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A widespread application of 3D bioprinting in basic and translational research requires accessibility to affordable printers able to produce physiologically relevant tissue models. To facilitate the use of bioprinting as a standard technique in biology, an open-source device based on a consumer-grade 3D stereolithography apparatus (SLA) printer is developed. This SLA bioprinter can produce complex constructs that preserve cell viability and recapitulate the physiology of tissues. The detailed documentation of the modifications apported to the printer as well as a throughout performance analysis allow for a straightforward adoption of the device in other labs and its customization for specific applications. Given the low cost, several modified bioprinters could be simultaneously operated for a parallelized tissue production. To showcase the capability of the bioprinter, constructs consisting of patient-derived cholangiocarcinoma organoids encapsulated in a gelatin methacrylate (GelMA)/polyethylene glycol diacrylate (PEGDA) hydrogel are produced. A thorough characterization of different GelMA/PEGDA ratios reveals that the mechanical properties of the bioprinted tumor model can be accurately fine-tuned to mimic a specific tumor micro-environment. Immunofluorescence and gene expression analyses of tumor markers confirm that the bioprinted synthetic hydrogel provides a flexible and adequate replacement of animal-derived reconstituted extracellular matrix.
Species of the genus Blautia are typical inhabitants of the human gut and considered as beneficial gut microbes. However, their role in the gut microbiome and their metabolic features are poorly understood. Blautia schinkii was described as an acetogenic bacterium, characterized by a functional Wood–Ljungdahl pathway (WLP) of acetogenesis from H2 + CO2. Here we report that two relatives, Blautia luti and Blautia wexlerae do not grow on H2 + CO2. Inspection of the genome sequence revealed all genes of the WLP except genes encoding a formate dehydrogenase and an electron-bifurcating hydrogenase. Enzyme assays confirmed this prediction. Accordingly, resting cells neither converted H2 + CO2 nor H2 + HCOOH + CO2 to acetate. Carbon monoxide is an intermediate of the WLP and substrate for many acetogens. Blautia luti and B. wexlerae had an active CO dehydrogenase and resting cells performed acetogenesis from HCOOH + CO2 + CO, demonstrating a functional WLP. Bioinformatic analyses revealed that many Blautia strains as well as other gut acetogens lack formate dehydrogenases and hydrogenases. Thus, the use of formate instead of H2 + CO2 as an interspecies hydrogen and electron carrier seems to be more common in the gut microbiome.
Many metabolic pathways of eukaryotes are carried out in form of interconnected pathways, which take place in organelles. The organelle membrane separates the reaction compartments from each other, making it a key feature of organelle existence in the cell. To maintain cellular homeostasis, organelle positioning in and transport through the cell as well as organelle interaction are important for the organisms. In plants, organellar movement of peroxisomes, Golgi stacks and mitochondria was shown to be mediated by the actin-myosin machinery. The molecular mechanisms are not elucidated, but working models comprise classical movement mechanisms of motor proteins pulling their cargo on cytoskeletal filaments. In contrast, many mechanisms of chloroplasts movement, which are regulated by blue and red light, are deciphered but follow a different molecular mechanism. Plastidal relatives of the chloroplast have long been disregarded by scientific research but carry out important metabolic reactions to maintain cellular homeostasis. The cellular transport and movement mechanisms of root plastids have not been described in detail until now. Additionally, all plastid subspecies can form tubular structures, called stromules. Those are thought to be involved in the organelle communication and metabolite exchange. Since they are very mobile structures, they influence the organellar dynamic of plastids. This work aimed for an in-detail description of the cellular movements of root plastids in the plant Arabidopsis thaliana to elucidate underlying mechanisms of their movement. Additionally, the dynamics of root plastid stromules were investigated, led by the questions, if and how stromules are involved in the mediation of plastidal movement and their overall dynamics. Plastidal movement in Arabidopsis thaliana was captured using light sheet-based fluorescence microscopy. 4D image data was automatically analyzed using the program Arivis Vision 4D with subsequent manual correction. Additionally to the 4D approach, a manual 3D analysis of plastid and stromule dynamics was performed. The results of the semiautomated analysis displayed heterologous distribution of the plastidal movement. Using a combination of the vector length of each motion event and the angle in relation to previous motion vectors, the proportions of different movement patterns were determined. Main fractions of the data showed undirected motion of plastids, whereas small proportions displayed directed movement with speed up to 8.5 µm/sec. Directed motion was shown to be carried out on defined routes in the cell. Salt stress did not affect plastidal motion, whereas drought stress lead to its reduction. Sucrose depletion led to a drastic decrease of plastidal movement. Additionally, stromule dynamics were investigated using the acquired image data. Stromules were observed in high frequency mainly at stationary plastids giving them the opportunity of dynamic interaction in their cellular surrounding. Stromules reached lengths of up to 60 µm. Additionally, they displayed a variety of movement patterns that contributed greatly to the overall plastid dynamics. Stromule related motion events were captured reaching up to 3.2 µm/sec. Similar to determined plastid dynamics, stromule motions were reduced during drought stress and sucrose depletion, but also were negatively influenced by salt stress. Those results strongly favor an actin-myosin mediated movement machinery mediating the plastidal and stromule movement. This stands in contrast to previous results describing the movement mechanisms of light induced chloroplast movement.
In an additional approach, the molecular mechanisms underlying stromule formation were analyzed. Previous results describe that stromule formation can be induced at isolated chloroplasts of the plant Nicotiana benthamiana by mixing it with concentrated cell extract. During this work, a variation of the described assay was established using the plant Pisum sativum. It was shown that an unknown protein factor presumably undergoing protein-lipid interaction is responsible for in vitro stromule formation. Using a combination of sucrose gradient centrifugation and anion exchange chromatography, the desired factor could be enriched, while the majority of unwanted proteins could be reduced drastically. A following LC-MS analysis revealed a selection of proteins with membrane interaction- and unknown functions that might be involved in in vitro stromule formation.
An increasing number of voices highlight the need for science itself to transform and to engage in the co-production of knowledge and action, in order to enable the fundamental transformations needed to advance towards sustainable futures. But how can global sustainability-oriented research networks engage in co-production of knowledge and action? The present article introduces a strategic tool called the ‘network compass’ which highlights four generic, interrelated fields of action through which networks can strive to foster co-production. It is based on the networks’ particular functions and how these can be engaged for co-production processes. This tool aims to foster self-reflection and learning within and between networks in the process of (re)developing strategies and activity plans and effectively contributing to sustainability transformations.
Background: Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood–Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study.
Results: We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+–NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis.
Discussion: We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme.
Following severe population decline and local extinction due to massive habitat destruction and persecution, wildcats have recently reappeared in several parts of Germany’s low mountain region. It remains unknown how this reemergence occurred, specifically if local populations have been overlooked at low densities or if the species has successfully spread across the highly fragmented anthropogenic landscape. In the central German Rhön Mountains, for instance, wildcats were believed to be extinct during most of the twentieth century, however, the species was recently detected and subsequent genetic monitoring found the presence of a sizeable population. In this study, we used microsatellite and SNP genotypes from 146 wildcat individuals from 2008 to 2017 across a ~ 15,000 km2 area in the central German low mountain region to understand the population re-establishment of wildcats in the region. Bayesian clustering and subsequent analyses revealed that animals in the Rhön Mountains appear to be a mix from the two adjacent populations in the North and South of the area, suggesting a recent range expansion from two different directions. Both populations meet in the Rhön Biosphere Reserve, leading to an admixture of the northern, autochthonous, and the southern reintroduced wildcat population. While we cannot completely exclude the possibility of undetected population persistence, the high genetic homogeneity in the central German wildcat population and the lack of any signatures of past population decline in the Rhön favor a scenario of natural expansion. Our findings thus suggest that wildcats are well capable of rapid range expansion across richly structured landscape mosaics consisting of open land, settlements, and forest patches and document the potential of massive non-invasive genetic sampling when aiming to reconstruct the complex population and range dynamics of wildlife.
In situ burning (ISB) is discussed to be one of the most suitable response strategies to combat oil spills in extreme conditions. After burning, a highly viscous and sticky residue is left and may over time pose a risk of exposing aquatic biota to toxic oil compounds. Scientific information about the impact of burn residues on the environment is scarce. In this context, a comprehensive ISB field experiment with approx. 1000L IFO 180 was conducted in a fjord in Greenland. The present study investigated the toxicity of collected ISB residues to early life stages of zebrafish (Danio rerio) as a model for potentially exposed pelagic organisms. The toxicity of ISB residues on zebrafish embryos was compared with the toxicity of the initial (unweathered) IFO 180 and chemically dispersed IFO 180. Morphological malformations, hatching success, swimming behavior, and biomarkers for exposure (CYP1A activity, AChE inhibition) were evaluated in order to cover the toxic response on different biological organization levels. Across all endpoints, ISB residues did not induce greater toxicity in zebrafish embryos compared with the initial oil. The application of a chemical dispersant increased the acute toxicity most likely due to a higher bioavailability of dissolved and particulate oil components. The results provide insight into the adverse effects of ISB residues on sensitive life stages of fish in comparison with chemical dispersant application.
Acetogenic bacteria such as Acetobacterium woodii use the Wood–Ljungdahl pathway (WLP) for fixation of CO2 and energy conservation. This pathway enables conversion of diverse substrates to the main product of acetogenesis, acetate. Methyl group containing substrates such as methanol or methylated compounds, derived from pectin, are abundant in the environment and a source for CO2. Methyl groups enter the WLP at the level of methyltetrahydrofolic acid (methyl-THF). For methyl transfer from methanol to THF a substrate-specific methyltransferase system is required. In this study, we used genetic methods to identify mtaBC2A (Awo_c22760-Awo_c22740) as the methanol-specific methyltransferase system of A. woodii. After methyl transfer, methyl-THF serves as carbon and/or electron source and the respiratory Rnf complex is required for redox homeostasis if methanol + CO2 is the substrate. Resting cells fed with methanol + CO2, indeed converted methanol to acetate in a 4:3 stoichiometry. When methanol was fed in combination with other electron sources such as H2 + CO2 or CO, methanol was converted Rnf-independently and the methyl group was condensed with CO to build acetate. When fed in combination with alternative electron sinks such as caffeate methanol was oxidized only and resulting electrons were used for non-acetogenic growth. These different pathways for the conversion of methyl-group containing substrates enable acetogens to adapt to various ecological niches and to syntrophic communities.
Tick-borne diseases are a major health problem worldwide and could become even more important in Europe in the future. Due to changing climatic conditions, ticks are assumed to be able to expand their ranges in Europe towards higher latitudes and altitudes, which could result in an increased occurrence of tick-borne diseases.
There is a great interest to identify potential (new) areas of distribution of vector species in order to assess the future infection risk with vector-borne diseases, improve surveillance, to develop more targeted monitoring program, and, if required, control measures.
Based on an ecological niche modelling approach we project the climatic suitability for the three tick species Ixodes ricinus, Dermacentor reticulatus and Dermacentor marginatus under current and future climatic conditions in Europe. These common tick species also feed on humans and livestock and are vector competent for a number of pathogens.
For niche modelling, we used a comprehensive occurrence data set based on several databases and publications and six bioclimatic variables in a maximum entropy approach. For projections, we used the most recent IPCC data on current and future climatic conditions including four different scenarios of socio-economic developments.
Our models clearly support the assumption that the three tick species will benefit from climate change with projected range expansions towards north-eastern Europe and wide areas in central Europe with projected potential co-occurrence.
A higher tick biodiversity and locally higher abundances might increase the risk of tick-borne diseases, although other factors such as pathogen prevalence and host abundances are also important.
tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.
Entoloma (Agaricales, Basidiomycota) is a species-rich genus with approximately 2000 species known worldwide. In Central America, however, information about the species of this genus is sparse, despite the generally high biodiversity in this region. Recently, 124 specimens of Entoloma were collected in Panama, Chiriquí Province. In the present publication, the morphology of 20 species represented by more than one specimen is described and depicted with photographs, line drawings, and scanning electron micrographs. Molecular phylograms based on ITS or concatenated ITS and partial nc LSU rDNA sequences are provided. The taxonomic status of these species is evaluated and 17 species of Entoloma are described as new to science. Only one species could be assigned to an already known species, viz. Entoloma belouvense. Nolanea albertinae, described from Brazil, appeared similar and is combined in E. belouvense on varietal level. The identifications of two further species are uncertain. At least 30 other species, including potentially new species, cannot formally be described due to insufficient material. A preliminary key to the species of the genus Entoloma in Panama is provided. The spatial shape of the polyhedroid basidiospores of Entoloma spp. is discussed based on literature and the micrographs generated for the present study. Our re-evaluations indicate that the type of polyhedroid basidiospore and the structure of its base are not reliable as diagnostic characters for the delimitation of subgenera in Entoloma.
NAD is a coenzyme central to metabolism that was also found to serve as a 5’-terminal cap of bacterial and eukaryotic RNA species. The presence and functionality of NAD-capped RNAs (NAD-RNAs) in the archaeal domain remain to be characterized in detail. Here, by combining LC-MS and NAD captureSeq methodology, we quantified the total levels of NAD-RNAs and determined the identity of NAD-RNAs in the two model archaea, Sulfolobus acidocaldarius and Haloferax volcanii. A complementary differential RNA-Seq (dRNA-Seq) analysis revealed that NAD transcription start sites (NAD-TSS) correlate with well-defined promoter regions and often overlap with primary transcription start sites (pTSS). The population of NAD-RNAs in the two archaeal organisms shows clear differences, with S. acidocaldarius possessing more capped small non-coding RNAs (sncRNAs) and leader sequences. The NAD-cap did not prevent 5’→3’ exonucleolytic activity by the RNase Saci-aCPSF2. To investigate enzymes that facilitate the removal of the NAD-cap, four Nudix proteins of S. acidocaldarius were screened. None of the recombinant proteins showed NAD decapping activity. Instead, the Nudix protein Saci_NudT5 showed activity after incubating NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into RNAs and the regulation of ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells.
Importance: This study reports the first characterization of 5’-terminally modified RNA molecules in Archaea and establishes that NAD-RNA modifications, previously only identified in the other two domains of life, are also prevalent in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii. We screened for NUDIX hydrolases that could remove the NAD-RNA cap and showed that none of these enzymes removed NAD modifications, but we discovered an enzyme that hydrolyzes ADPR-RNA. We propose that these activities influence the stabilization of NAD and its thermal degradation to potentially toxic ADPR products at elevated growth temperatures.
Sphingolipids are not only structural components of cell membranes but can also act as signalling molecules in different pathways. Sphingolipid precursors, Ceramides (Cer), are synthesized de novo by six different synthases (CerS1-6) which generate Cer of different chain lengths. Cer can be further synthesized to glycosphingolipids and sphingomyelin. Cell membrane parts that are enriched in glycosphingolipids are so-called lipid rafts and can function as signalling platforms for different receptors, such like the T cell receptor (TCR). CD4+ T cells play a crucial role in the development of ulcerative colitis, a chronic inflammatory disease of the colon. As CerS3 expression was increased in the white blood cells of human colitis patients, the role of CerS3 in the TCR signalling and colitis was investigated in this dissertation. By lenti-viral transduction of a CerS3-shRNA into a CD4+ Jurkat cell line, it was shown that CerS3 has an impact on activated T cells. A decrease of different sphingolipids after T cell activation via CD2/3/28 activation beads and IL2 treatment was observed that was accompanied by an inhibition of Zap70 phosphorylation, an important protein of the TCR signalling. The impaired TCR signalling led to a diminished NFAT1 translocation into the nucleus which subsequently led to a reduced NFAT1- dependent TNFα release. Downregulation of CerS3 in primary CD4+ T cells, obtained from the blood of healthy volunteers, also showed a reduced release of pro-inflammatory cytokines after activation. This dissertation demonstrates a pivotal role for CerS3 in T cell function and highlights CerS3 as potential new target for T cell driven colitis.
Subject of this thesis was the investigation of the actin-interacting and glucocorticoid-sensitive Protein DRR1 (or Fam107a) and its role in promoting stress resilience in the murine hippocampus.
We proposed the hypothesis that DRR1 through its actin-binding properties specifically modulates neuronal actin dynamics and promotes resilience through synaptic plasticity leading to subsequently improvement of cognitive performance and social behavior. The accompanied AMPA-receptor transport could create an efficient way regulating neural function and complex behavior during stress episodes.
By utilizing fluorescent immunohistochemistry, we showed basal expression of DRR1 primarily in the murine cerebellum and hippocampal CA3 and CA1 area. Co-staining with different cell marker proteins showed DRR1 expression in neurons, microglia and especially in astrocytic end-feet, which create contact to the brain vasculature.
To test whether DRR1 and AMPA receptor function correlate to modulate stress-associated consequences, primary hippocampal neuron cultures were transduced with adeno-associated virus (AAV) for overexpression or suppression of the protein. Western Blot analysis showed a positive correlation between the AMPA-receptor subunit GluR2 and DRR1 amounts. Further the application of the proximity ligation assay (PLA) in untreated neural cultures indicated interaction between DRR1 and the AMPA receptor subunit GluR2. To address whether DRR1 even affects AMPAR trafficking we performed the “newly inserted assay” after AAV-treatment of primary hippocampal neuron cultures. Suppression of DRR1 revealed less newly inserted GluR2 subunits as compared to controls. Inconclusive were the results upon DRR1 overexpression, however they point to no changes.
In the second part we correlated behavioral phenotypes originating from in vivo overexpression and suppression of DRR1 in the murine hippocampus with potential alterations in neuronal morphology. Therefore, in vitro analysis was performed utilizing AAV transduced primary hippocampal cultures overexpressing or suppressing DRR1. Synchronously the viral vector included a green fluorescent protein (GFP) being expressed throughout the complete neural cell. GFP staining was used to verify successful transfection and for reconstruction of dendritic arbors and dendritic stretches for spine classification. DRR1 suppression showed reduced total spine numbers especially evoked by reduced numbers of immature spine classes – namely long thin spines and filopodia. Whereas mature mushroom spines and stubby spines were unaffected. By overexpressing DRR1, tendencies inclined against higher total dendritic lengths, branch points and increased dendritic arbors in comparison to controls. In regard of spines, total numbers were unaffected. However, mature mushroom spines were significantly declined in numbers, but compensated by increased numbers of immature long thin spines and filopodia.
Chronic social defeat stress (CSDS) is widely used in mouse models to study the effects of stress and resilience. We exposed C57Bl/6J mice expressing GFP under the Thy1 promoter CSDS and categorized them into resilient (R+/-), susceptible (R-/-) and non-learning (R+/+) mice following a modified social interaction test (MSIT). We found alterations in CA1 spine compositions with resilient animals resembling the untreated phenotype. Stress susceptible and non-learning animals displayed reduced numbers in stubby spines with simultaneous increases in mature mushroom spines. In addition, we could detect a tendency towards more immature spines in susceptible animals and non-learners, mirroring our in vitro results.
Finally, we present a different investigative approach in this thesis. Sequenced acute stress was previously found to compromise cognition including spine loss.
We aimed to investigate the implication of acute stress on DRR1 levels and its occurrence in diverse cell types of the brain. We subjected one group of C57Bl/6J mice to acute stress and injected another group with the artificial glucocorticoid DEX. Six hours post stress, animals were perfused and brains were subsequently immunobiologically analyzed. We found DRR1 protein levels elevated in the hippocampus of stressed and DEX-treated animals compared to controls. Interestingly, DRR1 seemed was especially elevated in endothelial cells. This coincides with our investigations finding DRR1 present in astrocytic end-feet under basal conditions and might claim a participation of DRR1 in the blood-brain-barrier integrity.
Our results show DRR1 as actin-interacting and glucocorticoid-sensitive gene affecting structural plasticity of hippocampal spines. Moreover, DRR1 directly interacts with AMPA glutamate receptors and presumably is involved in AMPA trafficking to the postsynaptic membrane. In addition, this study could demonstrate that DRR1 is expressed by other cell types of the brain. Of special interest is DRR1’s occurrence in astrocytic end-feet and endothelial cells suggesting a role as integrator of cell-cell communication and to this end also acting as modifier of stress-induced consequences at the neurovascular unit.
In vivo data of chronically stressed mice displayed no phenotypic differences in hippocampal pyramidal neurons of resilient animals as compared to unstressed mice. Morphological alterations of spine structures were particularly visible in stress susceptible and non-learning animals. Integrating our findings with existing behavioral data, we can conclude that DRR1 plays a role in stress resilience whereby it needs to be expressed in a tightly managed homeostatic equilibrium.
Energy-conserving dimethyl sulfoxide reduction in the acetogenic bacterium Moorella thermoacetica
(2022)
Moorella thermoacetica is one of the well-studied thermophilic acetogenic bacteria. It grows by oxidation of organic substrates, CO or H2 coupled to CO2 reduction to acetate. Here, we describe that M. thermoacetica can also use dimethyl sulfoxide as terminal electron acceptor. Growth of M. thermoacetica on glucose or H2 + CO2 was stimulated by dimethyl sulfoxide (DMSO). Membranes showed a DMSO reductase activity, that was induced by growing cells in presence of DMSO. The enzyme used reduced anthraquinone-2,6-disulfonate, benzyl- and methyl viologen as electron donor, but not NAD(P)H. Activity was highest at pH 5 and 60°C, the Km for DMSO was 2.4 mM. Potential DMSO reductase subunits were identified by peptide mass fingerprinting; they are encoded in a genomic region that contains three potential dmsA genes, three dmsB genes and one dmsC gene. Transcriptome analysis revealed that two different dmsAB gene clusters were induced in the presence of DMSO. The function of these two and their predicted biochemical features are discussed. In addition, the data are in line with the hypothesis that M. thermoacetica can use DMSO alongside CO2 as electron acceptor and DMSO reduction is catalysed by an energy-conserving, membrane-bound electron transport chain with DMSO as final electron acceptor.
Thermoanaerobacter kivui is a thermophilic acetogen that can grow on carbon monoxide as sole carbon and energy source. To identify the gene(s) involved in CO oxidation, the genome sequence was analyzed. Two genes potentially encoding CO dehydrogenases were identified. One, cooS, potentially encodes a monofunctional CO dehydrogenase, whereas another, acsA, potentially encodes the CODH component of the CODH/ACS complex. Both genes were cloned, a His-tag encoding sequence was added, and the proteins were produced from a plasmid in T. kivui. His-AcsA copurified by affinity chromatography with AcsB, the acetyl-CoA synthase of the CO dehydrogenase/acetyl CoA synthase complex. His-CooS copurified with CooF1, a small iron-sulfur center containing protein likely involved in electron transport. Both protein complexes had CO:ferredoxin oxidoreductase as well as CO:methyl viologen oxidoreductase activity, but the activity of CooSF1 was 15-times and 231-times lower, respectively. To underline the importance of CooS, the gene was deleted in the CO-adapted strain. Interestingly, the ∆cooS deletion mutant did not grow on CO anymore. These experiments clearly demonstrated that CooS is essential for growth of T. kivui on CO. This is in line with the hypothesis that CooS is the CO-oxidizing enzyme in cells growing on CO.
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.
Bei den meisten erwachsenen Säugetieren führt ein Herzinfarkt zu Fibrose und Verlust von funktionellem Herzgewebe. Einige Wirbeltiere, wie der Zebrabärbling, besitzen jedoch die bemerkenswerte Fähigkeit, nach einer Schädigung ihres Herzgewebes verlorenes Gewebe zu regenerieren und so schädliche Folgen zu verhindern. Die lokale Immunantwort auf eine Verletzung wird zunehmend als eine wichtige Determinante für das regenerative Potential eines Gewebes gesehen. Das Komplementsystem ist Teil des humoralen Immunsystems. Historisch ist es als eine Sammlung von Protein bekannt, den Komplementkomponenten, die in der Leber synthetisiert werden und im Blutkreislauf zirkulieren. Bei Exposition gegenüber einem Auslöser, wie z. B. einem Pathogen, wird eine Komplementkomponentproteinspaltungskaskade initiiert, die dazu führen kann, dass Immunzellen rekrutiert werden, und, dass die Phagozytose erleichtert, ggf. die Zielzelle lysiert wird. Studien legen nahe, dass das Komplementsystem an zellulären Prozessen beteiligt sei, die für Entwicklungs- und Krankheitsprozesse entscheidend sind, wie etwa Proliferation und Dedifferenzierung. Es gibt Hinweise, dass das Komplementsystem eine Rolle bei Krebserkrankungen und bei regenerativen Prozessen spielen könnte. In verschiedenen Arten wurde eine lokale verletzungsinduzierte Expression von komplementkomponentkodierenden Genen in regenerierendem Gewebe beobachtet.
Einzelne Studien legen nahe, dass Funktionsverlust einzelner Komplementkomponenten regenerative Prozesse beeinträchtigt.
Offene Fragen bleiben jedoch: Ist die lokale Expression von mehreren komplementkomponentkodierenden Genen ein Merkmal von regenerierendem Gewebe, das sie von Geweben unterscheidet, welchem die Fähigkeit zur Regeneration fehlt? Und welche Rolle könnte das Komplementsystem und seine Komponenten während des regenerativen Prozesses spielen? Um diesen Fragen nachzugehen, wurde eine Expressionsanalyse von Zebrabärblingsgewebe nach Verletzung mittels RT-qPCR und in situ Hybridisierung durchgeführt: kardiale Kryoverletzung, Larvenrumpfamputation und Schwanzflossenamputation. Ich beobachtete, dass mehrere komplementkomponentkodierende Gene in diesen Geweben nach Verletzung induziert wurden. Die Interpretation veröffentlichter single cell RNAseq Datensätze legt nahe, dass diese komplementkomponentenkodierenden Gene von verschiedenen Zelltypen exprimiert werden, darunter Immunzellen, Epikardzellen und Fibroblasten. Um transkriptionelle Unterschiede zwischen regenerierendem und nicht regenerierendem Gewebe zu identifizieren, verwendete ich ein nicht regeneratives Zebrabärblingmodell, die il11ra- Mutante. Dieser Mutante fehlt die Fähigkeit, verschiedene Organe zu regenerieren, das ist der Fall beim Herzen, dem larvalen Rumpf, und der Schwanzflosse. Ich stellte fest, dass die Mehrheit der verletzungsinduzierten komplementkomponentkodierenden Gene il11ra nachgeschaltet war. Darüber hinaus zeigten Experimente unter Verwendung chemischer Inhibitoren, dass speziell die Expression der komplementkomponentkodierenden Gene c3a.1,
c4b und c7a im Larvenrumpfamputationsmodell durch den Il11-Stat3-Signalweg moduliert wird.
Zur Klärung der Frage, ob das Komplementsystem und/ oder seine Komponenten eine Rolle während der Regeneration spielen, wurden verschiede Funktionsverlustmodelle generiert und im larvalen Rumpfamputationsmodell auf mögliche Aberrationen getestet. Zum einen generierte ich Überexpressionslinien von endogenen Inhibitoren der Komplementproteinspaltungskaskade. Überexpression eines etablierten Komplementsysteminhibitors rca2.1/ tecrem führte zu einer im Vergleich zu Wildtyp- Geschwistern verringerten Regeneration des larvalen Rumpfs. Zum anderen generierte ich Funktionsverlustmutanten von individuellen Komplementkomponenten durch CRISPR/Cas9 vermittelter Mutagenese, und zwar für masp1, masp2, cfd, c1s, c4b, c5 und c9. Die larvale Rumpfregeneration war in diesen Mutanten unauffällig. Allerdings zeigten c4b Mutanten eine verringerte Kardiomyozytenproliferation und eine differenzielle Expression von einigen Markergenen, einschließlich einer erhöhten Expression von inflammatorischen Zytokinen.
Meine Studien führten zu neuen Einblicken in das Komplementsystem im Kontext der Regeneration. Ich fand heraus, dass mehrere komplementkomponentenkodierenden Gene in regenerierendem Zebrabärblinggewebe exprimiert werden, und zwar im Herzgewebe, im larvalen Rumpf und in der adulten Flosse. Darüber hinaus zeige ich, dass die verletzungsinduzierte Expression von komplementkodierenden Genen in regenerierendem Gewebe dem Regenerationsmasterregulator il11ra nachgeschaltet ist. Speziell c3a.1, c4b und c7a wurden durch il11/ stat3 reguliert...