New methods for fluorescence labelling of human rhinovirus in live cell imaging

Diese Thesis behandelt die Insertion eines RNA-Aptermers, genannt Spinach, welches mit dem kleinen Molekül DFHBI einen fluoreszierenden Komplex bildet, in das Genom des humanen Rhinovirus 14 (HRV-14). Damit soll es ermöglicht werden die virale RNA während eines gesamten Infektionszykluses in der Zelle zu beobachten um neue Erkenntnisse über die gesamte Replikation des Viruses zu erhalten, welcher am häufigsten mit der gemeinen Erkältung assoziiert ist. Die Sequenz des Aptamers musste in nicht kodierende Regionen des Genoms insertiert werden, wo auch bereits bekannte Schlüsselstrukturen wie „Cloverleaf“ und „IRES“ intakt bleiben mussten. Das reduzierte die Anzahl der möglichen Stellen auf 4 Positionen: In der 5‘-UTR jeweils vor und nach dem Cloverleaf und zwei in der 3‘-UTR. Um die Stabilität der Konstruktionen und um ungewünschte Strukturbildung vorherzusagen wurde die Vienna RNA Websuite verwendet. Die Einfügung des Aptamers resultierte in drei Fällen in einem defekten Virus, wobei das Ausmaß von der Position des Aptamers abhing. Das vierte Konstrukt zeigte Wachstumsraten vergleichbar mit dem nativen Virus, trug jedoch ein nicht fluoreszierendes Aptamer mit sich. Zwei der drei leuchtenden rekombinanten Viren konnten in HeLa-Zellen gezüchtet werden, zeigten sich jedoch genetisch instabil und deletierten das Aptamer interessanterweise punktgenau. Drei Konstrukte fluoreszierten in Gegenwart von DFHBI in vitro und in vivo transifiziert in HeLa Zellen. Die erste Passage von aus Plaques isolierten rekombinanten Viren zeigten ebenfalls Fluoreszenz 5 Stunden nach der Infektion. Als ein weiteres Projekt wurde die Einführung eines Tetra-Cystein-Tags in ein Kapsidprotein (entweder VP1 oder VP2) geplant. Wegen des beschränkten Zeitrahmens konnte dies jedoch nicht während der Masterarbeit fertig gestellt werden.We inserted an RNA aptamer called spinach which form a fluorescent complex with the small compound DFHBI into the genome of human rhinovirus 14 (HRV-14) aiming at facile dynamic fluorescent monitoring of its fate in order to gain new insight into the life-cycle of the viruses most frequently associated with the common cold. The sequence of the aptamer required insertion into non-coding regions of HRV-14, where the known key cis-regulatory elements forming conserved secondary structures like the cloverleaf or the IRES had to be kept intact. These constraints reduced the spectrum of available options to only 4 target sites: At the 5’-UTR after the two start Uracils – before the cloverleaf; at the 5’-UTR after the cloverleaf – before the IRES within the short polymorphic spacer region; or within the 3’-UTR between the end of the open reading frame and before the conserved stem-loop and between the stem-loop and poly A tract. The Vienna RNA Websuite was used to analyze for and rule out misfolding of the chimeric viral RNA as a result of aptamer tagging. We found that the insertion of spinach resulted in replication defective viruses in three instances, with severity dependent on the insertion site. The fourth construct was comparable to wild type but likely carried a misfolded inactive aptamer. Two recombinant viruses could be passaged on HeLa cells but [the virus is] proofed genetically unstable and deleted the aptamer, surprisingly without leaving behind any truncated sequences. Three chimeric RNA species became fluorescent in the presence of DFHBI in vitro and also in vivo when transfected into HeLa cells. First-generation plaque isolated recombinant viruses carrying the equivalent spinach-tagged genomic RNA gave rise to fluorescent signals in HeLa cells but only at the peak of infection (5 h post infection) which presently precludes dynamic analysis of early events such as endosomal RNA release. As a further project we also planned to insert a tetra-cystein-tag into a capsid protein, either VP1 or VP2. However, due to limitations in time this task could not be completed within this master thesis

Chytrids enhance Daphnia fitness by selectively retained chytrid‐synthesised stearidonic acid and conversion of short‐chain to long‐chain polyunsaturated fatty acids

International audienceChytrid fungal parasites convert dietary energy and essential dietary molecules, such as long-chain (LC) polyunsaturated fatty acids (PUFA), from inedible algal/cyanobacteria hosts into edible zoospores. How the improved biochemical PUFA composition of chytrid-infected diet may extend to zooplankton, linking diet quality to consumer fitness, remains unexplored. Here, we assessed the trophic role of chytrids in supporting dietary energy and PUFA requirements of the crustacean zooplankton Daphnia, when feeding on the filamentous cyanobacterium Planktothrix. Only Daphnia feeding on chytrid-infected Planktothrix reproduced successfully and had significantly higher survival and growth rates compared with Daphnia feeding on the sole Planktothrix diet. While the presence of chytrids resulted in a two-fold increase of carbon ingested by Daphnia, carbon assimilation increased by a factor of four, clearly indicating enhanced carbon transfer efficiency with chytrid presence. Bulk carbon (delta C-13) and nitrogen (delta N-15) stable isotopes did not indicate any treatment-specific dietary effects on Daphnia, nor differences in trophic position among diet sources and the consumer. Compound-specific carbon isotopes of fatty acids (delta C-13(FA)), however, revealed that chytrids bioconverted short-chain to LC-PUFA, making it available for Daphnia. Chytrids synthesised the omega-3 PUFA stearidonic acid de novo, which was selectively retained by Daphnia. Values of delta C-13(FA) demonstrated that Daphnia also bioconverted short-chain to LC-PUFA. We provide isotopic evidence that chytrids improved the dietary provision of LC-PUFA for Daphnia and enhanced their fitness. We argue for the existence of a positive feedback loop between enhanced Daphnia growth and herbivory in response to chytrid-mediated improved diet quality. Chytrids upgrade carbon from the primary producer and facilitate energy and PUFA transfer to primary consumers, potentially also benefitting upper trophic levels of pelagic food webs

Reproducible measurements of the δ2H composition of non-exchangeable hydrogen in complex organic materials using the UniPrep2 online static vapour equilibration and sample drying system

Non-exchangeable hydrogen-isotope (δ2Hn) measurements of complex organic samples are used in forensics to determine sample authenticity, traceability, and provenance. However, δ2Hn assays of organics are usually complicated by uncontrolled “exchangeable hydrogen” and residual moisture contamination; hence, δ2Hn assays are persistently incomparable amongst laboratories. We introduce a revised technical solution (UniPrep2) to control hydrogen-isotope exchange and for robust online sample drying and vapour equilibration. The UniPrep2 device is coupled to a high-temperature thermochemical elemental analyser and continuous-flow isotope-ratio mass spectrometer. This technical solution empowers isotope analysts to: • Conduct reproducible controlled vapour equilibrations of complex organic samples and standards to determine the δ2Hn values by controlling hydrogen-isotope exchange. • Conduct online vacuum-oven evacuation with extensive helium drying without exposure to air to reabsorb or exchange hydrogen with ambient water vapour.The protocol describes the operation of the UniPrep2 device and the step-by-step procedures needed to obtain accurate and precise δ2Hn values for a wide range of organic sample types. Two analytical approaches are described in detail; the Dual-Vapour Equilibration (DVE) approach, intended for determining δ2Hn for a complex organic environmental sample where matrix equivalent H isotope reference materials are not available, and the Comparative Equilibration (CE) approach, which is intended for routine high-throughput analyses of complex organic samples where at least two matrix-equivalent organic isotope reference materials with consensus δ2Hn values are being used. These standard operating procedures are envisioned to be a sound basis for advancing hydrogen-isotope analysis for different organic environmental matrices and studies

Chytrids alleviate the harmful effect of heat and cyanobacteria diet on Daphnia via PUFA-upgrading

Abstract Chytrid fungal parasites increase herbivory and dietary access to essential molecules, such as polyunsaturated fatty acids (PUFA), at the phytoplankton–zooplankton interface. Warming enhances cyanobacteria blooms and decreases algae-derived PUFA for zooplankton. Whether chytrids could support zooplankton with PUFA under global warming scenarios remains unknown. We tested the combined effect of water temperature (ambient: 18°C, heat: +6°C) and the presence of chytrids with Daphnia magna as the consumer, and Planktothrix rubescens as the main diet. We hypothesized that chytrids would support Daphnia fitness with PUFA, irrespective of water temperature. Heating was detrimental to the fitness of Daphnia when feeding solely on the Planktothrix diet. Chytrid-infected Planktothrix diet alleviated the negative impact of heat and could support Daphnia survival, somatic growth and reproduction. Carbon stable isotopes of fatty acids highlighted a ~3x more efficient n-3 than n-6 PUFA conversion by Daphnia feeding on the chytrid-infected diet, irrespective of temperature. The chytrid diet significantly increased eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6) retention in Daphnia. The EPA retention remained unaffected, while ARA retention increased in response to heat. We conclude that chytrids support pelagic ecosystem functioning under cyanobacteria blooms and global warming via chytrids-conveyed PUFA toward higher trophic levels

Simulation and Performance Optimization of an Amperometric Histamine Detection System

One of the most widely known biogenic amines is histamine, which plays an important role in the human immune system. Some people suffer from allergic reactions after a histamine-rich diet; this is called histamine intolerance. The aim of this work is to develop a quick and reliable method for the detection and quantification of histamine in food, based on an electrochemical approach. In presence of biogenic amines, a reduction cascade induces a current at the working electrode. Prior to chronoamperometric measurements, Finite Elemente simulations were performed. The results are presented in this work

Protocols for sample preparation and compound-specific stable-isotope analyses (δ2H, δ13C) of fatty acids in biological and environmental samples

Compound-specific stable-isotope analysis (CSIA) of fatty acids is a powerful tool to better understand the trophic transfer of fatty acids and their biochemical fate in and across ecosystems, including tracing animal migration and understanding physiological processes. The non-exchangeable nature of C–H bonds in acyl chains, hydrogen (2H) and carbon (13C) stable-isotope values of fatty acids (FA) provide independent information about the origins of fatty acids. Several technical obstacles must be overcome to ensure accurate and reproducible measurements of FA-CSIA can be made. This protocol describes the sample preparation process for successful stable-isotope analyses of fatty acids obtained from environmental and biological samples. Numerous techniques for the preanalytical processing of fatty acid samples are available, and these often have minimal impact on values. Here, we provide an in-depth guide detailing our well-established laboratory protocols, ranging from the initial sample preparation, lipid extraction, and transmethylation to the instrumental arrangement, data collection, and analysis. •Protocol from obtaining a sample to standardized fatty acid specific 2H and 13C values. •Separate GC analysis procedures for C and H are recommended for optimal performance.peerReviewe

Mass spectrometry imaging reveals the spatial distribution of essential lipids in <i>Daphnia magna –</i> potential implications for trophic ecology

Lipids and fatty acids are key dietary components for the nutrition of organisms at all trophic levels. They are required to build cellular structures, such as cell membranes, serve as energy storage and are taking part in signal transduction cascades. For decades, ecological research investigated how dietary fatty acid availability contributes to the fitness of individuals and their populations. The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) found particular interest, as its dietary availability determines the fitness of many aquatic consumers. Due to the small body size of zooplankton, only bulk tissue fatty acid analysis was so far performed and thus the tissue-specific importance of EPA for zooplankton remained elusive. Here we used matrix-assisted laser desorption ionization – mass spectrometry imaging (MALDI-MSI) to reveal the tissue-specific distribution of functional phospholipids in the herbivorous zooplankton Daphnia magna. We demonstrate several lipid species for heart, eggs, gut, gonads, somatic and neurological tissues of D. magna, including the compound eye, as well as the optical and cerebral ganglion. The compound eye revealed a large diversity in EPA-containing lipid species, which were also found in other neurological tissues and the eggs. Such knowledge of tissue-specific fatty acid requirements is essential to investigate how selective allocation of dietary fatty acids within this key grazer on a functional and molecular level affects processes from the individual to food web scales. This methodological advancement will allow investigations on how invertebrates’ physiology and behaviour adjust to changing environmental conditions and potentially affect food web structures including the trophic transfer of dietary fatty acids.</p

Decomposition rate and biochemical fate of carbon from natural polymers and microplastics in boreal lakes

Microbial mineralization of organic compounds is essential for carbon recycling in food webs. Microbes can decompose terrestrial recalcitrant and semi-recalcitrant polymers such as lignin and cellulose, which are precursors for humus formation. In addition to naturally occurring recalcitrant substrates, microplastics have been found in various aquatic environments. However, microbial utilization of lignin, hemicellulose, and microplastics as carbon sources in freshwaters and their biochemical fate and mineralization rate in freshwaters is poorly understood. To fill this knowledge gap, we investigated the biochemical fate and mineralization rates of several natural and synthetic polymer-derived carbon in clear and humic lake waters. We used stable isotope analysis to unravel the decomposition processes of different 13C-labeled substrates [polyethylene, polypropylene, polystyrene, lignin/hemicellulose, and leaves (Fagus sylvatica)]. We also used compound-specific isotope analysis and molecular biology to identify microbes associated with used substrates. Leaves and hemicellulose were rapidly decomposed compared to microplastics which were degraded slowly or below detection level. Furthermore, aromatic polystyrene was decomposed faster than aliphatic polyethylene and polypropylene. The major biochemical fate of decomposed substrate carbon was in microbial biomass. Bacteria were the main decomposers of all studied substrates, whereas fungal contribution was poor. Bacteria from the family Burkholderiaceae were identified as potential leaf and polystyrene decomposers, whereas polypropylene and polyethylene were not decomposed.peerReviewe

Parasitic Chytrids Upgrade and Convey Primary Produced Carbon During Inedible Algae Proliferation

International audienceMicrobial parasites have only recently been included in planktonic food web studies, but their functional role in conveying dietary energy still remains to be elucidated. Parasitic fungi (chytrids) infecting phytoplankton may constitute an alternative trophic link and promote organic matter transfer through the production of dissemination zoospores. Particularly, during proliferation of inedible or toxic algal species, such as large Cyanobacteria fostered by global warming, parasites can constitute an alternative trophic link providing essential dietary nutrients that support somatic growth and reproduction of consumers. Using phytoplankton-parasites associations grown under laboratory controlled conditions we assessed the fatty acids and biochemical composition of species with different nutritional quality and followed the metabolic pathway from the algal host and their parasites zoospores using compound specific stable isotope analysis. This study demonstrated that chytrids are trophic upgraders able to retain essential nutrients that can be transferred to upper trophic levels both in terms of organic matter quantity and nutritional quality. Through the production of zoospores, nutritionally important long-chain polyunsaturated fatty acids that can be consequently assimilated by consumers. We conclude that parasitism at the base of aquatic food webs may represent a crucial trophic link for dietary nutrients and essential biomolecules alternative to herbivory or bacterivory, which can be particularly crucial during the proliferation of inedible or nutritionally inadequate algal species fostered by climate change