Combining Flow Cytometry and Metagenomics Improves Recovery of Metagenome-Assembled Genomes in a Cell Culture from Activated Sludge

The recovery of metagenome-assembled genomes is biased towards the most abundant species in a given community. To improve the identification of species, even if only dominant species are recovered, we investigated the integration of flow cytometry cell sorting with bioinformatics tools to recover metagenome-assembled genomes. We used a cell culture of a wastewater microbial community as our model system. Cells were separated based on fluorescence signals via flow cytometry cell sorting into sub-communities: dominant gates, low abundant gates, and outer gates into subsets of the original community. Metagenome sequencing was performed for all groups. The unsorted community was used as control. We recovered a total of 24 metagenome-assembled genomes (MAGs) representing 11 species-level genome operational taxonomic units (gOTUs). In addition, 57 ribosomal operational taxonomic units (rOTUs) affiliated with 29 taxa at species level were reconstructed from metagenomic libraries. Our approach suggests a two-fold increase in the resolution when comparing sorted and unsorted communities. Our results also indicate that species abundance is one determinant of genome recovery from metagenomes as we can recover taxa in the sorted libraries that are not present in the unsorted community. In conclusion, a combination of cell sorting and metagenomics allows the recovery of MAGs undetected without cell sorting

DNA Origami-based fabrication of Palladium Nanostructures

Die DNA-Origami-Technologie zeigt großes Potenzial für die Bottom-up-Fertigung von nanoelektronischen Bauteilen. Insbesondere das auf Gussformen basierende Herstellungsverfahren ermöglichte die Synthese homogener metallischer Nanostrukturen mit kontrollierten Formen, Längen und Anordnungen. Allerdings war das Verfahren bisher auf die Elemente Gold und Silber beschränkt. In dieser Arbeit wurden die Möglichkeiten des auf DNA-Origami-Gussformen basierenden Herstellungsverfahrens erweitert. In einem ersten Schritt wurde ein Verfahren für das Wachstum von Palladium-Nanostrukturen in DNA-Origami-Gussformen entwickelt. Zu diesem Zweck wurde ein einfaches Syntheseverfahren für wasserdispergierte Palladium-Nanopartikel entwickelt. Diese wurden anschließend mit DNA-Einzelsträngen funktionalisiert und konnten mit hoher Effizienz in den DNA-Origami-Gussformen gebunden werden. Dort konnten sie erfolgreich als Nukleationskeime für eine kontrollierte Abscheidungsreaktion verwendet werden, was die Synthese von Palladium-Nanostrukturen mit designbaren Längen und Anordnungen ermöglichte. Die so hergestellten Palladium-Nanostrukturen wiesen eine körnige Morphologie auf. Eine anschließende Wärmebehandlung ermöglichte das Verschmelzen der Körner zu homogenen Strukturen und ein zusätzlicher Reduktionsschritt entfernte den in die Nanostrukturen eingebauten Sauerstoff. In einem zweiten Schritt wurde die Anwendbarkeit der auf DNA-Origami-Gussformen basierenden Methode zur Herstellung nanoelektronischer Bauteile auf harsche Reaktionsbedingungen erweitert, indem ein schwermetallbasiertes Stabilisierungsverfahren für DNA-Origami-Strukturen entwickelt wurde. Unter Ausnutzung der Bindungsaffinität von Pd2+ Ionen an die DNA-Basen konnten DNA-Origami-Strukturen unabhängig von ihrem Gittertyp gegen Temperaturen bis zu 100 °C, gegen Puffer mit niedriger Ionenstärke sowie gegen pH-Werte zwischen 4 und 12 stabilisiert werden. Zusätzlich konnten Multimere aus drei einzelnen DNA-Origami-Strukturen sowie angebundene Cargos mit hoher Effizienz stabilisiert werden. Die Anwendbarkeit der schwermetallbasierten Stabilisierung wurde durch Anwendung des Stabilisierungsprozesses auf das in dieser Arbeit entwickelte Palladium-Wachstumsverfahren demonstriert, wodurch die Reaktionszeit von 40 Minuten bei Raumtemperatur auf 30 Sekunden bei 90 °C reduziert werden konnte. Basierend auf den Ergebnissen dieser Arbeit, könnte in Zukunft das Wachstum von zwei verschiedenen Metallen in einer DNA-Origami-Gussform realisiert werden. Zusammen mit bereits existierenden Goldwachstumsprotokollen könnte die Implementierung von Palladium zur Herstellung eines selbstorganisierten Wasserstoffgassensors führen. Darüber hinaus erscheint zusammen mit dem schwermetallbasierten Stabilisierungsverfahren eine Synthese magnetischer Nickel- oder Kobalt-Nanostrukturen, welche harsche Reaktionsbedingungen erfordert, realistisch. Neben der DNA-Origami-basierten Bottom-up-Fertigung von nanoelektronischen Komponenten birgt das Stabilisierungsverfahren aufgrund seiner Einfachheit ein großes Potenzial für weitere Anwendungen, bei denen harsche Reaktionsbedingungen angewendet werden.:1 Introduction 2 Thesis Objectives 3 Theoretical Background 3.1 Structure and Thermodynamics of DNA 3.2 DNA-Metal Interactions 3.2.1 Interaction Sites 3.2.2 Binding Kinetics 3.3 The DNA Origami technique 3.4 Fabrication of Metallic Nanostructures based on DNA Origami 3.4.1 Classical Nucleation 3.4.2 Nanoparticle Growth Mechanisms 3.4.3 Secondary Nucleation 3.4.4 DNA Origami Metallization 3.5 Characterization of Nanoparticles using Electron Diffraction 3.5.1 Basic Principles of Crystal Lattices 3.5.2 Selective Area Electron Diffraction 4 DNA Mold-Based Fabrication of Palladium Nanostructures 4.1 Summary 4.2 Associated Publication 5 Heavy Metal Stabilization of DNA Origami Nanostructures 5.1 Summary 5.2 Associated Publication 6 Summary and Outlook Bibliography AppendixIn recent years, DNA origami technology has been shown to bear great potential in the bottom-up fabrication of nanoelectronic devices. In particular, the mold-based fabrication scheme has enabled the synthesis of homogeneous shape, pattern and length-controlled metal nanostructures. However, the approach has so far been limited to the elements gold and silver. In this thesis, the capabilities of the mold-based fabrication scheme were extended. Firstly, a process for the growth of palladium nanostructures inside DNA origami molds was developed. To this end, an easy synthesis procedure for water dispersible palladium nanoparticles was established. Subsequently, a protocol was developed that allowed for the tunable decoration of the palladium nanoparticles with single-stranded DNA. Once functionalized, the nanoparticles were loaded into the DNA origami molds with high efficiencies. There, they could successfully be used as nucleation centers for a seeded palladium deposition, which allowed for a pattern and length-controlled synthesis of palladium nanostructures. Resulting nanostructures exhibited a grainy morphology. A subsequent heat treatment enabled the annealing of the grains and an additional reduction step removed incorporated oxygen. Secondly, the mold-based fabrication scheme was expanded to work under harsh reaction conditions by developing a heavy metal-based stabilization procedure for DNA origami structures. Utilizing the binding affinity of Pd2+ ions to the DNA bases DNA origami structures independent of their lattice type could be stabilized against temperatures of up to 100 °C, low ionic strength buffers and pH values between 4 and 12. Additionally, DNA origami superstructures which consisted of three individual DNA origami structures as well as bound cargos could be stabilized with high yields. The applicability of the heavy metal-based stabilization was demonstrated by employing the stabilization process on the palladium growth procedure developed in this thesis, which reduced the reaction time from 40 minutes at room temperature to 30 seconds at 90 °C. The developments in this thesis have significantly expanded the capabilities of the DNA mold-based fabrication scheme of nanoelectronic components. In the future, the growth of two different metals in one mold superstructure could likely be realized. Additionally, together with already existing gold growth protocols the implementation of palladium could lead to the fabrication of a self-assembled hydrogen gas sensor. Furthermore, the growth of palladium lays the basis for the implementation of less noble metals. Together with the heavy metal stabilization procedure, an incorporation of the magnetic metals nickel or cobalt, which require harsh reaction conditions, seems realistic. Beyond that, due to its simplicity, the stabilization procedure bears great potential for application not only in the DNA origami-based bottom-up fabrication of nanoelectronic components, but also in other application in which harsh reaction conditions are applied.:1 Introduction 2 Thesis Objectives 3 Theoretical Background 3.1 Structure and Thermodynamics of DNA 3.2 DNA-Metal Interactions 3.2.1 Interaction Sites 3.2.2 Binding Kinetics 3.3 The DNA Origami technique 3.4 Fabrication of Metallic Nanostructures based on DNA Origami 3.4.1 Classical Nucleation 3.4.2 Nanoparticle Growth Mechanisms 3.4.3 Secondary Nucleation 3.4.4 DNA Origami Metallization 3.5 Characterization of Nanoparticles using Electron Diffraction 3.5.1 Basic Principles of Crystal Lattices 3.5.2 Selective Area Electron Diffraction 4 DNA Mold-Based Fabrication of Palladium Nanostructures 4.1 Summary 4.2 Associated Publication 5 Heavy Metal Stabilization of DNA Origami Nanostructures 5.1 Summary 5.2 Associated Publication 6 Summary and Outlook Bibliography Appendi

Association between TGFb1 levels in cord blood and weight progress in the first year of life

Transforming growth factor beta-1 (TGFβ1) is an adipokine secreted by adipose tissue, placental tissue, and immune cells, playing a role in cell proliferation, apoptosis, and angiogenesis. However, its role in pregnancy and child growth, as well as the primary source of cord TGFβ1, remains unclear. This study aimed to investigate the relationship between TGFβ1 levels and intrauterine growth parameters, as well as child growth during the first year of life, while also determining whether its primary origin is fetal or maternal. Serum samples and anthropometric measurements were collected from 79 healthy mother–child pairs within the LIFE Child cohort. TGFβ1 concentrations were measured using enzyme-linked immunosorbent assays (ELISA), and statistical analyses—including the Mann–Whitney U-test, correlation analyses, and linear regression—were performed using GraphPad Prism and R. Results showed that TGFβ1 levels were significantly higher in cord serum compared to maternal serum, suggesting a predominantly fetal origin. Multivariate regression analyses identified a strong positive association between cord TGFβ1 levels at birth and child weight at U6. Additionally, cord TGFβ1 levels were significantly correlated with child weight at approximately one year of age. An increase of 10,000 pg/mL in cord TGFβ1 at birth was associated with a 201 g higher body weight at one year, after adjusting for sex.:Introduction Paper Manuscript Manuscript Summary References Supplementary Materials Presentation of own Contributions Appendice

WARS1 and SARS1: Two tRNA synthetases implicated in autosomal recessive microcephaly

Aminoacylation of transfer RNA (tRNA) is a key step in protein biosynthesis, carried out by highly specific aminoacyl‐tRNA synthetases (ARSs). ARSs have been implicated in autosomal dominant and autosomal recessive human disorders. Autosomal dominant variants in tryptophanyl‐tRNA synthetase 1 (WARS1) are known to cause distal hereditary motor neuropathy and Charcot‐Marie‐Tooth disease, but a recessively inherited phenotype is yet to be clearly defined. Seryl‐tRNA synthetase 1 (SARS1) has rarely been implicated in an autosomal recessive developmental disorder. Here, we report five individuals with biallelic missense variants in WARS1 or SARS1, who presented with an overlapping phenotype of microcephaly, developmental delay, intellectual disability, and brain anomalies. Structural mapping showed that the SARS1 variant is located directly within the enzyme's active site, most likely diminishing activity, while the WARS1 variant is located in the N‐terminal domain. We further characterize the identified WARS1 variant by showing that it negatively impacts protein abundance and is unable to rescue the phenotype of a CRISPR/Cas9 wars1 knockout zebrafish model. In summary, we describe two overlapping autosomal recessive syndromes caused by variants in WARS1 and SARS1, present functional insights into the pathogenesis of the WARS1‐ related syndrome and define an emerging disease spectrum: ARS‐related developmental disorders with or without microcephaly

Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation

Introduction: Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. Methods: We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Results and discussion: Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels

Das CXCL12-System kontrolliert die Migration humaner Tumorzellen über EGFR-abhängige Signalwege

Das Chemokin CXCL12 (stromal cell derived-factor, SDF-1) bindet an die Rezeptoren CXCR4 und CXCR7 und kontrolliert damit unter anderem das Wachstum sowie die Metastasierung von Tumoren in unterschiedlichen Organen. Interessanterweise werden diese protumorogenen Einflüsse abhängig vom Zelltyp und der jeweiligen zellulären Antwort entweder von CXCR4, CXCR7 oder der Kombination beider Rezeptoren vermittelt. CXCR4 ist ein klassischer G-Protein-gekoppelter Rezeptor, der nach Ligandenbindung zur Aktivierung von Gi, Gq sowie G12/G13 führt. Demgegenüber steht der CXCR7, welcher primär als Scavenger Rezeptor betrachtet wird und über Bildung eines extrazellulären CXCL12-Gradienten vor allem die CXCR4-abhängige Zellmigration kontrolliert. Neuere Arbeiten belegen allerdings, dass auch CXCR7 zu einem eigenständigen Signalverhalten in der Lage ist und ähnlich wie CXCR4 die Migration, Proliferation sowie Invasion von (Tumor)zellen beeinflusst. Die Aktivierung der beteiligten Signalwege erfolgt dabei im Regelfall über Arrestine. Ausnahmen bilden bislang Astrozyten und bestimmte Gliomzellen, in denen CXCR7 als klassischer G-Protein-gekoppelter Rezeptor wirkt. Eine weitere, bislang vor allem für CXCR4 gezeigte Möglichkeit, zelluläre Signalwege zu aktivieren, ist über die Transaktivierung des EGFRs (engl.: epidermal growth factor receptor). Diese Arbeit zeigt, dass die Src-Kinase-vermittelte und G-Protein-abghängige Transaktivierung des EGFRs einen generellen Mechanismus darstellt, über den CXCL12 die Migration in den Tumorzelllinien steuert und damit zur Tumormetastasierung führen kann. Untersucht wurden dabei die Tumorzelllinien A549 (nichtkleinzelliges Lungenkarzinom), C33A (Zervixkarzinom), DLD‑1 (kolorektales Karzinom), MDA‑MB‑231 (Mammakarzinom) und PC‑3 (Prostatakarzinom), in denen die CXCL12-induzierte Zellmigration entweder über CXCR4, CXCR7 oder beide Rezeptoren vermittelt wird. Tatsächlich unterblieb die CXCL12-abhängige Migration aller Tumorzellen in Anwesenheit sowohl des Src-Kinase-Inhibitors Src-I1 als auch des EGFR-Inhibitors AG1478, nicht aber nach Hemmung der Arrestin-Expression mittels RNA-Interferenz. Das zelluläre Migrationsverhalten wurde mittels der Boyden-Kammer analysiert. Die Expression des EGFRs sowie β-Arrestin 1/2 wurde im Western Blot nachgewiesen. Die bereits früher gezeigte unterschiedliche Verwendung von CXCR4 und/oder CXCR7 für die Vermittlung des CXCL12-Signals in den verschiedenen Tumorzellen wurde unter Zuhilfenahme spezifischer Rezeptor-Antagonisten (AMD3100, CCX771) bestätigt. Die erhaltenen Befunde identifizieren den EGFR als einen möglichen therapeutischen Ansatzpunktpunkt zur Unterbindung der von CXCL12 induzierten Metastasierung von Tumoren. Im Vergleich zu CXCR4- oder CXCR7-Rezeptorantagonisten hat dieser Ansatz den Vorteil, dass keine Kenntnis darüber notwendig ist, über welchen CXCL12-Rezeptor das Metastasierungsverhalten des jeweiligen Tumors gesteuert wird

Bridging the Causality Gap: Insights into the Spatiotemporal Dynamics of Language Comprehension

Language comprehension is a fascinating and complex ability unique to humans. Its neurobiology has yielded profound insights through decades of electrophysiological and neuroimaging studies (Chapter 1). Despite these significant advances on the neurobiology of speech comprehension, understanding the precise timing and causal relevance of brain regions within the human language network remains one of the key challenges. This dissertation addresses this challenge by combining transcranial magnetic stimulation (TMS) with electroencephalography (EEG) measurements, providing a means to identify the causal relevance of particular brain regions with high temporal precision. By focusing on the causality of both the time and place of comprehension, this innovative approach offers new insights for developing adequate functional-anatomical models of the neurobiology of language. This dissertation reports data from three distinct studies. Going beyond correlative ev idence, Study 1 (Chapter 2) describes a set of three concurrent TMS-EEG experiments investigating the bottom-up and top-down information flow within the core language network while German native speakers listen to single sentences. Together, the findings of these three experiments indicated that speech comprehension is subserved by the temporally-coordinated interplay between the left inferior frontal and posterior temporal cortex, which may be supported by the left inferior parietal cortex. Going beyond the core system, Study 2 (Chapter 3) describes two EEG experiments that investigated the neurocognitive mechanisms involved in multi-sentence discourse comprehension. Their findings showed that a distinction between a set of positive-going event-related potentials (ERP) reflect general processing principles relevant for interpreting speech in everyday communitive settings. Going beyond this ERP-based timing information, Study 3 (Chapter 4) describes a sequential TMS-EEG probing the causal relevance of brain regions of the semantic (meaning-related) network in the processing of discourse contexts. The findings of this study highlights that the critical contribution of the left inferior parietal cortex in achieving a higher-level interpretation of speech input. In the General Discussion (Chapter 5), the findings of these studies are placed in a wider perspective, addressing their limitations and providing a number of future directions for an even finer-grained understanding of the neurobiology of speech comprehension