Experimental approaches to unravel proximate mechanisms of parasitoid searching and patch leaving behaviour

Animals exploit complex environments in an optimal way, often with limited brain capacities. Interestingly, it is largely unknown, how they do so. This thesis comprises five studies investigating proximate mechanisms modulating the searching behaviour of parasitoid wasps. These organisms serve as excellent organisms for such questions due to their tight link of searching success and fitness. While the first study assumed a simple motor response to serve as a heuristic, yet effective, mechanism, the remaining studies focussed on the role of octopamine [OA] and dopamine [DA]. Both substances being essential in the assessment of reward and aversive stimuli, respectively. Neither the assumed motor response could be met nor did OA or DA reveal any consistent effects with respect to the assessment of rewards and costs. DA slightly impacted the movement pattern. Treatment with OA revealed numerous effects, in total indicating an influence on stress level. Both is in line with studies on other species. Yet, although OA significantly influences searching behaviour, the underlying mechanism is considerably more complex than initially assumed. Last, it was shown that a generalisation on the basis of a few studies and stimuli with respect to the role of OA in the integration of rewards is a simplification

Monitoring the dynamics of Src activity in response to anti-invasive dasatinib treatment at a subcellular level using dual intravital imaging

Optimising response to tyrosine kinase inhibitors in cancer remains an extensive field of research. Intravital imaging is an emerging tool, which can be used in drug discovery to facilitate and fine-tune maximum drug response in live tumors. A greater understanding of intratumoural delivery and pharmacodynamics of a drug can be obtained by imaging drug target-specific fluorescence resonance energy transfer (FRET) biosensors in real time. Here, we outline our recent work using a Src-FRET biosensor as a readout of Src activity to gauge optimal tyrosine kinase inhibition in response to dasatinib treatment regimens in vivo. By simultaneously monitoring both the inhibition of Src using FRET imaging, and the modulation of the surrounding extracellular matrix using second harmonic generation (SHG) imaging, we were able to show enhanced drug penetrance and delivery to live pancreatic tumors. We discuss the implications of this dual intravital imaging approach in the context of altered tumor-stromal interactions, while summarising how this approach could be applied to assess other combination strategies or tyrosine kinase inhibitors in a preclinical setting

Mode-of-Action studies on cytostatic compounds from myxobacteria : antimitotic pretubulysins and actin-targeting chondramides

Myxobacteria have proven to be highly valuable as sources of natural products since they produce a variety of secondary metabolites with unique structures. These natural products often exhibit astonishing biological activities with a new mode-of-action. This thesis provides new insights into the mode-of-action of the myxobacterial metabolite pretubulysin. Synthetic pretubulysin and some of its derivatives, which are available in gram-scale quantities, were characterized and bioactivity studies clearly showed the compounds’ ability to depolymerize microtubules. Pretubulysin treatment consequently leads to mitotic arrest and the induction of apoptosis in cultured mammalian cells. Furthermore, important structure-activity relationships were elucidated by comparing several synthetic analogs. Another topic of this thesis was the implementation of high-content screening for bioactivity-guided isolation of secondary metabolites from myxobacteria. For this purpose, Chondromyces sp. extracts were subjected to several in-depth cell-based assays, which resulted in the discovery of over 30 new chondramide derivatives. Compounds of this class are described to target actin filaments with a phalloidin-like binding mode. In addition to structure-activity relationship insights, the biological evaluation of purified compounds provided evidence for a putative second target for some brominated analogs of this exciting class of natural products.Myxobakterien haben sich als sehr nützliche Naturstoffproduzenten erwiesen, da sie eine Vielzahl von Sekundärmetaboliten mit einzigartigen Strukturen liefern. Diese Naturstoffe besitzen darüber hinaus häufig biologische Aktivität und weisen neuartige Wirkmechanismen auf. Diese Arbeit vermittelt erste Einblicke in den Wirkmechanismus des myxobakteriellen Metabolits Prätubulysin. Synthetisches Prätubulysin und einige Derivate davon sind in größeren Mengen verfügbar und wurden im Zuge dieser Arbeit charakterisiert. Durch diese Bioaktivitätsstudien konnte eindeutig gezeigt werden, dass Prätubulysine zu einer Depolymerisation von Mikrotubuli führen und im Weiteren einen mitotischen Arrest und Apotose in kultivierten Säugerzellen induzieren. Durch vergleichende Studien mit mehreren synthetischen Analoga konnten darüber hinaus wichtige Struktur-Aktivitäts-Beziehungen hergeleitet werden. Ein weiteres Thema dieser Arbeit war die Implementierung von High-Content-Screening für die bioaktivitätsgeleitete Aufreinigung von Sekundärmetaboliten aus Myxobakterien. Im Laufe der Untersuchungen wurden so über 30 neue Chondramid-Derivate in Extrakten von Chondromyces sp. gefunden und zum Teil isoliert. Die biologische Evaluierung der Reinstoffe, die eine Polymerisation von G-Aktin induzieren, erbrachte wichtige Struktur-Aktivitäts-Beziehungen. Außerdem lieferten zellbasierte und in vitro-Studien im Falle von bromierten Analoga Hinweise auf ein vermeintliches zweites zelluläres Target

Analyse der PSMA Expression in Tumorendothelien des kolorektalen Karzinoms

Das kolorektale Karzinom zählt zu den häufigsten Tumorerkrankungen. Prognose und Therapiemanagement basieren vor allem auf klinischen Kriterien wie Tumordifferenzierung, lokaler Invasion und Lymphknotenmetastasierung, einen wichtigen Anteil nehmen präventive Maßnahmen ein, die jedoch größtenteils aufwändig, kostenintensiv und zu wenig sensitiv sind. Das Interesse an einem molekularen prognostischen Marker und einem Angriffspunkt für zielgerichtete Therapien ist groß. PSMA, das Prostata-spezifische Membran Antigen, ist bereits als Zielantigen beim Prostatakarzinom etabliert, es wird in der Nuklearmedizin zur Tumor- und Metastasenlokalisation angewendet. Ziel dieser Arbeit war die detaillierte Analyse der PSMA-Expression in Tumorepithelien und Tumorgefäßendothelien an einer kliniko-pathologisch charakterisierten Kohorte kolorektaler Karzinome (n=243) und die Klärung der Prävalenz positiver Fälle, der Assoziation mit dem Tumorstadium, dem Tumorgrad sowie der prognostischen Bedeutung von PSMA im kolorektalen Karzinom. Formalin-fixiertes, paraffin-eingebettetes, zu einem Microarray zusammengefügtes Resektionsmaterial wurde immunhistochemisch mit einem PSMA- und einem CD34-Antikörper gefärbt. In 72% der untersuchten Fälle kolorektaler Karzinome wurden PSMA-positive Tumorendothelien nachgewiesen. Es konnte eine reziproke und statistisch signifikante Assoziation zwischen PSMA-Expression und V-Kategorie nachgewiesen werden (p= 0,003). Zudem zeigte sich ein Trend bezüglich des TNM-Stadiums, es fanden sich seltener als erwartet PSMA-negative KRK im TNM-Stadium T2 und häufiger als erwartet im Stadium T4. Für keinen der weiteren untersuchten klinisch-pathologischen Parameter konnte eine Korrelation nachgewiesen werden. In der Betrachtung von Vollwandschnittpräparaten von Kolonkarzinomen, welche der Validierung der TMA-Methodik dienten, konnte ein signifikanter Zusammenhang (p=0,024) zwischen der Häufigkeit der PSMA-Expression in TMA-Stanzbiopsien und Vollwandanschnitten belegt werden. Dessen ungeachtet, würde eine Untersuchung einer noch größeren Kohorte kolorektaler Karzinome eine detailliertere Untersuchung der intratumoralen Heterogenität ermöglichen

Optimization of Mass Spectrometry Imaging for Drug Metabolism and Distribution Studies in the Zebrafish Larvae Model: A Case Study with the Opioid Antagonist Naloxone

Zebrafish (ZF; Danio rerio) larvae have emerged as a promising in vivo model in drug metabolism studies. Here, we set out to ready this model for integrated mass spectrometry imaging (MSI) to comprehensively study the spatial distribution of drugs and their metabolites inside ZF larvae. In our pilot study with the overall goal to improve MSI protocols for ZF larvae, we investigated the metabolism of the opioid antagonist naloxone. We confirmed that the metabolic modification of naloxone is in high accordance with metabolites detected in HepaRG cells, human biosamples, and other in vivo models. In particular, all three major human metabolites were detected at high abundance in the ZF larvae model. Next, the in vivo distribution of naloxone was investigated in three body sections of ZF larvae using LC-HRMS/MS showing that the opioid antagonist is mainly present in the head and body sections, as suspected from published human pharmacological data. Having optimized sample preparation procedures for MSI (i.e., embedding layer composition, cryosectioning, and matrix composition and spraying), we were able to record MS images of naloxone and its metabolites in ZF larvae, providing highly informative distributional images. In conclusion, we demonstrate that all major ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters, as part of in vivo pharmacokinetic studies, can be assessed in a simple and cost-effective ZF larvae model. Our established protocols for ZF larvae using naloxone are broadly applicable, particularly for MSI sample preparation, to various types of compounds, and they will help to predict and understand human metabolism and pharmacokinetics

Drug Administration Routes Impact the Metabolism of a Synthetic Cannabinoid in the Zebrafish Larvae Model

Zebrafish (Danio rerio) larvae have gained attention as a valid model to study in vivo drug metabolism and to predict human metabolism. The microinjection of compounds, oligonucleotides, or pathogens into zebrafish embryos at an early developmental stage is a well-established technique. Here, we investigated the metabolism of zebrafish larvae after microinjection of methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-dimethylbutanoate (70N-5F-ADB) as a representative of recently introduced synthetic cannabinoids. Results were compared to human urine data and data from the in vitro HepaRG model and the metabolic pathway of 70N-5F-ADB were reconstructed. Out of 27 metabolites detected in human urine samples, 19 and 15 metabolites were present in zebrafish larvae and HepaRG cells, respectively. The route of administration to zebrafish larvae had a major impact and we found a high number of metabolites when 70N-5F-ADB was microinjected into the caudal vein, heart ventricle, or hindbrain. We further studied the spatial distribution of the parent compound and its metabolites by mass spectrometry imaging (MSI) of treated zebrafish larvae to demonstrate the discrepancy in metabolite profiles among larvae exposed through different administration routes. In conclusion, zebrafish larvae represent a superb model for studying drug metabolism, and when combined with MSI, the optimal administration route can be determined based on in vivo drug distribution

Pupil Dilation Is Sensitive to Semantic Ambiguity and Acoustic Degradation

© The Author(s) 2020. Speech comprehension is challenged by background noise, acoustic interference, and linguistic factors, such as the presence of words with more than one meaning (homonyms and homophones). Previous work suggests that homophony in spoken language increases cognitive demand. Here, we measured pupil dilation—a physiological index of cognitive demand—while listeners heard high-ambiguity sentences, containing words with more than one meaning, or well-matched low-ambiguity sentences without ambiguous words. This semantic-ambiguity manipulation was crossed with an acoustic manipulation in two experiments. In Experiment 1, sentences were masked with 30-talker babble at 0 and +6 dB signal-to-noise ratio (SNR), and in Experiment 2, sentences were heard with or without a pink noise masker at –2 dB SNR. Speech comprehension was measured by asking listeners to judge the semantic relatedness of a visual probe word to the previous sentence. In both experiments, comprehension was lower for high- than for low-ambiguity sentences when SNRs were low. Pupils dilated more when sentences included ambiguous words, even when no noise was added (Experiment 2). Pupil also dilated more when SNRs were low. The effect of masking was larger than the effect of ambiguity for performance and pupil responses. This work demonstrates that the presence of homophones, a condition that is ubiquitous in natural language, increases cognitive demand and reduces intelligibility of speech heard with a noisy background

Combating pancreatic cancer with PI3K pathway inhibitors in the era of personalised medicine

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly solid tumours. This is due to a generally late-stage diagnosis of a primarily treatment-refractory disease. Several large-scale sequencing and mass spectrometry approaches have identified key drivers of this disease and in doing so highlighted the vast heterogeneity of lower frequency mutations that make clinical trials of targeted agents in unselected patients increasingly futile. There is a clear need for improved biomarkers to guide effective targeted therapies, with biomarker-driven clinical trials for personalised medicine becoming increasingly common in several cancers. Interestingly, many of the aberrant signalling pathways in PDAC rely on downstream signal transduction through the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways, which has led to the development of several approaches to target these key regulators, primarily as combination therapies. The following review discusses the trend of PDAC therapy towards molecular subtyping for biomarker-driven personalised therapies, highlighting the key pathways under investigation and their relationship to the PI3K pathway

Induction of Liver Size Reduction in Zebrafish Larvae by the Emerging Synthetic Cannabinoid 4F-MDMB-BINACA and Its Impact on Drug Metabolism

Zebrafish (ZF; Danio rerio) larvae have become a popular in vivo model in drug metabolism studies. Here, we investigated the metabolism of methyl 2-[1-(4-fluorobutyl)-1H-indazole-3-carboxamido]- 3,3-dimethylbutanoate (4F-MDMB-BINACA) in ZF larvae after direct administration of the cannabinoid via microinjection, and we visualized the spatial distributions of the parent compound and its metabolites by mass spectrometry imaging (MSI). Furthermore, using genetically modified ZF larvae, the role of cannabinoid receptor type 1 (CB1) and type 2 (CB2) on drug metabolism was studied. Receptor-deficient ZF mutant larvae were created using morpholino oligonucleotides (MOs), and CB2-deficiency had a critical impact on liver development of ZF larva, leading to a significant reduction of liver size. A similar phenotype was observed when treating wild-type ZF larvae with 4F-MDMB-BINACA. Thus, we reasoned that the cannabinoid-induced impaired liver development might also influence its metabolic function. Studying the metabolism of two synthetic cannabinoids, 4F-MDMB-BINACA and methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3- dimethylbutanoate (70N-5F-ADB), revealed important insights into the in vivo metabolism of these compounds and the role of cannabinoid receptor binding