TY - JOUR A1 - Yu, Sung-Huan A1 - Vogel, Jörg A1 - Förstner, Konrad U. T1 - ANNOgesic: a Swiss army knife for the RNA-seq based annotation of bacterial/archaeal genomes JF - GigaScience N2 - To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/. KW - genome annotation KW - RNA-seq KW - transcriptomics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-178942 VL - 7 ER - TY - JOUR A1 - Reuter, Christian A1 - Hauf, Laura A1 - Imdahl, Fabian A1 - Sen, Rituparno A1 - Vafadarnejad, Ehsan A1 - Fey, Philipp A1 - Finger, Tamara A1 - Jones, Nicola G. A1 - Walles, Heike A1 - Barquist, Lars A1 - Saliba, Antoine-Emmanuel A1 - Groeber-Becker, Florian A1 - Engstler, Markus T1 - Vector-borne Trypanosoma brucei parasites develop in artificial human skin and persist as skin tissue forms JF - Nature Communications N2 - Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly’s bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites’ development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals. KW - mechanisms of disease KW - parasitology KW - transcriptomics Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-358142 VL - 14 ER - TY - JOUR A1 - Remes, Bernhard A1 - Berghoff, Bork A. A1 - Förstner, Konrad U. A1 - Klug, Gabriele T1 - Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides JF - BMC Genomics N2 - Background: High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the alpha-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen. Results: One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation. Conclusion: Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a "core iron response" that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides. KW - oxidative stress KW - Rhodobacter sphaeroides KW - RNAseq KW - OxyR KW - iron limitation KW - transcriptomics KW - dependent gene-expression KW - hydrogen-peroxide KW - escherichia coli Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115357 SN - 1471-2164 VL - 15 IS - 794 ER - TY - THES A1 - Iosip, Anda-Larisa T1 - Molecular Mechanosensing Mechanisms of the Carnivorous Plant \(Dionaea\) \(muscipula\) T1 - Molekulare Mechanismen der Mechanoperzeption in der fleischfressenden Pflanze \(Dionaea\) \(muscipula\) N2 - Plants are able to sense mechanical forces in order to defend themselves against predators, for instance by synthesizing repellent compounds. Very few plants evolved extremely sensitive tactile abilities that allow them to perceive, interpret and respond by rapid movement in the milliseconds range. One such rarity is the charismatic Venus flytrap (Dionaea muscipula) - a carnivorous plant which relies on its spectacular active trapping strategy to catch its prey. The snapping traps are equipped with touch-specialised trigger hairs, that upon bending elicit an action potential (AP). This electrical signal originates within the trigger hairs’ mechanosensory cells and further propagates throughout the whole trap, alerting the plant of potential prey. Two APs triggered within thirty seconds will set off the trap and more than five APs will initiate the green stomach formation for prey decomposition and nutrient uptake. Neither the molecular components of the plant’s AP nor the Venus flytrap’s fast closure mechanism have been fully elucidated yet. Therefore, the general objective of this study is to expound on the molecular basis of touch perception: from AP initiation to trap closure and finally to stomach formation. The typical electrical signal in plants lasts for minutes and its shape is determined by the intensity of the mechanical force applied. In contrast, the Venus flytrap’s one-second AP is of all-or-nothing type, similar in shape to the animal AP. In order to gain more insight into the molecular components that give rise to the Venus flytrap’s emblematic AP, the transcriptomic landscape of its unique mechanotransducer - the trigger hair – was compared to the rest of the non-specialised tissues and organs. Additionally, the transcriptome of the electrically excitable fully-developed adult trap was compared to non-excitable juvenile traps that are unable to produce sharp APs. Together, the two strategies helped with the identification of electrogenic channels and pumps for each step of the AP as follows: (1) the most specific to the trigger hair was the mechanosensitive channel DmMSL10, making up the best candidate for the initial AP depolarization phase, (2) the K+ outward rectifier DmSKOR could be responsible for repolarisation, (3) further, the proton pump DmAHA4, might kick in during repolarisation and go on with hyperpolarisation and (4) the hyperpolarization- and acid-activated K+ inward rectifier KDM1 might contribute to the re-establishment of electrochemical gradient and the resting potential. Responsible for the AP-associated Ca2+ wave and electrical signal propagation, the glutamate-like receptor DmGLR3.6 was also enriched in the trigger hairs. Together, these findings suggest that the reuse of genes involved in electrical signalling in ordinary plants can give rise to the Venus flytrap’s trademark AP. The Venus flytrap has been cultivated ever since its discovery, generating more than one hundred cultivars over the years. Among them, indistinguishable from a normal Venus flytrap at first sight, the ’ERROR’ cultivar exhibits a peculiar behaviour: it is unable to snap its traps upon two APs. Nevertheless, it is still able to elicit normal APs. To get a better understanding of the key molecular mechanisms and pathways that are essential for a successful trap closure, the ’ERROR’ mutant was compared to the functional wild type. Timelapse photography led to the observation that the ’ERROR’ mutants were able to leisurely half close their traps when repeated mechanostimulation was applied (10 minutes after 20 APs, 0.03 Hz). As a result of touch or wounding in non-carnivorous plants, jasmonic acid (JA) is synthesized, alerting the plants of potential predators. Curiously, the JA levels were reduced upon mechanostimulation and completely impaired upon wounding in the ’ERROR’ mutant. In search of genes accountable for the ’ERROR’ mutant’s defects, the transcriptomes of the two phenotypes were compared before and after mechanostimulation (1h after 10 APs, 0.01 Hz). The overall dampened response of the mutant compared to the wild type, was reflected at transcriptomic level as well. Only about 50% of wild type’s upregulated genes after touch stimulation were differentially expressed in ’ERROR’ and they manifested only half of the wild type’s expression amplitude. Among unresponsive functional categories of genes in ’ERROR’ phenotype, there were: cell wall integrity surveilling system, auxin biosynthesis and stress-related transcription factors from the ethylene-responsive AP2/ERF and C2H2-ZF families. Deregulated Ca2+-decoding as well as redox-related elements together with JA-pathway components might also contribute to the malfunctioning of the ’ERROR’ mutant. As the mutant does not undergo full stomach formation after mechanical treatment, these missing processes represent key milestones that might mediate growth-defence trade-offs under JA signalling. This confirms the idea that carnivory has evolved by recycling the already available molecular machineries of the ubiquitous plant immune system. To better understand the mutant’s defect in the trap snapping mechanism, the ground states (unstimulated traps) of the two phenotypes were compared. In this case, many cell wall-related genes (e.g. expansins) were downregulated in the ’ERROR’ mutant. For the first time, these data point to the importance of a special cell wall architecture of the trap, that might confer the mechanical properties needed for a functional buckling system - which amplifies the speed of the trap closure. This study provides candidate channels for each of the AP phases that give rise to and shape the sharp Venus flytrap-specific AP. It further underlines the possible contribution of the cell wall architecture to the metastable ready-to-snap configuration of the trap before stimulation - which might be crucial for the buckling-dependent snapping. And finally, it highlights molecular milestones linked to defence responses that ensure trap morphing into a green stomach after mechanostimulation. Altogether, these processes prove to be interdependent and essential for a successful carnivorous lifestyle. N2 - Pflanzen sind in der Lage, mechanische Einflüsse zu spüren, um sich gegen Fressfeinde zu verteidigen, indem sie zum Beispiel abweisende Verbindungen synthetisieren. Nur sehr wenige Pflanzen haben extrem sensible taktile Fähigkeiten entwickelt, die es ihnen ermöglichen, schnelle Bewegungen im Millisekundenbereich wahrzunehmen, zu interpretieren und darauf zu reagieren. Eine solche Rarität ist die charismatische Venusfliegenfalle (Dionaea muscipula) - eine fleischfressende Pflanze, die sich auf ihre spektakuläre aktive Fallenstrategie verlässt, um ihre Beute zu fangen. Die Schnappfallen sind mit berührungssensitiven Auslösehaaren ausgestattet, die beim Biegen ein Aktionspotenzial (AP) auslösen. Dieses elektrische Signal entsteht in den mechanosensorischen Zellen der Auslösehaare und breitet sich in der gesamten Falle aus, wodurch die Pflanze auf potenzielle Beute aufmerksam gemacht wird. Zwei APs, die innerhalb von dreißig Sekunden ausgelöst werden, lösen die Falle aus, und mehr als fünf APs leiten die Bildung des grünen Magens ein, der die Beute zersetzt und die Nährstoffe aufnimmt. Weder die molekularen Komponenten des AP der Pflanze noch der Schnellverschlussmechanismus der Venusfliegenfalle sind bisher vollständig geklärt. Daher besteht das allgemeine Ziel dieser Studie darin, die molekularen Grundlagen der Berührungswahrnehmung zu erforschen: von der Initiierung des AP bis zum Schließen der Falle und schließlich zur Magenbildung. Das typische elektrische Signal in Pflanzen dauert Minuten und seine Form wird durch die Intensität der angewandten mechanischen Kraft bestimmt. Im Gegensatz dazu ist das einsekündige AP der Venusfliegenfalle vom Alles-oder-Nichts-Typ und ähnelt in seiner Form dem tierischen AP. Um mehr Einblick in die molekularen Komponenten zu erhalten, die das emblematische AP der Venusfliegenfalle hervorbringen, wurde das Transkriptom ihres einzigartigen Mechanosensors - des Triggerhaars - mit den übrigen nicht spezialisierten Geweben und Organen verglichen. Darüber hinaus wurde das Transkriptom der elektrisch erregbaren, voll entwickelten adulten Falle mit nicht erregbaren juvenilen Fallen verglichen, die keine scharfen APs erzeugen können. Beide Strategien zusammen halfen bei der Identifizierung von elektrogenen Kanälen und Pumpen für jeden Schritt des AP: (1) Am spezifischsten für die Triggerhaare war der mechanosensitive Kanal DmMSL10, der der beste Kandidat für die anfängliche AP-Depolarisationsphase war, (2) der K+-Auswärtsgleichrichter DmSKOR könnte für die Repolarisation verantwortlich sein, (3) ferner, die H+-Pumpe DmAHA4, könnte während der Repolarisation einsetzen und mit der Hyperpolarisation fortfahren und (4) der durch Hyperpolarisation und Säure aktivierte K+-Einwärtsgleichrichter KDM1 könnte zur Wiederherstellung des elektrochemischen Gradienten und des Ruhepotentials beitragen. Der möglicherweise für die AP-assoziierte Ca2+-Welle und die elektrische Signalausbreitung verantwortliche Glutamatrezeptor DmGLR3.6 war ebenfalls in den Triggerhaaren angereichert. Zusammengenommen deuten diese Ergebnisse darauf hin, dass die Wiederverwendung von Genen, die an der elektrischen Signalübertragung in gewöhnlichen Pflanzen beteiligt sind, zu dem für die Venusfliegenfalle typischen AP führen kann. Die Venusfliegenfalle wird seit ihrer Entdeckung kultiviert und hat im Laufe der Jahre mehr als hundert Kultivare hervorgebracht. Die Sorte "ERROR", die auf den ersten Blick nicht von einer normalen Venusfliegenfalle zu unterscheiden ist, weist ein besonderes Verhalten auf: Sie ist nicht in der Lage, ihre Fallen nach dem Auslösen von 2 APs zu schließen. Dennoch ist sie in der Lage, normale APs auszulösen. Um ein besseres Verständnis der molekularen Schlüsselmechanismen und -wege zu erhalten, die für ein erfolgreiches Schließen der Fallen notwendig sind, wurde die "ERROR"-Mutante mit dem funktionalen Wildtyp verglichen. Zeitrafferaufnahmen führten zu der Beobachtung, dass die ’ERROR’-Mutanten in der Lage waren, ihre Fallen bei wiederholter mechanischer Stimulation (10 Minuten nach 20 APs, 0,03 Hz) sehr langsam etwa zur Hälfte zu schließen. Bei nicht karnivoren Pflanzen wird infolge von Berührungen oder Verletzungen Jasmonsäure (JA) synthetisiert, die die Pflanzen vor potenziellen Fressfeinden warnt. Merkwürdigerweise waren die JA-Spiegel bei mechanischer Stimulation reduziert und bei Verwundung in der "ERROR"-Mutante im Gegensatz zum WT überhaupt nicht erhöht. Auf der Suche nach Genen, die für die Defekte der "ERROR"-Mutante verantwortlich sind, wurden die Transkriptome der beiden Phänotypen vor und nach der Mechanostimulation (1 Stunde nach 10 APs, 0,01 Hz) verglichen. Die insgesamt gedämpfte Reaktion der Mutante im Vergleich zum Wildtyp spiegelte sich auch auf transkriptomischer Ebene wider. Nur etwa 50 % der nach Berührungsstimulation hochregulierten Gene des Wildtyps wurden in "ERROR" unterschiedlich exprimiert, und sie wiesen nur die Hälfte der Expressionsamplitude des Wildtyps auf. Zu den nicht reagierenden funktionellen Genkategorien gehörten: das System zur Überwachung der Zellwandintegrität, die Auxin-Biosynthese und stressbezogene Transkriptionsfaktoren aus den auf Ethylen reagierenden AP2/ERF- und C2H2-ZF-Familien. Deregulierte Ca2+-decodierende sowie redoxbezogene Elemente könnten zusammen mit Komponenten des JA-Signalwegs ebenfalls zur Fehlfunktion der "ERROR"-Mutante beitragen. Da die Mutante nach mechanischer Behandlung keine vollständige Magenbildung durchläuft, stellen diese fehlenden Prozesse wichtige Meilensteine dar, die bei der JA-Signalübertragung einen Kompromiss zwischen Wachstum und Verteidigung vermitteln könnten. Dies bestätigt die Idee, dass sich Karnivorie durch die Wiederverwertung bereits vorhandener Signalwege und -komponenten entwickelt hat. Um den Defekt der Mutante im Fallenschnappmechanismus besser zu verstehen, wurden die Grundzustände (unstimulierte Fallen) der beiden Phänotypen verglichen. In diesem Fall waren viele zellwandbezogene Gene (z. B. Expansine) in der "ERROR"-Mutante herunterreguliert. Diese Daten weisen zum ersten Mal auf die Bedeutung einer speziellen Zellwandarchitektur der Falle hin, die möglicherweise die mechanischen Eigenschaften für ein Umklappen der Fallenhälften verleiht, was wiederum die Geschwindigkeit des Fallenschlusses erhöht. Diese Studie liefert Kandidatenkanäle für jede der AP-Phasen, die das scharfe Venusfliegenfallen-spezifische AP hervorbringen und formen. Sie unterstreicht außerdem den möglichen Beitrag der Zellwandarchitektur zur metastabilen, schnappbereiten Konfiguration der Falle vor der Stimulation - die für das durch das Umklappen der Fallenhälften bedingte Zuschnappen der Falle entscheidend sein könnte. Und schließlich werden molekulare Meilensteine hervorgehoben, die mit Abwehrreaktionen verbunden sind und dafür sorgen, dass sich die Falle nach mechanischer Stimulation in einen grünen Magen verwandelt. Insgesamt erweisen sich diese Prozesse als voneinander abhängig und wesentlich für eine erfolgreiche fleischfressende Lebens-weise. KW - carnivorous plants KW - action potential KW - trap closure KW - jasmonic acid KW - mechanosensation KW - touch KW - molecular pathways KW - wounding KW - defence mechanisms KW - transcriptomics KW - Venusfliegenfalle KW - Dionaea muscipula Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-287649 ER - TY - JOUR A1 - Habenstein, Jens A1 - Schmitt, Franziska A1 - Liessem, Sander A1 - Ly, Alice A1 - Trede, Dennis A1 - Wegener, Christian A1 - Predel, Reinhard A1 - Rössler, Wolfgang A1 - Neupert, Susanne T1 - Transcriptomic, peptidomic, and mass spectrometry imaging analysis of the brain in the ant Cataglyphis nodus JF - Journal of Neurochemistry N2 - Behavioral flexibility is an important cornerstone for the ecological success of animals. Social Cataglyphis nodus ants with their age‐related polyethism characterized by age‐related behavioral phenotypes represent a prime example for behavioral flexibility. We propose neuropeptides as powerful candidates for the flexible modulation of age‐related behavioral transitions in individual ants. As the neuropeptidome of C. nodus was unknown, we collected a comprehensive peptidomic data set obtained by transcriptome analysis of the ants’ central nervous system combined with brain extract analysis by Q‐Exactive Orbitrap mass spectrometry (MS) and direct tissue profiling of different regions of the brain by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. In total, we identified 71 peptides with likely bioactive function, encoded on 49 neuropeptide‐, neuropeptide‐like, and protein hormone prepropeptide genes, including a novel neuropeptide‐like gene (fliktin). We next characterized the spatial distribution of a subset of peptides encoded on 16 precursor proteins with high resolution by MALDI MS imaging (MALDI MSI) on 14 µm brain sections. The accuracy of our MSI data were confirmed by matching the immunostaining patterns for tachykinins with MSI ion images from consecutive brain sections. Our data provide a solid framework for future research into spatially resolved qualitative and quantitative peptidomic changes associated with stage‐specific behavioral transitions and the functional role of neuropeptides in Cataglyphis ants. KW - brain KW - MALDI imaging KW - neuropeptides KW - neuropeptidomics KW - social insect KW - transcriptomics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239917 VL - 158 IS - 2 SP - 391 EP - 412 ER - TY - JOUR A1 - Geisinger, Adriana A1 - Rodríguez-Casuriaga, Rosana A1 - Benavente, Ricardo T1 - Transcriptomics of Meiosis in the Male Mouse JF - Frontiers in Cell and Developmental Biology N2 - Molecular studies of meiosis in mammals have been long relegated due to some intrinsic obstacles, namely the impossibility to reproduce the process in vitro, and the difficulty to obtain highly pure isolated cells of the different meiotic stages. In the recent years, some technical advances, from the improvement of flow cytometry sorting protocols to single-cell RNAseq, are enabling to profile the transcriptome and its fluctuations along the meiotic process. In this mini-review we will outline the diverse methodological approaches that have been employed, and some of the main findings that have started to arise from these studies. As for practical reasons most studies have been carried out in males, and mostly using mouse as a model, our focus will be on murine male meiosis, although also including specific comments about humans. Particularly, we will center on the controversy about gene expression during early meiotic prophase; the widespread existing gap between transcription and translation in meiotic cells; the expression patterns and potential roles of meiotic long non-coding RNAs; and the visualization of meiotic sex chromosome inactivation from the RNAseq perspective. KW - meiosis KW - transcriptomics KW - RNAseq KW - meiotic prophase KW - spermatogenesis KW - lncRNAs KW - MSCI KW - spermatogenic cell sorting Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231032 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Esken, Jens A1 - Goris, Tobias A1 - Gadkari, Jennifer A1 - Bischler, Thorsten A1 - Förstner, Konrad U. A1 - Sharma, Cynthia M. A1 - Diekert, Gabriele A1 - Schubert, Torsten T1 - Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two‐component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies JF - MicrobiologyOpen N2 - Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE‐respiring representatives of the genus, uncovered the genetic inactivation of a two‐component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low‐level expression of the TCS operon in fumarate‐adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR‐family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter‐binding assays. The RR bound a cis‐regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the −35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine‐tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide‐respiring bacteria. KW - genomics KW - organohalide respiration KW - RNA sequencing KW - tetrachloroethene KW - transcriptomics KW - two‐component system Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225754 VL - 9 IS - 12 ER - TY - JOUR A1 - Buga, Ana Maria A1 - Margaritescu, Claudiu A1 - Scholz, Claus Jürgen A1 - Radu, Eugen A1 - Zelenak, Christine A1 - Popa-Wagner, Aurel T1 - Transcriptomics of Post-Stroke Angiogenesis in the Aged Brain JF - Frontiers in Aging Neuroscience N2 - Despite the obvious clinical significance of post-stroke angiogenesis in aged subjects, a detailed transcriptomic analysis of post-stroke angiogenesis has not yet been undertaken in an aged experimental model. In this study, by combining stroke transcriptomics with immunohistochemistry in aged rats and post-stroke patients, we sought to identify an age-specific gene expression pattern that may characterize the angiogenic process after stroke. We found that both young and old infarcted rats initiated vigorous angiogenesis. However, the young rats had a higher vascular density by day 14 post-stroke. “New-for-stroke” genes that were linked to the increased vasculature density in young animals included Angpt2, Angptl2, Angptl4, Cib1, Ccr2, Col4a2, Cxcl1, Lef1, Hhex, Lamc1, Nid2, Pcam1, Plod2, Runx3, Scpep1, S100a4, Tgfbi, and Wnt4, which are required for sprouting angiogenesis, reconstruction of the basal lamina (BL), and the resolution phase. The vast majority of genes involved in sprouting angiogenesis (Angpt2, Angptl4, Cib1, Col8a1, Nrp1, Pcam1, Pttg1ip, Rac2, Runx1, Tnp4, Wnt4); reconstruction of a new BL (Col4a2, Lamc1, Plod2); or tube formation and maturation (Angpt1, Gpc3, Igfbp7, Sparc, Tie2, Tnfsf10), had however, a delayed upregulation in the aged rats. The angiogenic response in aged rats was further diminished by the persistent upregulation of “inflammatory” genes (Cxcl12, Mmp8, Mmp12, Mmp14, Mpeg1, Tnfrsf1a, Tnfrsf1b) and vigorous expression of genes required for the buildup of the fibrotic scar (Cthrc1, Il6ra, Il13ar1, Il18, Mmp2, Rassf4, Tgfb1, Tgfbr2, Timp1). Beyond this barrier, angiogenesis in the aged brains was similar to that in young brains. We also found that the aged human brain is capable of mounting a vigorous angiogenic response after stroke, which most likely reflects the remaining brain plasticity of the aged brain. KW - aging KW - stroke KW - transcriptomics KW - angiogenesis Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120700 VL - 6 IS - 44 ER - TY - JOUR A1 - Boulos, Joelle C. A1 - Saeed, Mohamed E. M. A1 - Chatterjee, Manik A1 - Bülbül, Yagmur A1 - Crudo, Francesco A1 - Marko, Doris A1 - Munder, Markus A1 - Klauck, Sabine M. A1 - Efferth, Thomas T1 - Repurposing of the ALK inhibitor crizotinib for acute leukemia and multiple myeloma cells JF - Pharmaceuticals N2 - Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G\(_2\)M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib's ability to prevent mitotic exit. However, cells accumulated in the sub-G\(_0\)G\(_1\) fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma. KW - acute myeloid leukemia KW - drug repurposing KW - multiple myeloma KW - network pharmacology KW - transcriptomics KW - tyrosine kinase inhibitors Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250258 SN - 1424-8247 VL - 14 IS - 11 ER -