Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323–785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.

T-Zell-aktivierende Formate, wie BiTE (bispecific T-cell engagers) Antikörper und CAR T Zellen haben in den vergangen Jahren die Therapiemöglichkeiten für Tumorpatienten erweitert. Diese Therapeutika verknüpfen T-Zellen mit malignen Zellen über je ein spezifisches Oberflächenmolekül und initiieren, über eine T-Zell-vermittelte Immunantwort, die Lyse der Tumorzelle. Tumorspezifische Antigene sind jedoch selten. Häufig werden Proteine adressiert, die neben den Tumorzellen auch auf gesunden Zellen exprimiert werden. Die Folgen sind toxische Effekte abseits der Tumorzellen auf Antigen-positiven gesunden Zellen (on target/off tumor), welche nicht nur die Dosis des Therapeutikums und dessen Effektivität limitieren, sondern zu geringen bis letalen Begleiterscheinungen führen können. Der Bedarf an effektiven Therapieformen mit geringen Nebenwirkungen ist folglich immer noch sehr hoch. Diese Lücke soll durch ein neues Antikörperformat, sogenannten Hemibodies, geschlossen werden. Hemibodies sind eine neue Klasse von T-Zell-aktivierenden Antikörpern, die sich gegen eine Antigenkombination und nicht einzelne Antigene auf Tumorzellen richten. Sie bestehen aus zwei komplementären Molekülen mit je einer Antigen-bindenden Sequenz, die entweder mit der leichten (VL) oder der schweren (VH) Kette eines T-Zell-aktivierenden anti CD3 Antikörpers fusioniert ist. Nur wenn beide Hemibody-Fragmente gleichzeitig in unmittelbarer Nähe an ihr jeweiliges Antigenepitop auf der Tumorzelle binden, komplementieren die beiden Antikörperkonstrukte über das geteilte anti-CD3 und bilden einen trivalenten T Zell aktivierenden Komplex aus. Diese funktionale Einheit rekrutiert T-Zellen zur Tumorzelle und induzierte die T-Zell-vermittelte Lyse der malignen Zelle. Im Rahmen der vorliegenden Arbeit wurden geeignete Antigenkombinationen identifiziert und die erste effektive und spezifische Hemibody-basierte Immuntherapie gegen das Multiple Myelom (MM), ohne Nebenwirkungen auf Antigen-einfach-positiven gesunden Zellen, entwickelt. Basierend auf einer umfangreichen Analyse von Kandidaten-Antigenen wurden Kombinationen aus bekannten MM Zielmolekülen, wie BCMA, CD38, CD138, CD229 und SLAMF7, und für das MM unbekannte Oberflächenmolekülen, wie CHRM5 und LAX1, untersucht. Gegen die vielversprechendsten Antigene wurden Hemibodies entwickelt und produziert. Im Zusammenhang mit Analysen zur Produzierbarkeit sowie biochemischen und funktionalen Charakterisierungen, konnte aus 75 initialen Hemibody-Kombinationen drei Kombinationen mit geeigneten Eigenschaften identifiziert werden. Die Bindung von zwei Hemibody-Partnern auf der Oberfläche der MM Zelle führte zur Ausbildung eines trivalenten T-Zell-rekrutierenden Komplexes. Dieser initiierte nachfolgend über eine T-Zell-vermittelte Immunantwort die spezifische Lyse der malignen Zellen, ohne die Viabilität von Antigen-einfach-positiven gesunden Körper- oder Effektor-Zellen zu beeinflussen. Zusätzlich führte eine Hemibody-Therapie in vivo in einem NOD SCID MM-Mausmodel innerhalb von 7 Tagen zur kompletten Remission der MM Zellen. Diese Daten zeigten Hemibodies als ein neues, sehr vielversprechendes Antikörperformat für eine effektive und tumorspezifische Immuntherapie mit potentiell geringen Nebenwirkungen.

Indoor house dust is a blend of organic and inorganic materials, upon which diverse microbial communities such as viruses, bacteria and fungi reside. Adequate moisture in the indoor environment helps microbial communities multiply fast. The outdoor air and materials that are brought into the buildings by airflow, sandstorms, animals pets and house occupants endow the indoor dust particles with extra features that impact human health. Assessment of the health effects of indoor dust particles, the type of indoor microbial inoculants and the secreted enzymes by indoor insects as allergens merit detailed investigation. Here, we discuss the applications of next generation sequencing (NGS) technology which is used to assess microbial diversity and abundance of the indoor dust environments. Likewise, the applications of NGS are discussed to monitor the gene expression profiles of indoor human occupants or their surrogate cellular models when exposed to aqueous solution of collected indoor dust samples. We also highlight the detection methods of dust allergens and analytical procedures that quantify the chemical nature of indoor particulate matter with a potential impact on human health. Our review is thus unique in advocating the applications of interdisciplinary approaches that comprehensively assess the health effects due to bad air quality in built environments.

A fine balance of regulatory (T\(_{reg}\)) and conventional CD4\(^+\) T cells (T\(_{conv}\)) is required to prevent harmful immune responses, while at the same time ensuring the development of protective immunity against pathogens. As for many cellular processes, sphingolipid metabolism also crucially modulates the T\(_{reg}\)/T\(_{conv}\) balance. However, our understanding of how sphingolipid metabolism is involved in T cell biology is still evolving and a better characterization of the tools at hand is required to advance the field. Therefore, we established a reductionist liposomal membrane model system to imitate the plasma membrane of mouse T\(_{reg}\) and T\(_{conv}\) with regards to their ceramide content. We found that the capacity of membranes to incorporate externally added azide-functionalized ceramide positively correlated with the ceramide content of the liposomes. Moreover, we studied the impact of the different liposomal preparations on primary mouse splenocytes in vitro. The addition of liposomes to resting, but not activated, splenocytes maintained viability with liposomes containing high amounts of C\(_{16}\)-ceramide being most efficient. Our data thus suggest that differences in ceramide post-incorporation into T\(_{reg}\) and T\(_{conv}\) reflect differences in the ceramide content of cellular membranes.

Optogenetics is a powerful technique that utilizes light to precisely regulate physiological activities of neurons and other cell types. Specifically, light-sensitive ion channels, pumps or enzymes are expressed in cells to enable their regulation by illumination, thus allowing for precise control of biochemical signaling pathways. The first part of my study involved the construction, optimization, and characterization of two optogenetic tools, KCR1 and NCR1. Elena Govorunova et al. discovered a lightgated potassium channel, KCR1, in the protozoan Hyphochytrium catenoides. Traditional potassium ion channels are classified as either ligand-gated or voltage-gated and possess conserved pore-forming domains and K+ -selective filters. However, KCR1 is unique in that it does not contain the signature sequence of previously known K+ channels and is a channelrhodopsin. We synthesized the KCR1 plasmid according to the published sequence and expressed it in Xenopus oocytes. Due to the original KCR1 current being too small, I optimized it into KCR1 2.0 to improve its performance by fusing LR (signal peptide LucyRho, enhances expression) at the N-terminal and T (trafficking signal peptide) and E (ER export signal peptide) at the C-terminal. Additionally, I investigated the light sensitivity, action spectrum, and kinetics of KCR1 2.0 in Xenopus oocytes. The potassium permeability of KCR1 2.0, PK/Pna  24, makes KCR1 2.0 a powerful hyperpolarizing tool that can be used to inhibit neuronal firing in animals. Inspired by KCR1, we used the KCR1 sequence as a template for gene sequence alignment with the sequences in H. catenoides. We found that NCR1 and KCR1 have similar gene sequences. NCR1 was characterized by us as a light-gated sodium channel. This NCR1 was also characterized and published by Govorunova et al. very recently, with the name HcCCR. Due to the original NCR1 current being too small, I optimized it into NCR1 2.0 to improve its performance by fusing LR at the N-terminal and T and E at the C-terminal, which significantly improved the expression level and greatly increased the current amplitude of NCR1. Full-length NCR1 2.0 contains 432 amino acids. To test whether the number of amino acids changes the characteristics of NCR1 2.0, we designed NCR1 2.0 (330), NCR1 2.0 (283), and NCR1 2.0 (273) by retaining the number of amino acids at 330, 280, and 273 in NCR1 2.0, respectively. As the number of amino acids decreased, the current in NCR1 2.0 increased. I also investigated the light sensitivity, action spectrum, and kinetics of NCR1 2.0 (273) in the Xenopus Abstract 2 oocytes. We performed four point mutations at amino acid positions 133 and 116 of NCR1 2.0 and analyzed the reversal potentials of the mutants. The mutations were as follows: NCR1 2.0 (273 D116H), NCR1 2.0 (273 D116E), NCR1 2.0 (283 V133H), and NCR1 2.0 (283 D116Q). The second part of this study focuses on light-induced water transport using optogenetic tools. We explored the use of optogenetic tools to regulate water flow by changing the osmolarity in oocytes. Water flux through AQP1 is driven by the osmotic gradient that results from concentration differences of small molecules or ions. Therefore, we seek to regulate ion concentrations, using optogenetic tools to regulate the flux of water noninvasively. To achieve this, I applied the light-gated cation channels XXM 2.0 and NCR1 2.0 to regulate the concentration of Na+ , while K + channel KCR1 2.0 was used to regulate K + concentration. As Na+ flows into the Xenopus oocytes, the membrane potential of the oocytes becomes positive, and Clcan influx through the light-gated anion channel GtACR1. By combining these optogenetic tools to regulate NaCl or KCl concentrations, I can change the osmolarity inside the oocytes, thus regulating the flux of water. I co-expressed AQP1 with optogenetic tools in the oocytes to accelerate water flux. Overall, I designed three combinations (1: AQP1, XXM 2.0 and GtACR1. 2: AQP1, NCR1 2.0 and GtACR1. 3: AQP1, KCR1 2.0 and GtACR1) to regulate the flow of water in oocytes. The shrinking or swelling of the oocytes can only be achieved when AQP1, light-gated cation channels (XXM 2.0/NCR1 2.0/KCR1 2.0), and light-gated anion channels (GtACR1) are expressed together. The illumination after expression of either or both alone does not result in changes in oocyte morphology. In sum, I demonstrated a novel strategy to manipulate water movement into and out of Xenopus oocytes, non-invasively through illumination. These findings provide a new avenue to interfere with water homeostasis as a means to study related biological phenomena across cell types and organisms.