Synthese basenmodifizierter Nukleosidtriphosphate und ihre enzymatische Polymerisation zu funktionalisierter DNA

Im Rahmen dieser Arbeit wurden zahlreiche Nukleosidtriphosphate synthetisiert, die mit zusätzlichen funktionellen Gruppen versehen sind. Diese wurden als Ersatz für natürliche dNTPs enzymatisch in DNA eingebaut, um so eine hochgradig funktionalisierte DNA (fDNA) zu generieren. Weiterhin wurden alle notwendigen Methoden etabliert, um auf Basis von fDNA in vitro-Selektionen funktionaler Nukleinsäuren durchführen zu können. Nachdem der Versuch scheiterte, verschiedene 8-modifizierte dATP-Derivate als Substrat für die PCR zu verwenden, wurden fünf 7-modifizierte N-7-Deaza-2'-desoxyadenosintri­phosphate sowie ein 7-modifiziertes N-7-Deaza-2'-desoxyguanosin­triphosphat hergestellt. Während das Adenosinderivat 40 von der DNA-Polymerase aus Thermus thermophilus (Tth) gut eingebaut und sogar der gleichzeitige Austausch von TTP mit Verbindung 50 realisiert werden konnte, war das Guanosinderivat 47 nur mit mäßigem Erfolg zu verwenden. In einem alternativen Ansatz wurde die fDNA durch templatgesteuerte Primer-Verlängerung 7-modifizierter N-7-Deazapurine und C-5-modifizierter Pyrimidine generiert. Dazu wurden einige kommerziell erhältliche DNA-Polymerasen getestet, zunächst an einem einfachen Modelltemplat, später an ausgesuchten Templaten mit anspruchsvollen Sequenzmotiven und an einem teilweise randomisierten Templat. Mit der VentR® (exo-) DNA-Polymerase wurde ein Enzym identifiziert, das nicht nur alle Nukleotide, sondern auch an allen Templaten mit guter Ausbeute die entsprechende fDNA generieren kann. Anschließend wurden DNA-Polymerasen auf ihre Fähigkeit getestet, fDNA als Templat für eine PCR mit natürlichen dNTPs zu verwenden. Die DNA-Polymerase aus dem hyperthermophilen Organismus Pyrococcus woesei (Pwo) war schließlich nach Optimierung der Reaktions-Parameter in der Lage, fDNA in DNA zu retransformieren und zu amplifizieren. Der gesamte Prozess aus Generierung und Amplifikation lief sequenzspezifisch, wie durch Sequenzierung der PCR-Produkte nachgewiesen werden konnte. Abschließend wurde versucht, mit Hilfe von Kraftfeldrechnungen das unterschiedliche Einbauverhalten der 8- und 7-modifizierten Adenosinderivate zu erklären. Es ließen sich konformative Unterschiede der Nukleotide nachweisen, doch konnte dies die dramatischen Unterschiede bezüglich des enzymatischen Einbaus nur bedingt erklären. Weitere Untersuchungen deuteten darauf hin, dass der Hauptgrund für dieses Verhalten in der Verwendung des Alkinlinkers an Position C-8 liegt, da das entsprechend modifizierte Nukleotid in dem aktiven Zentrum des Enzyms nicht mehr die für den Einbau notwendige Konformation einnehmen kann

Fermentative oxidation of butane in bubble column reactors

To date the use of alkanes as starting materials for selective activation in chemical industry is very challenging. For this task the biocatalytic selective activation offers a number of advantages. The activation starts with C-H functionalization by a sequence of oxidation steps via alcohols, aldehydes/ ketones and carboxylic acids. All these derivatives are bulk-scale products, which are produced with standard chemical methods using high pressures and temperatures. In contrast, microorganisms are able to convert alkanes to various organic compounds at ambient pressure and temperature.[1] For the selective and efficient functionalization of alkanes appropriate fermentation of cells is required. Process engineering is required for a high yielding butane oxidation as well as reactor design. In this context it is essential to investigate the parameters of cell growth and to establish control of the fermentation conditions for production of the hydroxylated target compounds. At first a suitable reactor set up in accordance to the safety regulations required for handling a flammable gas like butane had to be installed to enable reaction engineering studies of the cell and reactor system. Specialized bubble column reactors are developed on lab scale and characterized in view of the implementation at industrial scale.[2] Additionally, a suitable control system was designed to monitor as well as control standard parameters and to simplify the implementation of further equipment. The mass transfer of the gaseous starting materials into the fermentation media is the key limiting factor for reaching sufficient productivities. The process window is mainly restricted by the requirements of the microorganisms and the flammability region of the substrates. Please click Additional Files below to see the full abstract

DGK and DZHK position paper on genome editing: basic science applications and future perspective

For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems

Functional architecture of reward learning in mushroom body extrinsic neurons of larval Drosophila.

The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. Here we use a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron. Our results reveal the identity and morphology of almost all of these 44 neurons in stage 3 Drosophila larvae. Upon an initial screen, functional analyses focusing on the mushroom body medial lobe uncover sparse and specific functions of its dopaminergic MBINs, its MBONs, and of the GABAergic APL neuron across three behavioral tasks, namely odor preference, taste preference, and associative learning between odor and taste. Our results thus provide a cellular-resolution study case of how brains organize behavior

The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure

Serum MicroRNA-21 as Marker for Necroinflammation in Hepatitis C Patients with and without Hepatocellular Carcinoma

Background: MicroRNA-21 (miR-21) is up-regulated in tumor tissue of patients with malignant diseases, including hepatocellular carcinoma (HCC). Elevated concentrations of miR-21 have also been found in sera or plasma from patients with malignancies, rendering it an interesting candidate as serum/plasma marker for malignancies. Here we correlated serum miR-21 levels with clinical parameters in patients with different stages of chronic hepatitis C virus infection (CHC) and CHC-associated HCC. Methodology/Principal Findings: 62 CHC patients, 29 patients with CHC and HCC and 19 healthy controls were prospectively enrolled. RNA was extracted from the sera and miR-21 as well as miR-16 levels were analyzed by quantitative real-time PCR; miR-21 levels (normalized by miR-16) were correlated with standard liver parameters, histological grading and staging of CHC. The data show that serum levels of miR-21 were elevated in patients with CHC compared to healthy controls (P<0.001); there was no difference between serum miR-21 in patients with CHC and CHC-associated HCC. Serum miR-21 levels correlated with histological activity index (HAI) in the liver (r = −0.494, P = 0.00002), alanine aminotransferase (ALT) (r = −0.309, P = 0.007), aspartate aminotransferase (r = −0.495, P = 0.000007), bilirubin (r = −0.362, P = 0.002), international normalized ratio (r = −0.338, P = 0.034) and γ-glutamyltransferase (r = −0.244, P = 0.034). Multivariate analysis revealed that ALT and miR-21 serum levels were independently associated with HAI. At a cut-off dCT of 1.96, miR-21 discriminated between minimal and mild-severe necroinflammation (AUC = 0.758) with a sensitivity of 53.3% and a specificity of 95.2%. Conclusions/Significance: The serum miR-21 level is a marker for necroinflammatory activity, but does not differ between patients with HCV and HCV-induced HCC

Targeted Manipulation of Serotonergic Neurotransmission Affects the Escalation of Aggression in Adult Male Drosophila melanogaster

Dopamine (DA) and serotonin (5HT) are reported to serve important roles in aggression in a wide variety of animals. Previous investigations of 5HT function in adult Drosophila behavior have relied on pharmacological manipulations, or on combinations of genetic tools that simultaneously target both DA and 5HT neurons. Here, we generated a transgenic line that allows selective, direct manipulation of serotonergic neurons and asked whether DA and 5HT have separable effects on aggression. Quantitative morphological examination demonstrated that our newly generated tryptophan hydroxylase (TRH)-Gal4 driver line was highly selective for 5HT-containing neurons. This line was used in conjunction with already available Gal4 driver lines that target DA or both DA and 5HT neurons to acutely alter the function of aminergic systems. First, we showed that acute impairment of DA and 5HT neurotransmission using expression of a temperature sensitive form of dynamin completely abolished mid- and high-level aggression. These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships. We showed next that manipulation of either 5HT or DA neurotransmission failed to duplicate this phenotype. Selective disruption of 5HT neurotransmission yielded flies that fought, but with reduced ability to escalate fights, leading to fewer dominance relationships. Acute activation of 5HT neurons using temperature sensitive dTrpA1 channel expression, in contrast, resulted in flies that escalated fights faster and that fought at higher intensities. Finally, acute disruption of DA neurotransmission produced hyperactive flies that moved faster than controls, and rarely engaged in any social interactions. By separately manipulating 5HT- and DA- neuron systems, we collected evidence demonstrating a direct role for 5HT in the escalation of aggression in Drosophila

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

The Open Anchoring Quest Dataset: Anchored Estimates from 96 Studies on Anchoring Effects

People’s estimates are biased toward previously considered numbers (anchoring). We have aggregated all available data from anchoring studies that included at least two anchors into one large dataset. Data were standardized to comprise one estimate per row, coded according to a wide range of variables, and are available for download and analyses online (https://metaanalyses.shinyapps.io/OpAQ/). Because the dataset includes both original and meta-data it allows for fine-grained analyses (e.g., correlations of estimates for different tasks) but also for meta-analyses (e.g., effect sizes for anchoring effects)