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Institute
- Biowissenschaften (199) (remove)
Chloroplasts are difficult to assemble because of the presence of large inverted repeats. At the same time, correct assemblies are important, as chloroplast loci are frequently used for biogeography and population genetics studies. In an attempt to elucidate the orientation of the single-copy regions and to find suitable loci for chloroplast single nucleotide polymorphism (SNP)-based studies, circular chloroplast sequences for the ultra-centenary reference individual of European Beech (Fagus sylvatica), Bhaga, and an additional Polish individual (named Jamy) was obtained based on hybrid assemblies. The chloroplast genome of Bhaga was 158,458 bp, and that of Jamy was 158,462 bp long. Using long-read mapping on the configuration inferred in this study and the one suggested in a previous study, we found an inverted orientation of the small single-copy region. The chloroplast genome of Bhaga and of the individual from Poland both have only two mismatches as well as three and two indels as compared to the previously published genome, respectively. The low divergence suggests low seed dispersal but high pollen dispersal. However, once chloroplast genomes become available from Pleistocene refugia, where a high degree of variation has been reported, they might prove useful for tracing the migration history of Fagus sylvatica in the Holocene.
The Southern Ocean (SO) is one of the most pristine regions of our Planet, characterised by high levels of biodiversity (5% of the global diversity) (David and Saucède 2015) and hosting a unique fauna (up to 90% of SO species are endemic) (De Broyer and Danis 2011; Chown et al. 2015). Yet, the knowledge on SO biodiversity is still far from being completed. In addition, the knowledge on the impact that changing environments have on SO species-richness is very little and for some groups, it is still totally unknown. For instance, most of studies generally focus on one single species such as Antarctic krill (Kawaguchi et al. 2011), Clio pyramidata Linnaeus, 1767 (Orr et al. 2005), Globigerina bulloides d'Orbigny, 1826 (Moy et al. 2009), or only on a high taxonomic level (e.g. phylum, class): Echinodermata, Crustacea, Mollusca, Porifera, Bryozoa, Brachiopoda, Hydrozoa, Ascidiacea, Holoturoidea
(Barnes 1999; Rowden et al. 2015; Post et al. 2017; Gutt et al. 2019; Vause et al. 2019; Pineda-Metz et al. 2020). Ultimately, the influence of sea-ice coverage on benthic species diversity was totally unknown prior to this study. In light of this, the objectives of the thesis are:
1. To expand the knowledge on shelf and deep-sea peracarid assemblage structure and abundance on a small regional (Weddell Sea) and on a large regional (Atlantic sector of the SO and South Atlantic Ocean) geographic scale.
2. To assess the environmental variables driving peracarid assemblage structure and abundance from the above mentioned areas.
3. To investigate SO benthic isopod species diversity from the Atlantic sector of the SO and assess the influence of environmental variables on their species-richness and composition.
4. To describe new possible peracarid species by means of integrative taxonomy, using morphological descriptions and whole genome sequencing analyses to support the species identification.
Objective outcomes: The present thesis provides new information on the abundance and assemblage structure based on 64766 peracarid crustaceans from different 28 locations within the Atlantic sector of the SO continental shelf and deep sea (Chapters I-II). These locations are characterised by different environmental conditions, for instance different sea-ice concentrations. Results from Chapters I-II confirmed the dominance of peracarid assemblages in the benthos, with amphipods being the most abundant group, followed by isopods. Sea ice was identified as the main driver shaping benthic peracarid assemblage structure (Chapter I). On a larger geographic scale and wider bathymetric range (e.g. including sampling locations from previous studies performed in the South Atlantic Ocean
and at a depth range from 160 to ~6000 m), depth was the main physical variable driving peracarid assemblage structure (Chapter III). In addition, 16157 isopod specimens from the Atlantic sector of the SO were identified to species level at a smaller scale (Chapter IV). In this case, sea ice was identified as the main physical driver affecting isopod diversity and composition among sampling locations (Chapter IV). Reduced concentration of sea ice
causes a decrease in isopod biodiversity, thus climate change was identified as a huge threat for this taxon and for SO benthos in general. During the identification process, two new isopod species were discovered (Chapter V). The two new species (Notopais sp.1 n. sp. and Notopais sp.2 n. sp.) were accurately described and identified by means of integrative taxonomy. This provided the first whole genome sequencing of benthic isopods from the SO and the first complete mitochondrial genome of the genus Notopais (Chapter V). Thanks to the collaboration with the University of Genoa (Dipartimento di Scienze della Terra dell'Ambiente e della Vita, DISTAV, Italy) and the National Antarctic Museum (MNA) in Genoa, two new SO species of the suborder Valvifera G. O. Sars, 1883 were described by means of classical taxonomy. In this case, a molecular approach could not be used because both new species were represented by a single specimen, therefore it was important to preserve the integrity of the holotypes (Chapters VI-VII).
Die Studien im Rahmen dieser Arbeit wurden am Modellorganismus Anabaena sp. PCC 7120 (Anabaena) durchgeführt, einem filamentösen Süßwasser-Cyanobakterium. Cyanobakterien sind photosynthetische, Gram-negative Organismen. Sie besitzen eine das Zytosol begrenzende Plasmamembran und eine Äußere Membran. TonB-abhängige Transporter (TBDTs) und Porine der Äußeren Membran bewerkstelligen und regulieren die Aufnahme von Nährstoffen. Typischerweise wenig abundante Substrate für den TBDT-vermittelten, aktiven Transport sind beispielsweise eisenhaltige Siderophore oder VitaminB12. Kleinere gelöste und abundante Stoffe wie Salze oder andere Ionen gelangen hingegen passiv durch Porine in das Periplasma.
In Anabaena wurden neun putative Porine identifiziert. Sieben hiervon wiesen eine porinspezifische Domänenstruktur auf (Alr0834, Alr2231, All4499, Alr4550, Alr4741, All5191 und All7614), und wurden im Rahmen dieser Arbeit näher betrachtet. Die Expression dieser sieben Gene wurde vergleichend untersucht, nachdem der Wildtyp in Standardmedium oder in Medium indem jeweils Mangan, Eisen, Kupfer oder Zink fehlte angezogen wurde. Außerdem wurde das Wachstum der einzelnen Porinmutanten im Vergleich zum Wildtyp auf Festmedium mit hohen Konzentrationen von Salzen, Antibiotika oder anderen Stoffen analysiert. Hierbei konnten den einzelnen Mutanten teilweise spezifische phänotypische Eigenschaften zugeschrieben werden. Zusammengefasst kann anhand der Analysenergebnisse vermutet werden, dass Alr4550 eine besondere Rolle in der Wahrung der Zellhüllenstabilität oder -integrität spielt, wohingegen das Fehlen von Alr5191 auf unbekannte Weise die Fixierung von Stickstoff zu erschweren scheint. Die alr2231-Mutante zeigte eine Resistenz gegenüber hohen Zinkkonzentrationen, was die Vermutung zulässt, dass Zink ein Substrat von Alr2231 darstellt. Für weitere Porine kann ebenfalls ein Zusammenhang zum Transport von Kupfer oder Mangan vermutet werden.
Neben Porinen wurden ebenfalls TonB-ähnliche Proteine in Anabaena untersucht. TonB ist ein plasmamembranständiges Protein, das in Komplex mit ExbB und ExbD die Energie für Transportprozesse über die Äußere Membran bereitstellt. Hierfür bindet TonB C-terminal an TBDTs und induziert dort Strukturänderungen, welche den Substratimport ins Periplasma ermöglichen. Als Energiequelle wird der Protonengradient genutzt, der über die Plasmamembran besteht. In Anabaena wurden vier putative TonB Proteine identifiziert, die sich jeweils in Länge und Domänenstruktur unterscheiden. Im Rahmen dieser Arbeit konnte durch Substrattransport-Experimente und Wachstumsanalysen gezeigt werden, dass TonB3 an der Aufnahme zweier Siderophore (Schizokinen und dem Xenosiderophor Ferrichrom) beteiligt ist, da die entsprechende Mutante sich als unfähig erwies diese zu als Eisenquelle nutzbar zu machen. Daneben wies TonB3 weitere Merkmale auf, die auch TonB-Proteinen anderer Organismen zugeschrieben wurden (Wachstumsdefizit der Mutante unter Eisenmangel, eisenabhängiges Expressionsprofil). Interessanterweise zeigte sich, dass das Siderophor Ferrichrom ebenfalls nicht als Eisenquelle für die tonB4-Mutante zur Verfügung stand, was zum Beispiel auf eine Beteiligung von TonB4 an dessen Transport hinweisen könnte.
TonB1, welches sich durch ein inkomplettes TBDT-Interaktionsmotiv auszeichnet, und TonB2 konnte keine Beteiligung am Siderophoretransport zugeschrieben werden, jedoch zeigten Mutanten der einzelnen Gene spezifische phänotypische Eigenschaften. Die tonB1-Mutante stach hervor durch ein vergleichsweise stark verzögertes Wachstum unter diazotrophen Bedingungen. Es konnte gezeigt werden, dass sowohl die Nitrogenaseaktivität als auch die expression vermindert war im tonB1-Mutantenstamm. Außerdem zeigten die Heterozysten dieser Mutante, die auf die Stickstoffixierung spezialisierten Zellen, eine abnormale Morphologie. Da die Expression von tonB1 jedoch nach dem Überführen von Wildypzellen in stickstoffreies Medium nicht erhöht war, kann eine direkte Beteiligung von TonB1 an der Heterozystendifferenzierung als unwahrscheinlich betrachtet werden. Die Zelleinschnürungen zwischen Heterozysten und vegetativen Zellen waren in I-tonB1 weniger ausgeprägt als im Wildtyp, was durch eine Anfärbung der Zellwand mit einem Fluoreszenzmarker gezeigt werden konnte. Ebenfalls konnte anhand des fluoreszierenden Markers Calcein gezeigt werden, dass die molekulare Diffusionsgeschwindigkeit zwischen Heterozysten und vegetativen Zellen, und auch zwischen zwei benachbarten vegetativen Zellen, in der tonB1-Mutante erhöht ist. Deswegen kann hier vermutlich vermehrt die Nitrogenase schädigender Sauerstoff in Heterozysten eindringen. Die aufgezählten Ergebnisse deuten auf eine Funktion von SjdR im Aufbau der Septumsstrukturen hin, beispielsweise durch Regulation der Peptidoglykansynthese oder -verteilung, weswegen TonB1 umbenannt wurde in SjdR (Septal junction disc regulator).
Die Untersuchung der tonB2-Mutante zeigte bei dieser eine veränderte Pigmentierung, eine vermehrte Lipopolysaccharidproduktion und Filamentaggregation sowie eine erhöhte Resistenz gegenüber bestimmten Antibiotika oder Detergenzien. Letzteres könnte auf die ebenfalls in der tonB2-Mutante beobachtete verringerte Porinexpression zurückgeführt werden. Es wurde außerdem eine vermehrte Anreicherung von Kupfer und Molybdän in der Mutante gemessen, was ein Grund für die Veränderte Pigmentierung sein könnte und ebenfalls die Porinexpression beeinflussen könnte. Insgesamt scheint sich das Fehlen von TonB2 auf die Integrität der Äußeren Membran auszuwirken. Daher kann für TonB2, eine Funktion in Anlehnung an das Tol-system vermutet werden.
Studium der Lebewesen : im Masterprogramm »Ökologie und Evolution« wird Diversität großgeschrieben
(2021)
The abyssal seafloor is a mosaic of highly diverse habitats that represent the least known marine ecosystems on Earth. Some regions enriched in natural resources, such as polymetallic nodules in the Clarion-Clipperton Zone (CCZ), attract much interest because of their huge commercial potential. Since nodule mining will be destructive, baseline data are necessary to measure its impact on benthic communities. Hence, we conducted an environmental DNA and RNA metabarcoding survey of CCZ biodiversity targeting microbial and meiofaunal eukaryotes that are the least known component of the deep-sea benthos. We analyzed two 18S rRNA gene regions targeting eukaryotes with a focus on Foraminifera (37F) and metazoans (V1V2), sequenced from 310 surface-sediment samples from the CCZ and other abyssal regions. Our results confirm huge unknown deep-sea biodiversity. Over 60% of benthic foraminiferal and almost a third of eukaryotic operational taxonomic units (OTUs) could not be assigned to a known taxon. Benthic Foraminifera are more common in CCZ samples than metazoans and dominated by clades that are only known from environmental surveys. The most striking results are the uniqueness of CCZ areas, both datasets being characterized by a high number of OTUs exclusive to the CCZ, as well as greater beta diversity compared to other abyssal regions. The alpha diversity in the CCZ is high and correlated with water depth and terrain complexity. Topography was important at a local scale, with communities at CCZ stations located in depressions more diverse and heterogeneous than those located on slopes. This could result from eDNA accumulation, justifying the interim use of eRNA for more accurate biomonitoring surveys. Our descriptions not only support previous findings and consolidate our general understanding of deep-sea ecosystems, but also provide a data resource inviting further taxon-specific and large-scale modeling studies. We foresee that metabarcoding will be useful for deep-sea biomonitoring efforts to consider the diversity of small taxa, but it must be validated based on ground truthing data or experimental studies.
Using walls to navigate the room: egocentric representations of borders for spatial navigation
(2021)
Spatial navigation forms one of the core components of an animal’s behavioural repertoire. Good navigational skills boost survival by allowing one to avoid predators, to search successfully for food in an unpredictable world, and to be able to find a mating partner. As a consequence, the brain has dedicated many of its resources to the processing of spatial information. Decades of seminal work has revealed how the brain is able to form detailed representations of one’s current position, and use an internal cognitive map of the environment to traverse the local space. However, what is much less understood is how neural computations of position depend on distance information of salient external locations such as landmarks, and how these distal places are encoded in the brain.
The work in this thesis explores the role of one brain region in particular, the retrosplenial cortex (RSC), as a key area to implement distance computations in relation to distal landmarks. Previous research has shown that damage to the RSC results in losses of spatial memory and navigation ability, but its exact role in spatial cognition remains unclear. Initial electrophysiological recordings of single cells in the RSC during free exploration behaviour of the animal resulted in the discovery of a new population of neurons that robustly encode distance information towards nearby walls throughout the environment. Activity of these border cells was characterized by high firing rates near all boundaries of the arena that were available to the animal, and sensory manipulation experiments revealed that this activity persisted in the absence of direct visual or somatosensory detection of the wall.
It quickly became apparent that border cell activity was not only modulated by the distance to walls, but was contingent on the direction the animal was facing relative to the boundary. Approximately 40% of neurons displayed significant selectivity to the direction of walls, mostly in the hemifield contra-lateral to the recorded hemisphere, such that a neuron in left RSC is active whenever a wall occupies proximal space on the right side of the animal. Using a cue-rotation paradigm, experiments initially showed that this egocentric direction information was invariant to the physical rotation of the arena. Yet this rotation elicited a corresponding shift in the preferred direction of local head-direction cells, as well as a rotation in the firing fields of spatially-tuned cells in RSC. As a consequence, position and direction encoding in RSC must be bound together, rotating in unison during the environmental manipulations, as information about allocentric boundary locations is integrated with head-direction signals to form egocentric border representations.
It is known that the RSC forms many anatomical connections with other parts of the brain that encode spatial information, like the hippocampus and para-hippocampal areas. The next step was to establish the circuit mechanisms in place for RSC neurons to generate their activity in respect to the distance and direction of walls. A series of inactivation experiments revealed how RSC activity is inter-dependent with one of its communication partners, the medial entorhinal cortex (MEC). Together they form a wider functional network that encodes precise spatial information of borders, with information flowing from the MEC to RSC but not vice versa. While the conjunction between distance and heading direction relative to the outer walls was the main driver of neural activity in RSC, border cells displayed further behavioural correlates related to movement trajectories. Spiking activity in either hemisphere tended to precede turning behaviour on a short time-scale in a way that border cells in the right RSC anticipated right-way turns ~300 ms into the future.
The interpretation of these results is that the RSC’s primary role in spatial cognition is not necessarily on the early sensory processing stage as suggested by previous studies. Instead, it is involved in computations related to the generation of motion plans, using spatial information that is processed in other brain areas to plan and execute future actions. One potential function of the RSC’s role in this process could be to act correctly in relation to the nearby perimeter, such that border cells in one hemisphere are involved in the encoding of walls in the contralateral hemifield, after which the animal makes an ipsilateral turn to avoid collision. Together this supports the idea that the MEC→RSC pathway links the encoding of space and position in the hippocampal system with the brain’s motor action systems, allowing animals to use walls as prominent landmarks to navigate the room.
Organismic aging is known to be controlled by genetic and environmental traits. Pathways involved in the control of cellular metabolism play a crucial role. Previously, we identified a role of PaCLPP, a mitochondrial matrix protease, in the control of the mitochondrial energy metabolism, aging, and lifespan of the fungal aging model Podospora anserina. Most surprisingly, we made the counterintuitive observation that the ablation of this component of the mitochondrial quality control network leads to lifespan extension. In the current study, we investigated the role of energy metabolism of P. anserina. An age-dependent metabolome analysis of the wild type and a PaClpP deletion strain verified differences and changes of various metabolites in cultures of the PaClpP mutant and the wild type. Based on these data, we generated and analyzed a PaSnf1 deletion mutant and a ΔPaSnf1/ΔPaClpP double mutant. In both mutants PaSNF1, the catalytic α-subunit of AMP-activated protein kinase (AMPK) is ablated. PaSNF1 was found to be required for the development of fruiting bodies and ascospores and the progeny of sexual reproduction of this ascomycete and impact mitochondrial dynamics and autophagy. Most interestingly, while the single PaSnf1 deletion mutant is characterized by a slight lifespan increase, simultaneous deletion of PaSnf1 and PaClpP leads to a pronounced lifespan extension. This synergistic effect is strongly reinforced in the presence of the mating-type “minus”-linked allele of the rmp1 gene. Compared to the wild type, culture temperature of 35°C instead of the standard laboratory temperature of 27°C leads to a short-lived phenotype of the ΔPaSnf1/ΔPaClpP double mutant. Overall, our study provides novel evidence for complex interactions of different molecular pathways involved in mitochondrial quality control, gene expression, and energy metabolism in the control of organismic aging.
Non-ribosomal peptide synthetase docking domains : structure, function and engineering strategies
(2021)
Non-ribosomal peptide synthetases (NRPSs) are known for their capability to produce a wide range of natural compounds and some of them possess interesting bioactivities relevant for clinical application like antibiotics, anticancer, and immunosuppressive drugs. The diverse bioactivity of non-ribosomal peptides (NRPs) originates from their structural diversity, which results not only from the incorporation of non-proteinogenic amino acids into the growing peptide chain, but also the formation of heterocycles or further peptide modifications like methylation, hydroxylation and acetylation.
The biosynthesis of NRPs is achieved via the orchestrated interplay of distinct catalytic domains, which are grouped to modules that are located on one or more polypeptide chains. Each cycle starts with the selection and activation of a specific amino acid by the adenylation (A) domain, which catalyzes the aminoacyl adenylate formation under ATP consumption. This activated amino acid is then bound via a thioester bond to the 4’-phosphopantetheine cofactor (PPant-arm) of the following thiolation (T) domain. Before substrate loading, the PPant-arm is post-translationally added to the T domain by a phosphopantetheinyl transferase (PPTase), which converts the inactive apo-T domain in its active holo-form. In the last step of the catalytic cycle, two T domain bound peptide building blocks are connected by the condensation (C) domain, resulting in peptide bond formation and transfer of the nascent peptide chain to the following module. Each catalytic cycle is performed by a C-A-T elongation module until the termination module with a C-terminal thioesterase (TE) domain is reached. Here, the peptide product is released by hydrolysis or intramolecular cyclisation.
In comparison to single-protein NRPSs, where all modules are encoded on a single polypeptide chain, multi-protein NRPS systems must also maintain a specific module order during the peptide biosynthesis. Therefore, small C-terminal and N-terminal communication-mediating (COM) domains/docking domains (DD) were identified in the C- and N-terminal regions of multi-protein NRPSs. It was shown that these domains mediate specific and selective non-covalent protein-protein interaction, even though DD interactions are generally characterized by low affinities.
The first publication of this work focuses on the Peptide-Antimicrobial-Xenorhabdus peptide-producing NRPS called PaxS, which consists of the three proteins PaxA, PaxB and PaxC. Here, in particular the trans DD interface between the C-terminal attached DD of PaxB and N-terminal attached DD of PaxC was structurally investigated and thermodynamically characterized by isothermal titration calorimetry (ITC), yielding a dissociation constant (KD) of ~25 µM, which is a DD typical affinity known from further characterized DD pairs. The artificial linking of the PaxB/C C/NDD pair via a glycine-serine (GS) linker facilitated the structure determination of the DD complex by solution nuclear magnetic resonance (NMR) spectroscopy. In comparison to known docking domain structures, this DD complex assembles in a completely new fold which is characterized by a central α-helix of PaxC NDD wrapped in two V-shaped α-helices of PaxB CDD.
The first manuscript of this work focuses on the application of synthetic zippers (SZ) to mimic natural docking domains, enabling the easy assembly of NRPS building blocks encoded on different plasmids in a functional way. Here, the high-affinity interaction of SZs unambiguously defines the order of the synthetases derived from single-protein NRPSs in the engineered NRPS system and allows the recombination in a plug-and-play manner. Notably, the SZ engineering strategy even facilitates the functional assembly of NRPSs derived from Gram-positive and Gram-negative bacteria. Furthermore, the functional incorporation of SZs into NRPS modules is not limited to a specific linker region, so we could introduce them within all native NRPS linker regions (A-T, T-C, C-A).
The second publication and the second manuscript of this thesis again focus on the multi-protein PaxS, in particular on the trans interface between the proteins PaxA and PaxB on a molecular level by solution NMR. Therefore, the PaxA CDD adjacent T domain was included into the structural investigation besides the native interaction partner PaxB NDD. Before a three-dimensional structure could be obtained from NMR data, the NH groups located in the peptide bonds had to be assigned to the respective amino acids of the proteins (backbone assignment). Based on these backbone assignments, the secondary structure of PaxA T1-CDD and PaxB NDD in the absence and presence of the respective interaction partner were predicted.
The structural and functional characterization of the PaxA T1-CDD:PaxB NDD complex is summarized in manuscript two. The thermodynamic analysis of this complex by ITC determined a KD value of ~250 nM, whereas the discrete DDs did not interact at all. The high-affinity interaction allowed to determine the solution NMR structure of the PaxA T1-CDD:PaxB NDD complex without the covalent linkage of the interaction partners and an extended docking domain interface could be determined. This interface comprises on the one hand α-helix 4 of the PaxA T1 domain together with the α-helical CDD, and on the other hand the PaxB NDD, which is composed of two α-helices separated by a sharp bend.
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Plastid DNA sequence data have been traditionally widely used in plant phylogenetics because of the high copy number of plastids, their uniparental inheritance, and the blend of coding and non-coding regions with divergent substitution rates that allow the reconstruction of phylogenetic relationships at different taxonomic ranks. In the present study, we evaluate the utility of the plastome for the reconstruction of phylogenetic relationships in the pantropical plant family Ochnaceae (Malpighiales). We used the off-target sequence read fraction of a targeted sequencing study (targeting nuclear loci only) to recover more than 100 kb of the plastid genome from the majority of the more than 200 species of Ochnaceae and all but two genera using de novo and reference-based assembly strategies. Most of the recalcitrant nodes in the family’s backbone were resolved by our plastome-based phylogenetic inference, corroborating the most recent classification system of Ochnaceae and findings from a phylogenomic study based on nuclear loci. Nonetheless, the phylogenetic relationships within the major clades of tribe Ochnineae, which comprise about two thirds of the family’s species diversity, received mostly low support. Generally, the phylogenetic resolution was lowest at the infrageneric level. Overall there was little phylogenetic conflict compared to a recent analysis of nuclear loci. Effects of taxon sampling were invoked as the most likely reason for some of the few well-supported discords. Our study demonstrates the utility of the off-target fraction of a target enrichment study for assembling near-complete plastid genomes for a large proportion of samples.
Photorhabdus and Xenorhabdus are Gram-negative, entomopathogenic bacteria, living in endosymbiosis with the soil-dwelling nematode of the genera Steinernema and Heterorhabditis. The life cycle of these nematodes consists of non-feeding infective juvenile (IJ) stage, which actively searches for insects in the soil. After penetrating the insect prey, Photorhabdus and Xenorhabdus bacteria are released from the nematode gut. The bacteria proliferate and produce toxins to kill the insect. Photorhabdus and Xenorhabdus support nematode development throughout the life cycle and to get rid of food competitors by providing a wide variety of specialized metabolites (SMs). However, little is known about which SMs function as so called “food signals” to trigger the development process.
The IJs develop into adult, self-fertilizing hermaphrodites in a process called recovery, while feeding on cadaver and bacterial biomass. Heterorhabditis and Steinernema proceed to breed until nutrients are exhausted. Next generation IJs (NG-IJs) develop and leave the cadaver to search for another insect prey.
Photorhabdus and Xenorhabdus can be cultivated in defined medium under laboratory conditions. By placing IJs on a plate containing their respective bacterial symbiont, the complete life cycle of the nematodes can be observed in vitro. The in vitro nematode bioassay was used as a tool to investigate the development of the nematode.
The aim of this study was to find the food signals responsible for nematode development. Different Photorhabdus deletion strains unable to produce one or several SMs were co-cultivated with nematodes in the nematode bioassay. Subsequently, two aspects of the life cycle were investigated: recovery and NG-IJ development.
As isopropyl stilbene (IPS) is postulated to function as a food signal to support nematode recovery, it was used as a starting point for investigations. This study was focused on the biosynthetic pathway of IPS, including intermediates, side products and derivatives to investigate which one is in fact responsible for supporting nematode development.
The biosynthesis of IPS requires two precursors, phenylalanine and leucine (Figure 5). The first topic was focused on the phenylalanine derived pathway. Photorhabdus laumondii deletion mutants, defective in intermediate steps of this pathway, were created. The deletion of the genes coding for the phenylalanine ammonium lyase (stlA), converting phenylalanine into cinnamic acid (CA), the coenzyme A (CoA) ligase (stlB) and the operon coding for a ketosynthase and aromatase (stlCDE), were used. These strains were used for nematode bioassay including complementation of mutant phenotypes by feeding experiments. Recovery of nematodes grown on the deletion strains was always lower than recovery of nematodes grown on wild type bacteria. Feeding IPS to a deletion strain did not restore wild type level nematode recovery, thus IPS cannot be the food signal. Instead, the food signal must be another compound derived from this part of biosynthetic pathway. Lumiquinone and 2,5-dihydrostilbene are suggested to function as food signals and need to be investigated in future work.
The second part of this study was focused on the leucine derived pathway, which involved the Bkd complex forming the iso-branched part of IPS. A deletion of bkd was created and phenotypically analysed, subsequently performed with the nematode bioassay. Not only IPS but also other branched SMs, like photopyrones and phurealipids are synthetised by the Bkd complex. Deletions strains defective in producing photopyrones and phurealipids were also performed in nematode bioassays to investigate effects of these SMs individually. Branched SMs did not have an impact on nematode development, but nematodes grown on the ΔbkdABC strain showed a reduced nematode recovery and almost diminished NG-IJs development. As the Bkd complex also produces branched chain fatty acids (BCFAs), feeding experiments were performed with lipid extracts of wild type and mutant strain. All lipid extracts improved recovery, but only wild type lipids could complement NG-IJ development. This strongly indicates that BCFAs play an important role in NG-IJ development, which needs to be proven with purified BCFA feeding. This is an interesting finding, which could improve nematode production for biocontrol agent usage.
The role of IPS derived to epoxy stilbene (EPS) for nematode development, was another focus in the nematode life cycle. Recently it was demonstrated that EPS does not support nematode development. However, EPS forms adducts with amino acids. In my thesis, novel adducts containing the amino acid phenylalanine or a tetrapeptide were characterized. Another adduct, most likely being an EPS dimer, was also characterized. The biological role of such adducts was discussed to be potentially important for insect weakening and the structure of the novel compounds need to be structure elucidated and tested for bioactivity.
FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms—that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3′ region of FAD1 mRNA was analyzed by 3′RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3′UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3′UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3′UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.
Human GLUT2 and GLUT3, members of the GLUT / SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.
The combined behaviours of individuals within insect societies determine the survival and development of the colony. For the western honey bee (Apis mellifera), individual behaviours include nest building, foraging, storing and ripening food, nursing the brood, temperature regulation, hygiene and defence. However, the various behaviours inside the colony, especially within the cells, are hidden from sight, and until recently, were primarily described through texts and line drawings, which lack the dynamics of moving images. In this study, we provide a comprehensive source of online video material that offers a view of honey bee behaviour within comb cells, thereby providing a new mode of observation for the scientific community and the general public. We analysed long-term video recordings from longitudinally truncated cells, which allowed us to see sideways into the cells in the middle of a colony. Our qualitative study provides insight into worker behaviours, including the use of wax scales and existing nest material to remodel combs, storing pollen and nectar in cells, brood care and thermoregulation, and hygienic practices, such as cannibalism, grooming and surface cleaning. We reveal unique processes that have not been previously published, such as the rare mouth-to-mouth feeding by nurses to larvae as well as thermoregulation within cells containing the developing brood. With our unique video method, we are able to bring the processes of a fully functioning social insect colony into classrooms and homes, facilitating ecological awareness in modern times. We provide new details and images that will help scientists test their hypotheses on social behaviours. In addition, we encourage the non-commercial use of our material to educate beekeepers, the media and the public and, in turn, call attention to the general decline of insect biomass and diversity.
Octanoic acid (C8 FA) is a medium-chain fatty acid which, in nature, mainly occurs in palm kernel oil and coconuts. It is used in various products including cleaning agents, cosmetics, pesticides and herbicides as well as in foods for preservation or flavoring. Furthermore, it is investigated for medical treatments, for instance, of high cholesterol levels. The cultivation of palm oil plants has surged in the last years to satisfy an increasing market demand. However, concerns about extensive monocultures, which often come along with deforestation of rainforest, have driven the search for more environmentally friendly production methods. A biotechnological production with microbial organisms presents an attractive, more sustainable alternative.
Traditionally, the yeast Saccharomyces cerevisiae has been utilized by mankind in bread, wine, and beer making. Based on comprehensive knowledge about its metabolism and genetics, it can nowadays be metabolically engineered to produce a plethora of compounds of industrial interest. To produce octanoic acid, the cytosolic fatty acid synthase (FAS) of S. cerevisiae was utilized and engineered. Naturally, the yeast produces mostly long-chain fatty acids with chain lengths of C16 and C18, and only trace amounts of medium-chain fatty acids, i.e. C8-C14 fatty acids. To generate an S. cerevisiae strain that produces primarily octanoic acid, a mutated version of the FAS was generated (Gajewski et al., 2017) and the resulting S. cerevisiae FASR1834K strain was utilized in this work as a starting strain.
The goal of this thesis was to develop and implement strategies to improve the production level of this strain. The current mode of quantification of octanoic acid includes labor-intensive, low-throughput sample preparation and measurement – a main obstacle in generating and screening for improved strain variants. To this end, a main objective of this thesis was the development of a biosensor. The biosensor was based on the pPDR12 promotor, which is regulated by the transcription factor War1. Coupling pPDR12 to GFP as the reporter gene on a multicopy plasmid allowed in vivo detection via fluorescence intensity. The developed biosensor enabled rapid and facile quantification of the short- and medium-chain fatty acids C6, C7 and C8 fatty acids (Baumann et al., 2018). This is the first biosensor that can quantify externally supplied octanoic acid as well as octanoic acid present in the culture supernatant of producer strains with a high linear and dynamic range. Its reliability was validated by correlation of the biosensor signal to the octanoic acid concentrations extracted from culture supernatants as determined by gas chromatography. The biosensor’s ability to detect octanoic acid in a linear range of 0.01-0.75 mM (≈1-110 mg/L), which is within the production range of the starting strain, and a response of up to 10-fold increase in fluorescence after activation was demonstrated.
A high-throughput FACS (fluorescence-activated cell sorting) screening of an octanoic acid producer strain library was performed with the biosensor to detect improved strain variants (Baumann et al., 2020a). For this purpose, the biosensor was genomically integrated into an octanoic acid producer strain, resulting in drastically reduced single cell noise. The additional knockout of FAA2 successfully prevented medium-chain fatty acid degradation. A high-throughput screening protocol was designed to include iterative enrichment rounds which decreased false positives. The functionality of the biosensor on single cell level was validated by adding octanoic acid in the range of 0-80 mg/L and subsequent flow cytometric analysis. The biosensor-assisted FACS screening of a plasmid overexpression library of the yeast genome led to the detection of two genetic targets, FSH2 and KCS1, that in combined overexpression enhanced octanoic acid titers by 55 % compared to the parental strain. This was the first report of an effect of FSH2 and KCS1 on fatty acid titers. The presented method can also be utilized to screen other genetic libraries and is a means to facilitate future engineering efforts.
In growth tests, the previously reported toxicity of octanoic acid on S. cerevisiae was confirmed. Different strategies were harnessed to create more robust strains. An adaptive laboratory evolution (ALE) experiment was conducted and several rational targets including transporter- (PDR12, TPO1) and transcription factor-encoding genes (PDR1, PDR3, WAR1) as well as the mutated acetyl-CoA carboxylase encoding gene ACC1S1157A were overexpressed or knocked out in producer or non-producer strains, respectively. Despite contrary previous reports for other strain backgrounds, an enhanced robustness was not observable. Suspecting that the utilized laboratory strains have a natively low tolerance level, four industrial S. cerevisiae strains were evaluated in growth assays with octanoic acid and inherently more robust strains were detected, which are suitable future production hosts.
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Taxa under scrutiny in this thesis are Halophytophthora-like oomycetes. The genus Halophytophthora, proposed in 1990, is an assemblage of unrelated species grouped together on the basis habitat preference, i.e. the mangrove or saltmarsh biome, and morphological similarity to Phytophthora. The premise “Phytophthora-like species from the mangrove environment” became the genus concept for Halophytophthora and lasted for almost 2 decades which resulted to the addition of several species (i.e. H. elongata, H. exoprolifera, H. porrigovesica, H. kandeliae, H. masteri, and H. tartarea). At the onset of molecular phylogenetics, Halophytophthora was inferred as a highly polyphyletic taxon and the genus concept was found to be unsuitable. This thesis adds to this, since six Phytophthora spp. were isolated from the mangrove environment, two of which were found in the Philippines (Phytophthora elongata and Phytophthora insolita). After a thorough assessment of the morphologic and phylogenetic data of taxa included in this thesis, several taxonomic novelties were introduced – a new family (Salispinaceae), a new genus (Calycofera), new species (Calycofera cryptica, Phytopythium dogmae, Phytopythium leanoi, Salisapilia coffeyi, and Salispina hoi), and new combinations (Calycofera operculata, Salisapilia bahamensis, S. elongata, S. epistomia, S. masteri, S. mycoparasitica). In addition, Salisapiliaceae and Salisapilia were emended.
The Mediterranean realm, comprising the Mediterranean and Macaronesian regions, has long been recognized as one of the world’s biodiversity hotspots, owing to its remarkable species richness and endemism. Several hypotheses on biotic and abiotic drivers of species diversification in the region have been often proposed but rarely tested in an explicit phylogenetic framework. Here, we investigate the impact of both species-intrinsic and -extrinsic factors on diversification in the species-rich, cosmopolitan Limonium, an angiosperm genus with center of diversity in the Mediterranean. First, we infer and time-calibrate the largest Limonium phylogeny to date. We then estimate ancestral ranges and diversification dynamics at both global and regional scales. At the global scale, we test whether the identified shifts in diversification rates are linked to specific geological and/or climatic events in the Mediterranean area and/or asexual reproduction (apomixis). Our results support a late Paleogene origin in the proto-Mediterranean area for Limonium, followed by extensive in situ diversification in the Mediterranean region during the late Miocene, Pliocene, and Pleistocene. We found significant increases of diversification rates in the “Mediterranean lineage” associated with the Messinian Salinity Crisis, onset of Mediterranean climate, Plio-Pleistocene sea-level fluctuations, and apomixis. Additionally, the Euro-Mediterranean area acted as the major source of species dispersals to the surrounding areas. At the regional scale, we infer the biogeographic origins of insular endemics in the oceanic archipelagos of Macaronesia, and test whether woodiness in the Canarian Nobiles clade is a derived trait linked to insular life and a biotic driver of diversification. We find that Limonium species diversity on the Canary Islands and Cape Verde archipelagos is the product of multiple colonization events followed by in situ diversification, and that woodiness of the Canarian endemics is indeed a derived trait but is not associated with a significant shift to higher diversification rates. Our study expands knowledge on how the interaction between abiotic and biotic drivers shape the uneven distribution of species diversity across taxonomic and geographical scales.
Neuroligin-3 (Nlgn3), a neuronal adhesion protein implicated in autism spectrum disorder (ASD), is expressed at excitatory and inhibitory postsynapses and hence may regulate neuronal excitation/inhibition balance. To test this hypothesis, we recorded field excitatory postsynaptic potentials (fEPSPs) in the dentate gyrus of Nlgn3 knockout (KO) and wild-type mice. Synaptic transmission evoked by perforant path stimulation was reduced in KO mice, but coupling of the fEPSP to the population spike was increased, suggesting a compensatory change in granule cell excitability. These findings closely resemble those in neuroligin-1 (Nlgn1) KO mice and could be partially explained by the reduction in Nlgn1 levels we observed in hippocampal synaptosomes from Nlgn3 KO mice. However, unlike Nlgn1, Nlgn3 is not necessary for long-term potentiation. We conclude that while Nlgn1 and Nlgn3 have distinct functions, both are required for intact synaptic transmission in the mouse dentate gyrus. Our results indicate that interactions between neuroligins may play an important role in regulating synaptic transmission and that ASD-related neuroligin mutations may also affect the synaptic availability of other neuroligins.
Despite constant progress in basic and translational research, cancer is still one of the leading cause of death. In particular, tumors of the central nervous system (CNS) are usually associated with dismal prognosis. Although about 100 distinct subtypes of primary CNS tumors have been classified molecularly, metastases derived from primaries outside the CNS (= brain metastases, BrM) are more frequently observed across brain tumor patients. It is estimated that approximately 20 - 40 % of all cancer patients will develop BrM during their course of disease, and basically every tumor type is able to metastasize to the brain. Nevertheless, BrM are most frequently derived from primaries of the lung, breast, and skin (melanoma). Treatment options for patients with BrM are very limited, and standard of care therapies include surgery, ionizing radiation (e.g. whole brain radio-therapy, WBRT), and some systemic and immuno-therapeutic approaches.
The brain represents a unique organ, which in part is due to the presence of the blood-brain barrier, a unit of the neuro-vascular interface ensuring tightly regulated exchange of nutrients, molecules, and cells. Furthermore, apart from microglia the brain parenchyma does not harbor other immune cells. Those cells however can be found at the borders of the CNS residing in the meninges, for instance. Based on recent insight on the immune landscape in the CNS, a paradigm shift occurred after which the brain is no longer regarded as immune-privileged but rather immune distinct. The phenomenon of immune cell infiltration has been described before in the context of neurological disorders including Multiple Sclerosis, as well as in brain tumors.
Since the development of immune-therapeutic approaches for tumors outside the CNS that aim to evoke sustainable anti-tumor effects, it became increasingly interesting to understand and harness the immune landscape (= tumor microenvironment, TME) of brain tumors, as well. Interestingly, most of the knowledge about the TME is based on studies of primary brain tumors. However, it is known that BrM compared to primary brain tumors induce a different TME like e.g. the recruitment of much more lymphocytes, which is one of the reasons primary brain tumors are considered immunologically “cold” and poorly respond to immuno-therapies. Previous insight into the functional contribution of tumor-associated cells in BrM progression revealed for example that brain-resident cell types (e.g. astrocytes or microglia) promote BrM development and outgrowth. However, until recently a comprehensive view on the cellular composition and functional role of the brain metastases-associated TME was missing and little was known how it changes during tumor progression or standard therapy.
Hence, within this thesis it was sought to describe novel aspects of the TME of preclinical BrM models, which include two xenograft and one syngeneic mouse model. BrM was induced via intra-cardiac injection of tumor cells with a high brain tropism. Both xenograft models were based on immuno-compromised nude mice (Balb/c nude) and included the melanoma-to-brain (M2B) model H1_DL2, and the lung-to-brain (L2B) model H2030. In addition the breast-to-brain model 99LN-BrM was used in wild-type mice (BL6), and therefore represented an immuno-competent, syngeneic model. First BrMs could be detected in the xenograft models at 3 weeks after injection, whereas first 99LN BrMs were detected at 5 weeks. BrM development and progression were monitored by bioluminescence imaging once per week in the xenograft models. Tumor progression in the 99LN model was examined by magnetic resonance imaging. Based on the measurement methods, and for further histologic and cytometric experiments, mice were stratified into groups with small or large BrMs, respectively. Some initial immuno-stainings confirmed previous findings, showing that brain-resident cells like astrocytes and microglia become activated in the presence of tumor cells, whereas neurons for example rather give the impression of passive bystanders. Importantly, an accumulation of IBA1+ cells was observed during BrM progression. IBA1 is a pan-macrophage marker that stains all tumor-associated macrophages (TAMs). However previous work suggested that the TAM population consists of at least two main subpopulations in BrM as well: the resident-infiltrating microglia (MG, TAM-MG), as well as the peripheral and monocytic-derived macrophages (TAM-MDM). Since both cell types within the tumor share morphological traits, and due to the lack of markers to distinguish them, an exact discrimination of both cell types was complicated in the past. Recently, an integrative lineage-tracing-based study identified the integrin CD49d as MDM-specific in the context of brain tumor-associated myeloid cells, hence enabling a reliable dissection of both TAM populations in e.g. flow cytometric experiments.
One of the main aims of this thesis was to dissect the myeloid TME in the three different BrM models during tumor progression. Using a 5-marker flow cytometry (FCM) (CD45/CD11b/Ly6C/Ly6G/CD49d) approach, the following cell populations were examined in more detail: granulocytes, inflammatory monocytes, MDM, and MG.
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Background: Understanding the processes that lead to hybridization of wolves and dogs is of scientific and management importance, particularly over large geographical scales, as wolves can disperse great distances. However, a method to efficiently detect hybrids in routine wolf monitoring is lacking. Microsatellites offer only limited resolution due to the low number of markers showing distinctive allele frequencies between wolves and dogs. Moreover, calibration across laboratories is time-consuming and costly. In this study, we selected a panel of 96 ancestry informative markers for wolves and dogs, derived from the Illumina CanineHD Whole-Genome BeadChip (174 K). We designed very short amplicons for genotyping on a microfluidic array, thus making the method suitable also for non-invasively collected samples.
Results: Genotypes based on 93 SNPs from wolves sampled throughout Europe, purebred and non-pedigree dogs, and suspected hybrids showed that the new panel accurately identifies parental individuals, first-generation hybrids and first-generation backcrosses to wolves, while second- and third-generation backcrosses to wolves were identified as advanced hybrids in almost all cases. Our results support the hybrid identity of suspect individuals and the non-hybrid status of individuals regarded as wolves. We also show the adequacy of these markers to assess hybridization at a European-wide scale and the importance of including samples from reference populations.
Conclusions: We showed that the proposed SNP panel is an efficient tool for detecting hybrids up to the third-generation backcrosses to wolves across Europe. Notably, the proposed genotyping method is suitable for a variety of samples, including non-invasive and museum samples, making this panel useful for wolf-dog hybrid assessments and wolf monitoring at both continental and different temporal scales.
Microplastics (MPs) are ubiquitous and persistent pollutants, and have been detected in a wide variety of media, from soils to aquatic systems. MPs, consisting primarily of polyethylene, polypropylene, and polyacrylamide polymers, have recently been found in 12% of samples of honey collected in Ecuador. Recently, MPs have also been identified in honey bees collected from apiaries in Copenhagen, Denmark, as well as nearby semiurban and rural areas. Given these documented exposures, assessment of their effects is critical for understanding the risks of MP exposure to honey bees. Exposure to polystyrene (PS)-MPs decreased diversity of the honey bee gut microbiota, followed by changes in gene expression related to oxidative damage, detoxification, and immunity. As a result, the aim of this perspective was to investigate whether wide-spread prevalence of MPs might have unintended negative effects on health and fitness of honey bees, as well as to draw the scientific community’s attention to the possible risks of MPs to the fitness of honey bees. Several research questions must be answered before MPs can be considered a potential threat to bees.
Macrophages not only represent an integral part of innate immunity but also critically contribute to tissue and organ homeostasis. Moreover, disease progression is accompanied by macrophage accumulation in many cancer types and is often associated with poor prognosis and therapy resistance. Given their critical role in modulating tumor immunity in primary and metastatic brain cancers, macrophages are emerging as promising therapeutic targets. Different types of macrophages infiltrate brain cancers, including (i) CNS resident macrophages that comprise microglia (TAM-MG) as well as border-associated macrophages and (ii) monocyte-derived macrophages (TAM-MDM) that are recruited from the periphery. Controversy remained about their disease-associated functions since classical approaches did not reliably distinguish between macrophage subpopulations. Recent conceptual and technological advances, such as large-scale omic approaches, provided new insight into molecular profiles of TAMs based on their cellular origin. In this review, we summarize insight from recent studies highlighting similarities and differences of TAM-MG and TAM-MDM at the molecular level. We will focus on data obtained from RNA sequencing and mass cytometry approaches. Together, this knowledge significantly contributes to our understanding of transcriptional and translational programs that define disease-associated TAM functions. Cross-species meta-analyses will further help to evaluate the translational significance of preclinical findings as part of the effort to identify candidates for macrophage-targeted therapy against brain metastasis.
Ribosome assembly is an essential and carefully choreographed cellular process. In eukaryotes, several 100 proteins, distributed across the nucleolus, nucleus, and cytoplasm, co-ordinate the step-wise assembly of four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins (RPs) into the mature ribosomal subunits. Due to the inherent complexity of the assembly process, functional studies identifying ribosome biogenesis factors and, more importantly, their precise functions and interplay are confined to a few and very well-established model organisms. Although best characterized in yeast (Saccharomyces cerevisiae), emerging links to disease and the discovery of additional layers of regulation have recently encouraged deeper analysis of the pathway in human cells. In archaea, ribosome biogenesis is less well-understood. However, their simpler sub-cellular structure should allow a less elaborated assembly procedure, potentially providing insights into the functional essentials of ribosome biogenesis that evolved long before the diversification of archaea and eukaryotes. Here, we use a comprehensive phylogenetic profiling setup, integrating targeted ortholog searches with automated scoring of protein domain architecture similarities and an assessment of when search sensitivity becomes limiting, to trace 301 curated eukaryotic ribosome biogenesis factors across 982 taxa spanning the tree of life and including 727 archaea. We show that both factor loss and lineage-specific modifications of factor function modulate ribosome biogenesis, and we highlight that limited sensitivity of the ortholog search can confound evolutionary conclusions. Projecting into the archaeal domain, we find that only few factors are consistently present across the analyzed taxa, and lineage-specific loss is common. While members of the Asgard group are not special with respect to their inventory of ribosome biogenesis factors (RBFs), they unite the highest number of orthologs to eukaryotic RBFs in one taxon. Using large ribosomal subunit maturation as an example, we demonstrate that archaea pursue a simplified version of the corresponding steps in eukaryotes. Much of the complexity of this process evolved on the eukaryotic lineage by the duplication of ribosomal proteins and their subsequent functional diversification into ribosome biogenesis factors. This highlights that studying ribosome biogenesis in archaea provides fundamental information also for understanding the process in eukaryotes.
Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While “exitrons” are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed “falsitrons,” that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.
Fungi and prokaryotes are dominant colonizers of wood and mediate its decomposition. Much progress has been achieved to unravel these communities and link them to specific wood properties. However, comparative studies considering both groups of organisms and assessing their relationships to wood resources are largely missing. Bipartite interaction networks provide an opportunity to investigate this colonizer-resource relationship more in detail and aim to directly compare results between different biotic groups. The main questions were as follows. Are network structures reflecting the trophic relationship between fungal and prokaryotic colonizers and their resources? If so, do they reflect the critical role of these groups, especially that of fungi, during decomposition? We used amplicon sequencing data to analyze fungal and prokaryotic interaction networks from deadwood of 13 temperate tree species at an early to middle stage of decomposition. Several diversity- and specialization-related indices were determined and the observed network structures were related to intrinsic wood traits. We hypothesized nonrandom bipartite networks for both groups and a higher degree of specialization for fungi, as they are the key players in wood decomposition. The results reveal highly modular and specialized interaction networks for both groups of organisms, demonstrating that many fungi and prokaryotes are resource-specific colonizers. However, as the level of specialization of fungi significantly surpassed that of prokaryotes, our findings reflect the strong association between fungi and their host. Our novel approach shows that the application of bipartite interaction networks is a useful tool to explore, quantify, and compare the deadwood-colonizers relationship based on sequencing data.
IMPORTANCE Deadwood is important for our forest ecosystems. It feeds and houses many organisms, e.g., fungi and prokaryotes, with many different species contributing to its decomposition and nutrient cycling. The aim of this study was to explore and quantify the relationship between these two main wood-inhabiting organism groups and their corresponding host trees. Two independent DNA-based amplicon sequencing data sets (fungi and prokaryotes) were analyzed via bipartite interaction networks. The links in the networks represent the interactions between the deadwood colonizers and their deadwood hosts. The networks allowed us to analyze whether many colonizing species interact mostly with a restricted number of deadwood tree species, so-called specialization. Our results demonstrate that many prokaryotes and fungi are resource-specific colonizers. The direct comparison between both groups revealed significantly higher specialization values for fungi, emphasizing their strong association to respective host trees, which reflects their dominant role in exploiting this resource.
Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, partial and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and their severe discomfort.
Plastics contain a complex mixture of chemicals including polymers, additives, starting substances and side-products of processing. These plastic chemicals are prone to leach into the packaged goods, in the case of food contact materials (FCMs), or into the natural environment, in the case of plastic debris. Thus, plastics represent an exposure source of chemicals for humans and wildlife alike. While it is widely known that individual plastic chemicals, such as bisphenol A and phthalates, are hazardous, little is known on the overall chemical composition and toxicity of plastics. When fragmented into smaller particles, referred to as microplastics (< 5 mm), the plastic itself can be ingested by many species. It is well established that microplastic ingestion can have negative consequences for a wide range of organisms including invertebrates, but the contribution of plastic chemicals to the toxicity of microplastics is unclear.
Given the above, the present thesis aimed at a comprehensive toxicological, ecotoxicological and chemical characterization of everyday plastics. For a comparative evaluation, 77 plastic products were selected covering 16 material types (e.g., polyethylene) made from petroleum or renewable feedstocks. These products included biodegradable products, FCMs and non-FCMs, as well as raw materials and final products, respectively. In the first two studies, the chemical mixtures contained in the 77 products were extracted with methanol and extracts were analyzed in a set of four in vitro bioassays and by non-target high-resolution gas or liquid chromatography mass spectrometry. Since an exposure only occurs if chemicals actually leach under realistic conditions, in a third study migration experiments with water were conducted for 24 out of the 77 products. The aqueous migrates were assessed in the same way as the methanolic extracts. In addition, the freshwater invertebrate Daphnia magna was exposed chronically to microplastics made of polyvinylchloride (PVC), polyurethane (PUR) and polylactic acid (PLA) to investigate the contribution of chemicals in microplastic toxicity, in a fourth study.
The experimental findings demonstrate that a wide variety of chemicals is present in plastics. A single plastic product can contain up to several thousand chemical features, most of which unique to that product and at the same time unknown. The results also indicate that the majority of these chemical mixtures are toxic in vitro. Accordingly, 65% of the plastic extracts induced baseline toxicity and 42% an oxidative stress response, while 25% had an antiandrogenic and 6% an estrogenic activity. This implies that chemicals causing unspecific toxicity are more prevalent in plastics than such with endocrine effects. These chemicals can also leach from plastics under realistic conditions. Between 17 and 8936 chemical features were detected in a single migrate sample and all 24 tested migrates induced in vitro toxicity. This means that humans and wildlife can actually be exposed to toxic plastic chemicals under realistic conditions. Generally, each product has its individual toxicological and chemical fingerprint. Thus, neither material type, feedstock, biodegradability nor the food contact suitability of a product can serve as a predictor for the toxicity, the chemical composition or complexity of a product. Likewise, this means that bio-based and biodegradable materials are not superior to their petroleum-based counterparts from a toxicological perspective despite being promoted as sustainable alternatives to conventional plastics.
Moreover, the present thesis demonstrates that plastic chemicals can be the main driver for microplastic toxicity. Irregular microplastics made of PVC, PUR and PLA adversely affected life-history traits of D. magna in a polymer type- and endpoint-dependent manner at concentrations between 100 and 500 mg L-1 and with a higher efficiency than natural kaolin particles. While the toxicity of PVC was triggered by the chemicals used in the material, the effects of PUR and PLA were induced by the physical properties of the particle.
In addition, in the fifth study, results and observations made during this thesis were integrated inter- and transdisciplinarily with the perspectives of a social scientist and a product manufacturer. This elucidated that knowledge on plastic ingredients is often concealed, is lacking or not applicable in practice. These intransparencies hinder the safety evaluation of plastic products as well as the choice and sale of the least toxic packaging material.
Overall, the present thesis highlights that the chemical safety of plastics and their bio-based and biodegradable alternatives is currently not ensured. Thus, chemicals require more consideration in the toxicity and risk assessment of plastics and microplastics. Product-specific and complex chemical compositions, including unknown compounds, pose a challenge here. Two essential steps towards non-toxic products are to increase transparency along the product life cycle and to reduce the chemical complexity of plastics by communication and regulation. The results of the present thesis indicate that products exist which do not contain toxic chemicals. These can serve to direct the design of safer plastics. Since toxicity and chemical complexity seem to increase with processing, the integration of toxicity testing during the production steps would further support the safe and sustainable production and use of plastic products.
First-principle metabolic modelling holds potential for designing microbial chassis that are resilient against phenotype reversal due to adaptive mutations. Yet, the theory of model-based chassis design has rarely been put to rigorous experimental test. Here, we report the development of Saccharomyces cerevisiae chassis strains for dicarboxylic acid production using genome-scale metabolic modelling. The chassis strains, albeit geared for higher flux towards succinate, fumarate and malate, do not appreciably secrete these metabolites. As predicted by the model, introducing product-specific TCA cycle disruptions resulted in the secretion of the corresponding acid. Adaptive laboratory evolution further improved production of succinate and fumarate, demonstrating the evolutionary robustness of the engineered cells. In the case of malate, multi-omics analysis revealed a flux bypass at peroxisomal malate dehydrogenase that was missing in the yeast metabolic model. In all three cases, flux balance analysis integrating transcriptomics, proteomics and metabolomics data confirmed the flux re-routing predicted by the model. Taken together, our modelling and experimental results have implications for the computer-aided design of microbial cell factories.
In order to form an organ, cells need to take up specialized functions and tasks. Cellular specialization is guided by an interplay of chemical signals and physical forces, where one influences the other. One aspect in cellular identity is its shape, which e.g. defines how susceptible the cell may be to intercellular signaling or in which section of the cell cycle it is and therefore can tell us about its current state. Shape changes are introduced by motor proteins that are controlled and activated in a locally confined manner. For my thesis, I was interested to understand better how cellular shape and geometry impacts downstream cell and organ development. What happens if a cell cant transition to a specific shape? How does it affect tissue structure? How does it affect further development?
One regulator of motor proteins like non-muscle myosin is Shroom3, which recently has been been shown to be expressed and involved in the development of the zebrafish lateral line organ (1 ). Development of the lateral line occurs through a migrating cluster of initially about 150 cells, the posterior lateral line primordium (pLLP), which migrates from the anterior (head) to the posterior (tail) while depositing cell clusters in a regular pattern. Literature on development of the lateral line suggests that in order for a cell cluster to be deposited from the pLLP, rosette formation is a key requirement. Therefore our expectation from the shroom3 mutant was that the number of clusters deposited was significantly reduced. To our surprise, when we first inspected the end of migration lateral line phenotype we found many individuals with a significant increase in cell clusters deposited.
This made us re-think the role of Shroom3 during rosette assembly and the processes its involved in.
To study the effects of Shroom3 on lateral line development, a mutant line was generated and crossed with various transgenic lines which express fluorescently labeled proteins that locate to organelles such as the plasmamembrane or the nucleus. Following, the mutant with its fluorescent labels was microscopically imaged under different conditions to quantify and analyze various cell-morphometric features. Even though the zebrafish is a popular model organism and its perfectly suited for developmental biology and advanced microscopy, there were no methods that would allow for a standardized and more automated pipeline of data acquisition and processing.
Therefore, in order to accurately quantify the morphogenic processes Shroom3 is involved in, I developed a new toolset that significantly improved and facilitated my research. The toolset consists of (1) a new sample mounting method that is based on a 3D agarose gel that increases the number of embryos that can be mounted and imaged at once and speeds up the imaging process significantly (2) for subseqent image analysis I developed four programs that automate the process and therefore make the results much more reproducible and the analysis much more efficient. The first program is used for end of migration analyses, to deduce the pattern, count and size of Lateral Line cell clusters. The second is used not for end of migration, but for migration analyses (on timelapse recordings). Besides this it also prepares the images for more advanced downstream migration analyses and allows to analyse fluorescence signal on a second channel. The third program is used to analyse the pLLP only at high spatial resolution and to deduce the cell count, 3D cell morphometrics (like the volume) and cell orientation. The fourth program finally is used downstream of the second and third program and is capable of detecting and comparing them with the look of wildtype rosettes.
Here I show that in absence of Shroom3 rosette formation in the migrating pLLP is destabilized leading to facilitated cell cluster deposition and I show how this might be related to traction forces due to a possible interdependence of pLLP acceleration and speed of migration. Furthermore I show that apical constriction and rosette formation is not blocked in Shroom3 deficient embryos, but that larger rosettes are fragmented into many smaller ones. Finally, I give an outlook on how the absense of Shroom3 and hence the absense of morphological changes may deregulate gene transcription by elevating the levels Atoh1a, a transcription factor necessary for hair cell development.
My results and methodology demonstrate the importance of morphology in guiding developmental processes and how rather small morphological changes on the cellular level can impact further development significantly. My work also shows how powerful modern genetics, imaging and image analysis are and how diverse they are in terms of range of questions they are capable of answering. The methods and tools I developed prepare the ground for at least three quarters of the analyses I carried out and together with the documentation and data I provide, they are highly reproducible. In that regard I am especially happy that one of my developments, an improved sample preparation method, is already used by many different labs all over the world helping them to make their results more reproducible.
Locating a vocalizing animal can be useful in many fields of bioacoustics and behavioral research, and is often done in the wild, covering large areas. In zoos, however, the application of this method becomes particularly difficult, because, on the one hand, the animals are in a relatively small area and, on the other hand, reverberant environments and background noise complicate the analysis. Nevertheless, by localizing and analyzing animal sounds, valuable information on physiological state, sex, subspecies, reproductive state, social status, and animal welfare can be gathered. Therefore, we developed a sound localization software that is able to estimate the position of a vocalizing animal precisely, making it possible to assign the vocalization to the corresponding individual, even under difficult conditions. In this study, the accuracy and reliability of the software is tested under various conditions. Different vocalizations were played back through a loudspeaker and recorded with several microphones to verify the accuracy. In addition, tests were carried out under real conditions using the example of the giant otter enclosure at Dortmund Zoo, Germany. The results show that the software can estimate the correct position of a sound source with a high accuracy (median of the deviation 0.234 m). Consequently, this software could make an important contribution to basic research via position determination and the associated differentiation of individuals, and could be relevant in a long-term application for monitoring animal welfare in zoos.
Correction to: Apidologie (2020) 51:1182–1198
https://doi.org/10.1007/s13592-020-00796-9
The article Insights into Ethiopian honey bee diversity based on wing geomorphometric and mitochondrial DNA analyses, written by Hailu, T.G., D’Alvise, P., Tofilski, A. et al., was originally published Online First without Open Access. After publication in volume 51, issue 6, page 1182-1198, the author decided to opt for Open Choice and to make the article an Open Access publication. Therefore, the copyright of the article has been changed to © The Author(s) 2020 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution, and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article is included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Open Access funding enabled and organized by Projekt DEAL.
Acetogenic bacteria are a polyphyletic group of organisms that fix carbon dioxide under anaerobic, non-phototrophic conditions by reduction of two mol of CO2 to acetyl-CoA via the Wood–Ljungdahl pathway. This pathway also allows for lithotrophic growth with H2 as electron donor and this pathway is considered to be one of the oldest, if not the oldest metabolic pathway on Earth for CO2 reduction, since it is coupled to the synthesis of ATP. How ATP is synthesized has been an enigma for decades, but in the last decade two ferredoxin-dependent respiratory chains were discovered. Those respiratory chains comprise of a cytochrome-free, ferredoxin-dependent respiratory enzyme complex, which is either the Rnf or Ech complex. However, it was discovered already 50 years ago that some acetogens contain cytochromes and quinones, but their role had only a shadowy existence. Here, we review the literature on the characterization of cytochromes and quinones in acetogens and present a hypothesis that they may function in electron transport chains in addition to Rnf and Ech.
Nematodes represent a diverse and ubiquitous group of metazoans in terrestrial environments. They feed on bacteria, fungi, plants, other nematodes or parasitize a variety of animals and hence may be considered as active members of many food webs. Deadwood is a structural component of forest ecosystems which harbors many niches for diverse biota. As fungi and bacteria are among the most prominent decomposing colonizers of deadwood, we anticipated frequent and diverse nematode populations to co-occur in such ecosystems. However, knowledge about their ability to colonize this habitat is still limited. We applied DNA-based amplicon sequencing (metabarcoding) of the 18S rRNA gene to analyze nematode communities in sapwood and heartwood of decaying logs from 13 different tree species. We identified 247 nematode ASVs (amplicon sequence variants) from 27 families. Most of these identified families represent bacterial and fungal feeders. Their composition strongly depended on tree species identity in both wood compartments. While pH and water content were the only wood properties that contributed to nematodes’ distribution, co-occurring fungal and prokaryotic (bacteria and archaea) α- and β-diversities were significantly related to nematode communities. By exploring thirteen different tree species, which exhibit a broad range of wood characteristics, this study provides first and comprehensive insights into nematode diversity in deadwood of temperate forests and indicates connectivity to other wood-inhabiting organisms.
Extremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural transformation plays an important role in bacterial adaptation. This mode of DNA transfer permits the transfer of genetic information between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal gene transfer (HGT), significantly contributes to genome plasticity over evolutionary history and is a driving force for the spread of fitness-enhancing functions including virulence genes and antibiotic resistances. In particular, HGT has played an important role for adaptation of bacteria to extreme environments. Here, we present a survey of the natural transformation systems in bacteria that live under extreme conditions: the thermophile Thermus thermophilus and two desiccation-resistant members of the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter baumannii. The latter is an opportunistic pathogen and has become a world-wide threat in health-care institutions. We highlight conserved and unique features of the DNA transporter in Thermus and Acinetobacter and present tentative models of both systems. The structure and function of both DNA transporter are described and the mechanism of DNA uptake is discussed.
The effect of the extreme summer drought and heatwave 2018 in Central Europe on wood properties of oaks at four sandy valley river sites (Quercus robur L.) and one south-exposed schist slope (Qu. petraea (Matt.) Liebl.) in the middle Rhine and lower Main valley were studied and compared to well-watered trees from a riparian stand. While properties of the 2018 tree rings mostly resembled those of the previous (wet) year, significant decreases in Δ13C, wood density and ring width occurred in 2019 at most drought-prone sites. In the sandy sites, ring widths correlated with previous-year precipitation from June to August over a 20-year period. In organs formed in 2018, in general, decreasing Δ13C values were obtained in the order leaves, twigs, wood and acorns, with the values from acorns often resembling those from 2019-year rings. The observed changes indicated an increased intrinsic water use efficiency and lack of starch reserve formation during the unprecedented hot and dry summer 2018. Qu. petraea revealed quite different values from Qu. robur (lower Δ13C, wider and denser year rings), but qualitatively showed the same reaction to the drought in 2018, except for an enhanced formation of tyloses in recent-year tree rings.
he ubiquity of microplastics in aquatic ecosystems has raised concerns over their interaction with biota. However,microplastics research on freshwater species, especially mollusks, is still scarce. We, therefore, investigated the factorsaffecting microplastics ingestion in the freshwater musselDreissena polymorpha. Using polystyrene spheres (5, 10, 45,90μm), we determined the body burden of microplastics in the mussels in relation to 1) exposure and depuration time, 2)body size, 3) food abundance, and 4) microplastic concentrations.D. polymorpharapidly ingested microplastics and ex-creted most particles within 12 h. A few microplastics were retained for up to 1 wk. Smaller individuals had a higher relativebody burden of microplastics than larger individuals. The uptake of microplastics was concentration‐dependent, whereas anadditional food supply (algae) reduced it. We also compared the ingestion of microplastics byD. polymorphawith 2 otherfreshwater species (Anodonta anatina,Sinanodonta woodiana), highlighting that absolute and relative uptake depends onthe species and the size of the mussels. In addition, we determined toxicity of polystyrene fragments (≤63μm,6.4–100 000 p mL–1) and diatomite (natural particle, 100 000 p mL–1)inD. polymorphaafter 1, 3, 7, and 42 d of exposure,investigating clearance rate, energy reserves, and oxidative stress. Despite ingesting large quantities, exposure to poly-styrene fragments only affected the clearance rate ofD. polymorpha. Further, results of the microplastic and diatomiteexposure did not differ significantly. Therefore,D. polymorphais unaffected by or can compensate for polystyrene fragmenttoxicity even at concentrations above current environmental levels.Environ Toxicol Chem2021;40:2247–2260. © 2021 TheAuthors.Environmental Toxicology and Chemistrypublished by Wiley Periodicals LLC on behalf of SETAC.Keywords:Microplastics; Toxic effects; Mollusk toxicity
Genetic and genomic tools have provided researchers with the opportunity to address fundamental questions regarding the reintroduction of species into their historical range with greater precision than ever before. Reintroduction has been employed as a conservation method to return locally extinct species to their native range for decades. However, it remains unknown how genetic factors may impact population establishment and persistence at the population and metapopulation level in the short- and long-term. Genetic methods are capable of producing datasets from many individuals, even when only low quality DNA can be collected. These methods offer an avenue to investigate unanswered questions in reintroduction biology, which is vital to provide evidence based management strategies for future projects. The Eurasian lynx (Lynx lynx) and European wildcat (Felis silvestris) are elusive carnivores native to Eurasia and have been the subject of multiple reintroduction attempts into their native range. During the 19th and 20th century, the Eurasian lynx was extirpated from West and Central Europe due to increasing habitat fragmentation and persecution. Similarly, the European wildcat was the subject of human persecution, residing in a few refugia in West and Central Europe. After legal protection in the 1950s, subsequent reintroduction projects of both species began in the 1970s and 1980s and continue to the present. Despite this large focus on species conservation, little attention has been given to the consequences these reintroductions have on the genetic composition of the reintroduced populations and if the populations have a chance of persisting in the long term. These species have not yet benefited from the large range of genetic and genomic techniques currently available to non-model organisms, leaving many fundamental aspects of their reintroduction poorly understood. In my dissertation, I investigate demography, population structure, genetic diversity and inbreeding at the population and metapopulation level in both species. In the introduction, which lays the foundation for the subsequent chapters of this PHD, I provide background on reintroduction, its role in conservation and the genetic consequences on populations, especially populations of apex and mesocarnivores. In Publication I, I investigated the reemergence of the European wildcat in a low mountain region in Germany using fine-scale spatial analysis. I found that the reintroduced population has persisted and merged with an expanding natural population. The reintroduced population showed no genetic differentiation from the natural population suggesting there is a good chance this population has retained sufficient genetic diversity despite reintroduction. In Publication II, I tracked population development and genetic diversity over 15 years in a reintroduced lynx population to determine the genetic ramifications on a temporal scale. I found slow genetic erosion after a period of outbreeding, which fits in line with other reintroduced taxa sharing similar demographic histories. I also found the number of genetic founders to be a fraction of the total released individuals, indicating that reintroduced populations of elusive carnivores may have fewer founder individuals than previously thought. In Publication III, I sampled all surviving lynx reintroductions in West and Central Europe as well as 11 natural populations to compare levels of genetic diversity and inbreeding across the species distribution. I found that all reintroduced populations have lower genetic variability and higher inbreeding than natural populations, which urgently requires further translocations to mitigate possible negative consequences. These translocations could stem from other reintroduced populations or from surrounding natural populations. The results contribute to a growing body of evidence indicating that inbreeding is likely to be more prevalent in wild populations than previously understood. Finally, in the discussion I explore how genetic methods can be applied to post-reintroduction monitoring of felid species to illuminate questions relating to genetic composition after release. The methods employed in these studies and in future work will be highly dependent on the research questions posed. Additionally, I investigate the drivers of the observed genetic patterns including founder size, source population, environmental factors, and population growth. I found that genetic diversity loss patterns across these two felid species are not clearly defined, however, management actions can be taken to mitigate the negative effects of reintroductions. These management actions include further translocation, introducing a sufficient number of released individuals and situating reintroductions adjacent to natural populations. All of these actions can minimize genetic drift and inbreeding, two factors which negatively impact small populations. This thesis further supports mounting evidence that genetic considerations should be assessed before releasing individuals, which allows for incorporation of scientific evidence into the planning process thereby increasing the overall success of reintroduction projects. Ultimately, the resources developed during this dissertation provide a solid baseline and foundation for future work regarding the consequences of reintroductions. This is especially important as an increasing number of species are at risk of extinction and reintroductions of both the European wildcat and Eurasian lynx, as well as many others, are planned in the coming years.
The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.
Accurate determination of the evolutionary relationships between genes is a foundational challenge in biology. Homology—evolutionary relatedness—is in many cases readily determined based on sequence similarity analysis. By contrast, whether or not two genes directly descended from a common ancestor by a speciation event (orthologs) or duplication event (paralogs) is more challenging, yet provides critical information on the history of a gene. Since 2009, this task has been the focus of the Quest for Orthologs (QFO) Consortium. The sixth QFO meeting took place in Okazaki, Japan in conjunction with the 67th National Institute for Basic Biology conference. Here, we report recent advances, applications, and oncoming challenges that were discussed during the conference. Steady progress has been made toward standardization and scalability of new and existing tools. A feature of the conference was the presentation of a panel of accessible tools for phylogenetic profiling and several developments to bring orthology beyond the gene unit—from domains to networks. This meeting brought into light several challenges to come: leveraging orthology computations to get the most of the incoming avalanche of genomic data, integrating orthology from domain to biological network levels, building better gene models, and adapting orthology approaches to the broad evolutionary and genomic diversity recognized in different forms of life and viruses.
Toxicogenomic differentiation of functional responses to fipronil and imidacloprid in Daphnia magna
(2021)
Active substances of pesticides, biocides or pharmaceuticals can induce adverse side effects in the aquatic ecosystem, necessitating environmental hazard and risk assessment prior to substance registration. The freshwater crustacean Daphnia magna is a model organism for acute and chronic toxicity assessment representing aquatic invertebrates. However, standardized tests involving daphnia are restricted to the endpoints immobility and reproduction and thus provide only limited insights into the underlying modes-of-action. Here, we applied transcriptome profiling to a modified D. magna Acute Immobilization test to analyze and compare gene expression profiles induced by the GABA-gated chloride channel blocker fipronil and the nicotinic acetylcholine receptor (nAChR) agonist imidacloprid. Daphnids were expose to two low effect concentrations of each substance followed by RNA sequencing and functional classification of affected gene ontologies and pathways. For both insecticides, we observed a concentration-dependent increase in the number of differentially expressed genes, whose expression changes were highly significantly positively correlated when comparing both test concentrations. These gene expression fingerprints showed virtually no overlap between the test substances and they related well to previous data of diazepam and carbaryl, two substances targeting similar molecular key events. While, based on our results, fipronil predominantly interfered with molecular functions involved in ATPase-coupled transmembrane transport and transcription regulation, imidacloprid primarily affected oxidase and oxidoreductase activity. These findings provide evidence that systems biology approaches can be utilized to identify and differentiate modes-of-action of chemical stressors in D. magna as an invertebrate aquatic non-target organism. The mechanistic knowledge extracted from such data will in future contribute to the development of Adverse Outcome Pathways (AOPs) for read-across and prediction of population effects.
Young trees of deciduous Quercus robur and Q. petraea and evergreen Q. ilex were grown together in a competition lysimeter experiment to assess i) – whether the observed growth differences between evergreen Q. ilex and the deciduous Q. robur and Q. petraea on sandy soil in the field and ii) – whether the different natural distribution of Q. robur and Q. petraea could be attributed to physiological differences between the species under experimental drought stress (DS). Half of the plants were subjected to long-term DS in two consecutive years and monitored for physiological and growth parameters. In the first year, water withholding for more than three months did not lead to significant drought stress, probably because of a sufficient residual water volume in the lysimeter for the relatively small plants. However, in the second year, 2018, which was warmer, the bigger plants now competed for the residual water and clear drought stress symptoms developed for more than two months in all trees in the DS lysimeter basin. Growth was only moderately (and mostly not significantly) affected by the DS in the second year, except for a smaller total leaf area in DS Q. ilex as compared to DS Q. robur and Q. petraea and smaller root collar diameter in DS Q. ilex compared to DS Q. robur. Under DS, the deciduous species revealed significant decreases in ΔVIP, indicating a negative effect on electron transport through PS I. Pn, PIabs and water relations parameters (ΨPD and LWC) all decreased to various extents under DS in all three species, leading to clear separation of the deciduous from the evergreen species by PCA. However, PCA did not separate the two deciduous species from each other. It is concluded that longer root growth in the two deciduous species as compared to Q. ilex ameliorates DS effects in Q. robur and Q. petraea and may be the key to understand the better performance of deciduous oaks on sandy soil in the field.
RATIONALE: RBPs (RNA-binding proteins) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP SRSF4 (serine/arginine-rich splicing factor 4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. OBJECTIVE: To investigate the role of SRSF4 in the heart. METHODS AND RESULTS: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long noncoding RNAs, including GAS5 (growth arrest-specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide- resolution cross-linking and immuno-precipitation. GAS5 is a repressor of the GR (glucocorticoid receptor) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. CONCLUSIONS: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in patients with Cushing syndrome.
My PhD work employed genetic and pharmacological manipulations, coupled with highresolution live imaging, to understand intercellular communications during zebrafish cardiovascular development. The heart is the first organ to form, and it is composed of several tissues, among which interactions are crucial. I identified two important interactions between muscular and non-muscular tissues in poorly characterized contexts, and the molecules required for the signalling. First, I discovered an important cellular and molecular crosstalk orchestrating the development of the cardiac outflow tract (i.e., the aortic root in mammals).
Endothelial-derived TGF-beta signalling controls the generation of the local extracellular matrix (ECM). The ECM in turn affects endothelial proliferation as well as smooth muscle cell organization (Boezio et al, 2020; Bensimon-Brito*, Boezio* et al, 2020). In my second project, I investigated the crosstalk between the epicardial layer and the myocardial wall. By generating epicardial-impairment models, I identified a novel role for the epicardium in regulating cardiomyocyte volume during heart development (Boezio et al, 2021). Ultimately, this research contributed to our understanding of how paracrine signalling controls the multicellular interactions integral to organogenesis.
Plastic products leach chemicals that induce in vitro toxicity under realistic use conditions
(2021)
Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies.
In the published article, there was an error regarding the affiliation for Diana Abondano Almeida. As well as having affiliation 2, they should also have Department of Wildlife-/Zoo-Animal-Biology and Systematics, Faculty of Biological Sciences, Goethe Universität, Frankfurt, Germany.
The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.
The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis
(2021)
The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T-cell-directed therapies fail to elicit effective anti-tumor immune responses. Here, we seek to evaluate the applicability of radio- immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapy- induced immune modulation resulted in an increase in cytotoxic T-cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experi- mental breast-to-brain metastasis. However, long-term efficacy was not observed. Recurrent brain metastases showed accumula- tion of blood-borne PD-L1+ myeloid cells after radio-immunother- apy indicating the establishment of an immune suppressive environment to counteract re-activated T-cell responses. This finding was further supported by transcriptional analyses indicat- ing a crucial role for monocyte-derived macrophages in mediating immune suppression and regulating T-cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases.
Thermoanaerobacter kivui is an acetogenic model organism that reduces CO2 with electrons derived from H2 or CO, or from organic substrates in the Wood–Ljugdahl pathway (WLP). For the calculation of ATP yields, it is necessary to know the electron carriers involved in coupling of the oxidative and reductive parts of metabolism. Analyses of key catabolic oxidoreductases in cell-free extract (CFE) or with purified enzymes revealed the physiological electron carriers involved. The glyceraldehyde-3-phosphate dehydrogenase (GA3P-DH) assayed in CFE was NAD+-specific, NADP+ was used with less than 4% and ferredoxin (Fd) was not used. The methylene-THF dehydrogenase was NADP+-specific, NAD+ or Fd were not used. A Nfn-type transhydrogenase that catalyzes reduced Fd-dependent reduction of NADP+ with NADH as electron donor was also identified in CFE. The electron carriers used by the potential electron-bifurcating hydrogenase (HydABC) could not be unambiguously determined in CFE for technical reasons. Therefore, the enzyme was produced homologously in T. kivui and purified by affinity chromatography. HydABC contained 33.9 ± 4.5 mol Fe/mol of protein and FMN; it reduced NADP+ but not NAD+. The methylene-THF reductase (MetFV) was also produced homologously in T. kivui and purified by affinity chromatography. MetFV contained 7.2 ± 0.4 mol Fe/mol of protein and FMN; the complex did neither use NADPH nor NADH as reductant but only reduced Fd. In sum, these analysis allowed us to propose a scheme for entire electron flow and bioenergetics in T. kivui.