bedeutung. Im Zuge der Durchführung der Hybridomtechnik werden tausende Antikörper-sezernierende und nicht-sezernierende Zellen generiert. Die Selektion der wenigen antigenselektiven Hybridomzellen zählt dabei zu den herausforderndsten Schritten für die Antikörpergewinnung. Bisherige Selektionsverfahren, wie die Limiting-Dilution-Klonierung in Verbindung mit Enzyme-linked Immunosorbent Assays (ELISAs), garantieren keine Monoklonalität und erlauben nur das Screening von einigen wenigen Zellklonen. Hingegen ermöglichen Hochdurchsatz-Selektionsmethoden, wie die Fluoreszenz-aktivierte Zellsortierung (FACS), einen sehr hohen Probendurchsatz. Eine Einzelzellablage garantiert hierbei Monoklonalität. Jedoch sind die dafür erforderlichen Zellmarkierungen oftmals zellschädigend oder aufwendig zu generieren. Auch ist bisher noch keine Markierungsmethode bekannt, die es ermöglicht, Hapten-selektive Hybridomzellen durchflusszytometrisch zu analysieren und eine FACS-Selektion durchzuführen. Aus diesem Grund wurden in dieser Arbeit zwei Zellmarkierungsmethoden entwickelt, die dies ermöglichen sollten. Die membranständigen Antikörper von Hybridomzellen sollten entweder direkt oder indirekt immunfluoreszenz-markiert und dadurch für die Durchflusszytometrie und FACS-Selektion zugänglich gemacht werden. Die direkte Markierung wurde mittels eines Hapten-Fluorophor-Konjugats durchgeführt. Sie ermöglichte erstmalig den Anteil an Haptenselektiven Hybridomzellen in einer Hybridomzelllinie zu überprüfen. Dies konnte für zwei Hapten-selektive Hybridomzelllinien, die Antikörper gegen das Hormon 17β-Estradiol und das Cardenolid Digoxigenin bilden, gezeigt werden. Durchflusszytometrie und ELISAs lieferten vergleichbare Ergebnisse. Zellen, die Hapten-selektiv markiert werden konnten, sezernierten ebenfalls Hapten-selektive Antikörper. Des Weiteren konnte die direkte Markierung dazu genutzt werden, zwei Mykotoxin-selektive Hybridomzelllinien, welche Antikörper gegen Aflatoxin und Zearalenon bilden, auf Monoklonalität zu testen. Dies ist mittels ELISA nicht möglich. Die Markierungsmethode eignete sich jedoch nur für fixierte Hybridomzellen. Eine Markierung von lebenden Zellen konnte weder durchflusszytometrisch noch mittels konfokaler Laser-Scanning-Mikroskopie gezeigt werden. Dies gelang erst mit einer neu entwickelten indirekten Immunfluoreszenzmarkierung. Dabei wurden die Zellen zunächst mit einem Hapten-Peroxidase-Konjugat inkubiert, gefolgt von einem Fluorophor-markierten anti-HRP-Antikörper-Konjugat. Dies wurde für zwei Analyten, das Hormon Estron und das Antiepileptikum Carbamazepin, gezeigt. Die indirekte Markierung wurde erfolgreich dazu verwendet, Carbamazepin-selektive Hybridomzellen aus einem Fusionsansatz für die monoklonale Antikörperproduktion auszusortieren. Damit wurde erstmalig eine Zellmarkierungsmethode entwickelt, die eine Hochdurchsatz-Selektion lebender Hybridomzellen aus einem Fusionsansatz ermöglicht. Sie ist nicht zellschädigend und kann zusätzlich zur Selektion Hapten-selektiver Plasmazellen verwendet werden. N2 - The ability to create monoclonal antibodies has allowed great strides to be made in immunoassay development for biochemical research and clinical diagnostics. Particularly for small molecular weight analytes, haptens, the need of selective antibodies has increased. The hybridoma technique generates thousands of fused antibody-secreting and non-secreting cells, with the majority being irrelevant. The subsequent screening and subcloning process in order to identify and isolate the very few hybrids that are secreting antibodies of the desired selectivity is a major concern. The traditional limiting dilution technique followed by enzymelinked immunosorbent assays (ELISAs) is inefficient and monoclonality is not guaranteed. Often the number of clones that can be screened is limited. High-throughput techniques such as fluorescence-activated cell sorting (FACS) provide an efficient tool to increase the number of cells to be screened. Furthermore, a single-cell deposition of cells would ensure monoclonality. However, antigen-selective cell labeling techniques are often cell damaging or laborious. The purpose of this study was to explore a cell labeling technique enabling the hapten-selective analysis and isolation of hybridoma cells via FACS. This would reduce much of the effort that has currently to be employed in hybridoma generation. For this reason, a direct and indirect hapten-selective labeling technique was developed. For the direct labeling, a haptenfluorophore conjugate was generated. The conjugate was used to tag membrane-bound immunoglobulin G of hybridoma cells and thereby enabling flow cytometric analysis. Using this kind of conjugate, it was possible to examine the selective antibody expression of hybridoma cell lines producing antibodies against the hormone estradiol and the steroid digoxigenin. Flow cytometric analysis and ELISAs showed comparable results: Cells, which were tagged with the corresponding hapten-fluorophore conjugate also secreted hapten-selective antibodies. Furthermore, it was possible to check hybridoma cell lines producing antibodies against the mycotoxins aflatoxin and zearalenone for monoclonality, which is not possible with ELISA. However, the direct labeling technique was only applicable to fixed cells. Successful labeling of living cells could neither be detected by flow cytometry nor by confocal laser scanning microscopy. On the contrary, using the newly developed indirect labeling technique, flow cytometric analysis and selection of living cells by FACS was possible. Here, the cells were first incubated with a hapten-peroxidase conjugate followed by a fluorophore-conjugated anti-peroxidase antibody. The technique was established on a hybridoma cell line selective for the hormone estrone. Furthermore, this labeling technique enabled for the first time the sorting of hybridoma cells producing selective antibodies against the medication carbamazepine out of a fusion mixture with high efficiency. The selected clones were used for monoclonal antibody production. The indirect labeling is harmless for cells and could also be applied on haptenselective plasma cells. KW - Durchflusszytometrie KW - Haptene KW - monoklonale Antikörper KW - Hybridom KW - Immunfluoreszenz KW - flow cytometry KW - hapten KW - monoclonal antibodies KW - hybridoma KW - immunofluorescence Y1 - 2017 ER - TY - JOUR A1 - Dolotovskaya, Sofya A1 - Bordallo, Juan Torroba A1 - Haus, Tanja A1 - Noll, Angela A1 - Hofreiter, Michael A1 - Zinner, Dietmar A1 - Roos, Christian T1 - Comparing mitogenomic timetrees for two African savannah primate genera (Chlorocebus and Papio) JF - Zoological Journal of the Linnean Society N2 - Complete mitochondrial (mtDNA) genomes have proved to be useful in reconstructing primate phylogenies with higher resolution and confidence compared to reconstructions based on partial mtDNA sequences. Here, we analyse complete mtDNA genomes of African green monkeys (genus Chlorocebus), a widely distributed primate genus in Africa representing an interesting phylogeographical model for the evolution of savannah species. Previous studies on partial mtDNA sequences revealed nine major clades, suggesting several cases of para- and polyphyly among Chlorocebus species. However, in these studies, phylogenetic relationships among several clades were not resolved, and divergence times were not estimated. We analysed complete mtDNA genomes for ten Chlorocebus samples representing major mtDNA clades to find stronger statistical support in the phylogenetic reconstruction than in the previous studies and to estimate divergence times. Our results confirmed para- and polyphyletic relationships of most Chlorocebus species, while the support for the phylogenetic relationships between the mtDNA clades increased compared to the previous studies. Our results indicate an initial west-east division in the northern part of the Chlorocebus range with subsequent divergence into north-eastern and southern clades. This phylogeographic scenario contrasts with that for another widespread African savannah primate genus, the baboons (Papio), for which a dispersal from southern Africa into East and West Africa was suggested. KW - African green monkeys KW - baboons KW - mitochondrial genomes KW - phylogeny KW - phylogeography Y1 - 2017 U6 - https://doi.org/10.1093/zoolinnean/zlx001 SN - 0024-4082 SN - 1096-3642 VL - 181 IS - 2 SP - 471 EP - 483 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Duncan, Susan A1 - Rosa, Stefanie Nunes T1 - Gaining insight into plant gene transcription using smFISH JF - Transcription N2 - Single molecule RNA fluorescent in situ hybridization (smFISH) enables gene transcription to be assessed at the cellular level. In this point of view article, we describe our recent smFISH research in the model plant Arabidopsis thaliana and discuss how this technique could further knowledge of plant gene transcription in the future. KW - Arabidopsis KW - lncRNA KW - mRNA Quantification KW - RNA Imaging KW - smFISH Y1 - 2017 U6 - https://doi.org/10.1080/21541264.2017.1372043 SN - 2154-1264 SN - 2154-1272 VL - 9 IS - 3 SP - 166 EP - 170 PB - Taylor & Francis Group CY - Philadelphia ER - TY - JOUR A1 - Eckert, Ester M. A1 - Di Cesare, Andrea A1 - Kettner, Marie Therese A1 - Arias-Andres, Maria A1 - Fontaneto, Diego A1 - Grossart, Hans-Peter A1 - Corno, Gianluca T1 - Microplastics increase impact of treated wastewater on freshwater microbial community JF - Environmental pollution N2 - Plastic pollution is a major global concern with several million microplastic particles entering every day freshwater ecosystems via wastewater discharge. Microplastic particles stimulate biofilm formation (plastisphere) throughout the water column and have the potential to affect microbial community structure if they accumulate in pelagic waters, especially enhancing the proliferation of biohazardous bacteria. To test this scenario, we simulated the inflow of treated wastewater into a temperate lake using a continuous culture system with a gradient of concentration of microplastic particles. We followed the effect of microplastics on the microbial community structure and on the occurrence of integrase 1 (intl), a marker associated with mobile genetic elements known as a proxy for anthropogenic effects on the spread of antimicrobial resistance genes. The abundance of intl increased in the plastisphere with increasing microplastic particle concentration, but not in the water surrounding the microplastic particles. Likewise, the microbial community on microplastic was more similar to the original wastewater community with increasing microplastic concentrations. Our results show that microplastic particles indeed promote persistence of typical indicators of microbial anthropogenic pollution in natural waters, and substantiate that their removal from treated wastewater should be prioritised. (C) 2017 Elsevier Ltd. All rights reserved. KW - Microplastics KW - Anthropogenic pollution KW - Treated wastewater KW - Freshwater microbial communities KW - Integrase 1 KW - Biofilm Y1 - 2017 U6 - https://doi.org/10.1016/j.envpol.2017.11.070 SN - 0269-7491 SN - 1873-6424 VL - 234 SP - 495 EP - 502 PB - Elsevier CY - Oxford ER -