Neue Werkzeuge für Hydra zur Untersuchung von Rho- und PtdInsP- vermittelten Signalwegen

In Vorarbeiten der Arbeitsgruppe konnte gezeigt werden, dass die Inhibition unterschiedlicher Komponenten des FGFR-Rho-ROCK-Myosin II- Signalweges jeweils den gleichen Phänotyp bei Hydra erzeugt und das Ablösen der Knospe verhindert. Dabei konnte deutlich gemacht werden, dass die Knospung ein komplexer, zeitlich exakt abgestimmter Prozess ist, der final in einer Akkumulation des Aktins an der Knospenbasis resultiert, die mit der Phosphorylierung von Myosin II korreliert (Holz et al., 2017; Holz et al., 2020). Ein Hauptregulator der Aktindynamik und der Aktomyosin-Kontraktion sind die kleinen Rho- GTPasen (Fagotto, 2014; Fagotto et al., 2013; Menke und Giehl, 2012). Durch eine Datenbankanalyse mittels BLAST der humanen RhoA- GTPase konnten vier homologe Rho- GTPasen in Hydra identifiziert werden. Phylogenetische Untersuchungen zeigten, dass sich Hv_Rho1-3 deutlich innerhalb der RhoABC- Subfamilie gruppieren, während sich Hv_Rho4 basal zu der Subfamilie einordnet. In-situ-Hybridisierungen zeigten, dass die vier Rho- GTPasen in Hydra an morphogenetisch aktiven Geweberegionen exprimiert werden, wobei vor allem Hv_Rho1 und Hv_Rho2 vermutlich an zellulären Prozessen an der Knospenbasis beteiligt sind. Um aktives RhoA nachzuweisen kann neben der immunhistochemischen Antikörperfärbung das Fusionsprotein RBD-GFP genutzt werden, welches durch die Rho-Binde-Domäne (RBD) spezifisch an aktives RhoA bindet (Berger et al., 2009). Das exprimierte und aufgereinigte Fusionsprotein RBD-GFP konnte als neue Methode zur Detektion von aktiven RhoA- homologen Rho- GTPasen in fixiertem Hydra-Gewebe etabliert werden. Dadurch konnten aktive RhoA- homologe Rho- GTPasen in evaginierenden- (Knospe oder Tentakel) und kontrahierenden Geweben (Knospenbasis und Regeneration) nachgewiesen werden. Die in-silico Analyse der Rho- GEFs Kalirin und Trio verdeutlichte eine komplexe und evolutionär interessante Entwicklung der Rho- GEFs, bezüglicher dynamischer Verluste und Gewinne von spezifischen Proteindomänen. In Hydra konnte interessanterweise lediglich eine Kalirin- homologe Sequenz identifiziert werden, welches möglicherweise die Verbindung zwischen membranständigen Lipiden und Rho- GTPasen vermittelt. Neben dem Rho-ROCK-Myosin II- Signalweg wurde die Beteiligung der Phosphatidylinositolphosphate (PtdInsP) an morphogenetischen Prozessen in Hydra untersucht. Erzeugte transgene Hydra- Linien dienen als wertvolles Werkzeug, um die Aktivität der Phopholipase C (PLC) und der PI3- Kinase live und in vivo beobachten zu können. So konnte die Aktivität der PLC an der Knospenbasis während des Knospungsprozesses sowie die Aktivität der PI3- Kinase an der Schnittstelle während der Regeneration nachgewiesen werden. Weiterhin konnte mit erzeugten PtdInsP-GFP- Sensor Transgenen die apikale Lokalisation des PtdIns(4,5)P2 sowie die basale Lokalisation des PtdIns(3,4,5)P3 nachgewiesen werden. Zur detaillierten in vivo Beobachtung und vor allem zur in vivo Mikroskopie der zellulären Lokalisation der PtdInsP-GFP- Sensoren wurden unterschiedliche Relaxantien getestet, wobei lediglich Linalool und Benzocain eine effektive Immobilisierung bei Hydra erzielten. Beide Relaxantien hatten keinen signifikanten Einfluss auf den Prozess der Knospung oder auf den Prozess des Wundverschlusses nach einem Einschnitt. Die Analyse der Aktinfasern verdeutlichte jedoch, dass Linalool im Gegensatz zu Benzocain, das Aktinzytoskelett des Polypen störte, indem es die Faserlänge deutlich verkürzte und zu einer fehlerhaften Faserorientierung führte. Die Daten der vorliegenden Arbeit zeigen, dass mit dem Fusionsprotein RBD-GFP und mit den PtdInsP-GFP- Sensor transgenen Hydren basierend auf existierenden Werkzeugen für die Analyse der Morhphogenese bei Vertebrata und Fliege, zwei interessante methodische Werkzeuge für Hydra etabliert werden konnten. Erstmals konnte dadurch die Aktivität der Rho- GTPasen an der Knospenbasis während der Knospung sowie die zelluläre Lokalisation von PtdIns(4,5)P2 und PtdIns(3,4,5)P3 in den ektodermalen Epithelmuskelzellen nachgewiesen werden. Die etablierten Methoden können zukünftig weiterhin der detaillierten Untersuchung der Rho- GTPasen, sowie der PLC- und PI3- Kinase- Signalwege und deren Funktionen während morphogenetischer Prozesse in Hydra dienen. Zusätzlich konnte für das Relaxans Benzocain ein Protokoll zur effektiven und Aktinfaser- schonenden Immobilisierung und Relaxierung von Hydra erarbeitet werden. Dieses kann zukünftig zur in vivo Mikroskopie, aber beispielsweise auch als Relaxans zur mechanischen Manipulation, wie dem gezielten Einschneiden des Polypen zur Regeneration, genutzt werden

What lies beneath: Hydra provides cnidarian perspectives into the evolution of FGFR docking proteins

Across the Bilateria, FGF/FGFR signaling is critical for normal development, and in both Drosophila and vertebrates, docking proteins are required to connect activated FGFRs with downstream pathways. While vertebrates use Frs2 to dock FGFR to the RAS/MAPK or PI3K pathways, the unrelated protein, downstream of FGFR (Dof/stumps/heartbroken), fulfills the corresponding function in Drosophila. To better understand the evolution of the signaling pathway downstream of FGFR, the available sequence databases were screened to identify Frs2, Dof, and other key pathway components in phyla that diverged early in animal evolution. While Frs2 homologues were detected only in members of the Bilateria, canonical Dof sequences (containing Dof, ankyrin, and SH2/SH3 domains) were present in cnidarians as well as bilaterians (but not in other animals or holozoans), correlating with the appearance of FGFR. Although these data suggested that Dof coupling might be ancestral, gene expression analysis in the cnidarian Hydra revealed that Dof is not upregulated in the zone of strong FGFRa and FGFRb expression at the bud base, where FGFR signaling controls detachment. In contrast, transcripts encoding other, known elements of FGFR signaling in Bilateria, namely the FGFR adaptors Grb2 and Crkl, which are acting downstream of Dof (and Frs2), as well as the guanyl nucleotide exchange factor Sos, and the tyrosine phosphatase Csw/Shp2, were strongly upregulated at the bud base. Our expression analysis, thus, identified transcriptional upregulation of known elements of FGFR signaling at the Hydra bud base indicating a highly conserved toolkit. Lack of transcriptional Dof upregulation raises the interesting question, whether Hydra FGFR signaling requires either of the docking proteins known from Bilateria

Cooperative Extension and Sustainability Outreach: Programmatic Successes, Administrative Support, and Areas for Improvement

According to the 2018 Intergovernmental Panel on Climate Change, without urgent global changes, climate catastrophe caused by warming of greater than 1.5°C will occur by 2030, endangering the planet\u27s capacity to sustain human populations and livelihoods. The National Network for Sustainable Living Education administered a national survey in January 2017 to assess how well-positioned Extension is to address sustainability in the communities the organization serves. Educators from 40 states responded, and 1,395 usable surveys were received. Survey results will help Extension employees discover opportunities for innovation and relevancy in their programming

Higher measured than modeled ozone production at increased NOx levels in the Colorado Front Range

Abstract. Chemical models must correctly calculate the ozone formation rate, P(O3), to accurately predict ozone levels and to test mitigation strategies. However, air quality models can have large uncertainties in P(O3) calculations, which can create uncertainties in ozone forecasts, especially during the summertime when P(O3) is high. One way to test mechanisms is to compare modeled P(O3) to direct measurements. During summer 2014, the Measurement of Ozone Production Sensor (MOPS) directly measured net P(O3) in Golden, CO, approximately 25 km west of Denver along the Colorado Front Range. Net P(O3) was compared to rates calculated by a photochemical box model that was constrained by measurements of other chemical species and that used a lumped chemical mechanism and a more explicit one. Median observed P(O3) was up to a factor of 2 higher than that modeled during early morning hours when nitric oxide (NO) levels were high and was similar to modeled P(O3) for the rest of the day. While all interferences and offsets in this new method are not fully understood, simulations of these possible uncertainties cannot explain the observed P(O3) behavior. Modeled and measured P(O3) and peroxy radical (HO2 and RO2) discrepancies observed here are similar to those presented in prior studies. While a missing atmospheric organic peroxy radical source from volatile organic compounds co-emitted with NO could be one plausible solution to the P(O3) discrepancy, such a source has not been identified and does not fully explain the peroxy radical model–data mismatch. If the MOPS accurately depicts atmospheric P(O3), then these results would imply that P(O3) in Golden, CO, would be NOx-sensitive for more of the day than what is calculated by models, extending the NOx-sensitive P(O3) regime from the afternoon further into the morning. These results could affect ozone reduction strategies for the region surrounding Golden and possibly other areas that do not comply with national ozone regulations. Thus, it is important to continue the development of this direct ozone measurement technique to understand P(O3), especially under high-NOx regimes

Atmospheric Acetaldehyde: Importance of Air-Sea Exchange and a Missing Source in the Remote Troposphere.

We report airborne measurements of acetaldehyde (CH3CHO) during the first and second deployments of the National Aeronautics and Space Administration (NASA) Atmospheric Tomography Mission (ATom). The budget of CH3CHO is examined using the Community Atmospheric Model with chemistry (CAM-chem), with a newly-developed online air-sea exchange module. The upper limit of the global ocean net emission of CH3CHO is estimated to be 34 Tg a-1 (42 Tg a-1 if considering bubble-mediated transfer), and the ocean impacts on tropospheric CH3CHO are mostly confined to the marine boundary layer. Our analysis suggests that there is an unaccounted CH3CHO source in the remote troposphere and that organic aerosols can only provide a fraction of this missing source. We propose that peroxyacetic acid (PAA) is an ideal indicator of the rapid CH3CHO production in the remote troposphere. The higher-than-expected CH3CHO measurements represent a missing sink of hydroxyl radicals (and halogen radical) in current chemistry-climate models

Transgressive segregation reveals mechanisms of Arabidopsis immunity to Brassica-infecting races of white rust (Albugo candida)

Arabidopsis thaliana accessions are universally resistant at the adult leaf stage to white rust (Albugo candida) races that infect the crop species Brassica juncea and Brassica oleracea. We used transgressive segregation in recombinant inbred lines to test if this apparent species-wide (nonhost) resistance in A. thaliana is due to natural pyramiding of multiple Resistance (R) genes. We screened 593 inbred lines from an Arabidopsis multiparent advanced generation intercross (MAGIC) mapping population, derived from 19 resistant parental accessions, and identified two transgressive segregants that are susceptible to the pathogen. These were crossed to each MAGIC parent, and analysis of resulting F2 progeny followed by positional cloning showed that resistance to an isolate of A. candida race 2 (Ac2V) can be explained in each accession by at least one of four genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. An additional gene was identified that confers resistance to an isolate of A. candida race 9 (AcBoT) that infects B. oleracea. Thus, effector-triggered immunity conferred by distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to these crop pathogens

Sources and characteristics of summertime organic aerosol in the Colorado Front Range: perspective from measurements and WRF-Chem modeling

Abstract. The evolution of organic aerosols (OAs) and their precursors in the boundary layer (BL) of the Colorado Front Range during the Front Range Air Pollution and Photochemistry Éxperiment (FRAPPÉ, July–August 2014) was analyzed by in situ measurements and chemical transport modeling. Measurements indicated significant production of secondary OA (SOA), with enhancement ratio of OA with respect to carbon monoxide (CO) reaching 0.085±0.003 µg m−3 ppbv−1. At background mixing ratios of CO, up to  ∼  1.8 µg m−3 background OA was observed, suggesting significant non-combustion contribution to OA in the Front Range. The mean concentration of OA in plumes with a high influence of oil and natural gas (O&G) emissions was  ∼  40 % higher than in urban-influenced plumes. Positive matrix factorization (PMF) confirmed a dominant contribution of secondary, oxygenated OA (OOA) in the boundary layer instead of fresh, hydrocarbon-like OA (HOA). Combinations of primary OA (POA) volatility assumptions, aging of semi-volatile species, and different emission estimates from the O&G sector were used in the Weather Research and Forecasting model coupled with Chemistry (WRF-Chem) simulation scenarios. The assumption of semi-volatile POA resulted in greater than a factor of 10 lower POA concentrations compared to PMF-resolved HOA. Including top-down modified O&G emissions resulted in substantially better agreements in modeled ethane, toluene, hydroxyl radical, and ozone compared to measurements in the high-O&G-influenced plumes. By including emissions from the O&G sector using the top-down approach, it was estimated that the O&G sector contributed to  <  5 % of total OA, but up to 38 % of anthropogenic SOA (aSOA) in the region. The best agreement between the measured and simulated median OA was achieved by limiting the extent of biogenic hydrocarbon aging and consequently biogenic SOA (bSOA) production. Despite a lower production of bSOA in this scenario, contribution of bSOA to total SOA remained high at 40–54 %. Future studies aiming at a better emissions characterization of POA and intermediate-volatility organic compounds (IVOCs) from the O&G sector are valuable

Transgressive segregation reveals mechanisms of Arabidopsis immunity to Brassica-infecting races of white rust (Albugo candida)

Arabidopsis thaliana accessions are universally resistant at the adult leaf stage to white rust (Albugo candida) races that infect the crop species Brassica juncea and Brassica oleracea. We used transgressive segregation in recombinant inbred lines to test if this apparent species-wide (nonhost) resistance in A. thaliana is due to natural pyramiding of multiple Resistance (R) genes. We screened 593 inbred lines from an Arabidopsis multiparent advanced generation intercross (MAGIC) mapping population, derived from 19 resistant parental accessions, and identified two transgressive segregants that are susceptible to the pathogen. These were crossed to each MAGIC parent, and analysis of resulting F 2 progeny followed by positional cloning showed that resistance to an isolate of A. candida race 2 (Ac2V) can be explained in each accession by at least one of four genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. An additional gene was identified that confers resistance to an isolate of A. candida race 9 (AcBoT) that infects B. oleracea. Thus, effector-triggered immunity conferred by distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to these crop pathogens