Prediction of designer-recombinases for DNA editing with generative deep learning

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. To accelerate the development of designer-recombinases I evaluated two sequencing approaches and gathered the sequence information of over two million Cre-like recombinase sequences evolved for 89 different target sites. With this information I first investigated the sequence compositions and residue changes of the recombinases to further our understanding of their target site selectivity. The complexity of the data led me to a generative deep learning approach. Using the sequence data I trained a conditional variational autoencoder called RecGen (Recombinase Generator) that is capable of generating novel recombinases for a given target site. With computational evaluation of the sequences I revealed that known recombinases functional on the desired target site are generally more similar to the RecGen predicted recombinases than other recombinase libraries. Additionally, I could experimentally show that predicted recombinases for known target sites are at least as active as the evolved recombinases. Finally, I also experimentally show that 4 out of 10 recombinases predicted for novel target sites are capable of excising their respective target sites. As a bonus to RecGen I also developed a new method capable of accurate sequencing of recombinases with nanopore sequencing while simultaneously counting DNA editing events. The data of this method should enable the next development iteration of RecGen

Metaanalyse zur genetischen Prädisposition bei Harnblasenkarzinomen anhand von Polymorphismen

Das Harnblasenkarzinom ist eine maligne Tumorerkrankung, die in den meisten Fällen von den Urothelzellen der Harnblase ausgeht. Männer sind von dieser Krebsart häufiger betroffen als Frauen. In Deutschland ist es die vierthäufigste Tumorlokalisation beim Mann und auf Platz 14 in der Krebsstatistik der Frau. Das relative 5-Jahres-Überleben beträgt bei Männern 77 % und bei Frauen 72 % (2013). Das Urothelkarzinom ist eine Erkrankung mit multifaktorieller Genese. Der Hauptrisikofaktor für Blasenkrebs ist Tabakkonsum mit einem signifikanten Einfluss auf Entstehung, Progression und Mortalität. Etwa 50–65 % der männlichen und 20–30 % der weiblichen Fälle sind auf Tabakkonsum zurückzuführen. Einen weiteren Risikofaktor stellt die Exposition gegenüber aromatischen Aminen und polyzyklischen aromatischen Kohlenwasserstoffen dar, die zwischen 7,1 % und 20 % der Erkrankungsfälle bedingen. Außerdem werden etwa 31 % der Blasentumoren durch genetische Risikofaktoren erklärt. In zahlreichen Studien wurde der Einfluss von Einzelnukleotid-Polymorphismen auf das Erkrankungsrisiko für das Harnblasenkarzinom untersucht. Dabei kam es teils zu widersprüchlichen Ergebnissen. Im Rahmen dieser Dissertation wurde daher eine qualitative Auswertung aller bis zum 07.04.2020 auf PubMed veröffentlichten Fall-Kontroll-Studien durchgeführt. Anschließend wurden die Arbeiten zu XRCC1 Arg399Gln (rs25487), ERCC2/XPD Lys751Gln (rs13181), TP53 Arg72Pro (rs1042522), XRCC1 Arg194Trp (rs1799782), XRCC3 Thr241Met (rs861539) und GSTP1 Ile105Val (rs1695) näher beleuchtet und deren Ergebnisse quantitativ in einer Metaanalyse zusammengefasst. Zusätzlich wurden Subgruppenanalysen nach Ethnie und Art der Kontrollpopulation durchgeführt, um die Vergleichbarkeit der Studien zu steigern. Es wurde jeweils die Odds Ratio und das 95 %-Konfidenzintervall berechnet. Für GSTP1 Ile105Val wurde im rezessiven Modell eine Odds Ratio von 1,340 mit einem 95 %-Konfidenzintervall von 1,038–1,730 berechnet. Weiterhin konnte in den Sensitivitätsanalysen und verschiedenen Subgruppen bei den Polymorphismen XRCC1 Arg399Gln, TP53 Arg72Pro und XRCC1 Arg194Trp teilweise statistische Signifikanz erreicht werden. Jedoch besteht diese Signifikanz nur für einzelne Ethnien oder Studiendesigns und lässt keine generelle Aussage über die genetische Prädisposition zu. Für ERCC2/XPD Lys751Gln und XRCC3 Thr241Met zeigte sich in keinem der berechneten Modelle ein statistisch signifikantes Ergebnis. Die Ergebnisse deuten darauf hin, dass Träger des GGGenotyps (Val105) ein erhöhtes Risiko besitzen, an einem Harnblasenkarzinom zu erkranken. Die Ergebnisse verdeutlichen die komplexe Ätiologie des Harnblasenkarzinoms als multifaktorielle Erkrankung im Hinblick auf dessen genetische Prädisposition und Interaktion mit zahlreichen Umweltfaktoren.Bladder cancer is a malignant tumour disease, which primarily originates from the urothelial cells of the bladder. Men are more frequently affected by this type of cancer than women, but the latter have a worse prognosis. In Germany it is the fourth most common tumour location in men and 14th in the cancer statistics for women. The relative 5-year survival rate is 77 % for men and 72 % for women (2013). Urinary bladder cancer is a disease with a multifactorial genesis. Tobacco consumption is the main risk factor for bladder cancer with a significant impact on its development, progression and mortality. About 50–65 % of male and 20–30 % of female cases are caused by smoking. The exposition to aromatic amines and polycyclic aromatic hydrocarbons represents another risk factor, which causes between 7,1 % and 20 % of cases. Furthermore, approximately 31 % of bladder tumours are explained by genetic risk factors. Numerous studies have investigated the influence of different single nucleotide polymorphisms on bladder cancer risk, with sometimes contradicting results. Therefore, a qualitative analysis of all case-control studies published on PubMed until the 7th of April 2020 was conducted in the context of this thesis. Subsequently, the publications concerning XRCC1 Arg399Gln (rs25487), ERCC2/XPD Lys751Gln (rs13181), TP53 Arg72Pro (rs1042522), XRCC1 Arg194Trp (rs1799782), XRCC3 Thr241Met (rs861539) and GSTP1 Ile105Val (rs1695) were further examined and quantitatively summarized in a meta-analysis. Additionally, subgroup analysis by ethnicity and source of controls were performed, to enhance the comparability of the studies. In each case, the odds ratio and the 95 %-confidence interval were calculated. For GSTP1 Ile105Val an odds ratio of 1,340 with a 95 %-confidence interval from 1,038 to 1,730 was calculated in the recessive model. Furthermore, statistical significance was partially achieved in the sensitivity analyses and various subgroups for the polymorphisms XRCC1 Arg399Gln, TP53 Arg72Pro and XRCC1 Arg194Trp. However, this significance only consists in several ethnicities or study designs and does not allow a general statement about genetic predisposition. No significant results were found in any of the calculated models for ERCC2/XPD Lys751Gln and XRCC3 Thr241Met. The findings indicate that carriers of the GG genotype (Val105) have an increased risk of developing bladder cancer. The results illustrate the complex etiology of bladder cancer as a multifactorial disease in terms of its genetic predisposition and interaction with numerous environmental factors

Positioning urban labs – a new form of smart governance?

In the current era, in which cities are considered key arenas for coping with a number of societal challenges, there is also renewed interest in the mobilisation of experimental practices within urban planning. A growing interest in innovative intiatives emphasising co-creation, exploration, experimentation, and evaluation, such as urban living labs must also be understood in relation to the uncertatinity regarding the modern growth paradigm and its institutional arrangements: “the pragmatist heritage of urban laboratories gains renewed strength in the current era in which the belief in modernity, progress and development is in crisis” (Karvonen & van Heur, 2014, p. 387). This paper provides a sympathetic critique of the notion of urban living labs and related expeimental practices from an urban planning and governance persepective. In this light, we argue that the core principels of urban living labs (i.e. co-creation, exploration, experimentation, and evaluation) offer a useful theortical frame to understand and position different informal self-organizing initiatives in contemporary urban development. Furthermore, we assert that, considered as a planning practice (or methodology), urban living labs can be construed as a temporary mode of soft governance which include a number of merits in terms of definig new innovative pathways for urban planning beyond business as usual thinking. However, caution must be taken due to the urban living lab’s inherent shortcomings in terms of demorcractic legitimacy, tendencies towards exclusiveness, and extreme temporality. In conclusion, we argue that urban living labs can be an environment for exploring new forms of smart urban governance through critical engagments with communicative planning theory and an explict focus on actor-relations

Urban Planning through Exhibition and Experimentation in Stockholm

In this paper we discuss findings of our case study on the making and implementation of the exhibition 'Experiment Stockholm' in 2015, which, based on artistic exhibits as well as a number of forums, aimed at generating creative narratives for the sustainable urban future in the Swedish capital city-region. Our analytical framework is informed by the emerging notion of 'urban living labs' across Europe as well as 'communicative' and 'actor-relational' planning theory', which is discussed in another paper within the poceedings of this conference (cf. Schmitt et al. 2016). We argue that the exhibition 'Experiment Stockholm' and the activities around it can be characterised as a soft mode of urban governance that can help to unlock creativity and to open up avenues for experimentation and alternative solutions in urban planning. However, caution must be taken to not overvalue such approaches, as our example implies a rather exclusive expert forum instead of a a mode of governance that might be associated with openness and wider engagement. In addition, our example illustrates the significance of suitable and unconventional methods, which otherwise considerably limits the innovative capacity of the participating stakeholders and their search for alternative solutions

Graphical smalltalk with my optimization system for urban planning tasks

Based on the description of a conceptual framework for the representation of planning problems on various scales, we introduce an evolutionary design optimization system. This system is exemplified by means of the generation of street networks with locally defined properties for centrality. We show three different scenarios for planning requirements and evaluate the resulting structures with respect to the requirements of our framework. Finally the potentials and challenges of the presented approach are discussed in detail

Adjektivgebrauch in Bildbeschreibungen – Eine korpuslinguistische Untersuchung zum Gebrauch von Adjektiven in Texten von Schülerinnen und Schülern

In this article, written descriptions by students in the third to sixth grade (N = 288) are examined with regard to the use of syntactically differently integrated and differently complex adjectives. Descriptively, attributive adjectives were used most frequently and participially attributive adjectives were used least frequently. The use of different adjectives correlated positively with each other almost universally across the sample. The use of attributive adjectives increased significantly with increasing age. The use of participial attributive, adverbial, compositional, and derivative adjectives was significantly associated with increasing grade level. The use of compositional and derivative adjectives was significantly associated with increasing grade level. Predicative adjective use did not differ significantly between grades. The results provide important insights into the language acquisition process of adjectives and give indications for didactic starting points for teaching text-sort-specific word types

Laser powder bed fusion recoater selection guide: comparison of resulting powder bed properties and part quality

Spreading devices used to create powder layers in the laser powder bed fusion of metals (PBF-LB/M) were found to have a significant impact on the additive manufacturing process. However, previous research primarily focused on theoretical investigations, including recoater concepts that are not available on the market, while no comprehensive comparison of commercially available spreading devices currently exists. The aim of this study is therefore to examine the powder bed properties and part qualities that can be achieved with the three most common types of recoater: carbon fiber brushes, polymer lips, and high speed steel (HSS) blades. Identical build jobs were produced using each of the spreading devices. Their capabilities were assessed by nine evaluation criteria, including dimensional, metallurgical, and mechanical properties and criticality of particles abraded from the spreading devices. Based on these quantitative findings, a spreading device selection guide was compiled for the benefit of PBF-LB/M practitioners. All recoaters yielded processes with high stability and part properties that were on a par with or even outperformed the nominal values from the literature. However, the HSS blade was found to provide higher accuracy and stability in steady-state processes. In turn, the brush and lip are better suited for parameter development and design studies. Additionally, the lip was found to have economic benefits over the brush, while the brush was deemed an effective all-rounder

ADJEKTIVE IN BILDBESCHREIBUNGEN. Eine Korpuslinguistische Untersuchung zum Adjektivgebrauch von bilingual und monolingual aufwachsenden Schülerinnen und Schülern

Im vorliegenden Beitrag werden schriftliche Bildbeschreibungen von monolingual und bilingual aufwachsenden Schüler*innen der dritten bis sechsten Klasse (N = 288) hinsichtlich ihres Adjektivgebrauchs untersucht. Deskriptiv zeigt sich, dass monolinguale Schüler*innen mehr Adjektive in der Freitextproduktion verwenden als bilinguale Schüler*innen. Mit Ausnahme der prädikativen und wertenden Adjektive, die prädikativ und attributiv vorkommen, zeigt sich ein vermehrter Adjektivgebrauch bei monolingual Lernenden im Vergleich zu bilingual Lernenden. Für die prädikativen Adjektive zeigen sich keine signifikanten Unterschiede; für die wertenden Adjektive zeigt sich ein Mehrgebrauch der bilingualen Schüler*innen. Die gefundenen Effekte können als klein bis mittel klassifiziert werden. Es zeigt sich keine Evidenz für das Vorliegen einer Interaktion zwischen Spracherwerb und Klassenstufe in der Verwendung attributiver und wertender Adjektive. Die Auswertung liefert wichtige Erkenntnisse zum Sprachaneignungsprozess von Adjektiven und zeigt schulischen Handlungsbedarf auf

Prediction of designer-recombinases for DNA editing with generative deep learning

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designerrecombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. Here we present RecGen (Recombinase Generator), an algorithm for the intelligent generation of designerrecombinases. We gather the sequence information of over one million Crelike recombinase sequences evolved for 89 different target sites with whichwe train Conditional Variational Autoencoders for recombinase generation. Experimental validation demonstrates that the algorithm can predict recombinase sequences with activity on novel target-sites, indicating that RecGen is useful to accelerate the development of future designer-recombinases

Cross-Linking Strategies for Fluorine-Containing Polymer Coatings for Durable Resistant Water- and Oil-Repellency

Functional coatings for application on surfaces are of growing interest. Especially in the textile industry, durable water and oil repellent finishes are of special demand for implementation in the outdoor sector, but also as safety-protection clothes against oil or chemicals. Such oil and chemical repellent textiles can be achieved by coating surfaces with fluoropolymers. As many concerns exist regarding (per)fluorinated polymers due to their high persistence and accumulation capacity in the environment, a durable and resistant coating is essential also during the washing processes of textiles. Within the present study, different strategies are examined for a durable resistant cross-linking of a novel fluoropolymer on the surface of fibers. The monomer 2-((1,1,2- trifluoro-2-(perfluoropropoxy)ethyl)thio)ethyl acrylate, whose fluorinated side-chain is degradable by treatment with ozone, was used for this purpose. The polymers were synthesized via free radical polymerization in emulsion, and different amounts of cross-linking reagents were copolymerized. The final polymer dispersions were applied to cellulose fibers and the cross-linking was induced thermally or by irradiation with UV-light. In order to investigate the cross-linking efficiency, tensile elongation studies were carried out. In addition, multiple washing processes of the fibers were performed and the polymer loss during washing, as well as the effects on oil and water repellency were investigated. The cross-linking strategy paves the way to a durable fluoropolymer-based functional coating and the polymers are expected to provide a promising and sustainable alternative to functional coatings