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The European Community has set a milestone in the European water policy in 2000: all water directives and policies were united into one comprehensive document – the European Water Framework Directive (EU WFD). The EU WFD requires the monitoring of 45 priority substances, primarily in the water phase, which is not related to a substantial amount of chemicals available on the market worldwide (about 50,000). About 60% of these are human and environmentally toxic. Hence, the currently monitored 45 priority substances are not even close to being sufficient to provide a comprehensive picture of the actual chemical pollution in the aquatic environment.
Furthermore, the EU WFD in its original shape paid less attention to sediments as an important source and sink for chemical contamination. Under stable hydrological conditions, polluted old sediments are covered by less polluted younger sediments preventing erosion of deeper sediment layers and, therefore, the release of particle-bound contaminants. However, urbanization, deforestation, flooding, dredging, riverbed renaturation, and stormwater overflow basin releases can lead to an unpredictable release of particle-bound pollutants. Therefore, in 2008, sediments were added to the EU WFD as a monitoring matrix for substances that tend to accumulate there. As a result, after 18 years of the EU WFD, less than half of all European waterbodies reached a good ecological (40%) and chemical (38%) status.
One of the primary pollution sources in aquatic ecosystems are wastewater treatment plants (WWTPs). Advanced wastewater treatment by ozonation is promising to remove most micropollutants. However, the knowledge about the possible improvement of the receiving waterbody is rare. The latter aspects were the main reasons for the start of the DemO3AC project in 2014. The study area was located in the federal state of North Rhine-Westphalia (Germany). The study area included the Wurm River and its tributary, the Haarbach River. Both waterbodies act as receiving waterbodies for WWTPs. One of them is the Aachen-Soers WWTP (receiving waterbody: Wurm River), upgraded by full stream ozonation as an advanced effluent treatment. Therefore, the extensive investigation program within the DemO3AC project included an investigation of the ecological and chemical status of both receiving waterbodies and the investigation of a possible improvement of the Wurm River after implementing advanced effluent treatment.
The current study was a part of the DemO3AC project and covered the sediment toxicity and a possible impact of the ozonation on aquatic organisms in the receiving waterbody. Time-resolved sampling campaigns allowed investigations under different hydrological conditions, mainly determined by the weather. The first sampling campaign took place in June 2017 during a prolonged dry period with low water flow in the receiving waterbodies. The second sampling campaign was performed exactly one year later (June 2018) after a long rainy period and corresponding high-water levels. Full-stream ozonation at the Aachen-Soers WWTP had been in operation for half a year. Furthermore, a wide range of organic micropollutants was investigated in the effluent of the studied WWTPs to assess a possible hazard emerging from contaminants released into the receiving waterbody.
The study design was developed based on the holistic approach to assessing the ecotoxicological pollution of surface waterbodies. It included the detection of chemical compounds combined with effect-based methods to identify possible drivers of toxicity. The sediment's ecotoxicological assessment included studies on endocrine-disrupting activity, genotoxic and embryotoxic potentials. These endpoints were evaluated using in vitro and in vivo bioassays. In addition, sediments’ chemical profiling was performed using modern analytical chemistry techniques.
The genotoxic potential was investigated using the Ames fluctuation assay with Salmonella typhimurium bacterial strains TA98, TA100, YG1041, and YG1042, sensitive to different classes of compounds, and the Micronucleus assay as a eukaryotic assay with mammalian cells. A unique feature of the present study was the implementation of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay. Moreover, a comprehensive genotoxicity ranking of chemical compounds identified in sediments was used and combined with statistical analysis to identify the drivers of genotoxicity. The results of this study were published in Shuliakevich et al. (2022a) (see also Annex 1), describing the mutagenic potential of all sampling sites, which was primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. In addition, the rainwater overflow basin was identified as a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay to assess the different classes of mutagenic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.
The sediments' endocrine-disruptive activity was investigated using the cell-based reporter gene CALUX® assay. A simultaneous launch of the full-scale effluent ozonation at the Aachen-Soers WWTP was used for investigation of the entrance of the ozonated effluent into the Wurm River and the endocrine-disrupting activity in the water phase. A particular focus of the present study was the unique investigation of PAHs as possible drivers of the endocrine-disrupting activity in sediments of the Wurm River. The results of this study were laid down in the publication by Shuliakevich et al. (2022b) (see also Annex 2), describing variations in endocrine-disrupting activity in the Wurm River under different weather conditions. Briefly, under stable hydrological conditions in June 2017, the estrogenic and the antiandrogenic activities in sediments of the Wurm River were within the range of 0.03-0.1 ng E2 equivalents (eq.)/g dry weight sediment equivalents (dw SEQ) and 3.0-13.9 µg Flu eq./g dw SEQ, respectively. After extensive rain events in June 2018, the sediments' estrogenic and antiandrogenic activities were detected within the range of 0.06-0.2 ng E2 eq./g dw SEQ and 1.7-39.2 µg Flu eq./g de SEQ, respectively. Increased endocrine-disruptive activity (up to 0.2 ng E2 eq./g dw SEQ in ERα- and 39.2 µg Flu eq./g dw SEQ in anti-AR-CALUX® assays) in sediments downstream of the rainwater overflow basin suggested it as a possible source of pollution. A unique result of the second study was finding a positive correlation between measured particle-bound antiandrogenic activity and detected polyaromatic hydrocarbons (PAHs) ...
Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish
(2022)
The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring.
To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish.
Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish.
To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively.
On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling.
Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.
The heart is the first functional organ that develops in the embryo. To become a functional organ, it undergoes several morphogenetic processes. These morphogenetic events involve different cell types, that interact with each other and respond to the surrounding extracellular matrix, as well as intrinsic and extrinsic mechanical forces, assuming different behaviors. Additionally, transcription factor networks, conserved among vertebrates, control the development.
To have a better understanding of cell behavior during development, it is necessary to find a model system that allows the investigation in vivo and at single-cell resolution. Thanks to the common evolutionary origin of the different cardiac structures, together with the conserved molecular pathways, the two-chambered zebrafish heart offers many advantages to study cell behavior during cardiac morphogenesis. Here, using the zebrafish heart as a model system, I uncovered the cell behavior behind two of the main cardiac morphogenetic events: cardiac wall maturation and cardiac valve formation.
In the first part of this study, I investigated how the cardiac wall is maintained at the molecular level. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required for myocardial wall integrity. Global loss of snai1b leads to the extrusion of CMs away from the cardiac lumen, a process we show is dependent on cardiac contractility. Examining CM junctions in snai1b mutants, we observed that N-cadherin localization was compromised, thereby likely weakening cell-cell adhesion. In addition, extruding CMs exhibit increased actomyosin contractility basally, as revealed by the specific enrichment of canonical markers of actomyosin tension - phosphorylated myosin light chain (active myosin) and the α-catenin epitope α-18. By comparing the transcriptome of wild-type and snai1b mutant hearts at the early stages of CM extrusion, we found the dysregulation of intermediate filament genes in mutants including the upregulation of desmin b. We tested the role of desmin b in myocardial wall integrity and found that CM-specific desmin b overexpression led to CM extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 is a critical regulator of intermediate filament gene expression in CMs and that it maintains the integrity of the myocardial epithelium during embryogenesis, at least in part by repressing desmin b expression.
In the second part of this study, I focused on the behavior of valve cells during cardiac development. Using the zebrafish atrioventricular valve, I focus on the valve interstitial cells which confer biomechanical strength to the cardiac valve leaflets. We find that initially AV endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the extracellular matrix (ECM) between the two EC monolayers, undergo an endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a pro-migratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b, a well-known regulator of epithelial-to-mesenchymal transition. This study shows for the first time that Nfatc1 regulates zebrafish VICs formation regulating valve EMT in part by regulating twist1b expression. Moreover, it proposes the zebrafish valve as an excellent model to study the cellular and molecular process that regulate VIC development and dysfunction.
In conclusion, my work: 1) identified an unsuspected role of Snai1 in maintaining the integrity of the myocardial epithelium, opening new avenues in its role in regulating cellular contractility; 2) uncovered the function of Nfatc1 in the establishment of the VIC, establishing a new model to study valve development and function.
The mammalian frontal and auditory cortices are important for vocal behaviour. Here, using local field potential recordings, we demonstrate for the first time that the timing and spatial pattern of oscillations in the fronto-auditory cortical network of vocalizing bats (Carollia perspicillata) predict the purpose of vocalization: echolocation or communication. Transfer entropy analyses revealed predominantly top-down (frontal-to-auditory cortex) information flow during spontaneous activity and pre-vocal periods. The dynamics of information flow depended on the behavioural role of the vocalization and on the timing relative to vocal onset. Remarkably, we observed the emergence of predominantly bottom-up (auditory-to-frontal cortex) information transfer patterns specific echolocation production, leading to self-directed acoustic feedback. Electrical stimulation of frontal areas selectively enhanced responses to echolocation sounds in auditory cortex. These results reveal unique changes in information flow across sensory and frontal cortices, potentially driven by the purpose of the vocalization in a highly vocal mammalian model.
The mammalian frontal and auditory cortices are important for vocal behavior. Here, using local-field potential recordings, we demonstrate that the timing and spatial patterns of oscillations in the fronto-auditory network of vocalizing bats (Carollia perspicillata) predict the purpose of vocalization: echolocation or communication. Transfer entropy analyses revealed predominant top-down (frontal-to-auditory cortex) information flow during spontaneous activity and pre-vocal periods. The dynamics of information flow depend on the behavioral role of the vocalization and on the timing relative to vocal onset. We observed the emergence of predominant bottom-up (auditory-to-frontal) information transfer during the post-vocal period specific to echolocation pulse emission, leading to self-directed acoustic feedback. Electrical stimulation of frontal areas selectively enhanced responses to sounds in auditory cortex. These results reveal unique changes in information flow across sensory and frontal cortices, potentially driven by the purpose of the vocalization in a highly vocal mammalian model.
Im Rahmen dieser Arbeit wurden verschiedene metabolische Anpassungsmechanismen des humanpathogenen Bakteriums Acinetobacter baumannii an seinen Wirt untersucht. Im ersten Teil wurde die Rolle von verschiedenen Trimethylammoniumverbindungen (Cholin, Glycinbetain und Carnitin) und den zugehörigen Aufnahmesystemen, sowie ihren Stoffwechselwegen während dieses Prozesses analysiert. Für die Analyse der Transportsysteme wurde eine markerlose Vierfachmutante (Δbcct) von A. baumannii generiert, sodass alle bekannten Transportsysteme für die genannten Verbindungen deletiert vorlagen. Wachstumsversuche mit dieser Mutante zeigten, dass es in A. baumannii keine weiteren Transporter für die Aufnahme von Cholin gibt, jedoch weitere primär aktive oder sekundär aktive Transporter für die Aufnahme von Glycinbetain. Weiterhin konnten innerhalb dieser Arbeit die KM-Werte der Transporter bestimmt werden. Verschiedene Virulenz- und Infektionsanalysen führten zu dem Schluss, dass die Transporter keine Rolle bei der Virulenz von A. baumannii spielen. In Genomanalysen konnten die Gene, die für die Enzyme des Oxidationsweges von Cholin zu Glycinbetain kodieren identifiziert werden (Cholin-Dehydrogenase (betA), GlycinbetainAldehyd-Dehydrogenase (betB) und ein potenzieller Regulator (betI)). Es wurden Deletionsmutanten innerhalb dieses Genclusters generiert, mit dessen Hilfe gezeigt werden konnte, dass Cholin unter Salzstress ausschließlich als Vorläufer für das kompatible Solut Glycinbetain fungiert und nicht als kompatibles Solut von A. baumannii genutzt werden kann. Virulenz- und Infektionsstudien mit den Deletionsmutanten zeigten, dass der Cholin-Oxidationsweg keine Rolle bei der Virulenz von A. baumannii spielt.
Die Cholin-Dehydrogenase BetA wurde zusätzlich in E. coli produziert und anschließend mittels NiNTA-Affinitätschromatographie aufgereinigt. Die biochemische Charakterisierung des Enzyms zeigte, dass BetA membranständig ist und die höchste Aktivität bei einem pH-Wert von 9,0 hat. Salze wie NaCl oder KCl hatten keinen Effekt auf die Aktivität des Enzyms, während Glutamat die Aktivität stimulierte.
Weiterhin konnte FAD als Cofaktor identifiziert werden und der KM-Wert ermittelt werden. Zudem konnte gezeigt werden, dass die Oxidation von Cholin zu Glycinbetain unter isoosmotischen Bedingungen zu einem Anstieg der ATP-Konzentration in A. baumannii-Zellsuspensionen führt und damit, dass Cholin als alternative Energiequelle genutzt wird. Das Phospholipid Phosphatidylcholin konnte als natürliche Cholinquelle identifiziert werden. Eine Rolle der Phospholipasen D bei der Abspaltung der Cholin-Kopfgruppe des Phosphatidylcholins konnte ausgeschlossen werden. Die Gene für die Oxidation von Cholin zu Glycinbetain werden ausschließlich in Anwesenheit von Cholin exprimiert, jedoch unabhängig von der extrazellulären Salzkonzentration. Diese Studien zeigten, dass der Cholin-Oxidationsweg eine Rolle in der metabolischen Adaptation von A. baumannii an den Wirt spielt. Phosphatidylcholin kann hier als natürliche Cholinquelle im Wirt genutzt werden, da die Wirtsmembranen aus bis zu 70 % Phosphatidylcholin bestehen. Transportstudien mit Carnitin führten zu dem Schluss, dass der Transporter Aci01347 aus A. baumannii neben Cholin ebenfalls Carnitin transportiert. Wachstumsversuche mit einer aci01347-Mutante bestätigen, dass Aci01347 essenziell für die Aufnahme und anschließende Verwertung von Carnitin als Kohlenstoffquelle ist. Es konnte weiterhin gezeigt werden, dass das Transportergen mit essenziellen Genen für den Carnitin-Abbau in einem Operon liegt. Für die Analyse des Abbauweges von Carnitin wurden markerlose Deletionsmutanten innerhalb des Operons generiert. In Wachstumsstudien mit diesen Mutanten konnte der Abbauweg aufgeklärt werden und der Regulator des Operons identifiziert werden. Carnitin wird hier über Trimethylamin und Malat-Semialdehyd zu D-Malat umgewandelt und anschließend über Pyruvat in den TCA-Zyklus eingespeist. Der Regulator wurde zusätzlich in E. coli produziert und mittels Ni-NTA-Affinitätschromatographie aufgereinigt. Mithilfe von EMSA-Studien konnte die Bindestelle des Regulators auf eine 634 Bp lange DNA-Sequenz stromaufwärts des CarnitinOperons eingegrenzt werden. Durch Transkriptomanalysen konnte gezeigt werden, dass bei Wachstum mit Acetylcarnitin, Carnitin und D-Malat die Expression des Carnitin-Operons induziert wurde. Darüber hinaus wurden die Gene konservierter Aromatenabbauwege wie z. B. des Homogentisatweges, des Phenylacetatweges und des Protocatechuat-Abbaus, verstärkt exprimiert. In G. mellonellaVirulenzstudien konnte eine Rolle des Abbaus von Carnitin bei der Virulenz von A. baumannii nachgewiesen werden. Zusätzlich konnte dieser Effekt dem entstehenden Trimethylamin zugesprochen werden...
Die Vorläuferform der eukaryotischen mRNA (prä-mRNA) durchläuft, eine Reihe von Prozessierungs-Schritte, die schließlich zu der Synthese einer „reifen“ und Exportkompetenten mRNA führt. prä-mRNA Spleißen ist ein essentieller Teilschritt dieser Reifung bei der intragene Sequenzen, sogenannte Introns, von der prä-mRNA entfernt werden, während Exons legiert werden. Das prä-mRNA Spleißen wird durch das Spleißosom katalysiert. Dieser Mega-Dalton Komplex, besteht aus fünf Sub-Komplexen, die sich wiederum aus katalytisch aktiven „kleinen nukleären Ribonukleinsäuren“ (snRNAs) und einer Vielzahl von proteinogenen Faktoren zusammensetzen. Diese Subkomplexe, bezeichnet als snRNPs (small nuclear Ribonucleoprotein Particles), binden die prä-mRNA an charakteristischen Sequenzen und richten die prä-mRNA durch eine Reihe von Konformations-Änderungen so aus, dass benachbarte Exons in Kontakt treten und über eine biochemische Ligations-Reaktion verbunden werden können.
Die Exon- bzw Intronerkennung der snRNPs wird durch zahlreiche Spleißfaktoren reguliert. Eine Proteinfamilie, die essentiell für die Regulierung des Spleißens ist, sind Serin/Arginin-reiche Proteine (SR-Proteine). Diese binden vorzugsweise an das 3‘ oder 5’ Ende von Exons, rekrutieren snRNPs und stimulieren dadurch die Exon-Inklusion. Durch diese Stimulierung können Spleiß-Events reguliert und gezielt spezifische Exons ausgeschlossen oder eingeschlossen werden. Dieser Prozess, der als alternatives Spleißen (AS) bezeichnet wird, tritt in 95% des menschlichen Transkriptoms auf und erweitert die Diversität eines Organismus, da verschiedene Transkripte von demselben Gen erzeugt werden können und folglich die Translation unterschiedlicher Proteine mit distinkten Funktionen ermöglicht wird.
Darüber hinaus verfügt die Zelle durch das AS über eine weitere posttranskriptionale Genregulationsebene, die insbesondere unter zellulären Stressbedingungen zur Expression von alternativen Protein-Isoformen von der Zelle genutzt wird. Eine in medizinischer Hinsicht besonders relevante Stressbedingung ist die sogenannte Hypoxie, die eine Sauerstoff-Unterversorgung von Zellen oder Gewebebereichen beschreibt. Hypoxie bzw. hypoxische Bereiche finden sich in Krebszellen und treten in 90% aller soliden Tumoren auf. Als Teil der Hypoxie Stress-Antwort, verfügt die Zelle über einen Adaptations-Mechanismus, der durch Hypoxieinduzierbare Faktoren (HIF) vermittelt wird. Diese Faktoren induzieren die Transkription zahlreicher Gene und stimulieren die Expression von Stressfaktoren, die an der zellulären Adaption der Hypoxie beteiligt sind. Einer dieser Faktoren ist der vaskuläre endotheliale Wachstumsfaktor A (VEGFA), welcher unter hypoxischen Bedingungen sekretiert wird und dadurch die Proliferation von Endothelzellen, die Neubildung von Blutgefäßen und damit die Vaskularisation des hypoxischen Bereichs stimuliert.
Die zelluläre Anpassung ist jedoch nicht nur auf die transkriptionelle Regulation des HIF-vermittelten Hypoxie Signalwegs beschränkt, sondern wird auf multiplen Genexpressions-Ebenen reguliert. Obwohl bekannt ist, dass tausende Transkripte unter hypoxischen Bedingungen alternativ gespleißt werden, sind die Faktoren, die die zelluläre Stress-Antwort durch AS regulieren, sowie deren molekularer Mechanismus jedoch weitestgehend unbekannt.
Diese Arbeit umfasst die Identifizierung und Charakterisierung von AS Events, sowie den Einfluss und die Regulation von Spleißfaktoren auf AS unter hypoxischen Bedingungen. Hierzu führten wir globale Genexpressions- und AS-Analysen in HeLaKarzinomzelllinien unter Normoxie (21% O2) und Hypoxie (0.2% O2) durch und zeigen, dass 7962 Gene nach 24h Hypoxie unterschiedlich exprimiert werden. Über AS-Analysen konnten 4434 Transkripte identifiziert werden, die bei Hypoxie über AS reguliert sind. Dabei trat „Exon-Skipping“ als das am häufigsten auftretende AS-Events auf. Über PCR basierte Validierungs-Experimente konnten 5 regulierte Transkripte nachgewiesen werden. Dabei weisen Exon 3 und 4 in BORA, Exon 6 in MDM4 und Exon 4-5 in CSSP1 Exon-Skipping Events auf, während Exon-Inklusionen in CEP192 Exon 28 und in der 3’UTR von EIF4A2 validiert werden konnten.
Darüber hinaus wurde im Rahmen der AS-Analyse die Regulation des sogenannten „backsplicings“ bei Hypoxie untersucht. Im Gegensatz zum linearen Spleißens, wird beim backsplicing das 5’Ende und das 3’Ende von Exons verbunden, was die Bildung von sogenannten zirkulären RNAs (circRNAs) zufolge hat. Obwohl nur wenige Funktionen dieser RNA-Klasse bekannt sind, wurde die Regulation von circRNAs während der Zell-Differenzierung sowie in diversen Krebszellen beschrieben. Dabei können circRNAs als microRNA- oder Protein-Schwämme fungieren oder dienen als Protein-Interaktion Plattform und regulieren dabei die Genexpression.
Im Rahmen dieser Dissertation wurden unterschiedliche Aspekte der Verbreitung der Vertreter des Pseudoterranova decipiens Komplexes betrachtet und Fragestellungen zur Ökologie und Humanpathogenität der Parasiten bearbeitet. Sie basiert auf drei (ISI-) Fachartikeln, in denen die Nutzung von Fischparasitengemeinschaften als ökologische Indikatoren für entlegene Ökosysteme des Südpolarmeeres (I), die Modellierung geeigneter Verbreitungsgebiete für Arten mit geringen Vorkommensdaten am Beispiel des P. decipiens Komplexes (II) und das Vorkommen potentiell humanpathogener P. bulbosa in unterschiedlichen Mikrohabitaten in Atlantischem Kabeljau (III) thematisiert wurde.
Die Parasitengemeinschaften der in Studie I untersuchten, nahverwandten Antarktisdorsche (Nototheniinae) Nototheniops larseni (n=40), N. nudifrons (n=40) und Lepidonotothen squamifrons (n=49) unterschieden sich hauptsächlich hinsichtlich seltener Parasitenarten. Pseudoterranova decipiens E zählte zu den häufigsten Parasiten der drei betrachteten Wirtsarten. Die Analyse der Wirtsspektren der auf Artebene bestimmten Parasiten zeigte eine geringe Spezifität antarktischer Fischparasiten im Larven- (z.B. Pseudoterranova decipiens E) und Adultstadium (z.B. Elytrophalloides oatesi). Für eine Nutzung als Bioindikatoren ergibt sich die Empfehlung, nicht auf einzelne Parasitenarten, sondern die Zusammensetzung von Parasitenfaunen zurückzugreifen und Parameter wie Abundanz oder Intensität zu berücksichtigen. Vergleiche mit Literaturdaten legten nahe, dass ein Studiendesign, das den periodischen Vergleich der Parasitierungsmuster von Nototheniinae ermöglichen soll, Standorteffekte berücksichtigen sollte. Da es sich bei der Probennahme demersaler Fische um ein aufwändiges und einschneidendes Verfahren handelt, sollten alternative Samplingmethoden vorangetrieben und eine Datenbasis dafür geschaffen werden.
Um die Belastung von Speisefischen mit potentiell humanpathogenen Parasiten in bestimmten Fanggebieten abzuschätzen, kann anhand von Vorkommens- und Umweltdaten mittels statistischer Modelle die Habitateignung für den Parasiten bestimmt werden. Eine Voraussetzung für eine verlässliche Modellierung bilden die Wahl eines geeigneten Algorithmus und die Qualität der Eingangsdaten. Für die Modellierung geeigneter Verbreitungsgebiete für die sechs Arten des P. decipiens Komplexes wurde im Rahmen von Studie II erstmalig ein biotischer Deskriptor herangezogen. Dem Ansatz lag die Annahme zugrunde, dass das Vorkommen geeigneter Endwirte der entscheidende, limitierende Faktor für die Verbreitung eines Parasiten ist, da nur so der Lebenszyklus geschlossen werden kann. Als Hypothesentest dienten Vergleiche der ökologischen Nischen von Parasiten und ihren spezifischen Endwirten im Nischenraum. Anhand der Endwirtdistanz wurde eine Verbesserung der Modellierungsergebnisse mit MaxEnt, gegenüber der ausschließlich auf abiotischen Prädiktoren basierenden Modellierung, für alle Pseudoterranova Arten, insbesondere jene mit einer geringen Anzahl Fundpunkte, erzielt. Grundsätzlich ist der Ansatz auf marine Parasitenarten, deren spezifische Endwirte verlässliche Vorkommensdaten aufweisen, übertragbar. Die Methode stellt jedoch keinen Ersatz für die Erhebung von Vorkommensdaten dar, weshalb die genetische Bestimmung schwer zu identifizierender Taxa sowie die Angabe von Metadaten in jeder parasitologischen Studie obligatorisch sein sollten.
Die Verteilung potentiell humanpathogener Parasitenstadien in für den menschlichen Verzehr vorgesehenen Fischen kann ein entscheidender Faktor für die Übertragung sein. Im Rahmen von Studie III wurde mit dem Referenztranskriptom von P. bulbosa das erste Transkriptom für eine Art den P. decipiens Komplexes erstellt. Anhand einer differentiellen Genexpressionsanalyse wurde untersucht, was die Verteilung der Parasiten auf unterschiedliche Mikrohabitate beeinflusst haben könnte. Dabei wurden siebzig differentiell exprimierte Gene identifiziert, die in aus Leber (32 Gene) und Viscera (38 Gene) von Atlantischem Kabeljau (Gadus morhua) isolierten Proben von P. bulbosa hochreguliert waren. Eine Erklärung für diesen subtilen Unterschied könnte ein Dauerstadium der P. bulbosa Larven zum Zeitpunkt der Probennahmen sein. Ob sich bestimmte Mikrohabitate innerhalb des Wirtes begünstigend auf den Parasiten auswirken, muss mit Hilfe experimenteller Studien gezeigt werden. Erste in Studie III erhobene Daten zum allergenen Potential von P. bulbosa sollten in serologischen Studien getestet werden. Als Grundlage für die Bewertung des pathogenen Potentials von P. bulbosa, sowie der weiteren Arten des P. decipiens Komplexes, sollten in experimentellen Studien NGS-Daten erhoben werden.
Im Rahmen dieser Dissertation wurde in drei methodisch unterschiedlichen Studien ein Bedarf besserer Referenzdaten aufgezeigt. Bestreben diese Datenlücken zu schließen, um das Potential der Methoden besser ausschöpfen zu können, müssen zukünftig noch weiter verstärkt werden.
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
Fungi play pivotal roles in ecosystem functioning, but little is known about their global patterns of diversity, endemicity, vulnerability to global change drivers and conservation priority areas. We applied the high-resolution PacBio sequencing technique to identify fungi based on a long DNA marker that revealed a high proportion of hitherto unknown fungal taxa. We used a Global Soil Mycobiome consortium dataset to test relative performance of various sequencing depth standardization methods (calculation of residuals, exclusion of singletons, traditional and SRS rarefaction, use of Shannon index of diversity) to find optimal protocols for statistical analyses. Altogether, we used six global surveys to infer these patterns for soil-inhabiting fungi and their functional groups. We found that residuals of log-transformed richness (including singletons) against log-transformed sequencing depth yields significantly better model estimates compared with most other standardization methods. With respect to global patterns, fungal functional groups differed in the patterns of diversity, endemicity and vulnerability to main global change predictors. Unlike α-diversity, endemicity and global-change vulnerability of fungi and most functional groups were greatest in the tropics. Fungi are vulnerable mostly to drought, heat, and land cover change. Fungal conservation areas of highest priority include wetlands and moist tropical ecosystems.
The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.
Tilletia caries and T. laevis, which are the causal agents of common bunt, as well as T. controversa, which causes dwarf bunt of wheat, threaten especially organic wheat farming. The three closely related fungal species differ in their teliospore morphology and partially in their physiology and infection biology. The gene content as well as intraspecies variation in these species and the genetic basis of their separation is unknown. We sequenced the genome of four T. caries, five T. controversa, and two T. laevis and extended this dataset with five publicly available ones. The genomes of the three species displayed microsynteny with up to 94.3% pairwise aligned regions excluding repetitive regions. The majority of functionally characterized genes involved in pathogenicity, life cycle, and infection of corn smut, Ustilago maydis, were found to be absent or poorly conserved in the draft genomes and the biosynthetic pathway for trimethylamine in Tilletia spp. could be different from bacteria. Overall, 75% of the identified protein-coding genes comprising 84% of the total predicted carbohydrate utilizing enzymes, 72.5% putatively secreted proteins, and 47.4% of effector-like proteins were conserved and shared across all 16 isolates. We predicted nine highly identical secondary metabolite biosynthesis gene clusters comprising in total 62 genes in all species and none were species-specific. Less than 0.1% of the protein-coding genes were species-specific and their function remained mostly unknown. Tilletia controversa had the highest intraspecies genetic variation, followed by T. caries and the lowest in T. laevis. Although the genomes of the three species are very similar, employing 241 single copy genes T. controversa was phylogenetically distinct from T. caries and T. laevis, however these two could not be resolved as individual monophyletic groups. This was in line with the genome-wide number of single nucleotide polymorphisms and small insertions and deletions. Despite the conspicuously different teliospore ornamentation of T. caries and T. laevis, a high degree of genomic identity and scarcity of species-specific genes indicate that the two species could be conspecific.
Highlights
• Enables immunostaining and visualization of epitopes deep within brain slices
• Utilizes expansion microscopy to increase imaging resolution
• Optimized for brain organotypic slice cultures and tested in acute brain slices
• Analysis workflow for protein distribution (surface vs. intracellular pool) using Imaris
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Summary
Assessing protein distribution with super-resolution in tissue is often complicated and restrictive. Here, we describe a protocol for immunostaining and expansion microscopy imaging of mouse brain organotypic slice cultures. We detail an Imaris analysis workflow to analyze the surface vs intracellular distribution of AMPA receptors at super-resolution during homeostatic plasticity. We have optimized the protocol for brain organotypic slice culture and tested in acute brain slices. This protocol is suitable to study protein distribution under multiple plasticity paradigms.
he most basic behavioural states of animals can be described as active or passive. While high-resolution observations of activity patterns can provide insights into the ecology of animal species, few methods are able to measure the activity of individuals of small taxa in their natural environment. We present a novel approach in which a combination of automatic radiotracking and machine learning is used to distinguish between active and passive behaviour in small vertebrates fitted with lightweight transmitters (<0.4 g).
We used a dataset containing >3 million signals from very-high-frequency (VHF) telemetry from two forest-dwelling bat species (Myotis bechsteinii [n = 52] and Nyctalus leisleri [n = 20]) to train and test a random forest model in assigning either active or passive behaviour to VHF-tagged individuals. The generalisability of the model was demonstrated by recording and classifying the behaviour of tagged birds and by simulating the effect of different activity levels with the help of humans carrying transmitters. The model successfully classified the activity states of bats as well as those of birds and humans, although the latter were not included in model training (F1 0.96–0.98).
We provide an ecological case-study demonstrating the potential of this automated monitoring tool. We used the trained models to compare differences in the daily activity patterns of two bat species. The analysis showed a pronounced bimodal activity distribution of N. leisleri over the course of the night while the night-time activity of M. bechsteinii was relatively constant. These results show that subtle differences in the timing of species' activity can be distinguished using our method.
Our approach can classify VHF-signal patterns into fundamental behavioural states with high precision and is applicable to different terrestrial and flying vertebrates. To encourage the broader use of our radiotracking method, we provide the trained random forest models together with an R package that includes all necessary data processing functionalities. In combination with state-of-the-art open-source automated radiotracking, this toolset can be used by the scientific community to investigate the activity patterns of small vertebrates with high temporal resolution, even in dense vegetation.
The production of ribosomes is a complicated multistep, that is susceptible to changes occurring within the cell and its environment. The process itself requires many proteins, known as ribosome biogenesis factors (RBFs) and many non-coding RNAs like the small nucleolar RNAs (snoRNAs). While RBFs are required for the accurate processing of the pre-rRNA into mature rRNAs, the snoRNAs act to coordinate and guide enzymes for post-transcriptional modifications, chiefly 2´-O-ribose methylation and pseudouridylation. While ribosome biogenesis is mostly described in human and yeast model eucaryotes, similar detailed studies in the model plant Arabidopsis thaliana are far less explored and understood. Furthermore, for many experimentally confirmed modification sites the according snoRNAs and for many pre-rRNA processing steps the responsible RBFs are missing. Therefore, it is expected that a high number of snoRNAs and RBFs are not identified till yet. For this reason, RNA-deep sequencing was performed in order to identify novel snoRNAs and MS analysis data of nucleoli and nuclei of A. thaliana from a former PhD student were used in order to find new proteins involved in pre-rRNA processing.
In here, it is shown that with RNA deep-sequencing still new snoRNAs and snRNAs can be identified and that detection of predicted snoRNAs can be fulfilled with a) antisense oligonucleotides tagged with fluorescence dyes and b) with radioactive labeled antisense probes. Furthermore, a secondary structure map of the 60S and 40S subunit highlighting the predicted and moreover verified modification sites in 5.8S, 25S and 18S rRNA was created. Especially, the correlation between the modification sites and the guiding snoRNA is highlighted further shedding light on overview about current pre-rRNA modification sites and corresponding guiding snoRNAs. The next chapter reveals the complex and multi-layered existence of the 5.8S rRNA and its numerous precursors. The mutant prp24 (also known as seap1) encoding AtPRP24, is recognized as factor being important for splicing as it is promoting the recruitment of the U4 and U6 snRNAs to the spliceosome. In here, it was found that AtPRP24 is involved in processing of 5.8S rRNA precursors, recognizable by precursors that are over accumulating in the mutant. Moreover, it could be shown for the first time that the plant-specific precursor 5´-5.8S is exported to the cytoplasm, where final cleavage steps of 5.8S rRNA takes place. In the prp24.2 mutant, this precursor is exported at an increased rate to the cytoplasm, where it can be detected in the actively translating ribosomes (polysomes). A lower sensitivity of the mutant seeds to cycloheximide (CHX) suggests that due to the extension at the 5´-end of 5.8S, the structure of the 60S subunit has altered CHX binding. In conclusion, this work highlights the importance and complexity of 5.8S rRNA and its precursors for ribosome biogenesis and displays new insights into pre-rRNA processing in A. thaliana.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components in S. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.
Patients harboring mutations in the gene DEPDC5 often display variations of neurological diseases including epilepsy, autism spectrum disorders (ASD) and other neuro-architectural alterations. DEPDC5 protein has been identified as an amino acid sensor responsible for negatively regulating the mechanistic target of rapamycin (mTOR), a central regulator in cell growth and cell homeostasis. Often, mutations of the DEPDC5 protein result in mTOR hyperactivity leading to abnormal neuronal phenotypes and the generation of excitatory/inhibitory imbalances in animal models. Complete knockout (KO) of DEPDC5 results in death shortly after birth, while inhibition of mTOR activity recovers postnatal death (Marsan et al. 2016). However, heterozygous DEPDC5-KOs in animals have been variable in their disease phenotypes during adulthood indicating developmental differences between subspecies and early development mechanisms which could be impactful on the outcome of the diseases.
To understand the mechanisms underlying DEPDC5 mutations during early development, a novel primary human neural progenitor cell line extracted from fetal tissue was characterized during proliferation and differentiation. CRISPR-Cas9 induced mutations of the DEPDC5 gene resulted in hyperphosphorylation of mTOR signaling processes and rapid expansion of the neuronal population during differentiation. Analysis of transcriptome data identified deregulation amongst p53 signaling, ribosome biogenesis, nucleotide and lipid synthesis as well as protein degradation pathways due to loss of DEPDC5. Disease gene datasets identified a correlation between Tuberous Sclerosis mutations as being more closely associated with DEPDC5 mutations while also finding overlap with some ASD and epilepsy genes. By using the mTOR inhibitor rapamycin, a substantial amount of the deregulated gene network was recovered while also reversing rapid neuronal differentiation caused by loss of DEPDC5. Though we saw increased dendritic arborization and subsequent decreases in dendrite lengths and soma sizes, rapamycin failed to recover these effects suggesting mTOR independent processes produced by DEPDC5-KO. This study provides new insights on the relationship between mutations in DEPDC5 and the functional, genomic and deregulatory networks it intertwines in humans and highlights that the DEPDC5 associated pathomechanisms are not fully related to mTOR hyperactivation, but include independent processes. This also sheds light on the question why rapamycin treatment only partially restores DEPDC5 related phenotypes and gives insight on treatments for DEPDC5 patients.
Abstract
Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being higher in warmer regions and lower in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylation regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.
Author Summary: Variation within species is an important level of biodiversity, and it is key for future adaptation. Besides variation in DNA sequence, plants also harbour heritable variation in DNA methylation, and we want to understand the evolutionary significance of this epigenetic variation, in particular how much of it is under genetic control, and how much is associated with the environment. We addressed these questions in a high-resolution molecular analysis of 207 lines of the common plant field pennycress (Thlaspi arvense), which we collected across Europe, propagated under standardized conditions, and sequenced for their genetic and epigenetic variation. We found large geographic variation in DNA methylation, associated with both DNA sequence and climate of origin. Genetic variation was generally the stronger predictor of DNA methylation variation, but the strength of environmental association varied between different sequence contexts. Climate-of-origin was the strongest predictor in about one third of the differentially methylated regions in the CHH context, which suggests that epigenetic variation may play a role in the short-term climate adaptation of pennycress. As pennycress is currently being domesticated as a new biofuel and winter cover crop, our results may be relevant also for agriculture, particularly in changing environments.
Tree bark constitutes an ideal habitat for microbial communities, because it is a stable substrate, rich in micro-niches. Bacteria, fungi, and terrestrial microalgae together form microbial communities, which in turn support more bark-associated organisms, such as mosses, lichens, and invertebrates, thus contributing to forest biodiversity. We have a limited understanding of the diversity and biotic interactions of the bark-associated microbiome, as investigations have mainly focused on agriculturally relevant systems and on single taxonomic groups. Here we implemented a multi-kingdom metabarcoding approach to analyze diversity and community structure of the green algal, bacterial, and fungal components of the bark-associated microbial communities of beech, the most common broadleaved tree of Central European forests. We identified the most abundant taxa, hub taxa, and co-occurring taxa. We found that tree size (as a proxy for age) is an important driver of community assembly, suggesting that environmental filtering leads to less diverse fungal and algal communities over time. Conversely, forest management intensity had negligible effects on microbial communities on bark. Our study suggests the presence of undescribed, yet ecologically meaningful taxa, especially in the fungi, and highlights the importance of bark surfaces as a reservoir of microbial diversity. Our results constitute a first, essential step toward an integrated framework for understanding microbial community assembly processes on bark surfaces, an understudied habitat and neglected component of terrestrial biodiversity. Finally, we propose a cost-effective sampling strategy to study bark-associated microbial communities across large spatial or environmental scales.
Lifespan Extension of Podospora anserina Mic60-Subcomplex Mutants Depends on Cardiolipin Remodeling
(2022)
Function of mitochondria largely depends on a characteristic ultrastructure with typical invaginations, namely the cristae of the inner mitochondrial membrane. The mitochondrial signature phospholipid cardiolipin (CL), the F1Fo-ATP-synthase, and the ‘mitochondrial contact site and cristae organizing system’ (MICOS) complex are involved in this process. Previous studies with Podospora anserina demonstrated that manipulation of MICOS leads to altered cristae structure and prolongs lifespan. While longevity of Mic10-subcomplex mutants is induced by mitohormesis, the underlying mechanism in the Mic60-subcomplex deletion mutants was unclear. Since several studies indicated a connection between MICOS and phospholipid composition, we now analyzed the impact of MICOS on mitochondrial phospholipid metabolism. Data from lipidomic analysis identified alterations in phospholipid profile and acyl composition of CL in Mic60-subcomplex mutants. These changes appear to have beneficial effects on membrane properties and promote longevity. Impairments of CL remodeling in a PaMIC60 ablated mutant lead to a complete abrogation of longevity. This effect is reversed by supplementation of the growth medium with linoleic acid, a fatty acid which allows the formation of tetra-octadecanoyl CL. In the PaMic60 deletion mutant, this CL species appears to lead to longevity. Overall, our data demonstrate a tight connection between MICOS, the regulation of mitochondrial phospholipid homeostasis, and aging of P. anserina.
Three of the four species of giraffe are threatened, particularly the northern giraffe (Giraffa camelopardalis), which collectively have the smallest known wild population estimates. Among the three subspecies of the northern giraffe, the West African giraffe (Giraffa camelopardalis peralta) had declined to 49 individuals by 1996 and only recovered due to conservation efforts undertaken in the past 25 years, while the Kordofan giraffe (Giraffa camelopardalis antiquorum) remains at <2300 individuals distributed in small, isolated populations over a large geographical range in Central Africa. These combined factors could lead to genetically depauperated populations. We analyzed 119 mitochondrial sequences and 26 whole genomes of northern giraffe individuals to investigate their population structure and assess the recent demographic history and current genomic diversity of West African and Kordofan giraffe. Phylogenetic and population structure analyses separate the three subspecies of northern giraffe and suggest genetic differentiation between populations from eastern and western areas of the Kordofan giraffe’s range. Both West African and Kordofan giraffe show a gradual decline in effective population size over the last 10 ka and have moderate genome-wide heterozygosity compared to other giraffe species. Recent inbreeding levels are higher in the West African giraffe and in Kordofan giraffe from Garamba National Park, Democratic Republic of Congo. Although numbers for both West African and some populations of Kordofan giraffe have increased in recent years, the threat of habitat loss, climate change impacts, and illegal hunting persists. Thus, future conservation actions should consider close genetic monitoring of populations to detect and, where practical, counteract negative trends that might develop.
Vampire bats are the only mammals that feed exclusively on blood. To uncover genomic changes associated with this dietary adaptation, we generated a haplotype-resolved genome of the common vampire bat and screened 27 bat species for genes that were specifically lost in the vampire bat lineage. We found previously unknown gene losses that relate to reduced insulin secretion (FFAR1 and SLC30A8), limited glycogen stores (PPP1R3E), and a unique gastric physiology (CTSE). Other gene losses likely reflect the biased nutrient composition (ERN2 and CTRL) and distinct pathogen diversity of blood (RNASE7) and predict the complete lack of cone-based vision in these strictly nocturnal bats (PDE6H and PDE6C). Notably, REP15 loss likely helped vampire bats adapt to high dietary iron levels by enhancing iron excretion, and the loss of CYP39A1 could have contributed to their exceptional cognitive abilities. These findings enhance our understanding of vampire bat biology and the genomic underpinnings of adaptations to blood feeding.
Die akute myeloische Leukämie (AML) ist eine aggressive Erkrankung des Knochenmarks, welche die Hämatopoese beeinträchtigt und zu Knochenmarksversagen führt. Trotz des Fortschritts in der AML-Therapie bleibt die Prognose für die meisten Patienten schlecht, sodass neue Therapieansätze für die Behandlung dringend benötigt werden. Autophagie, ein kataboler Abbauprozess von zellulären Komponenten, ist nachweislich an der Entstehung von AML beteiligt. Als zentraler Regulator von Zellüberleben, Homöostase und Stoffwechsel, dient die Autophagie als Nährstoffquelle durch die Wiederverwertung von Makromolekülen während begrenzter Energieversorgung. AML-Zellen benötigen ein konstantes Nährstoff- und Energieniveau, um ihre Vermehrung aufrechtzuerhalten. Dies wird durch eine Umstellung von Stoffwechselwegen, insbesondere des mitochondrialen Stoffwechsels einschließlich der oxidativen Phosphorylierung (OXPHOS) und des Tricarbonsäurezyklus (TCA), erreicht.
Mehrere Studien haben die Hemmung der Autophagie für die Behandlung von Krebs als vielversprechenden Ansatz vorgestellt. Doch eine Monotherapie mit Autophagie-Inhibitoren erzielte nur eine geringfügige Wirksamkeit. Eine mögliche Erklärung hierfür ist die Entstehung von Kompensationsmechanismen, die zum Ausgleich der Autophagie-Hemmung in Krebszellen entstehen. Bis heute sind diese Kompensationsmechanismen kaum untersucht. Ziel dieser Arbeit ist es, ein geeignetes Autophagie-Gen zu identifizieren, mit dem sich die Rolle der Autophagie-Hemmung für das Überleben von AML-Zellen untersuchen lässt. Zusätzlich sollen die kompensatorischen Mechanismen, die durch die Autophagie-Hemmung in AML-Zellen entstehen können, untersucht werden, um neue metabolische Angriffspunkte zu identifizieren, die für Kombinationstherapien genutzt werden können.
Zu Beginn der Arbeit wurde ein gezielter CRISPR/Cas9 Screen in zwei humanen AML-Zelllinien durchgeführt, um Autophagie-Gene zu identifizieren, deren Verlust eine Proliferationsstörung in AML-Zellen verursacht, welche überwunden werden kann. Validierungsexperimente zeigten, dass der Verlust von ATG3 das Zellwachstum signifikant verminderte. Außerdem zeigte die Messung des Autophagie-Fluxes, dass der Verlust von ATG3 die Autophagie stark beeinträchtigte. Dies wurde durch eine Western-Blot-Analyse, die eine beeinträchtigte LC3-Lipidierung zeigte, und durch eine Immunfluoreszenzanalyse der Autophagosomen-Bildung mittels konfokaler Mikroskopie, die eine geringere Anzahl von Autophagosomen in ATG3-defizienten Zellen ergab, bestätigt. Deshalb wurde der Knockdown von ATG3 in AML Zellen verwendet, um die Mechanismen, die zum Ausgleichen der Autophagie-Hemmung entstehen, zu untersuchen. Zuerst wurde die Zellproliferation in fünf verschiedenen AML Zelllinien über sieben Tage betrachtet. In allen Zellenlinien führte der Verlust von ATG3 mittels small hairpin RNA zu verminderter Zellproliferation. Diese Ergebnisse zeigen die wichtige Rolle von ATG3 in der Autophagie und dass Autophagie-Hemmung durch ATG3-Verlust das Wachstum von AML-Zellen beeinträchtigt.
Da der Verlust von ATG3 die Proliferation von AML-Zellen beeinträchtigte, wurde eine Zellzyklusanalyse durchgeführt. Eine reduzierte S-Phase bestätigte die verminderte Proliferation in ATG3-depletierten AML-Zellen, doch der Zellzyklus war grundsätzlich nicht gestoppt. Darüber hinaus ergab die Analyse der Apoptose, dass diese unter dem Verlust von ATG3 erhöht war, aber etwa 50% der Zellen blieben vital. Diese Beobachtungen deuten darauf hin, dass AML-Zellen trotz des Verlusts der ATG3-abhängigen Autophagie weiter proliferieren können.
Um die Mechanismen zur Kompensation der Autophagie-Hemmung zu untersuchen, wurden die Auswirkungen des ATG3-Verlusts auf die mitochondriale Homöostase untersucht. Die Mitophagie sowie das mitochondriale Membranpotenzial und die Masse unterschieden sich zwischen Kontroll- und ATG3-depletierten AML-Zellen nicht, was darauf hindeutet, dass die mitochondriale Homöostase durch den Verlust von ATG3 nicht beeinträchtigt ist. Als nächstes wurde die mitochondriale Funktion durch Messung des ATP-Spiegels und der OXPHOS untersucht. Die ATP-Level und die OXPHOS waren nach dem Verlust von ATG3 in AML-Zellen erhöht, was auf eine gesteigerte mitochondriale Aktivität bei Autophagie-Defizienz hinweist.
Due to their sessile nature, plants are constantly exposed to an everchanging environment. When these changes exceed certain limits, they can significantly impact plant growth and development, which, in case of crop plants, has consequences on food security. Exposure to high temperatures causes heat stress (HS), one of the most devastating stresses that plants can face. The survival and recovery from HS are dependent on the activation of the HS response (HSR), a collection of molecular mechanisms conferring HS tolerance by maintaining the cellular homeostasis. Stress responses follow a strictly orchestrated network of signal perception and -transduction, ultimately resulting in an adaptive cellular output. Thereby, the massive reshaping of the transcriptome plays a major part, in which heat stress transcription factors (HSFs) play the key role by inducing the expression of HS-responsive genes, including heat shock proteins and other transcription factors. Additionally, alternative splicing (AS), the selective usage of splice sites, contributes to the rapid adjustment of the transcriptome landscape by producing different mRNA variants from a single gene. Consequently, this results in the reduction of translatable transcripts by nonsense-mediated mRNA-decay or nuclear retention, but also enhances the proteome diversity by allowing the synthesis of protein isoforms with distinct functions. AS thereby modulates the activity of important regulatory factors like HSFA2 in Solanum lycopersicum (tomato). HSFA2 is the key factor of acquired thermotolerance (ATT), which enables the ability to survive a potentially lethal HS through pre-exposure to a preceding mild HS. Temperature-dependent AS leads to the synthesis of two HSFA2 protein variants, whereby inhibition of splicing ensures the synthesis of the stable isoform HSFA2-I that is required for ATT.
Transcriptome analysis of several plant species exposed to HS has highlighted the strong impact of high temperatures on the regulation of pre-mRNA splicing. Despite its importance, little is known about the molecular basis of the AS regulation in plants. Particularly for an economically important crop like tomato, understanding the regulation of HS-sensitive AS will contribute to the description of such an important regulatory mechanism but also might offer new insights for increasing HS resilience. Serine/arginine-rich proteins (SR proteins) are central regulators of constitutive and AS by modulating the splice site selection by the spliceosome. This study describes two members of the RS2Z subfamily of SR proteins in tomato, namely RS2Z35 and RS2Z36, which act as core regulators of AS under HS and consequently as central factors for thermotolerance. This study investigates the interaction of the two RS2Z proteins with the HSFA2 pre-mRNA and provides evidence for their function as splicing repressors in this particular AS event. Thereby, RS2Z proteins play an important role in the HSR by modulating the AS of the key factor of the ATT. Furthermore, based on global transcriptome analysis of knockout mutants of single or both RS2Z genes, it is demonstrated that RS2Z proteins are involved in the splicing of pre-mRNAs of almost 2000 genes. Moreover, RS2Z proteins act as splicing regulators and take part in a large portion of HS-induced AS events, thus playing a broader role in AS regulation. Furthermore, the HS-induced RS2Z36 is involved in basal thermotolerance (BTT), highlighting its importance for the basic HS resilience capacity of tomato. In addition, RNA sequencing demonstrates that RS2Z proteins–especially RS2Z36–regulate the expression of proteins involved in plant immunity. The study thereby provides experimental evidence for the important and essential role of SR proteins for plant thermotolerance and suggests the existence of RS2Z-mediated crossroads of different stress responses.
Abstract
Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being lower in colder regions and in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylated regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.
Author summary
Variation within species is an important level of biodiversity, and it is key for future adaptation. Besides variation in DNA sequence, plants also harbour heritable variation in DNA methylation, and we want to understand the evolutionary significance of this epigenetic variation, in particular how much of it is under genetic control, and how much is associated with the environment. We addressed these questions in a high-resolution molecular analysis of 207 lines of the common plant field pennycress (Thlaspi arvense), which we collected across Europe, propagated under standardized conditions, and sequenced for their genetic and epigenetic variation. We found large geographic variation in DNA methylation, associated with both DNA sequence and climate of origin. Genetic variation was generally the stronger predictor of DNA methylation variation, but the strength of environmental association varied between different sequence contexts. Climate-of-origin was the strongest predictor in about one third of the differentially methylated regions in the CHH context, which suggests that epigenetic variation may play a role in the short-term climate adaptation of pennycress. As pennycress is currently being domesticated as a new biofuel and winter cover crop, our results may be relevant also for agriculture, particularly in changing environments.
This work characterizes the post-PKS modifications of AQ-256. Additionally, the second part describes the establishment of an AQ production platform for electrolyte generation that can be utilized in redox-flow-batteries. Lastly, a silent BGC that encodes the genes for terpenoid biosynthesis was described and characterized with regards to product formation and putative ecological function.
Na+ homeostasis in Acinetobacter baumannii is facilitated via the activity of the Mrp antiporter
(2022)
The human opportunistic pathogen Acinetobacter baumannii is a global threat to healthcare institutions worldwide, since it developed very efficient strategies to evade host defence and to adapt to the different environmental conditions of the host. This work focused on the importance of Na+ homeostasis in A. baumannii with regards to pathobiological aspects. In silico studies revealed a homologue of a multicomponent Na+/H+ antiporter system. Inactivation of the Mrp antiporter through deletion of the first gene (mrpA′) resulted in a mutant that was sensitive to increasing pH values. Furthermore, the strain was highly sensitive to increasing Na+ and Li+ concentrations. Increasing Na+ sensitivity is thought to be responsible for growth impairment in human fluids. Furthermore, deletion of mrpA′ is associated with energetic defects, inhibition of motility and survival under anoxic and dry conditions.
The opportunistic human pathogen Acinetobacter baumannii can grow with carnitine but its metabolism, regulation and role in virulence remained elusive. Recently, we identified a carnitine transporter encoded by a gene closely associated with potential carnitine degradation genes. Among those is a gene coding for a putative d-malate dehydrogenase (Mdh). Deletion of the mdh gene led to a loss of growth with carnitine but not l-malate; growth with d-malate was strongly reduced. Therefore, it is hypothesized that d-malate is formed during carnitine oxidation and further oxidized to CO2 and pyruvate and, that not, as previously suggested, l-malate is the product and funnelled directly into the TCA cycle. Mutant analyses revealed that the hydrolase in this cluster funnels acetylcarnitine into the degradation pathway by deacetylation. A transcriptional regulator CarR bound in a concentration-dependent manner to the intergenic region between the mdh gene, the first gene of the carnitine catabolic operon and the carR gene in the presence and absence of carnitine. Both carnitine and d-malate induced CarR-dependent expression of the carnitine operon. Infection studies with Galleria mellonella larvae demonstrated a strong increase in virulence by addition of carnitine indicating that carnitine degradation plays a pivotal role in virulence of A. baumannii.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions, using signals from nanopore direct RNA sequencing. CHEUI processes observed and expected signals with convolutional neural networks to achieve high single-molecule accuracy and outperform other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
Understanding how species relate mechanistically to their environment via traits is a central goal in ecology. Many macroecological rules were found for macroorganisms, however, whether they can explain microorganismal macroecological patterns still requires investigation. Further, whether macroecological rules are also applicable in microclimates is largely unexplored. Here we use fruit body-forming fungi to understand both aspects better. A recent study showed first evidence for the thermal-melanism hypothesis (Bogert’s rule) in fruit body-forming fungi and relied on a continental spatial scale with large grid size. At large spatial extent and grid sizes, other factors like dispersal limitation or local microclimatic variability might influence observed patterns besides the rule of interest. Therefore, we test fungal assemblage fruit body color lightness along a local elevational gradient (mean annual temperature gradient of 7°C) while considering the vegetation cover as a proxy for local variability in microclimate. Using multivariate linear modeling, we found that fungal fruiting assemblages are significantly darker at lower mean annual temperatures supporting the thermal-melanism hypothesis. Further, we found a non-significant trend of assemblage color lightness with vegetation cover. Our results support Bogert’s rule for microorganisms with macroclimate, which was also found for macroorganisms.
Establishing management programs to preserve the benthic communities along the NW Pacific and the Arctic Ocean (AO) requires a deep understanding of the composition of communities and their responses to environmental stressors. In this study, we thus examine patterns of benthic community composition and patterns of species richness along the NW Pacific and Arctic Seas and investigate the most important environmental drivers of those patterns. Overall we found a trend of decreasing species richness toward higher latitudes and deeper waters, peaking in coastal waters of the eastern Philippines. The most dominant taxa along the entire study area were Arthropoda, Mollusca, Cnidaria, Echinodermata, and Annelida. We found that depth, not temperature, was the main driver of community composition along the NW Pacific and neighboring Arctic Seas. Depth has been previously suggested as a factor driving species distribution in benthic fauna. Following depth, the most influential environmental drivers of community composition along the NW Pacific and the Arctic Ocean were silicate, light, and currents. For example, silicate in Hexactinellida, Holothuroidea, and Ophiuroidea; and light in Cephalopoda and Gymnolaemata had the highest correlations with community composition. In this study, based on a combination of new samples and open-access data, we show that different benthic communities might respond differently to future climatic changes based on their taxon-specific biological, physiological, and ecological characteristics. International conservation efforts and habitat preservation should take an adaptive approach and apply measures that take the differences among benthic communities in responding to future climate change into account. This facilitates implementing appropriate conservation management strategies and sustainable utilization of the NW Pacific and Arctic marine ecosystems.
Out-of-school laboratories, also called student labs, are an advantageous opportunity to teach biological subjects. Particularly in the case of complex fields such as neurobiology, student labs offer the opportunity to learn about difficult topics in a practical way. Due to numerous advantages, digital student labs are becoming increasingly popular nowadays. In this study, we investigated the effect of an electrophysiological setup for a virtual experiment with and without hands-on elements on participant motivation and technology acceptance. For this purpose, 235 students were questioned during a student laboratory day. The surveyed students showed high motivation and technology acceptance for the virtual experiment. In the comparison, the electrophysiological setup with hands-on elements performs better in the intrinsic components than the setup without hands-on elements: Thus, the hands-on approach is rated as more interesting and the perceived enjoyment scores higher. Nevertheless, both experimental groups show high values, so that the results of the study support the positive influence of digital laboratory as well as a positive influence of hands-on elements.
Nanoplastics affect the inflammatory cytokine release by primary human monocytes and dendritic cells
(2022)
So far, the human health impacts of nano- and microplastics are poorly understood. Thus, we investigated whether nanoplastics exposure induces inflammatory processes in primary human monocytes and monocyte-derived dendritic cells. We exposed these cells in vitro to nanoplastics of different shapes (irregular vs. spherical), sizes (50–310 nm and polydisperse mixtures) and polymer types (polystyrene; polymethyl methacrylate; polyvinyl chloride, PVC) using concentrations of 30–300 particles cell−1. Our results show that irregular PVC particles induce the strongest cytokine release of these nanoplastics. Irregular polystyrene triggered a significantly higher pro-inflammatory response compared to spherical nanoplastics. The contribution of chemicals leaching from the particles was minor. The effects were concentration-dependent but varied markedly between cell donors. We conclude that nanoplastics exposure can provoke human immune cells to secrete cytokines as key initiators of inflammation. This response is specific to certain polymers (PVC) and particle shapes (fragments). Accordingly, nanoplastics cannot be considered one homogenous entity when assessing their health implications and the use of spherical polystyrene nanoplastics may underestimate their inflammatory effects.
Insects with aquatic life stages can transfer sediment and water pollutants to terrestrial ecosystems, which has been described for metals, polyaromatic hydrocarbons, and polychlorinated chemicals. However, knowledge of the transfer of aquatic micropollutants released by wastewater treatment plants is scarce despite some preliminary studies on their occurrence in riparian spiders. In our study, we address a major analytical gap focusing on the transfer of the micropollutant carbamazepine from the larvae to the adult midges of Chironomus riparius using an optimized QuEChERS extraction method and HPLC–MS/MS applicable to both life stages down to the level of about three individuals. We show that the uptake of carbamazepine by larvae is concentration-dependent and reduces the emergence rate. Importantly, the body burden remained constant in adult midges. Using this information, we estimated the daily exposure of insectivorous tree swallows as terrestrial predators to carbamazepine using the energy demand of the predator and the energy content of the prey. Assuming environmentally relevant water concentrations of about 1 μg/L, the daily dose per kilogram of body weight for tree swallows was estimated to be 0.5 μg/kg/day. At places of high water contamination of 10 μg/L, the exposure may reach 5 μg/kg/day for this micropollutant of medium polarity. Considering body burden changes upon metamorphosis, this study fills the missing link between aquatic contamination and exposure in terrestrial habitats showing that wastewater pollutants can impact birds’ life. Clearly, further analytical methods for biota analysis in both habitats are urgently required to improve risk assessment.
mRNA localization to subcellular compartments has been reported across all kingdoms of life and it is generally believed to promote asymmetric protein synthesis and localization. In striking contrast to previous observations, we show that in S. cerevisiae the B-type cyclin CLB2 mRNA is localized and translated in the yeast bud, while the Clb2 protein, a key regulator of mitosis progression, is concentrated in the mother nucleus. Using single-molecule RNA imaging in fixed (smFISH) and living cells (MS2 system), we show that the CLB2 mRNA is transported to the yeast bud by the She2-She3 complex, via an mRNA ZIP-code situated in the coding sequence. In CLB2 mRNA localization mutants, Clb2 protein synthesis in the bud is decreased resulting in changes in cell cycle distribution and genetic instability. Altogether, we propose that CLB2 mRNA localization acts as a sensor for bud development to couple cell growth and cell cycle progression, revealing a novel function for mRNA localization.
Bird-mediated seed dispersal is crucial for the regeneration and viability of ecosystems, often resulting in complex mutualistic species networks. Yet, how this mutualism drives the evolution of seed dispersing birds is still poorly understood. In the present study we combine whole genome re-sequencing analyses and morphometric data to assess the evolutionary processes that shaped the diversification of the Eurasian nutcracker (Nucifraga), a seed disperser known for its mutualism with pines (Pinus). Our results show that the divergence and phylogeographic patterns of nutcrackers resemble those of other non-mutualistic passerine birds and suggest that their early diversification was shaped by similar biogeographic and climatic processes. The limited variation in foraging traits indicates that local adaptation to pines likely played a minor role. Our study shows that close mutualistic relationships between bird and plant species might not necessarily act as a primary driver of evolution and diversification in resource-specialized birds.
A widespread application of 3D bioprinting in basic and translational research requires accessibility to affordable printers able to produce physiologically relevant tissue models. To facilitate the use of bioprinting as a standard technique in biology, an open-source device based on a consumer-grade 3D stereolithography apparatus (SLA) printer is developed. This SLA bioprinter can produce complex constructs that preserve cell viability and recapitulate the physiology of tissues. The detailed documentation of the modifications apported to the printer as well as a throughout performance analysis allow for a straightforward adoption of the device in other labs and its customization for specific applications. Given the low cost, several modified bioprinters could be simultaneously operated for a parallelized tissue production. To showcase the capability of the bioprinter, constructs consisting of patient-derived cholangiocarcinoma organoids encapsulated in a gelatin methacrylate (GelMA)/polyethylene glycol diacrylate (PEGDA) hydrogel are produced. A thorough characterization of different GelMA/PEGDA ratios reveals that the mechanical properties of the bioprinted tumor model can be accurately fine-tuned to mimic a specific tumor micro-environment. Immunofluorescence and gene expression analyses of tumor markers confirm that the bioprinted synthetic hydrogel provides a flexible and adequate replacement of animal-derived reconstituted extracellular matrix.
Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.
The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids.
Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1-based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1-based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1-based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B-based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.
Only a few studies on the nocturnal behavior of African ungulates exist so far, with mostly small sample sizes. For a comprehensive understanding of nocturnal behavior, the data basis needs to be expanded. Results obtained by observing zoo animals can provide clues for the study of wild animals and furthermore contribute to a better understanding of animal welfare and better husbandry conditions in zoos. The current contribution reduces the lack of data in two ways. First, we present a stand-alone open-source software package based on deep learning techniques, named Behavioral Observations by Videos and Images using Deep-Learning Software (BOVIDS). It can be used to identify ungulates in their enclosure and to determine the three behavioral poses “Standing,” “Lying—head up,” and “Lying—head down” on 11,411 h of video material with an accuracy of 99.4%. Second, BOVIDS is used to conduct a case study on 25 common elands (Tragelaphus oryx) out of 5 EAZA zoos with a total of 822 nights, yielding the first detailed description of the nightly behavior of common elands. Our results indicate that age and sex are influencing factors on the nocturnal activity budget, the length of behavioral phases as well as the number of phases per behavioral state during the night while the keeping zoo has no significant influence. It is found that males spend more time in REM sleep posture than females while young animals spend more time in this position than adult ones. Finally, the results suggest a rhythm between the Standing and Lying phases among common elands that opens future research directions.
The establishment and maintenance of protected areas(PAs) is viewed as a key action in delivering post-2020 biodiversity targets. PAs often need to meet a multitude of objectives, ranging from biodiversity protection to ecosystem service provision and climate change mitigation. As available land and conservation funding are limited, optimizing resources by selecting the most beneficial PAs is vital. Here we present a decision support tool that enables a flexible approach to PA selection on a global scale, allowing different conservation objectives to be weighted and prioritized according to user-specified preferences. We apply the tool across 1347 terrestrial PAs and highlight frequent trade-offs among different objectives, e.g., between biodiversity protection and ecosystem integrity. These results indicate that decision makers must usually decide among conflicting objectives. To assist this our decision support tool provides an explicitly value-based approach that can help resolve such conflicts by considering divergent societal and political demands and values.
Species of the genus Blautia are typical inhabitants of the human gut and considered as beneficial gut microbes. However, their role in the gut microbiome and their metabolic features are poorly understood. Blautia schinkii was described as an acetogenic bacterium, characterized by a functional Wood–Ljungdahl pathway (WLP) of acetogenesis from H2 + CO2. Here we report that two relatives, Blautia luti and Blautia wexlerae do not grow on H2 + CO2. Inspection of the genome sequence revealed all genes of the WLP except genes encoding a formate dehydrogenase and an electron-bifurcating hydrogenase. Enzyme assays confirmed this prediction. Accordingly, resting cells neither converted H2 + CO2 nor H2 + HCOOH + CO2 to acetate. Carbon monoxide is an intermediate of the WLP and substrate for many acetogens. Blautia luti and B. wexlerae had an active CO dehydrogenase and resting cells performed acetogenesis from HCOOH + CO2 + CO, demonstrating a functional WLP. Bioinformatic analyses revealed that many Blautia strains as well as other gut acetogens lack formate dehydrogenases and hydrogenases. Thus, the use of formate instead of H2 + CO2 as an interspecies hydrogen and electron carrier seems to be more common in the gut microbiome.
Many metabolic pathways of eukaryotes are carried out in form of interconnected pathways, which take place in organelles. The organelle membrane separates the reaction compartments from each other, making it a key feature of organelle existence in the cell. To maintain cellular homeostasis, organelle positioning in and transport through the cell as well as organelle interaction are important for the organisms. In plants, organellar movement of peroxisomes, Golgi stacks and mitochondria was shown to be mediated by the actin-myosin machinery. The molecular mechanisms are not elucidated, but working models comprise classical movement mechanisms of motor proteins pulling their cargo on cytoskeletal filaments. In contrast, many mechanisms of chloroplasts movement, which are regulated by blue and red light, are deciphered but follow a different molecular mechanism. Plastidal relatives of the chloroplast have long been disregarded by scientific research but carry out important metabolic reactions to maintain cellular homeostasis. The cellular transport and movement mechanisms of root plastids have not been described in detail until now. Additionally, all plastid subspecies can form tubular structures, called stromules. Those are thought to be involved in the organelle communication and metabolite exchange. Since they are very mobile structures, they influence the organellar dynamic of plastids. This work aimed for an in-detail description of the cellular movements of root plastids in the plant Arabidopsis thaliana to elucidate underlying mechanisms of their movement. Additionally, the dynamics of root plastid stromules were investigated, led by the questions, if and how stromules are involved in the mediation of plastidal movement and their overall dynamics. Plastidal movement in Arabidopsis thaliana was captured using light sheet-based fluorescence microscopy. 4D image data was automatically analyzed using the program Arivis Vision 4D with subsequent manual correction. Additionally to the 4D approach, a manual 3D analysis of plastid and stromule dynamics was performed. The results of the semiautomated analysis displayed heterologous distribution of the plastidal movement. Using a combination of the vector length of each motion event and the angle in relation to previous motion vectors, the proportions of different movement patterns were determined. Main fractions of the data showed undirected motion of plastids, whereas small proportions displayed directed movement with speed up to 8.5 µm/sec. Directed motion was shown to be carried out on defined routes in the cell. Salt stress did not affect plastidal motion, whereas drought stress lead to its reduction. Sucrose depletion led to a drastic decrease of plastidal movement. Additionally, stromule dynamics were investigated using the acquired image data. Stromules were observed in high frequency mainly at stationary plastids giving them the opportunity of dynamic interaction in their cellular surrounding. Stromules reached lengths of up to 60 µm. Additionally, they displayed a variety of movement patterns that contributed greatly to the overall plastid dynamics. Stromule related motion events were captured reaching up to 3.2 µm/sec. Similar to determined plastid dynamics, stromule motions were reduced during drought stress and sucrose depletion, but also were negatively influenced by salt stress. Those results strongly favor an actin-myosin mediated movement machinery mediating the plastidal and stromule movement. This stands in contrast to previous results describing the movement mechanisms of light induced chloroplast movement.
In an additional approach, the molecular mechanisms underlying stromule formation were analyzed. Previous results describe that stromule formation can be induced at isolated chloroplasts of the plant Nicotiana benthamiana by mixing it with concentrated cell extract. During this work, a variation of the described assay was established using the plant Pisum sativum. It was shown that an unknown protein factor presumably undergoing protein-lipid interaction is responsible for in vitro stromule formation. Using a combination of sucrose gradient centrifugation and anion exchange chromatography, the desired factor could be enriched, while the majority of unwanted proteins could be reduced drastically. A following LC-MS analysis revealed a selection of proteins with membrane interaction- and unknown functions that might be involved in in vitro stromule formation.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
NAD is a coenzyme central to metabolism that was also found to serve as a 5’-terminal cap of bacterial and eukaryotic RNA species. The presence and functionality of NAD-capped RNAs (NAD-RNAs) in the archaeal domain remain to be characterized in detail. Here, by combining LC-MS and NAD captureSeq methodology, we quantified the total levels of NAD-RNAs and determined the identity of NAD-RNAs in the two model archaea, Sulfolobus acidocaldarius and Haloferax volcanii. A complementary differential RNA-Seq (dRNA-Seq) analysis revealed that NAD transcription start sites (NAD-TSS) correlate with well-defined promoter regions and often overlap with primary transcription start sites (pTSS). The population of NAD-RNAs in the two archaeal organisms shows clear differences, with S. acidocaldarius possessing more capped small non-coding RNAs (sncRNAs) and leader sequences. The NAD-cap did not prevent 5’→3’ exonucleolytic activity by the RNase Saci-aCPSF2. To investigate enzymes that facilitate the removal of the NAD-cap, four Nudix proteins of S. acidocaldarius were screened. None of the recombinant proteins showed NAD decapping activity. Instead, the Nudix protein Saci_NudT5 showed activity after incubating NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into RNAs and the regulation of ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells.
Importance: This study reports the first characterization of 5’-terminally modified RNA molecules in Archaea and establishes that NAD-RNA modifications, previously only identified in the other two domains of life, are also prevalent in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii. We screened for NUDIX hydrolases that could remove the NAD-RNA cap and showed that none of these enzymes removed NAD modifications, but we discovered an enzyme that hydrolyzes ADPR-RNA. We propose that these activities influence the stabilization of NAD and its thermal degradation to potentially toxic ADPR products at elevated growth temperatures.
In (eco-)toxicological studies the light/dark transition (LDT) test is one of the most frequently used behaviour assays with zebrafish eleutheroembryos. However, study results vary regarding data presentation and analysis and mostly focus on a limited amount of the recorded data. In this study, we investigated whether monitoring two behavioural outcomes (time and distance moved) together with analysing multiple parameters can improve test sensitivity and data interpretation. As a proof of principle 5-day old zebrafish (Danio rerio) eleutheroembryos exposed to either endocrine disruptors (EDs) or acetylcholine esterase (AChE) inhibitors were investigated. We analysed conventional parameters such as mean and sum and implemented additional endpoints such as minimum or maximum distance moved and new parameters assessing the bursting response of eleutheroembryos. Furthermore, changes in eleutheroembryonic behaviour during the moment of the light to dark transition were added. To improve data presentation control-normalised results were displayed in radar charts, enabling the simultaneous presentation of different parameters in relation to each other. This enabled us to identify parameters most relevant to a certain behavioural response. A cut off threshold using control data was applied to identify parameters that were altered in a biological relevant manner. Our approach was able to detect effects on different parameters that remained undetected when analysis was done using conventional bar graphs on - in most cases analysed - averaged, mean distance moved values. By combining the radar charts with additional parameters and by using control-based thresholds, we were able to increase the test sensitivity and promote a deeper understanding of the behaviour response of zebrafish eleutheroembryos in the LDT test and thereby increased its usability for behavioural toxicity studies.
In recent decades, zoos have been increasingly transformed into education centers with the goal of raising awareness about environmental issues and providing environmental education. Probably the simplest and most widespread environmental education program in the zoo is the guided tour. This study therefore aims to test whether a one hour zoo tour has an influence on the participants’ connection to nature and attitude towards species conservation. For this purpose, 269 people who had voluntarily registered for a zoo tour were surveyed before and after the tour. In addition to the regular zoo tour, special themed tours and tours with animal feedings were included. The results show a positive increase in connection to nature and a strengthening of positive attitudes towards species conservation for all tour types. For nature connectedness, in particular, people with an initial high connection to nature benefitted from the special themed tours and the tours, including animal feedings. For attitudes towards species conservation, no difference was found between the tour types. The results prove the positive influence of a very simple environmental education program, even for people with a preexisting high level of connection to nature and positive attitude towards species conservation.