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Hematopoietic differentiation is controlled by key transcription factors, which regulate stem cell functions and differentiation. TAL1 is a central transcription factor for hematopoietic stem cell development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation. Knowledge of the target genes controlled by a given transcription factor is important to understand its contribution to normal development and disease. To uncover direct target genes of TAL1 we used high affinity streptavidin/biotin-based chromatin precipitation (Strep-CP) followed by Strep-CP on ChIP analysis using ChIP promoter arrays. We identified 451 TAL1 target genes in K562 cells. Furthermore, we analysed the regulation of one of these genes, the catalytic subunit beta of protein kinase A (PRKACB), during megakaryopoiesis of K562 and primary human CD34+ stem cell/progenitor cells. We found that TAL1 together with hematopoietic transcription factors RUNX1 and GATA1 binds to the promoter of the isoform 3 of PRKACB (Cβ3). During megakaryocytic differentiation a coactivator complex on the Cβ3 promoter, which includes WDR5 and p300, is replaced with a corepressor complex. In this manner, activating chromatin modifications are removed and expression of the PRKACB-Cβ3 isoform during megakaryocytic differentiation is reduced. Our data uncover a role of the TAL1 complex in controlling differential isoform expression of PRKACB. These results reveal a novel function of TAL1, RUNX1 and GATA1 in the transcriptional control of protein kinase A activity, with implications for cellular signalling control during differentiation and disease.
Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.
Background: Butanol isomers are regarded as more suitable fuel substitutes than bioethanol. n-Butanol is naturally produced by some Clostridia species, but due to inherent problems with clostridial fermentations, industrially more relevant organisms have been genetically engineered for n-butanol production. Although the yeast Saccharomyces cerevisiae holds significant advantages in terms of scalable industrial fermentation, n-butanol yields and titers obtained so far are only low.
Results: Here we report a thorough analysis and significant improvements of n-butanol production from glucose with yeast via the acetoacetyl-CoA-derived pathway. First, we established an improved n-butanol pathway by testing various isoenzymes of different pathway reactions. This resulted in n-butanol titers around 15 mg/L in synthetic medium after 74 h. As the initial substrate of the n-butanol pathway is acetyl-coenzyme A (acetyl-CoA) and most intermediates are bound to coenzyme A (CoA), we increased CoA synthesis by overexpression of the pantothenate kinase coaA gene from Escherichia coli. Supplementation with pantothenate increased n-butanol production up to 34 mg/L. Additional reduction of ethanol formation by deletion of alcohol dehydrogenase genes ADH1-5 led to n-butanol titers of 71 mg/L. Further expression of a mutant form of an ATP independent acetylating acetaldehyde dehydrogenase, adhEA267T/E568K, converting acetaldehyde into acetyl-CoA, resulted in 95 mg/L n-butanol. In the final strain, the n-butanol pathway genes, coaA and adhE A267T/E568K, were stably integrated into the yeast genome, thereby deleting another alcohol dehydrogenase gene, ADH6, and GPD2-encoding glycerol-3-phosphate dehydrogenase. This led to a further decrease in ethanol and glycerol by-product formation and elevated redox power in the form of NADH. With the addition of pantothenate, this strain produced n-butanol up to a titer of 130 ± 20 mg/L and a yield of 0.012 g/g glucose. These are the highest values reported so far for S. cerevisiae in synthetic medium via an acetoacetyl-CoA-derived n-butanol pathway.
Conclusions: By gradually increasing substrate supply and redox power in the form of CoA, acetyl-CoA, and NADH, and decreasing ethanol and glycerol formation, we could stepwise increase n-butanol production in S. cerevisiae. However, still further bottlenecks in the n-butanol pathway must be deciphered and improved for industrially relevant n-butanol production levels.
The genome of S. cerevisae encodes at least twenty hexose transporter-like proteins. Despite extensive research, the functions of Hxt8-Hxt17 have remained poorly defined. Here, we show that Hxt13, Hxt15, Hxt16 and Hxt17 transport two major hexitols in nature, mannitol and sorbitol, with moderate affinities, by a facilitative mechanism. Moreover, Hxt11 and Hxt15 are capable of transporting xylitol, a five-carbon polyol derived from xylose, the most abundant pentose in lignocellulosic biomass. Hxt11, Hxt13, Hxt15, Hxt16 and Hxt17 are phylogenetically and functionally distinct from known polyol transporters. Based on docking of polyols to homology models of transporters, we propose the architecture of their active site. In addition, we determined the kinetic parameters of mannitol and sorbitol dehydrogenases encoded in the yeast genome, showing that they discriminate between mannitol and sorbitol to a much higher degree than the transporters.
We explored the characteristics and motivations of people who, having obtained their genetic or genomic data from Direct-To-Consumer genetic testing (DTC-GT) companies, voluntarily decide to share them on the publicly accessible web platform openSNP. The study is the first attempt to describe open data sharing activities undertaken by individuals without institutional oversight. In the paper we provide a detailed overview of the distribution of the demographic characteristics and motivations of people engaged in genetic or genomic open data sharing. The geographical distribution of the respondents showed the USA as dominant. There was no significant gender divide, the age distribution was broad, educational background varied and respondents with and without children were equally represented. Health, even though prominent, was not the respondents’ primary or only motivation to be tested. As to their motivations to openly share their data, 86.05% indicated wanting to learn about themselves as relevant, followed by contributing to the advancement of medical research (80.30%), improving the predictability of genetic testing (76.02%) and considering it fun to explore genotype and phenotype data (75.51%). Whereas most respondents were well aware of the privacy risks of their involvement in open genetic data sharing and considered the possibility of direct, personal repercussions troubling, they estimated the risk of this happening to be negligible. Our findings highlight the diversity of DTC-GT consumers who decide to openly share their data. Instead of focusing exclusively on health-related aspects of genetic testing and data sharing, our study emphasizes the importance of taking into account benefits and risks that stretch beyond the health spectrum. Our results thus lend further support to the call for a broader and multi-faceted conceptualization of genomic utility.
Ziel der vorliegenden Arbeit war es, vor- und nachbereitenden Unterricht zu Biodiversitätsführungen an den vier außerschulischen Lernorten Palmengarten, Senckenbergmuseum, Stadtwaldhaus und Zoo Frankfurt zu evaluieren. Durch den Unterricht mithilfe neu entwickelter Arbeitsmaterialien sollte die aktuelle Motivation der Schüler und weitere pädagogisch-psychologische Lernvariablen gefördert werden. Es stellte sich die Frage, ob so eine erhöhte Auseinandersetzung mit dem Themenkomplex Biodiversität erreicht werden kann und welche Einflussfaktoren dabei eine Rolle spielen.
Theoretische Grundlage war dabei das Risikowahlmodell der Leistungsmotivation nach Atkinson, das von Rheinberg zum handlungstheoretischen Modell der Motivation erweitert wurde (Rheinberg & Vollmeyer, 2012). Auf dieses bezieht sich der von Rheinberg et al. (2001) entwickelte und hier eingesetzte Fragebogen zur aktuellen Motivation (FAM).
Die Stichprobe setzte sich aus insgesamt 523 Schülern der Klassen 5 bis 9 zusammen. Davon nahm jeweils die Hälfte mit (Versuchsgruppe) und die andere ohne (Kontrollgruppe) vor- und nachbereitendem Unterricht an den Biodiversitätsführungen teil. Die Erhebung der aktuellen Motivation, des erworbenen Fachwissens und weiterer Variablen erfolgte in einem Pre/Post/Follow-Up-Design mit Fragebögen, deren Auswertung analytisch statistisch durgeführt wurde.
Es zeigte sich, dass in der Gesamtstichprobe die Teilnahme an der Biodiversitätsführung die aktuelle Motivation der Schüler erhöhte. Dauerhafte Lernparameter wie die Biologieeinstellung und die Interessenshandlung wurden jedoch nicht signifikant verändert. Ein eindeutiger Effekt der unterrichtlichen Vorbereitung konnte jedoch nicht ermittelt werden. Einzig beim gemessen Fachwissen zu den Führungsinhalten schnitt die Versuchsgruppe signifikant besser ab. Insgesamt wird angenommen, dass der Effekt des Besuchs des außerschulischen Lernortes an sich den Effekt der Vor- und Nachbereitung überdeckt oder vom Einfluss anderer Parameter beeinflusst wird. Hier stach besonders das Alter der Jugendlichen hervor, das vor allem in der hier evaluierten Schülergruppe bedingt durch die Pubertät eine große Rolle spielt. Weitere Einflussfaktoren waren die Biologieeinstellung und die Unterrichtsvariablen der Führung. In den Stichproben der einzelnen außerschulischen Lernorte zeigten sich leichte Abweichungen von der Gesamtstichprobe. Diese waren meist auf die leicht unterschiedliche Zusammensetzung der Stichproben zurückzuführen. Aber auch Besonderheiten der Lernorte hatten dabei ein bedeutendes Gewicht.
Bezüglich der Lernbedingungen für die Lernorte ließen sich aus den Ergebnissen vor allem zwei Komponenten ermitteln: Zum einen die Architektur/räumliche Struktur der Lernorte. Hier können Faktoren wie drinnen/ draußen, Größe und die räumliche Orientierung unterschieden werden. All dies hat Auswirkungen auf das physische Wohlbefinden der Schüler, was wiederum eine Voraussetzung für eine hohe Lernmotivation ist. Die andere Hauptkomponente ist das am Lernort behandelte Thema. Hier kann grob zwischen Pflanzen und Tieren unterschieden werden. Pflanzen wurden dabei in mehreren Studien von den Schülern als weniger attraktiv eingeschätzt. Trotzdem sollten aber die Möglichkeiten, auch botanische Themen außerhalb der Schule zu behandeln, von den Lehrkräften zur Vermittlung biologischer Vielfalt genutzt werden.
Als Konsequenz der Ergebnisse kann der Besuch eines außerschulischen Lernrotes im Biologieunterricht bezüglich der Förderung der Lernmotivation unbedingt empfohlen werden. Da kein klarer Effekt des vor- und nachbereitenden Unterrichts der Biodiversitätsführungen erkennbar war, wären hier weitere Untersuchungen vonnöten, um genauere Aussagen machen zu können. Hier böten sich Studien mit Schülern anderer Altersgruppen und der Vergleich nur zweier außerschulischer Lernorte an.
Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.
Objective: To determine the role of the AMPKα2 subunit in vascular repair.
Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice.
Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.
In China and other countries of East Asia, so-called Ling-zhi or Reishi mushrooms are used in traditional medicine since several centuries. Although the common practice to apply the originally European name ‘Ganoderma lucidum’ to these fungi has been questioned by several taxonomists, this is still generally done in recent publications and with commercially cultivated strains. In the present study, two commercially sold strains of ‘G. lucidum’, M9720 and M9724 from the company Mycelia bvba (Belgium), are compared for their fruiting body (basidiocarp) morphology combined with molecular phylogenetic analyses, and for their secondary metabolite profile employing an ultra-performance liquid chromatography–electrospray ionization mass spectrometry (UPLC–ESIMS) in combination with a high resolution electrospray ionization mass spectrometry (HR-ESI-MS). According to basidiocarp morphology, the strain M9720 was identified as G. lucidum s.str. whereas M9724 was determined as Ganoderma lingzhi. In molecular phylogenetic analyses, the M9720 ITS and beta-tubulin sequences grouped with sequences of G. lucidum s.str. from Europe whereas those from M9724 clustered with sequences of G. lingzhi from East Asia. We show that an ethanol extract of ground basidiocarps from G. lucidum (M9720) contains much less triterpenic acids than found in the extract of G. lingzhi (M9724). The high amount of triterpenic acids accounts for the bitter taste of the basidiocarps of G. lingzhi (M9724) and of its ethanol extract. Apparently, triterpenic acids of G. lucidum s.str. are analyzed here for the first time. These results demonstrate the importance of taxonomy for commercial use of fungi.
Parkinson's disease (PD) is a frequent neurodegenerative process in old age. Accumulation and aggregation of the lipid-binding SNARE complex component α-synuclein (SNCA) underlies this vulnerability and defines stages of disease progression. Determinants of SNCA levels and mechanisms of SNCA neurotoxicity have been intensely investigated. In view of the physiological roles of SNCA in blood to modulate vesicle release, we studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation) to identify effects of SNCA gain of function as potential disease biomarkers. Downregulation of complexin 1 (CPLX1) mRNA was correlated with genotype, but the expression of other Parkinson's disease genes was not. In global RNA-seq profiling of blood from presymptomatic PARK4 indviduals, bioinformatics detected significant upregulations for platelet activation, hemostasis, lipoproteins, endocytosis, lysosome, cytokine, Toll-like receptor signaling and extracellular pathways. In PARK4 platelets, stimulus-triggered degranulation was impaired. Strong SPP1, GZMH and PLTP mRNA upregulations were validated in PARK4. When analysing individuals with rapid eye movement sleep behavior disorder, the most specific known prodromal stage of general PD, only blood CPLX1 levels were altered. Validation experiments confirmed an inverse mutual regulation of SNCA and CPLX1 mRNA levels. In the 3′-UTR of the CPLX1 gene we identified a single nucleotide polymorphism that is significantly associated with PD risk. In summary, our data define CPLX1 as a PD risk factor and provide functional insights into the role and regulation of blood SNCA levels. The new blood biomarkers of PARK4 in this Turkish family might become useful for PD prediction.
50 years of amino acid hydrophobicity scales : revisiting the capacity for peptide classification
(2016)
Background: Physicochemical properties are frequently analyzed to characterize protein-sequences of known and unknown function. Especially the hydrophobicity of amino acids is often used for structural prediction or for the detection of membrane associated or embedded β-sheets and α-helices. For this purpose many scales classifying amino acids according to their physicochemical properties have been defined over the past decades. In parallel, several hydrophobicity parameters have been defined for calculation of peptide properties. We analyzed the performance of separating sequence pools using 98 hydrophobicity scales and five different hydrophobicity parameters, namely the overall hydrophobicity, the hydrophobic moment for detection of the α-helical and β-sheet membrane segments, the alternating hydrophobicity and the exact ß-strand score.
Results: Most of the scales are capable of discriminating between transmembrane α-helices and transmembrane β-sheets, but assignment of peptides to pools of soluble peptides of different secondary structures is not achieved at the same quality. The separation capacity as measure of the discrimination between different structural elements is best by using the five different hydrophobicity parameters, but addition of the alternating hydrophobicity does not provide a large benefit. An in silico evolutionary approach shows that scales have limitation in separation capacity with a maximal threshold of 0.6 in general. We observed that scales derived from the evolutionary approach performed best in separating the different peptide pools when values for arginine and tyrosine were largely distinct from the value of glutamate. Finally, the separation of secondary structure pools via hydrophobicity can be supported by specific detectable patterns of four amino acids.
Conclusion: It could be assumed that the quality of separation capacity of a certain scale depends on the spacing of the hydrophobicity value of certain amino acids. Irrespective of the wealth of hydrophobicity scales a scale separating all different kinds of secondary structures or between soluble and transmembrane peptides does not exist reflecting that properties other than hydrophobicity affect secondary structure formation as well. Nevertheless, application of hydrophobicity scales allows distinguishing between peptides with transmembrane α-helices and β-sheets. Furthermore, the overall separation capacity score of 0.6 using different hydrophobicity parameters could be assisted by pattern search on the protein sequence level for specific peptides with a length of four amino acids.
Bartonella Adhäsin A (BadA), das zur Gruppe der TAAs gehört, ist ein essentieller Pathogenitätsfaktor von B. henselae und übernimmt während des Infektionsverlaufs wichtige Funktion wie Autoagglutination, Adhärenz an ECM-Proteine und Endothelzellen. BadA weist die für die für die Proteinklasse der TAAs charakteristische modulare Architektur bestehend aus N-terminaler Kopf-Domäne, Stiel-Domäne, Hals-Domäne und C-terminaler Membrananker-Domäne auf. Der modulare Aufbau des Proteins deutet daraufhin, dass bestimmte Domänen mit bestimmten biologischen Funktionen des Proteins verknüpft sind. Zur Untersuchung dieser Hypothese wurden Deletionsmutanten des BadA generiert.
Die Generierung weiterer BadA-Deletionsmutanten wird durch das langsame Wachstum des Erregers und die geringe Auswahl an molekularbiologischen Werkzeugen zur genetischen Manipulation von B. henselae erschwert. Daher sollte in ersten Teil dieser Arbeit ein Expressionsmodell für Deletionsmutanten des BadA etabliert und charakterisiert werden. Dies sollte am Beispiel des trunkierten BadA, BadA HN23, durchgeführt werden. Hierzu sollten drei Hybrid-Varianten des BadA HN23 erstellt werden: (i) Austausch der BadA-Signalsequenz gegen die E. coli OmpA-Signalsequenz, (ii) Austausch der BadA-Membrananker-Domäne gegen die YadA-Membrananker-Domäne sowie (iii) Austausch von sowohl der BadA-Signalsequenz als auch der BadA-Membrananker-Domäne gegen die bereits genannten Elemente. Danach sollten die konstruierten BadA HN23 Hybride und das BadA HN23 in induzierbare Expressionsvektoren kloniert und spezielle E. coli-Expressionsstämme mit diesen Plasmiden transformiert werden. Bei erfolgreicher Expression sollten die optimalen Bedingungen für die Expression (Temperatur, Induktorkonzentration) ermittelt werden und an-schließend die biologische Funktion der heterolog exprimierten BadA HN23 Hybride überprüft werden.
Der erste Abschnitt der hier vorliegenden Arbeit zeigte folgende Ergebnisse:
1) Die beschrieben BadA HN23 Hybrid Konstrukte wurden durch Austausch von: (i) BadA-Signalsequenz gegen E. coli OmpA-Signalsequenz im BadA HN23,
(ii) BadA-Membrananker-Domäne gegen YadA-Membrananker-Domäne im BadA HN23 und
(iii) Austausch von BadA-Signalsequenz und BadA-Membrananker-Domäne gegen E. coli OmpA-Signalsequenz und YadA-Membrananker-Domäne im BadA HN23 generiert.
Die BadA HN23 Hybride und BadA HN23 wurden in Expressionsvektoren kloniert und E. coli Omp2, E. coli Omp8 und E. coli Omp8ΔdegP transformiert.
2) Alle BadA HN23 Hybrid-Konstrukte und BadA HN23 lagen in einer monomeren und trimeren Form vor.
3) Durch IFT und - Durchflusszytometrie-Untersuchungen wurde die Oberflächenexpression der einzelnen Konstrukte quantifiziert. Es zeigte sich, dass es deutliche Unterschiede in der Menge des auf der Zelloberfläche befindlichen jeweiligen BadA HN23 Proteins gab. Dabei wiesen die Konstrukte, die die YadA-Membrananker-Domäne besaßen (BadA HN23 Hybrid 2 und 3), die stärkste Oberflächenexpression auf.
4) Die biologische Funktion des BadA HN23 wurde mittels des E. coli Omp2 BadA HN23 Hybrid 3 charakterisiert. Heterolog exprimiertes BadA HN23 vermittelt Autoagglutination, die Adhärenz des Expressionsstammes an Kollagen G und Endothelzellen.
5) Die Expression des BadA HN23 führt zur signifikant verstärkten in-vivo-Pathogenität im Galleria mellonella-Infektionsmodell.
6) Das E. coli-Expressionsmodell lieferte keine Aussage über eventuelle immunodominate Funktionen des heterolog exprimierten BadA HN23, da auch mit im IFT als anti- B. henselae negativ eingestuften Patientenseren im WB ein BadA HN23 spezifisches Bandensignal detektiert wurde. Dot Blot-Experimente ermöglichten ebenfalls keine Aussage über eventuelle immunodominate Funktion des nativen BadA HN23, da das verwendete anti-B. henselae-positive Patientenserum unspezifische Reaktion gegenüber dem Kontrollstamm zeigte.
Für verschiedene TAAs ist beschrieben worden, dass sie die Serumresistenz der exprimierenden Spezies vermitteln. Daher sollte im zweiten Teil dieser Arbeit der Einfluss von BadA auf eventuelle Serumresistenz zweier B. henselae-Isolate untersucht werden. Dieser Teil lieferte folgende Ergebnisse:
1) B. henselae zeigte Sensitivität gegenüber normalem humanem Serum.
2) Sowohl BadA-positive als auch BadA-negative B. henselae-Isolate können Komplementinhibitoren wie Faktor H binden. Die dabei gebundene Menge ist relativ klein.
Die Expression von Deletionsmutanten des BadA in E. coli ist ein vielversprechendes Modell zur Analyse der Domänen-Funktionsbeziehung des BadA, da die meisten biologischen Funktionen einer homolog exprimierten BadA-Deletionsmutante reproduziert werden konnten und es sich bei E. coli um ein schnell wachsendes Bakterium, das sich leicht genetisch manipulieren lässt, handelt. Allerdings stellt das zytotoxische LPS des E. coli sowie das schnelle Wachstums der Bakterien eine Limitation des Expressionssystems dar, indem es Untersuchungen zum Einfluss der jeweiligen BadA-Deletionsmutante auf die Induktion der proangiogenetischen Wirtszellantwort verhindert oder Untersuchungen zum Einfluss der jeweiligen BadA-Deletionsmutante auf die Adhärenz an Endothelzellen deutlich erschwert. Außerdem kann eine mögliche Interaktion zwischen BadA bzw. BadA-Deletionsmutanten und dem TIVSS und zwischen BadA bzw. BadA-Deletionsmutanten und weiteren Adhäsinen (wie z.B. dem FHA) mit Hilfe dieses Expressionssystems nicht untersucht werden. Dies wäre nur im B. henselae Wildtyp-Stamm möglich.
This thesis describes the adaptation of Acinetobacter species to dry environments with the soil bacterium A. baylyi and the opportunistic hospital pathogen A. baumanii in its focus. The adaptation of A. baylyi and A. baumannii to osmotic stress was investigated. Compatible solutes that were uptaken from the environment or synthesized de novo to cope with the loss of water at high salinity were identified. The corresponding transporters and enzymes involved were characzerized. In addition, the desiccation resistance of A. baumannii was analyzed to elucidate its survival in hospital environments. The usage of compatible solutes during desiccation stress was analyzed and proteins that were produced were identified.
The availability of water is essential for bacterial life and if environmental conditions are awkward, bacteria have to cope with high salinitiy to prevent loss of water. In this thesis it was shown that A. baylyi synthesizes glutamate and mannitol de novo as compatible solutes in response to osmotic stress to balance the osmotic potential. The pathway for mannitol biosynthesis from Fructose-6-Phosphate (F-6-P) via Mannitol-1-Phosphate (Mtl-1-P) was elucidated and the isolation and characterization of a novel type of biofunctional enzyme was described. Interestingly, the unique bifunctional enzyme MtlD, acting as dehydrogenase and phosphatase, mediates both steps of the mannitol biosynthesis pathway. This enzyme catalyzes the reduction of F-6-P to Mtl-1-P with NADPH as reducing equivalent. The dehydrogenase activity of MtlD was salt dependent and the phosphatase activity was dependent on Mg2+ as cofactor. Phylogenetic analyses revealed that MtlD is broadly distributed among other Acinetobacter strains but not in other phylogenetic tribes.
In this thesis it is also described that, besides de novo synthesis of compatible solutes, A. baylyi takes up glycine betaine (GB) or its precursor choline by different transport systems and uses this solutes as osmoprotectants. The uptake of GB occurs via a secondary transporter (ACIAD3460) of the BCCT family. Choline is taken up as precursor and oxidized to GB by two dehydrogenases. The uptake and use of choline as GB precursor involves two transporters, whose genes are encoded in the bet cluster (BetT1, BetT2), two dehydrogenases (BetA, BetB) and a regulatory protein (BetI). Both transporters differ from each other in structure and function: BetT1 is osmo-independent and active independently of osmotic stress. BetT2 contains - in contrast to BetT1 - a long C-terminal domain for osmo-sensing and its activity highly increases in the presence of high osmolarity. The oxidation of choline occurs independently of the osmolarity of the medium but in the absence of salt stress, GB is exported. In contrast, in the presence of high salinity, GB is accumulated in the cytoplasm to balance the osmotic potential in order to prevent loss of water. The regulation of both transporters, the uptake of choline independently of the osmolarity and the export of GB under isoosmotic conditions are regulated by the transcriptional regulator BetI.
A. baumannii ATCC 19606 was also shown to cope with high salinity. Analogously to A. baylyi, A. baumannii ATCC19606 synthesizes glutamate and mannitol de novo in response to osmotic stress. The genes for the synthesis of these compatible solutes are identical to those found in A. baylyi. This suggests that the solute biosynthesis pathways of A. baumannii and A. baylyi are identical. A. baumannii was also able to take up GB and choline in response to osmotic stress and growth at high salinity was restored upon addition of GB and its precursor choline. The bet cluster was also present in the genome A. baumannii and also contains the two different choline transporters BetT1 and BetT2.
Our suggestion that choline or GB or the utilization of phosphatidylcholine as carbon source led to an increase in the survival under desiccation stress was not confirmed. However, 2D analysis of proteins produced during desiccation stress in A. baumannii led to elevated amounts of proteins implicated in biofilm formation, regulation, cell morphology and general stress response, such as Hsp60 or superoxide dismutase, both might play a role in general stress protection.
Soil fungal communities are an essential element in the terrestrial ecosystem, however their response to ongoing anthropogenic climate change is currently poorly understood. Fungi are one of the most abundant groups of microbes in soil, they are mainly responsible for the decomposition of organic matter (Baldrian et al., 2012; Buée et al., 2009). By binding carbon in soil, fungi thus maintain an important role in the global carbon cycle (Bardgett et al., 2008). Future climates are likely to influence the communities of belowground microbial organisms (Castro et al., 2010; Deacon et al., 2006). However, how these communities are affected in their diversity, composition, and function after environmental perturbation is insufficiently known.
Molecular techniques using high-throughput sequencing are presently revolutionizing the analysis of complex communities, such as soil fungi. High-throughput metabarcoding enables the recovery of DNA sequence data directly from environmental samples, and DNA sequences from entire communities present in these samples can be simultaneously recovered through massively parallel sequencing reactions (Bik et al., 2012; Taberlet et al., 2012b). This results in more accurate estimation of diversity and community composition and thus provides unprecedented insight into cryptic communities (Lindahl and Kuske, 2014). Yet, challenges associated with these novel techniques include the bioinformatic processing, and the ecological analyses of the large amount of sequence data generated. Most biologists without explicit training in bioinformatics spend a fair amount of time learning how to filter raw sequence data, and customize bioinformatics pipelines specific to their project. To improve the quality of data treatment, and decrease the time needed for the analyses, it is desirable to have bioinformatics pipelines that are easy to use, well explained to researchers not trained in bioinformatics, and adaptable to individual research needs...
The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.
Saccharomyces cerevisiae is a natural producer of isobutanol, which has more advantages as biofuel than ethanol, i.e. superior combustion energy, weaker corrosive action and reduced aqueous miscibility. Isobutanol is produced by the combination of the valine biosynthesis and the Ehrlich pathway. In this work, an industrial strain was employed for isobutanol production, in which the valine pathway was relocated into the cytosol. The valine pathway in yeast has a cofactor imbalance, since the glycolysis produces NADH, while Ilv5 employs NADPH for the reaction. Therefore, the cofactor specificity of the pathway was rebalanced with exchange of Ilv5 by an NADH-consuming mutant, IlvC6E6. Furthermore, Ilv6, which regulates the feed-back inhibition of the valine biosynthesis, was tested to boost isobutanol production; however, none of these Ilv6 alternatives could greatly enhance isobutanol production. Therefore, due to a still low production yield, the bottlenecks of the isobutanol pathway were deeper studied.
The major observed bottleneck concerned the conversion of DIV into KIV, since high concentrations of acetoin, 2,3-butandiol and, specially, DIV were observed in the fermentation supernatant, while neither KIV nor isobutyraldehyde were detected. This step is performed by the dihydroxy-acid dehydratase, Ilv3, which needs iron-sulfur clusters for its activity. Therefore, the first approach to circumvent this limitation was to increase the FeS assembly and its transference into the cytoplasm; however, Ilv3Δ19 activity was not improvement. Afterwards, Ilv3 alternatives were screened for substitution of Ilv3Δ19. Heterologous ILV3 orthologous with possible advantages were investigated, but Ilv3Δ19 was still the most promising alternative. Furthermore, sugar-acid enolases were tested as Ilv3Δ19 substitutes. These enolases also catalyze the dehydration of the substrate in the same way as Ilv3, but uses Mg2+ as cofactor. One of the employed enolases could complement valine auxotrophy; however, it allowed just a very slow growth of the Δilv3 strain and its activity could not be enhanced by mutagenesis studies.
Interestingly, we observed that once DIV is secreted out of the cell, it cannot be re-uptaken from the medium and this possibly further aggravates the pathway flux and Ilv3Δ19 activity. In order to suppress DIV waste, two strategies were formulated: the deletion of the possible DIV transporter, and the substrate channeling of DIV from IlvC6E6 to Ilv3Δ19. In order to find possible DIV export proteins, a transcriptome analysis of a strain producing high amounts of DIV against a strain producing no detected DIV were compared. Several transporters were found upregulated in the DIV producing strain, but, alone, none of these were responsible for the DIV efflux. For the substrate channeling, an artificial enzymatic net was constructed by the fusion of IlvC6E6 and Ilv319 with synthetic zippers, which have high affinity to each other, and as both enzymes are alone organized as oligomers. The use of this enzymatic net enhanced not only the isobutanol production in about 17%, but also 3-methyl-butanol production yield was 25% increased.
Nevertheless, together with bottlenecks arising from Ilv3 activity, the isobutanol production is limited by the ethanol production, which is the main product of S. cerevisiae. Therefore, in order to abolish ethanol production, PDC1 and PDC5 were deleted. Moreover, BDH1 and BDH2 were also deleted to create an NADH-driving force towards isobutanol production. However, the isobutanol yield of this mutant was even lower than that of the strain without the mentioned deletions. As a high production of isobutyric acid was observed, and it could be produced directly from KIV, different KIV decarboxylases and isobutanol dehydrogenases were investigated; but without improvement. Then, alternative pathways were abolished in other to favor isobutanol production, e.g. valine, leucine, isoleucine and panthotenate biosyntheses. Nevertheless, isobutanol yields were still low and the main byproducts were glycerol, acetoin, DIV and isobutyric acid. Despite the outcomes were not enough to enhance isobutanol production up to commercially required yields, these results help in the comprehension of the bottlenecks surrounding the isobutanol production pathway and serve as basis for further studies within the branched-chain amino acids biosynthesis and Ehrlich pathway.
Nearly 170 million people are chronically infected with HCV and thus at risk of developing liver cirrhosis and hepatocellular carcinoma. Although new and effective oral antiviral drugs are available, there is still the need for a preventive vaccine. In addition, in light of the high number of patients who are chronically infected with HCV the development of a therapeutic vaccine will present a support or even an alternative to the expensive medications.
To induce HCV-specific immune responses in a vaccine model, the HBV capsid is used as a carrier to deliver HCV antigens. Due to its icosahedral structure, the HBV capsid is highly immunogenic and helps to elicit a strong B cell response against the delivered antigens. In addition, the translocation motif (TLM) from the HBV surface protein is fused to the core protein. The TLM conveys membrane-permeability to the carrier capsid, enabling antigen transfer into the cytoplasm, and thus allows immunoproteasomal processing and MHC class I-mediated presentation of the antigen. To load the capsid with foreign antigens, a strep-Tag/streptavidin system is utilized. Recombinant capsids and antigens were purified from the E. coli production system. Detailed characterization of the carrier capsid demonstrated the proper assembly, adequate thermal stability and the successful loading of the foreign antigens onto the capsid surface.
As a further step, seven different HCV-derived proteins were produced and purified for the coupling on the surface of TLM-core particles. The characterization of their immunogenicity using this system is being performed.
Using ovalbumin as a model antigen, which is coupled to the carrier capsids via strep-Tag/streptavidin binding, shows that this system is suitable to efficiently deliver antigens into the cytoplasm of antigen-presenting cells (APCs), leading to the activation of APCs. This activation was assessed by measuring the secretion of IL-6 and TNF-α, in addition to the upregulation of activation markers (CD40, CD80, CD69, and MHC class I). Upon activation, the APCs were able to activate ova-specific CD8+ T cells measured by secreted IFN-γ, which was up to 20-folds more than IFN-γ secreted upon incubation with free ovalbumin. These data indicate that the TLM-capsid is suitable to serve as a carrier to deliver foreign antigens into the cytoplasm of APCs leading to MHC class I-mediated presentation and induction of an antigen-specific CTLs response.
In the adult mammalian brain stem cells within defined neurogenic niches retain the capacity for lifelong de novo generation of neurons. The subventricular zone (SVZ) of the lateral ventricles and the subgranular layer (SGL) of the hippocampal dentate gyrus (DG) have been identified as the two major sites of adult neurogenesis. Moreover, the third ventricle in the hypothalamus is emerging as a new neurogenic niche in the adult brain. Extracellular purine and pyrimidine nucleotides are involved in the control of both embryonic and adult neuro-genesis. These nucleotides act via ionotropic P2X or metabotropic P2Y receptors and studies of the adult SVZ and the DG provide strong evidence that ATP promotes progenitor cell proliferation in this stem cell rich regions. Previous studies have shown that the extracellular nucleotide-hydrolyzing enzyme NTPDase2 is highly expressed by adult neural stem and progenitor cells of the SVZ and the rostral migratory stream (RMS), the hippocampal SGL, and the third ventricle. NTPDase2 preferentially hydrolyzes extracellular nucleoside triphosphates (NTPs) and, to a lower extent, diphosphates, thus modulating their effect on nearby nucleotide receptors. Deletion of the enzyme increases extracellular NTP concentrations, and might indicate roles of purinergic signaling in adult neurogenesis. As shown by enzyme histochemistry, genetic deletion of NTPDase2 essentially eliminates ATPase activity in neurogenic niches but does not affect protein expression levels and activity of other ectonucleotidases. Lack of NTPDase2 leads to expansion of the hippocampal stem cell pool as well as of the inter-mediate progenitor type-2 cells. Cell expansion is lost at around type-3 stage, paralleled by increased labeling for caspase-3, indicating increased apoptosis, and decreased levels in CREB phosphorylation in doublecortin-expressing cells, diminishing survival in this cell population. In line with increased cell death, P2Y12 receptor-expressing microglia is enriched at the hilus orientated side of the granule cell layer. These data strongly suggest that NTPDase2 functions as central homeostatic regulator of nucleotide-mediated neural progenitor cell proliferation and expansion in the adult brain by balancing extracellular nucleotide concentrations and activation of purinergic receptors.
In order to further characterize the role of purinergic signaling in adult neurogenesis, the ADP-sensitive P2Y13 receptor was identified as a potential candidate whose activation might inhibit neurogenesis in the hippocampal dentate gyrus and the newly identified neurogenic niche at the third ventricle. Deletion of P2ry13 increased progenitor cell proliferation and long-term progenitor survival as well as new neuron formation in the hippocampal neurogenic niche. This was further paralleled by increased thickening of the granule cell layer, CREB phosphorylation, and expression of the neuronal activity marker c-Fos. Increased progenitor cell proliferation and progenitor survival persist in aged P2ry13 knockout animals. However, in the ventral dentate gyrus proliferation and expansion levels of progenitor cells did not differ significantly from the wild type. This study strongly supports the notion that extracellular nucleotides significantly contribute to the control of adult neurogenesis in the dentate gyrus in situ. Data in this work suggest that activation of the P2Y13 receptor dampens progenitor cell proliferation, new neuron formation, and neuronal activity. In contrast to several in vitro studies and studies in the SVZ in situ, a contribution of the ATP/ADP-sensitive P2Y1 receptor could not be confirmed in the dentate gyrus in vivo.
To unravel implications of purinergic signaling and P2Y13 receptor action in the control of adult hypothalamic neurogenesis a pilot study was performed. Mice null for P2ry13 revealed increased progenitor cell proliferation at the third ventricle as well as long-term progeny survival and new neuron formation in the hypothalamus. In contrast to results obtained in the dentate gyrus expression of the neuronal activity marker c-Fos was significantly decreased in hypothalamic nuclei, indicating increased inhibition of appetite-regulating neuronal circuits by surplus neurons in knockout animals. These data provide first evidence that extracellular nucleotide signaling contributes to the control of adult hypothalamic neurogenesis in situ. Activation of the P2Y13 receptor inhibits progenitor cell proliferation, long-term survival and neuron formation and therefore controls inhibition of appetite-regulating circuits in the adult rodent hypothalamus.
The baker’s yeast Saccharomyces cerevisiae is a valuable and increasingly important microorganism for industrial applications (Hong and Nielsen, 2012). Its robustness concerning process conditions like low pH, osmotic and mechanical stress as well as toxic compounds is an advantage. Moreover, S. cerevisiae is ‘generally regarded as safe’ (GRAS). The model organism has been studied intensively. The collected data, including genomic, proteomic and metabolic information, can be used to genetically modify and improve its metabolism. Fatty acids and fatty acid derivatives have wide applications as biofuels, biomaterials, and other biochemicals. Several studies have been dealing with the overproduction of fatty acids and derivatives thereof in S. cerevisiae. The fatty acid biosynthesis starting with acetyl-CoA requires two enzymes, the acetyl-CoA carboxylase (Acc1p) and the fatty acid synthase complex (FAS), to produce acyl-CoA esters with predominantly 16 to 18 carbon atoms chain length (Lynen et al., 1980). For the synthesis of monounsaturated fatty acids in S. cerevisiae the ER bound acyl-CoA desaturase, Ole1p is essential (Tamura et al., 1976; Certik and Shimizu, 1999).
Using S. cerevisiae, the first section of this work dealt with the heterologous characterization of potential ω1-desaturases. Due to the fact that unsaturated fatty compounds can be modified further by hydrosilylations, hydrovinylations, oxidations to epoxides, acids, aldehydes, ketones or metathesis reactions, the interest in ω1-fatty acids is tremendous (Behr and Gomes, 2010). With the intention to find enzymes in fungi, that have a terminal desaturase activity a search in different genome databases was performed. The sequences of Pex-Desat3 and Obr-TerDes were used as reference sequences. The analysed proteins from Schizophyllum commune (EFI94599.1), Schizosaccharomyces octosporus (EPX72095.1), Wallemia mellicola (EIM20316.1), Wallemia ichthyophaga (EOR00207.1) and Agaricus bisporus var. bisporus (EKV44635.1), however, finally turned out to be Δ9 desaturases. A fungal desaturase with ω1-activity could not be found. The Δ9 desaturase SCD1 from Mus musculus was crystallized by Bai et al. (2015) and the information for specific amino acids responsible for the substrate specificity or enzyme activity were allocated. In combination with sequence and enzyme activity data form ChDes1 from Calanus hyperboreus, Desat2 from Drosophila melanogaster, Pex-Desat3 from Planotortrix excessana and Obr-TerDes from Operophtera brumata single amino acid exchanges were performed in the Δ9 desaturase Ole1p from S. cerevisiae. For all mutants, only fatty acids (C16 - C18) with a double bond between carbon C9 and C10 could be found. This indicates, that all inserted amino acid exchanges do not affect the substrate specificity or the position of the introduced double bond.
In the second section the focus was in the development of a production system for fatty acids in S. cerevisiae with regard to the previously established procedures by metabolic engineering. The combination of cytosolic malate dehydrogenase (MDH3), cytosolic malate enzyme (MAE1) and a citrate- α-ketoglutarate- carrier (YHM2) should improve the availability of acetyl-CoA in the cytosol, which is an important precursor for the fatty acid biosynthesis. If the major pathway (acetyl-CoA carboxylase and fatty acid synthase) was already optimized by high expression levels than no positive effect on increased fatty acid synthesis was detectable. Only non-optimized strains, with the additional overexpression of ATP-citrate lyase and cytosolic malate dehydrogenase, lead to a 41 % (20 mg/g dcw) improvement of fatty acid synthesis. In order to increase the fatty acid content further, the additional overexpression of DGA1 and TGL3 was performed. Hence, the highest amount of fatty acids could be observed with the strain S. cerevisiae WRY1ΔFAA1ΔFAA4 (2.5 g/L ± 0.8 g/L). The additional elimination of acyl-CoA synthetase Fat1p did not improve the yield.
It was recently reported, that chain length control of the fatty acid synthesis of bacterial FAS can be changed by rational engineering (Gajewski et al., 2017a). The knowledge about bacterial FAS was transferred in this work to S. cerevisiae FAS. Mutating up to five amino acids in the FAS complex enabled S. cerevisiae to produce medium chain fatty acids (C6 - C12). Further improvement was done by metabolic pathway engineering (promoter of alcohol dehydrogenase II from S. cerevisiae (pADH2), deletion of acyl-CoA synthetase FAA2) and optimization of fermentation conditions (YEPD-bacto medium buffered with potassium phosphate). The production of medium chain fatty acids resulted in the highest yield of 464 mg/L (C6 to C12 fatty acids). Furthermore, strains were created specifically overproducing hexanoic acid (158 mg/L) and octanoic acid (301 mg/L). The characterization of transferases, which could be responsible for the de-esterification of CoA-bound fatty acids, was analysed in an additional approach. It could be shown, that the genes EHT1, EEB1 and MGL2 have an influence on the MCFA yield in the supernatant. Generally speaking, the data from the single and double deletion strains suggest that Eeb1p has a selective hydrolytic activity for hexanoic acid-CoA ester, while Eht1p shows selective hydrolytic activity for octanoic acid-CoA ester, which is in line with Saerens et al. (2006).
Retinal OFF bipolar cells show distinct connectivity patterns with photoreceptors in the wild-type mouse retina. Some types are cone-specific while others penetrate further through the outer plexiform layer (OPL) to contact rods in addition to cones. To explore dendritic stratification of OFF bipolar cells in the absence of rods, we made use of the ‘cone-full’ Nrl-/- mouse retina in which all photoreceptor precursor cells commit to a cone fate including those which would have become rods in wild-type retinas. The dendritic distribution of OFF bipolar cell types was investigated by confocal and electron microscopic imaging of immunolabeled tissue sections. The cells’ dendrites formed basal contacts with cone terminals and expressed the corresponding glutamate receptor subunits at those sites, indicating putative synapses. All of the four analyzed cell populations showed distinctive patterns of vertical dendritic invasion through the OPL. This disparate behavior of dendritic extension in an environment containing only cone terminals demonstrates type-dependent specificity for dendritic outgrowth in OFF bipolar cells: rod terminals are not required for inducing dendritic extension into distal areas of the OPL.
Background: Baker’s yeast, Saccharomyces cerevisiae, as one of the most often used workhorses in biotechnology has been developed into a huge family of application optimised strains in the last decades. Increasing numbers of strains render their characterisation highly challenging, even with the simple methods of growth-based analytics. Here we present a new sensor system for the automated, non-invasive and parallelisable monitoring of biomass in continuously shaken shake flask cultures, called CGQ (“cell growth quantifier”). The CGQ implements a dynamic approach of backscattered light measurement, allowing for efficient and accurate growth-based strain characterisation, as exemplarily demonstrated for the four most commonly used laboratory and industrial yeast strains, BY4741, W303-1A, CEN.PK2-1C and Ethanol Red.
Results: Growth experiments revealed distinct carbon source utilisation differences between the investigated S. cerevisiae strains. Phenomena such as diauxic shifts, morphological changes and oxygen limitations were clearly observable in the growth curves. A strictly monotonic non-linear correlation of OD600 and the CGQ’s backscattered light intensities was found, with strain-to-strain as well as growth-phase related differences. The CGQ measurements showed high resolution, sensitivity and smoothness even below an OD600 of 0.2 and were furthermore characterised by low background noise and signal drift in combination with high reproducibility.
Conclusions: With the CGQ, shake flask fermentations can be automatically monitored regarding biomass and growth rates with high resolution and parallelisation. This makes the CGQ a valuable tool for growth-based strain characterisation and development. The exceptionally high resolution allows for the identification of distinct metabolic differences and shifts as well as for morphologic changes. Applications that will benefit from that kind of automatized biomass monitoring include, amongst many others, the characterization of deregulated native or integrated heterologous pathways, the fast detection of co-fermentation as well as the realisation of rational and growth-data driven evolutionary engineering approaches.
The amyloid precursor protein (APP) was discovered in the 1980s as the precursor protein of the amyloid A4 peptide. The amyloid A4 peptide, also known as A-beta (Aβ), is the main constituent of senile plaques implicated in Alzheimer’s disease (AD). In association with the amyloid deposits, increasing impairments in learning and memory as well as the degeneration of neurons especially in the hippocampus formation are hallmarks of the pathogenesis of AD. Within the last decades much effort has been expended into understanding the pathogenesis of AD. However, little is known about the physiological role of APP within the central nervous system (CNS). Allocating APP to the proteome of the highly dynamic presynaptic active zone (PAZ) identified APP as a novel player within this neuronal communication and signaling network. The analysis of the hippocampal PAZ proteome derived from APP-mutant mice demonstrates that APP is tightly embedded in the underlying protein network. Strikingly, APP deletion accounts for major dysregulation within the PAZ proteome network. Ca2+-homeostasis, neurotransmitter release and mitochondrial function are affected and resemble the outcome during the pathogenesis of AD. The observed changes in protein abundance that occur in the absence of APP as well as in AD suggest that APP is a structural and functional regulator within the hippocampal PAZ proteome. Within this review article, we intend to introduce APP as an important player within the hippocampal PAZ proteome and to outline the impact of APP deletion on individual PAZ proteome subcommunities.
In der vorliegenden Arbeit werden funktionale Details der Okklusion während der Mastikation bei ausgewählten fossilen und rezenten Primaten quantitativ vergleichend untersucht. Dazu wurden die Okklusionsflächen von antagonistischen Molarenpaaren mit modernen virtuellen Verfahren eingescannt und anhand von 3D Kronenmodellen kartiert und funktional ausgewertet. Die in der Forschergruppe DFG FOR 771 entwickelte Software „Occlusal Fingerprint Analyser“ (OFA) kam erstmals bei einer großen Stichprobe von Primaten zum Einsatz.
Aus dem ursprünglichen tribosphenischen Molarentyp der frühen eozänen Primaten haben einige Nahrungsspezialisten im Laufe der Evolution Modifikationen entwickelt um ihre Nahrung mechanisch besser aufzubereiten. So sind neue Funktionselemente auf den Molaren entstanden, wie z.B. ein distolingualer Höcker (Hypoconus) auf den Oberkiefermolaren.
Die Auswertung der Parametermessungen, wie die Facettenlage und -größe, der Okklusale Kompass, der Mastikationskompass und die Messungen der Okklusionsreliefs ergaben, dass die basalen Primatenvertreter aus dem Eozän einen flachen Hypoconus als vergrößerte Fläche zum Quetschen der Nahrung genutzt haben. Das weist auf eine frugivore Nahrungspräferenz hin. Der distolinguale Höcker ist unter den rezenten Spezies mit insektivorer Nahrungspräferenz besonders häufig ausgebildet. Es konnte gezeigt werden, dass eine zweite Kauphase, die nach der maximalen Verzahnung mit der Öffnung des Kiefers eintritt, unter den rezenten Strepsirrhini mit Hypoconus nur sehr schwach ausgeprägt ist.
Eine weitere evolutionäre Modifikation sind buccolingual ausgerichtete komplementäre Kantenpaare auf den Molaren, die sogenannte Bilophodontie, die sich in der Familie der Cercopithecidae entwickelt hat. Die Unterfamilie der Colobinae zeigt eine besonders stark reliefgeführte Okklusion und hat deshalb während der Mastikation weniger Bewegungsspielraum als die der Cercopithecinae. Die zweite Kauphase der Cercopithecinae ist gegenüber den folivoren Colobinae zum Teil auffällig verlängert. Da die Colobinae Vormagenverdauer und die Cercopithecinae Monogastrier sind, kann vermutet werden, dass die Zahnmorphologie eng mit der entsprechenden chemischen Verdauungsweise verknüpft ist. Das dryopithecine Höckermuster der Hominoidea hat eine wesentlich flachere Höckermorphologie als die bilophodonten Molaren. Daher war ein höherer Bewegungsspielraum während der Mastikation beobachtbar. Es konnte gezeigt werden, dass steilere Facetten bei den folivoren Nahrungsspezialisten zu finden sind, wie den Colobinae bei den bilophodonten, oder den Gorillas unter dem dryopithecinen Molarentyp. Mit einem flacheren Molarenrelief kann auf ein breiteres Nahrungsspektrum zugegriffen werden.
Mit den OFA-Analysen und den Ergebnissen der Quantifizierung des Kronenreliefs von rezenten und fossilen Primatenzähnen konnte in der vorliegenden Untersuchung eine relevante Vergleichsbasis für ein funktionelles Verständnis der Evolution der vielfältigen Kronenformen bei Primaten erarbeitet werden. Für zukünftige Studien sollte die innerartliche Stichprobe erweitert werden um die Variabilität näher zu untersuchen.
Genetic data in studies of systematics of Amazonian amphibians frequently reveal that purportedly widespread single species in reality comprise species complexes. This means that real species richness may be significantly higher than current estimates. Here we combine genetic, morphological, and bioacoustic data to assess the phylogenetic relationships and species boundaries of two Amazonian species of the Dendropsophus leucophyllatus species group: D. leucophyllatus and D. triangulum. Our results uncovered the existence of five confirmed and four unconfirmed candidate species. Among the confirmed candidate species, three have available names: Dendropsophus leucophyllatus, Dendropsophus triangulum, and Dendropsophus reticulatus, this last being removed from the synonymy of D. triangulum. A neotype of D. leucophyllatus is designated. We describe the remaining two confirmed candidate species, one from Bolivia and another from Peru. All confirmed candidate species are morphologically distinct and have much smaller geographic ranges than those previously reported for D. leucophyllatus and D. triangulum sensu lato. Dendropsophus leucophyllatus sensu stricto occurs in the Guianan region. Dendropsophus reticulatus comb. nov. corresponds to populations in the Amazon basin of Brazil, Ecuador, and Peru previously referred to as D. triangulum. Dendropsophus triangulum sensu stricto is the most widely distributed species; it occurs in Amazonian Ecuador, Peru and Brazil, reaching the state of Pará. We provide accounts for all described species including an assessment of their conservation status.
Possible effects of clothianidin seed-treated oilseed rape on honey bee colonies were investigated in a large-scale monitoring project in Northern Germany, where oilseed rape usually comprises 25–33 % of the arable land. For both reference and test sites, six study locations were selected and eight honey bee hives were placed at each location. At each site, three locations were directly adjacent to oilseed rape fields and three locations were situated 400 m away from the nearest oilseed rape field. Thus, 96 hives were exposed to fully flowering oilseed rape crops. Colony sizes and weights, the amount of honey harvested, and infection with parasites and diseases were monitored between April and September 2014. The percentage of oilseed rape pollen was determined in pollen and honey samples. After oilseed rape flowering, the hives were transferred to an extensive isolated area for post-exposure monitoring. Total numbers of adult bees and brood cells showed seasonal fluctuations, and there were no significant differences between the sites. The honey, which was extracted at the end of the exposure phase, contained 62.0–83.5 % oilseed rape pollen. Varroa destructor infestation was low during most of the course of the study but increased at the end of the study due to flumethrin resistance in the mite populations. In summary, honey bee colonies foraging in clothianidin seed-treated oilseed rape did not show any detrimental symptoms as compared to colonies foraging in clothianidin-free oilseed rape. Development of colony strength, brood success as well as honey yield and pathogen infection were not significantly affected by clothianidin seed-treatment during this study.
This study was part of a large-scale monitoring project to assess the possible effects of Elado® (10 g clothianidin & 2 g β-cyfluthrin/kg seed)-dressed oilseed rape seeds on different pollinators in Northern Germany. Firstly, residues of clothianidin and its active metabolites thiazolylnitroguanidine and thiazolylmethylurea were measured in nectar and pollen from Elado®-dressed (test site, T) and undressed (reference site, R) oilseed rape collected by honey bees confined within tunnel tents. Clothianidin and its metabolites could not be detected or quantified in samples from R fields. Clothianidin concentrations in samples from T fields were 1.3 ± 0.9 μg/kg and 1.7 ± 0.9 μg/kg in nectar and pollen, respectively. Secondly, pollen and nectar for residue analyses were sampled from free flying honey bees, bumble bees and mason bees, placed at six study locations each in the R and T sites at the start of oilseed rape flowering. Honey samples were analysed from all honey bee colonies at the end of oilseed rape flowering. Neither clothianidin nor its metabolites were detectable or quantifiable in R site samples. Clothianidin concentrations in samples from the T site were below the limit of quantification (LOQ, 1.0 µg/kg) in most pollen and nectar samples collected by bees and 1.4 ± 0.5 µg/kg in honey taken from honey bee colonies. In summary, the study provides reliable semi-field and field data of clothianidin residues in nectar and pollen collected by different bee species in oilseed rape fields under common agricultural conditions.
Despite an increasing demand for Burgundy truffles (Tuber aestivum), gaps remain in our understanding of the fungus’ overall lifecycle and ecology. Here, we compile evidence from three independent surveys in Hungary and Switzerland. First, we measured the weight and maturity of 2,656 T. aestivum fruit bodies from a three-day harvest in August 2014 in a highly productive orchard in Hungary. All specimens ranging between 2 and 755 g were almost evenly distributed through five maturation classes. Then, we measured the weight and maturity of another 4,795 T. aestivum fruit bodies harvested on four occasions between June and October 2015 in the same truffière. Again, different maturation stages occurred at varying fruit body size and during the entire fruiting season. Finally, the predominantly unrelated weight and maturity of 81 T. aestivum fruit bodies from four fruiting seasons between 2010 and 2013 in Switzerland confirmed the Hungarian results. The spatiotemporal coexistence of 7,532 small-ripe and large-unripe T. aestivum, which accumulate to ~182 kg, differs from species-specific associations between the size and ripeness that have been reported for other mushrooms. Although size-independent truffle maturation stages may possibly relate to the perpetual belowground environment, the role of mycelial connectivity, soil property, microclimatology, as well as other abiotic factors and a combination thereof, is still unclear. Despite its massive sample size and proof of concept, this study, together with existing literature, suggests consideration of a wider ecological and biogeographical range, as well as the complex symbiotic fungus-host interaction, to further illuminate the hidden development of belowground truffle fruit bodies.
Die Wärme liebende Asiatische Tigermücke »Aedes albopictus« fühlt sich seit Jahrzehnten im Mittelmeerraum wohl. Sie ist Überträgerin gefährlicher, bisher in Europa nicht verbreiteter Viren. Wird sie sich aufgrund des Klimawandels und anderer Umweltfaktoren weiter nach Norden ausbreiten? Und werden andere eingeschleppte Arten ihr folgen? Das untersucht die Arbeitsgruppe von Prof. Dr. Sven Klimpel mithilfe der ökologischen Nischenmodellierung und genomischer Analysen.
Die meisten von Menschen in neue Habitate eingeschleppten Arten sind harmlos. Doch einige richten beträchtliche ökologische und ökonomische Schäden an. Rückgängig machen kann man den Prozess nicht, aber vorbeugen sollte man. Computermodelle ermitteln die gefährdeten Knotenpunkte im Handelsnetz und sagen die nächsten Invasoren im marinen Bereich inzwischen zuverlässig voraus.
Lauschangriff mit tödlichen Folgen : Signalmoleküle von Bakterien können fremden Arten schaden
(2016)
Eine der wichtigsten Fähigkeiten aller Lebewesen ist die Kommunikation. Ihre universelle Ausdrucksform findet sie im Austausch hoch spezifischer Signalmoleküle. Bei der Entschlüsselung der diversen »Sprachen« und »Dialekte« von Bakterien machen Forscher immer wieder neue und überraschende Entdeckungen, die auch eine Alternative zu Antibiotika versprechen.
Background: One aspect of premating isolation between diverging, locally-adapted population pairs is female mate choice for resident over alien male phenotypes. Mating preferences often show considerable individual variation, and whether or not certain individuals are more likely to contribute to population interbreeding remains to be studied. In the Poecilia mexicana-species complex different ecotypes have adapted to hydrogen sulfide (H2S)-toxic springs, and females from adjacent non-sulfidic habitats prefer resident over sulfide-adapted males. We asked if consistent individual differences in behavioral tendencies (animal personality) predict the strength and direction of the mate choice component of premating isolation in this system.
Results: We characterized focal females for their personality and found behavioral measures of ‘novel object exploration’, ‘boldness’ and ‘activity in an unknown area’ to be highly repeatable. Furthermore, the interaction term between our measures of exploration and boldness affected focal females’ strength of preference (SOP) for the resident male phenotype in dichotomous association preference tests. High exploration tendencies were coupled with stronger SOPs for resident over alien mating partners in bold, but not shy, females. Shy and/or little explorative females had an increased likelihood of preferring the non-resident phenotype and thus, are more likely to contribute to rare population hybridization. When we offered large vs. small conspecific stimulus males instead, less explorative females showed stronger preferences for large male body size. However, this effect disappeared when the size difference between the stimulus males was small.
Conclusions: Our results suggest that personality affects female mate choice in a very nuanced fashion. Hence, population differences in the distribution of personality types could be facilitating or impeding reproductive isolation between diverging populations depending on the study system and the male trait(s) upon which females base their mating decisions, respectively.
The fungal genus Pestalotiopsis s.l. contains approximately 300 described species and is globally distributed. The monotypic genus Pestalotia is considered the closest relative of Pestalotiopsis s.l. This study aims to investigate the diversity and systematics within Pestalotiopsis s.l. and its relation to Pestalotia. Therefore, an integrative approach is used considering molecular phylogeny methods as well as examination of morphological characters.
Recently, Pestalotiopsis s.l. was split into three genera with the addition of the newly erected Neopestalotiopsis and Pseudopestalotiopsis. The species of these genera are usually saprotrophic, phytoparasitic, or endophytic, and have been isolated from soil, air, and many kinds of anorganic material. The asexual fruiting bodies appear on infected plant material as black acervuli that release conidia. The conidia are important to examine for morphological taxon recognition. The number of conidial cells is the feature that distinguishes Pestalotiopsis s.l. spp. with five celled conidia, from Pestalotia pezizoides with six celled conidia. However, the significance of morphological characters is controversially discussed among mycologists. In recent years, 55 new species were described based on minor genetic distances and marginal or no morphological differences. Thus, the value of certain morphological characters and genetic markers need to be reconsidered.
In this study, 102 herbarium specimens of 26 described species, with an emphasis on plant pathogenic species from North America, have been morphologically examined and documented through drawings and photographs. Morphological examination was complemented with a comprehensive molecular dataset obtained from 191 cultures representing the genera Neopestalotiopsis, Pestalotia, Pestalotiopsis, Pseudopestalotiopsis, and Truncatella. One novelty of this work is that, besides the well-established markers ITS, TEF1, and ß-tubulin, the protein-coding genes MCM7 and TSR1 were successfully sequenced and included in the analyses. Phylogenies using Maximum Likelihood and Bayesian inference methods of single loci and the combined dataset were calculated. By comparison of these phylogenies, MCM7 was identified as the most powerful one in terms of phylogenetic resolution and statistical support of nodes and is proposed as an additional barcoding marker in Pestalotiopsis s.l.
In Pestalotiopsis, species delimitation was tested using the Baysian Phylogenetics and Phylogeography (BP&P) program that tests an existing species scenario against Bayesian inference methods under a multispecies coalescent model. The program supported only ten species out of the predetermined 19 species scenario. Measurements of conidia for species detected by BP&P were explored using a TukeyHSD-Test in the program R to find means that are significantly different from each other. This test revealed that combinations of morphological characters are required to distinguish between the ten species found by BP&P.
Another purpose of this work was to clarify the status of Pestalotia with regard to Pestalotiopsis s.l. Therefore, fresh epitypic material of Pestalotia pezizoides, was collected, isolated, and cultivated. The molecular analysis of a combined dataset of the gene regions ITS and LSU for species of Amphisphaeriales nested P. pezizoides in the genus Seiridium. Thus, synonymy of Pestalotia with Seiridium is proposed here. This is supported by morphology of the conidia. Further, an epitype is proposed for the type species of Pestalotiopsis, P. maculans. On the other hand, the recently proposed epitype of P. adusta is rejected here as it conflicts with the taxonomic hypothesis obtained in this study and its introduction is inconsistent with the formal requirements for epitypification. A new topotypic specimen is proposed instead. Additionally, several nomenclatural changes become necessary in many species examined. These include three new combinations and six synonyms of species of Pestalotiopsis s.l.
The conclusion of this work is that morphological data have potential as a valuable, inexpensive and easy way to recognize species. However, it is not the best method for species discovery and delimitation bearing in mind that in microfungi and many other organisms, individual plasticity and analogous structures are inadequately investigated. By phylogenetic analyses of molecular sequence data, it is possible to compare a great amount of equivalent characters and to delimit species that are morphologically cryptic. This is especially important since species of Pestalotiopsis s.l. mostly lack sexual structures that are helpful for morphological species delimitation in other groups of fungi. Thus, the Genealogical Concordance Species Concept (GCSC) finds its application in many fungal taxa. Conflicts in the genealogy between phylogenetic trees of different markers are interpreted as recombination of the genetic material within a linage. Accordingly, the change from conflict to congruence in a set of different phylogenetic trees can be seen as the species limit. It can be expected that increased application of the GCSC will lead to further approximation of described species numbers to the real number of species, especially in complicated groups like asexual microfungi.
This report provides a brief review of the 20th annual meeting of the German Language Branch of the Society of Environmental Toxicology and Chemistry (SETAC GLB) held from September 7th to 10th 2015 at ETH (Swiss Technical University) in Zurich, Switzerland. The event was chaired by Inge Werner, Director of the Swiss Centre for Applied Ecotoxicology (Ecotox Centre) Eawag-EPFL, and organized by a team from Ecotox Centre, Eawag, Federal Office of the Environment, Federal Office of Agriculture, and Mesocosm GmbH (Germany). Over 200 delegates from academia, public agencies and private industry of Germany, Switzerland and Austria attended and discussed the current state of science and its application presented in 75 talks and 83 posters. In addition, three invited keynote speakers provided new insights into scientific knowledge ‘brokering’, and—as it was the International Year of Soil—the important role of healthy soil ecosystems. Awards were presented to young scientists for best oral and poster presentations, and for best 2014 master and doctoral theses. Program and abstracts of the meeting (mostly in German) are provided as Additional file 1.
Autophagy can act either as a tumor suppressor or as a survival mechanism for established tumors. To understand how autophagy plays this dual role in cancer, in vivo models are required. By using a highly heterogeneous C. elegans germline tumor, we show that autophagy-related proteins are expressed in a specific subset of tumor cells, neurons. Inhibition of autophagy impairs neuronal differentiation and increases tumor cell number, resulting in a shorter life span of animals with tumors, while induction of autophagy extends their life span by impairing tumor proliferation. Fasting of animals with fully developed tumors leads to a doubling of their life span, which depends on modular changes in transcription including switches in transcription factor networks and mitochondrial metabolism. Hence, our results suggest that metabolic restructuring, cell-type specific regulation of autophagy and neuronal differentiation constitute central pathways preventing growth of heterogeneous tumors.
It is long known that Kasugamycin inhibits translation of canonical transcripts containing a 5’-UTR with a Shine Dalgarno (SD) motif, but not that of leaderless transcripts. To gain a global overview of the influence of Kasugamycin on translation efficiencies, the changes of the translatome of Escherichia coli induced by a 10 minutes Kasugamycin treatment were quantified. The effect of Kasugamycin differed widely, 102 transcripts were at least twofold more sensitive to Kasugamycin than average, and 137 transcripts were at least twofold more resistant, and there was a more than 100-fold difference between the most resistant and the most sensitive transcript. The 5’-ends of 19 transcripts were determined from treated and untreated cultures, but Kasugamycin resistance did neither correlate with the presence or absence of a SD motif, nor with differences in 5’-UTR lengths or GC content. RNA Structure Logos were generated for the 102 Kasugamycin-sensitive and for the 137 resistant transcripts. For both groups a short Shine Dalgarno (SD) motif was retrieved, but no specific motifs associated with resistance or sensitivity could be found. Notably, this was also true for the region -3 to -1 upstream of the start codon and the presence of an extended SD motif, which had been proposed to result in Kasugamycin resistance. Comparison of the translatome results with the database RegulonDB showed that the transcript with the highest resistance was leaderless, but no further leaderless transcripts were among the resistant transcripts. Unexpectedly, it was found that translational coupling might be a novel feature that is associated with Kasugamycin resistance. Taken together, Kasugamycin has a profound effect on translational efficiencies of E. coli transcripts, but the mechanism of action is different than previously described.
Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae’s 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.
Embryonale Stammzellen (ESCs) sind ein wichtiges Werkzeug zur Untersuchung der frühen embryonalen Entwicklung. ESCs können mit Hilfe neuer Technologien zur Modifikation von Genen (z.B. mit dem CRISPR/Cas9 System) genetisch manipuliert werden. Daraus resultierende „knockout“ ES Zelllinien können helfen, die physiologische Rolle von Proteinen während der Differenzierung zu verstehen.
Transkriptionsfaktoren, die schnell und spezifisch Signalwege regulieren, spielen während der Embryonalentwicklung und während der Differenzierung von ESCs in vielen verschiedenen Zelltypen eine essentielle Rolle. Der Transkriptionsregulator „Far Upstream Binding Protein 1“ (FUBP1) ist ein Protein, welches eine ganz bestimmte einzelsträngige DNA Sequenz, das „Far Upstream Sequenz Element“, erkennt, bindet, und dadurch Gene wie z.B c-myc oder p21 reguliert. Mit der Entwicklung zweier Fubp1 Genfallen Mausstämme (Fubp1 GT) sollte die Frage nach der physiologischen Funktion von FUBP1 beantwortet werden. Die homozygoten FUBP1-defizienten GT Embryonen sterben im Mutterleib ungefähr am Tag E15.5 der Embryonalentwicklung. Sie sind kleiner als Wildtypembryonen und zeigen ein anämisches Aussehen. Daher wurden diese Mausmodelle hinsichtlich der Hämatopoese untersucht, die zu diesem Zeitpunkt vor allem in der Leber stattfindet. Es konnte eine signifikante Reduktion der hämatopoetischen Stammzellen (HSCs) festgestellt werden und zusätzlich war die langfristige Repopulation der FUBP1-/--Stammzellen im Knochenmark in Transplantationsexperimenten reduziert.
In der vorliegenden Arbeit wurde die Rolle von FUBP1 in einem weiteren Stammzellsystem analysiert und gleichzeitig seine Bedeutung in anderen Zelltypen der frühen Embryonalentwicklung untersucht.
Die Quantifizierung der FUBP1 Expression in den ESCs und während der Differenzierung zu sogenannten `embryoid bodies` (EBs) zeigten eine starke Expression auf mRNA- und auf Proteinebene. Nach der erfolgreichen Optimierung der Differenzierung von murinen ESCs wurden Fubp1 „knockout“ (KO) ESC Klone mit Hilfe der CRISPR/Cas9 Technologie etabliert. Die molekularbiologische Analyse der ESCs zeigte eine signifikante Erhöhung der Oct4 mRNA-Expression, während Nanog und die Differenzierungsmarker Brachyury, Nestin und Sox17 unverändert und in vergleichbarer Menge zu den Kontrollen vorhanden waren. Während der Differenzierung der Fubp1 KO Klone zu EBs zeigte sich eine signifikante Reduktion mesodermaler Marker wie Flk-1, SnaiI, Snai2, Bmp4 und FgfR2. Mit Hilfe durchflusszytometrischer Analysen bestätigte sich die verzögerte Bildung mesodermaler Zellen (Brachyury- und Flk-1-exprimierender Zellen) in den Fubp1 KO Klonen der EBs an den Tagen 3, 4 und 5 nach Beginn der Differenzierung.
Die Anwendung einer Ko-Kultivierung auf OP9 Zellen zur Differenzierung der ESCs in hämatopoetische Linien sollte zeigen, ob der Fubp1 KO ESCs ein Defekt in der frühen Entwicklung hämatopoetischer Stammzellen zu beobachten ist. Erneut konnte am Tag 5 der ESC-Differenzierung in der OP9 Ko-Kultur eine signifikante Reduktion der mesodermalen (Flk-1+) Zellen festgestellt werden. Die weitere Differenzierung zu hämatopoetischen CD45+ Zellen zeigte jedoch keinen Unterschied im prozentualen Anteil CD45+ Zellen am Tag 12 der Differenzierung. Auch die gezielte Differenzierung zu erythroiden Zellen durch Zugabe des Zytokins EPO zum Medium zeigte keinen signifikanten Unterschied im Differenzierungsgrad der erythroiden Zellen zwischen Kontroll- und Fubp1 KO Klonen.
In weiteren Experimenten habe ich in dieser Arbeit die Expression von FUBP1 in WT Embryos an den Tagen E9.5 und E13.5 der Embryonalentwicklung untersucht. Hierbei zeigte sich in beiden Entwicklungsstadien eine immunhistochemische Anfärbung von FUBP1 in den meisten Zellen des Embryos. Die Annahme, dass die Abwesenheit von FUBP1 in der Embryonalentwicklung zu verstärkten apoptotischen Vorgängen führen könnte und gleichzeitig die massive Expansion von Zellen gestört sein könnte wurde mit Hilfe immunhistochemischer Färbung von „cleaved Caspase 3“ (Apoptosemarker) und „Ki-67“ (Proliferationsmarker) in den homozygoten Fubp1 GT Embryos an den Tagen E9.5 und E13.5 nicht bestätigt.
Die Ergebnisse dieser Arbeit lassen darauf schließen, dass die Regulation von Apoptose und Proliferation durch FUBP1 während der Embryonalentwicklung nicht die Hauptrolle von FUBP1 darstellt. Es zeigte sich jedoch, dass FUBP1 als Transkriptionsregulator wichtig für die mesodermale Differenzierung von ESCs ist. Zu beobachten war, dass es in den FUBP1-defizienten ESCs zu einer Verzögerung der mesodermalen Differenzierung kommt. Es konnte bereits gezeigt werden, dass FUBP1 essenziell für die Selbsterneuerung von HSCs ist. Dies macht deutlich, dass FUBP1 neben der Proliferation und Apoptose ein breiteres Spektrum an Signalwegen reguliert, die für Stammzellen und deren Differenzierung von Bedeutung sind.
Bei Cryptochromen handelt es sich um Blaulichtrezeptoren der Cryptochrom-Photolyase-Proteinfamilie (CPF). Mitglieder dieser Proteinfamilie sind in allen Domänen des Lebens zu finden und haben eine essentielle Rolle in der Reparatur der DNA sowie der lichtgesteuerten Regulation der Expression. Cryptochrome sind in der Regel keine DNA-reparierenden Proteine. Sie sind regulativ an der Steuerung der inneren Uhr und des Zellzyklus der Organismen beteiligt. In der Kieselalge Phaeodactylum tricornutum konnten bisher sechs phylogenetisch unterschiedliche Mitglieder der CPF identifiziert werden. Bei CryP handelt es sich um das einzige pflanzenähnliche Cryptochrom der photoautotrophen Diatomee. Für das Protein CryP konnte bereits ein blaulichtinduzierter Photozyklus durch die Absorption der Chromophore 5-Methenlytetrahydrofolat (MTHF) und Flavinadenindinukleotid (FAD) gezeigt werden. Außerdem ist eine regulative Wirkung des Proteins auf die Lichtsammelkomplexe (Lhc) der Diatomee bekannt. Für eine weitere Charakterisierung des CryPs wurde in dieser Arbeit zunächst das Absorptionsverhalten unter verschiedenen Wellenlängen beobachtet, um so einen Einblick in eine mögliche Aktivierung und Deaktivierung des Proteins durch Licht unterschiedlicher Wellenlängen zu erlangen. Es zeigte sich hierbei eine mit pflanzlichen Cryptochromen vergleichbare Anreicherung verschiedener Redoxzustände des FADs in Abhängigkeit von der Wellenlänge.
Für eine Aufklärung der Wirkungsweise des CryP-Proteins wurden verschiedene Hypothesen untersucht: Die phylogenetische Nähe und ein ähnliches Absorptionsverhalten des CryPs zu Cryptochromen mit Reparaturfähigkeit für einzelsträngige DNA (Cry-DASH) führte zu einer Untersuchung des Proteins als möglicher Transkriptionsfaktor. Hierfür konnte eine Kernlokalisation des Proteins nachgewiesen werden, was Rückschlüsse auf eine potentielle Regulation der Expression mittels DNA-Bindung zulässt. Außerdem wurde gezeigt, dass CryP DNA-Bindefähigkeit besitzt. Die bisher nachgewiesenen Bindungen waren jedoch unspezifischer Art. Dies konnte auch für die Promotersequenz eines der durch CryP regulierten Gene lhcf1 festgestellt werden. Auf Grund der unspezifischen DNA-Bindung wurde eine zweite Hypothese für CryP untersucht: CryP wirkt regulativ auf die Expression verschiedener Gene durch Protein-Protein-Interaktionen und ist Teil einer Reaktionskaskade zur Signalweiterleitung in P. tricornutum.
Durch die Untersuchung der zweiten Hypothese konnten drei Interaktionspartner für CryP identifiziert und eine Interaktion verifiziert werden. Hierbei handelt es sich um das Protein AAA mit einer bisher unbekannten Funktion und das Protein BolA, welches Teil der zuerst in Escherichia coli identifizierten BolA-like-Proteinfamilie ist. Außerdem konnte eine Interaktion mit dem Cold-Shock-Domänen-Protein CSDP gezeigt werden. Bei den Proteinen BolA und CSDP handelt es sich um potentiell regulierende Faktoren der Transkription und Translation, was Teil einer Reaktionskaskade sein kann. Die aus anderen Organismen bekannten Funktionen des BolA-Proteins überschneiden sich mit den in CryP-Knockdown-Mutanten beobachteten Effekten. Sie zeigen eine erhöhte Sensitivität für Stresssituationen wie abweichende Nährstoffkonzentration, Osmolaritäten und Temperaturen. Diese Beobachtungen stellen einen Zusammenhang der durch einen CryP-Knockdown beobachteten Effekte und der CryP-BolA-Interaktion her. Durch Homologien zu Cold-Shock-Proteinen aus Chlamydomonas reinhardtii gibt die CryP-Interaktion mit dem Protein CSDP Hinweise auf einen potentiellen Mechanismus zur Regulation der Lhc-Proteine, für welche zuvor ein CryP-abhängiger Effekt beschrieben war.
Über die Protein-Protein-Interaktionen hinaus wurde die Phosphorylierung des CryPs als Möglichkeit der Signalweiterleitung untersucht. Es konnte eine reversible Phosphorylierung des heterolog aus E. coli isolierten CryPs gezeigt werden. Diese zeigt Ähnlichkeiten zu bekannten Phosphorylierungen pflanzlicher Cryptochrome und gibt Hinweise auf einen Mechanismus der Signalweiterleitung.
Durch die Untersuchung der CryP-regulierten Transkription mit P. tricornutum CryP-Knockdown-Mutanten durch Next-Generation-Sequencing (NGS) konnte die Hypothese der regulativen Proteinkaskade und der Signalweiterleitung weiter bestätigt werden. Die Auswirkungen des CryPs auf die Transkription erwiesen sich als nicht auf einen Teilbereich des Metabolismus begrenzt, sondern sind in einem großen Teil der funktionellen Gengruppen in P. tricornutum zu sehen. Außerdem konnten drei Klassen CryP-regulierter Gene festgestellt werden. Kategorie 1: die ausschließlich unter Blaulicht regulierten Gene; Kategorie 2: die sowohl unter Blaulicht als auch im Dunkeln regulierten Gene und Kategorie 3: die ausschließlich im Dunkeln regulierten Gene. Ein im Dunkeln und unter Blaulicht jeweils unterschiedlicher regulativer Effekt deutet auf eine Doppelfunktion des CryPs hin. Möglicherweise hat das Cryptochrom unterschiedliche lichtabhängige und lichtunabhängige Funktionen.
Durch die Analyse der CryP-regulierten Genexpression konnte außerdem ein Zusammenhang zwischen CryP und weiteren Photorezeptoren gezeigt werden. Der CryP-Proteingehalt in der Zelle hat einen regulativen Einfluss auf das CPF1-Protein, eine Photolyase mit dualer Funktion aus der gleichen Proteinfamilie. Zusätzlich konnte auch ein Einfluss auf die Lichtsensitivität der Genexpression des Rotlichtrezeptors Phytochrom (DPH) durch CryP gezeigt werden. Vergleichbar mit höheren Pflanzen scheint ein regulatives Netzwerk der Photorezeptoren auch in der Diatomee P. tricornutum vorhanden zu sein.
Noise-induced hearing loss is one of the most common auditory pathologies, resulting from overstimulation of the human cochlea, an exquisitely sensitive micromechanical device. At very low frequencies (less than 250 Hz), however, the sensitivity of human hearing, and therefore the perceived loudness is poor. The perceived loudness is mediated by the inner hair cells of the cochlea which are driven very inadequately at low frequencies. To assess the impact of low-frequency (LF) sound, we exploited a by-product of the active amplification of sound outer hair cells (OHCs) perform, so-called spontaneous otoacoustic emissions. These are faint sounds produced by the inner ear that can be used to detect changes of cochlear physiology. We show that a short exposure to perceptually unobtrusive, LF sounds significantly affects OHCs: a 90 s, 80 dB(A) LF sound induced slow, concordant and positively correlated frequency and level oscillations of spontaneous otoacoustic emissions that lasted for about 2 min after LF sound offset. LF sounds, contrary to their unobtrusive perception, strongly stimulate the human cochlea and affect amplification processes in the most sensitive and important frequency range of human hearing.
BACKGROUND: Acetogenic bacteria are able to use CO2 as terminal electron acceptor of an anaerobic respiration, thereby producing acetate with electrons coming from H2. Due to this feature, acetogens came into focus as platforms to produce biocommodities from waste gases such as H2+CO2 and/or CO. A prerequisite for metabolic engineering is a detailed understanding of the mechanisms of ATP synthesis and electron-transfer reactions to ensure redox homeostasis. Acetogenesis involves the reduction of CO2 to acetate via soluble enzymes and is coupled to energy conservation by a chemiosmotic mechanism. The membrane-bound module, acting as an ion pump, was of special interest for decades and recently, an Rnf complex was shown to couple electron flow from reduced ferredoxin to NAD+ with the export of Na+ in Acetobacterium woodii. However, not all acetogens have rnf genes in their genome. In order to gain further insights into energy conservation of non-Rnf-containing, thermophilic acetogens, we sequenced the genome of Thermoanaerobacter kivui.
RESULTS: The genome of Thermoanaerobacter kivui comprises 2.9 Mbp with a G+C content of 35% and 2,378 protein encoding orfs. Neither autotrophic growth nor acetate formation from H2+CO2 was dependent on Na+ and acetate formation was inhibited by a protonophore, indicating that H+ is used as coupling ion for primary bioenergetics. This is consistent with the finding that the c subunit of the F1FO ATP synthase does not have the conserved Na+ binding motif. A search for potential H+-translocating, membrane-bound protein complexes revealed genes potentially encoding two different proton-reducing, energy-conserving hydrogenases (Ech).
CONCLUSIONS: The thermophilic acetogen T. kivui does not use Na+ but H+ for chemiosmotic ATP synthesis. It does not contain cytochromes and the electrochemical proton gradient is most likely established by an energy-conserving hydrogenase (Ech). Its thermophilic nature and the efficient conversion of H2+CO2 make T. kivui an interesting acetogen to be used for the production of biocommodities in industrial micobiology. Furthermore, our experimental data as well as the increasing number of sequenced genomes of acetogenic bacteria supported the new classification of acetogens into two groups: Rnf- and Ech-containing acetogens.
In recent years, there have been prominent calls for a new social contract that accords a more central role to citizens in health research. Typically, this has been understood as citizens and patients having a greater voice and role within the standard research enterprise. Beyond this, however, it is important that the renegotiated contract specifically addresses the oversight of a new, path-breaking approach to health research: participant-led research. In light of the momentum behind participant-led research and its potential to advance health knowledge by challenging and complementing traditional research, it is vital for all stakeholders to work together in securing the conditions that will enable it to flourish.
Particularly in savannas, termites are ecosystem engineers and a keystone group in ecology. For the understanding of the savanna vegetation, mound building termites are of particular interest. Due to their special soil chemistry and physical structure, termite mounds often host other plants than the surrounding savanna. As our knowledge of the specific contribution of mound-building termites to overall savanna diversity and ecosystem dynamics doubtlessly is not complete, this paper summarises the state of the art in order to stimulate further research. According to the research interest of the authors, focus is laid on the West African savanna and on the genus Macrotermes.
In West African savannas, human land use affects the density of woody species seedlings and saplings (juveniles) by altering the state of the physical, chemical, and biological characteristics of the land resulting in different land-cover types. We determined juvenile densities of 25 characteristic woody savanna species on non-arable sites, in fallows and in a protected area (in total 39 plots), and analyzed the influence of land use on juvenile densities. We further related the influence of land use on juvenile densities to 23 environmental parameters describing soil properties and vegetation structure. Soil acidity, particle size distribution of the soil, and vegetation structure differed between land-cover types. In terms of human impact, we detected five groups of species responding similarly to land use. Although we detected significant differences in soil properties, their direct effects on juvenile densities are less pronounced than their indirect effects. By altering the availability of resources, soil properties affect height and cover of all plants growing in the surrounding of a young woody plant, increasing the competition for light, water and nutrients during the establishment and initial growth. These effects are intensified by human land use and vary between land-cover types.
Non-Timber Forest Products (NTFPs) make a major contribution to the livelihoods and diets of rural households in the savanna ecosystems of West Africa. However, land use change and climatic variability might affect their availability in the future. Based on a survey among 227 households in Northern Benin, we investigated local substitution patterns for the seeds of the three socio-economically most important NTFP-species in the region, Vitellaria paradoxa, Adansonia digitata and Parkia biglobosa, being major sources for protein, fat, and micronutrients in local daily diets. Our study compared substitution patterns between, firstly, three income groups, to assess whether a households’ socio-economic status has an influence on the choice of surrogates (low cost vs. more costly options). Secondly, we compared substitution patterns between the five major ethnic groups in the study region (the Fulani, the Bariba, the Ditammarie, the Kabiyé and the Yom). The choice of substitutes differed significantly across income groups. However, the poorest households clearly show to be the most vulnerable: up to 30 % of the sampled households stated they would lack an adequate replacement for the NTFPs in question. Furthermore, ethnic affiliation showed to have a considerable impact on the preferred alternative products due to underlying cultural traditions of plant use. Subsequently, aiming at maintaining – and enhancing – the local supply of V. paradoxa, P. biglobosa and A. digitata in order to secure their contributions to local diets, local land use policy should have a particular focus on their ethnic-conditioned use and particularly the specific requirements of the poorest community members.
To improve data availability and exchange in the area of the WAP complex, West Africa’s largest continuous area of reserves, we set up a citizen science project on the iNaturalist platform, allowing contribution of observations, ideally documented by photographs and/or sounds. Along with the project we created a number of online field guides for the local flora. Within only two months, 852 observations of 312 species have been assembled. We expect this dataset to further grow in the future and complement existing data sets from scientific collections and surveys.
Biodiversity is unevenly distributed on Earth and hotspots of biodiversity are often associated with areas that have undergone orogenic activity during recent geological history (i.e. tens of millions of years). Understanding the underlying processes that have driven the accumulation of species in some areas and not in others may help guide prioritization in conservation and may facilitate forecasts on ecosystem services under future climate conditions. Consequently, the study of the origin and evolution of biodiversity in mountain systems has motivated growing scientific interest. Despite an increasing number of studies, the origin and evolution of diversity hotspots associated with the Qinghai-Tibetan Plateau (QTP) remains poorly understood. We review literature related to the diversification of organisms linked to the uplift of the QTP. To promote hypothesis-based research, we provide a geological and palaeoclimatic scenario for the region of the QTP and argue that further studies would benefit from providing a complete set of complementary analyses (molecular dating, biogeographic, and diversification rates analyses) to test for a link between organismic diversification and past geological and climatic changes in this region. In general, we found that the contribution of biological interchange between the QTP and other hotspots of biodiversity has not been sufficiently studied to date. Finally, we suggest that the biological consequences of the uplift of the QTP would be best understood using a meta-analysis approach, encompassing studies on a variety of organisms (plants and animals) from diverse habitats (forests, meadows, rivers), and thermal belts (montane, subalpine, alpine, nival). Since the species diversity in the QTP region is better documented for some organismic groups than for others, we suggest that baseline taxonomic work should be promoted.
The LPJ-GUESS dynamic vegetation model uniquely combines an individual- and patch-based representation of vegetation dynamics with ecosystem biogeochemical cycling from regional to global scales. We present an updated version that includes plant and soil N dynamics, analysing the implications of accounting for C-N interactions on predictions and performance of the model. Stand structural dynamics and allometric scaling of tree growth suggested by global databases of forest stand structure and development were well-reproduced by the model in comparison to an earlier multi-model study. Accounting for N cycle dynamics improved the goodness-of-fit for broadleaved forests. N limitation associated with low N mineralisation rates reduces productivity of cold-climate and dry-climate ecosystems relative to mesic temperate and tropical ecosystems. In a model experiment emulating free-air CO2 enrichment (FACE) treatment for forests globally, N-limitation associated with low N mineralisation rates of colder soils reduces CO2-enhancement of NPP for boreal forests, while some temperate and tropical forests exhibit increased NPP enhancement. Under a business-as-usual future climate and emissions scenario, ecosystem C storage globally was projected to increase by c. 10 %; additional N requirements to match this increasing ecosystem C were within the high N supply limit estimated on stoichiometric grounds in an earlier study. Our results highlight the importance of accounting for C-N interactions not only in studies of global terrestrial C cycling, but to understand underlying mechanisms on local scales and in different regional contexts.
The LPJ-GUESS dynamic vegetation model uniquely combines an individual- and patch-based representation of vegetation dynamics with ecosystem biogeochemical cycling from regional to global scales. We present an updated version that includes plant and soil N dynamics, analysing the implications of accounting for C–N interactions on predictions and performance of the model. Stand structural dynamics and allometric scaling of tree growth suggested by global databases of forest stand structure and development were well reproduced by the model in comparison to an earlier multi-model study. Accounting for N cycle dynamics improved the goodness of fit for broadleaved forests. N limitation associated with low N-mineralisation rates reduces productivity of cold-climate and dry-climate ecosystems relative to mesic temperate and tropical ecosystems. In a model experiment emulating free-air CO2 enrichment (FACE) treatment for forests globally, N limitation associated with low N-mineralisation rates of colder soils reduces CO2 enhancement of net primary production (NPP) for boreal forests, while some temperate and tropical forests exhibit increased NPP enhancement. Under a business-as-usual future climate and emissions scenario, ecosystem C storage globally was projected to increase by ca. 10%; additional N requirements to match this increasing ecosystem C were within the high N supply limit estimated on stoichiometric grounds in an earlier study. Our results highlight the importance of accounting for C–N interactions in studies of global terrestrial N cycling, and as a basis for understanding mechanisms on local scales and in different regional contexts.
Strong seasonal variability of hygric and thermal soil conditions are a defining environmental feature in Northern Australia. However, how such changes affect the soil–atmosphere exchange of nitrous oxide (N2O), nitric oxide (NO) and dinitrogen (N2) is still 5 not well explored. By incubating intact soil cores from four sites (3 savanna, 1 pasture) under controlled soil temperatures (ST) and soil moisture (SM) we investigated the release of the trace gas fluxes of N2O, NO and carbon dioxide (CO2). Furthermore, the release of N2 due to denitrification was measured using the helium gas flow soil core technique. Under dry pre-incubation conditions NO and N2O emission were very low (< 7.0± 5.0 μgNO-Nm−2 h−1; < 0.0± 1.4 μgN2O-Nm−2 h−1) or in case of N2O, even a net soil uptake was observed. Substantial NO (max: 306.5 μgNm−2 h−1) and relatively small N2O pulse emissions (max: 5.8±5.0 μgNm−2 h−1) were recorded following soil wetting, but these pulses were short-lived, lasting only up to 3 days. The total atmospheric loss of nitrogen was dominated by N2 emissions (82.4–99.3% of total N lost), although NO emissions contributed almost 43.2% at 50% SM and 30 °C ST. N2O emissions were systematically higher for 3 of 12 sample locations, which indicates substantial spatial variability at site level, but on average soils acted as weak N2O sources or even sinks. Emissions were controlled by SM and ST for N2O and CO2, ST and pH for NO, and SM and pH for N2.
Strong seasonal variability of hygric and thermal soil conditions are a defining environmental feature in northern Australia. However, how such changes affect the soil–atmosphere exchange of nitrous oxide (N2O), nitric oxide (NO) and dinitrogen (N2) is still not well explored. By incubating intact soil cores from four sites (three savanna, one pasture) under controlled soil temperatures (ST) and soil moisture (SM) we investigated the release of the trace gas fluxes of N2O, NO and carbon dioxide (CO2). Furthermore, the release of N2 due to denitrification was measured using the helium gas flow soil core technique. Under dry pre-incubation conditions NO and N2O emissions were very low (<7.0 ± 5.0 μg NO-N m−2 h−1; <0.0 ± 1.4 μg N2O-N m−2 h−1) or in the case of N2O, even a net soil uptake was observed. Substantial NO (max: 306.5 μg N m−2 h−1) and relatively small N2O pulse emissions (max: 5.8 ± 5.0 μg N m−2 h−1) were recorded following soil wetting, but these pulses were short lived, lasting only up to 3 days. The total atmospheric loss of nitrogen was generally dominated by N2 emissions (82.4–99.3% of total N lost), although NO emissions contributed almost 43.2% to the total atmospheric nitrogen loss at 50% SM and 30 °C ST incubation settings (the contribution of N2 at these soil conditions was only 53.2%). N2O emissions were systematically higher for 3 of 12 sample locations, which indicates substantial spatial variability at site level, but on average soils acted as weak N2O sources or even sinks. By using a conservative upscale approach we estimate total annual emissions from savanna soils to average 0.12 kg N ha−1 yr−1 (N2O), 0.68 kg N ha−1 yr−1 (NO) and 6.65 kg N ha−1 yr−1 (N2). The analysis of long-term SM and ST records makes it clear that extreme soil saturation that can lead to high N2O and N2 emissions only occurs a few days per year and thus has little impact on the annual total. The potential contribution of nitrogen released due to pulse events compared to the total annual emissions was found to be of importance for NO emissions (contribution to total: 5–22%), but not for N2O emissions. Our results indicate that the total gaseous release of nitrogen from these soils is low and clearly dominated by loss in the form of inert nitrogen. Effects of seasonally varying soil temperature and moisture were detected, but were found to be low due to the small amounts of available nitrogen in the soils (total nitrogen <0.1%).
Phylogenetic relationships of the primarily wingless insects are still considered unresolved. Even the most comprehensive phylogenomic studies that addressed this question did not yield congruent results. In order to get a grip on these problems, we here analyzed the sources of incongruence in these phylogenomic studies using an extended transcriptome dataset.Our analyses showed that unevenly distributed missing data can be severely misleading by inflating node support despite the absence of phylogenetic signal. In consequence, only decisive datasets should be used which exclusively comprise data blocks containing all taxa whose relationships are addressed. Additionally, we employed Four-cluster Likelihood-Mapping (FcLM) to measure the degree of congruence among genes of a dataset, as a measure of support alternative to bootstrap. FcLM showed incongruent signal among genes, which in our case is correlated with neither functional class assignment of these genes, nor with model misspecification due to unpartitioned analyses. The herein analyzed dataset is the currently largest dataset covering primarily wingless insects, but failed to elucidate their interordinal phylogenetic relationships. While this is unsatisfying from a phylogenetic perspective, we try to show that the analyses of structure and signal within phylogenomic data can protect us from biased phylogenetic inferences due to analytical artefacts.
Biomass burning impacts vegetation dynamics, biogeochemical cycling, atmospheric chemistry, and climate, with sometimes deleterious socio-economic impacts. Under future climate projections it is often expected that the risk of wildfires will increase. Our ability to predict the magnitude and geographic pattern of future fire impacts rests on our ability to model fire regimes, using either well-founded empirical relationships or process-based models with good predictive skill. While a large variety of models exist today, it is still unclear which type of model or degree of complexity is required to model fire adequately at regional to global scales. This is the central question underpinning the creation of the Fire Model Intercomparison Project (FireMIP), an international initiative to compare and evaluate existing global fire models against benchmark data sets for present-day and historical conditions. In this paper we review how fires have been represented in fire-enabled dynamic global vegetation models (DGVMs) and give an overview of the current state of the art in fire-regime modelling. We indicate which challenges still remain in global fire modelling and stress the need for a comprehensive model evaluation and outline what lessons may be learned from FireMIP.
In old and heavily weathered soils, the availability of P might be so small that the primary production of plants is limited. However, plants have evolved several mechanisms to actively take up P from the soil or mine it to overcome this limitation. These mechanisms involve the active uptake of P mediated by mycorrhiza, biotic de-occlusion through root clusters, and the biotic enhancement of weathering through root exudation. The objective of this paper is to investigate how and where these processes contribute to alleviate P limitation on primary productivity. To do so, we propose a process-based model accounting for the major processes of the carbon, water, and P cycles including chemical weathering at the global scale. Implementing P limitation on biomass synthesis allows the assessment of the efficiencies of biomass production across different ecosystems. We use simulation experiments to assess the relative importance of the different uptake mechanisms to alleviate P limitation on biomass production. We find that active P uptake is an essential mechanism for sustaining P availability on long timescales, whereas biotic de-occlusion might serve as a buffer on timescales shorter than 10 000 yr. Although active P uptake is essential for reducing P losses by leaching, humid lowland soils reach P limitation after around 100 000 yr of soil evolution. Given the generalized modelling framework, our model results compare reasonably with observed or independently estimated patterns and ranges of P concentrations in soils and vegetation. Furthermore, our simulations suggest that P limitation might be an important driver of biomass production efficiency (the fraction of the gross primary productivity used for biomass growth), and that vegetation on old soils has a smaller biomass production rate when P becomes limiting. With this study, we provide a theoretical basis for investigating the responses of terrestrial ecosystems to P availability linking geological and ecological timescales under different environmental settings.
Modelling short-term variability in carbon and water exchange in a temperate Scots pine forest
(2015)
The vegetation–atmosphere carbon and water exchange at one particular site can strongly vary from year to year, and understanding this interannual variability in carbon and water exchange (IAVcw) is a critical factor in projecting future ecosystem changes. However, the mechanisms driving this IAVcw are not well understood. We used data on carbon and water fluxes from a multi-year eddy covariance study (1997–2009) in a Dutch Scots pine forest and forced a process-based ecosystem model (Lund–Potsdam–Jena General Ecosystem Simulator; LPJ-GUESS) with local data to, firstly, test whether the model can explain IAVcw and seasonal carbon and water exchange from direct environmental factors only. Initial model runs showed low correlations with estimated annual gross primary productivity (GPP) and annual actual evapotranspiration (AET), while monthly and daily fluxes showed high correlations. The model underestimated GPP and AET during winter and drought events. Secondly, we adapted the temperature inhibition function of photosynthesis to account for the observation that at this particular site, trees continue to assimilate at very low atmospheric temperatures (up to daily averages of −10 °C), resulting in a net carbon sink in winter. While we were able to improve daily and monthly simulations during winter by lowering the modelled minimum temperature threshold for photosynthesis, this did not increase explained IAVcw at the site. Thirdly, we implemented three alternative hypotheses concerning water uptake by plants in order to test which one best corresponds with the data. In particular, we analyse the effects during the 2003 heatwave. These simulations revealed a strong sensitivity of the modelled fluxes during dry and warm conditions, but no single formulation was consistently superior in reproducing the data for all timescales and the overall model–data match for IAVcw could not be improved. Most probably access to deep soil water leads to higher AET and GPP simulated during the heatwave of 2003. We conclude that photosynthesis at lower temperatures than assumed in most models can be important for winter carbon and water fluxes in pine forests. Furthermore, details of the model representations of water uptake, which are often overlooked, need further attention, and deep water access should be treated explicitly.
Modelling short-term variability in carbon and water exchange in a temperate Scots pine forest
(2015)
The vegetation–atmosphere carbon and water exchange at one particular site can strongly vary from year to year, and understanding this interannual variability in carbon and water exchange (IAVcw) is a critical factor in projecting future ecosystem changes. However, the mechanisms driving this IAVcw are not well understood. We used data on carbon and water fluxes from a multi-year eddy covariance study (1997–2009) in a Dutch Scots pine forest and forced a process-based ecosystem model (Lund–Potsdam–Jena General Ecosystem Simulator; LPJ-GUESS) with local data to, firstly, test whether the model can explain IAVcw and seasonal carbon and water exchange from direct environmental factors only. Initial model runs showed low correlations with estimated annual gross primary productivity (GPP) and annual actual evapotranspiration (AET), while monthly and daily fluxes showed high correlations. The model underestimated GPP and AET during winter and drought events. Secondly, we adapted the temperature inhibition function of photosynthesis to account for the observation that at this particular site, trees continue to assimilate at very low atmospheric temperatures (up to daily averages of −10 °C), resulting in a net carbon sink in winter. While we were able to improve daily and monthly simulations during winter by lowering the modelled minimum temperature threshold for photosynthesis, this did not increase explained IAVcw at the site. Thirdly, we implemented three alternative hypotheses concerning water uptake by plants in order to test which one best corresponds with the data. In particular, we analyse the effects during the 2003 heatwave. These simulations revealed a strong sensitivity of the modelled fluxes during dry and warm conditions, but no single formulation was consistently superior in reproducing the data for all timescales and the overall model–data match for IAVcw could not be improved. Most probably access to deep soil water leads to higher AET and GPP simulated during the heatwave of 2003. We conclude that photosynthesis at lower temperatures than assumed in most models can be important for winter carbon and water fluxes in pine forests. Furthermore, details of the model representations of water uptake, which are often overlooked, need further attention, and deep water access should be treated explicitly.
Es wird davon ausgegangen, dass das ehemalige Larven-Mikrohabitat der Asiatische Tigermücke Aedes albopictus (synonym: Stegomyia albopicta) die Phytotelmata in den Waldgebieten von Südostasien darstellte. In den letzten vier Jahrzehnten adaptierte sich die Art jedoch an urbanere Regionen und ihre Antrotelmata. Dank ihrer Eigenschaft, Eier mit einer gewissen Trocken- und Kältetoleranz zu produzieren, verbreitete sich die Art zusammen mit den international gehandelten Waren weltweit. Zudem ist Ae. albopictus ein theoretischer Vektor für mindestens 27 Viren sowie Parasiten und spielt eine Hauptrolle bei der Übertragung von Dengue-Viren und Chikungunya-Viren und Zika-Vieren. Daher wird die Art als große Gefahr für die öffentliche Gesundheit betrachtet.
Die vorliegende Arbeit thematisiert drei Untersuchungen zum Anpassungs- und Etablierungs-potential der invasiven Asiatischen Tigermücke.
In einem ersten Ansatz wurde das Problem behandelt, dass es lediglich zwei standardisierte toxikologische Testverfahren für Culicidae gab. Daher wurde ein Dosis-Wirkungs-Testsystem entwickelt, das den Weg für weitere biologische Endpunkte und ihre integrativen Parameter freimachte und dadurch ein besseres Verständnis für die Wirkweisen von Insektiziden ermöglicht. Hierdurch konnte nun der Frage nachgegangen werden, ob es Unterschiede in der ökotoxikologischen Reaktion zwischen der invasiven tropisch-subtropischen Asiatischen Tigermücke und der einheimischen nördlichen Hausstechmücke Culex pipiens auf das Insektizid λ-Cyhalothrin gibt. Weiter wurde der Einfluss von Temperatur und die Verfügbarkeit von Nahrung auf die Insektizidsensitivitäten der Arten getestet. Schließlich konnte in einer Risikobewertung festgestellt werden, dass bei falsch angewendeten Bekämpfungsmaßnahmen höhere Temperaturen sowie der Ausfall von aquatischen Top-Prädatoren zu Fitnessvorteilen für die Art führen können.
In einer zweiten Untersuchung wurde der Mechanismus der Kältetoleranz der Eier (Kälteakklimatisierung und Diapause) näher untersucht, da dieser für die erfolgreiche Invasion in gemäßigten Breitengraden verantwortlich gemacht wird. Nachdem eine lang vorherrschende Hypothese verworfen wurde, dass die Einlagerung von Polyolen die Frosttoleranz bewirken würde, war der aktuelle Stand der Wissenschaft, dass eine Verdickung der Wachsschicht des Chorions dafür verantwortlich sei. Jedoch lag keine detaillierte Evaluierung von Stechmücken-Eihüllen vor. Mittels einer transmissionselektronenmikroskopischen Studie konnte gezeigt werden, dass nicht nur die Wachsschicht nicht in der Serosa-Cuticula zu verorten ist, sondern im Endochorion und sie zudem im Zuge der Diapause in der Mächtigkeit schrumpft. Daher wird auf Basis der gewonnenen Erkenntnisse auf eine Kompaktierung der Schicht geschlossen.
Die dritte Untersuchung schließlich hatte das hohe Adaptationspotential in gemäßigten Breiten zum Gegenstand. Eine Adaptation auf genetischen Level gilt als unwahrscheinlich, da Gründerpopulationen in den neu besiedelten Gebieten eine niedrige genetische Diversität aufwiesen und ein regelmäßiger Neueintrag von Allelen unwahrscheinlich ist. Jedoch bietet das Konzept der epigenetischen Temperatur-Adaptation einen Erklärungsansatz für dieses Phänomen. Daher wurde die Frage gestellt, ob es möglich ist, eine vererbbare Diversifizierung dieses kältetoleranten Phänotyps nach einer randomisierten epigenetischen Behandlung der DNA zu detektieren. Es wurde eine transgenerationale Untersuchung der Effekte von zwei epigenetischen Agenzien (und einem Lösemittel) auf die Kältetoleranz der Eier durchgeführt. Die Ergebnisse zeigten ein Korrelationsmuster, das den durch die Agenzien veränderten Methylierungsgrad der DNA mit der Forsttoleranz verband, was die gestellte Hypothese unterstützte.
In Folge dieser drei Untersuchungen wurde festgestellt, dass Ae. albopictus ein hohes Potential hat, in weiteren Ländern – vor allem in gemäßigten Breiten – ein Gesundheitsrisiko darzustellen. Da die Art einerseits Fitnessvorteile durch falsche Bekämpfungsmaßnahmen und andererseits möglicherweise eine hohes epigenetisches Adaptationspotential besitzt, kann zusammenfassend empfohlen werden, dass der Fokus für weitere Forschung maßgeblich auf der Entwicklung von Impfstoffen für die übertragenen Viren und Pathogene liegen sollte. Dadurch kann die Bevölkerung geschützt werden, ohne Ökosysteme und ihre Dienstleistungen zu gefährden, und dies wäre zudem ökonomisch gesehen die effektivere Lösung.
Background: Bacteria within the genus Photorhabdus maintain mutualistic symbioses with nematodes in complicated lifecycles that also involves insect pathogenic phases. Intriguingly, these bacteria are rich in biosynthetic gene clusters that produce compounds with diverse biological activities. As a basis to better understand the life cycles of Photorhabdus we sequenced the genomes of two recently discovered representative species and performed detailed genomic comparisons with five publically available genomes.
Results: Here we report the genomic details of two new reference Photorhabdus species. By then conducting genomic comparisons across the genus, we show that there are several highly conserved biosynthetic gene clusters. These clusters produce a range of bioactive small molecules that support the pathogenic phase of the integral relationship that Photorhabdus maintain with nematodes.
Conclusions: Photorhabdus contain several genetic loci that allow them to become specialist insect pathogens by efficiently evading insect immune responses and killing the insect host.
Premise of the study: Polymorphic microsatellite markers were developed for the lichen species Cetraria aculeata (Parmeliaceae) to study fine-scale population diversity and phylogeographic structure.
Methods and Results: Using Illumina HiSeq and MiSeq, 15 fungus-specific microsatellite markers were developed and tested on 81 specimens from four populations from Spain. The number of alleles ranged from four to 13 alleles per locus with a mean of 7.9, and average gene diversities varied from 0.40 to 0.73 over four populations. The amplification rates of 10 markers (CA01– CA10) in populations of C. aculeata exceeded 85%. The markers also amplified across a range of closely related species, except for locus CA05, which did not amplify in C. australiensis and C. "panamericana," and locus CA10 which did not amplify in C. australiensis.
Conclusions: The identified microsatellite markers will be used to study the genetic diversity and phylogeographic structure in populations of C. aculeata in western Eurasia.
Background: n-Butanol can serve as an excellent gasoline substitute. Naturally, it is produced by some Clostridia species which, however, exhibit only limited suitability for industrial n-butanol production. The yeast Saccharomyces cerevisiae would be an ideal host due to its high robustness in fermentation processes. Nevertheless, n-butanol yields and titers obtained so far with genetically engineered yeast strains are only low.
Results: In our recent work, we showed that n-butanol production via a clostridial acetoacetyl-CoA-derived pathway in engineered yeast was limited by the availability of coenzyme A (CoA) and cytosolic acetyl-CoA. Increasing their levels resulted in a strain producing up to 130 mg/L n-butanol under anaerobic conditions. Here, we show that under aerobic conditions. this strain can even produce up to 235 mg/L n-butanol probably due to a more efficient NADH re-oxidation. Nevertheless, expression of a bacterial water-forming NADH oxidase (nox) significantly reduced n-butanol production although it showed a positive effect on growth and glucose consumption. Screening for an improved version of an acetyl-CoA forming NAD+-dependent acetylating acetaldehyde dehydrogenase, adhEA267T/E568K/R577S, and its integration into n-butanol-producing strain further improved n-butanol production. Moreover, deletion of the competing NADP+-dependent acetaldehyde dehydrogenase Ald6 had a superior effect on n-butanol formation. To increase the endogenous supply of CoA, amine oxidase Fms1 was overexpressed together with pantothenate kinase coaA from Escherichia coli, and could completely compensate the beneficial effect on n-butanol synthesis of addition of pantothenate to the medium. By overexpression of each of the enzymes of n-butanol pathway in the n-butanol-producing yeast strain, it turned out that trans-2-enoyl-CoA reductase (ter) was limiting n-butanol production. Additional overexpression of ter finally resulted in a yeast strain producing n-butanol up to a titer of 0.86 g/L and a yield of 0.071 g/g glucose.
Conclusions: By further optimizing substrate supply and redox power in the form of coenzyme A, acetyl-CoA and NADH, n-butanol production with engineered yeast cells could be improved to levels never reached before with S. cerevisiae via an acetoacetyl-CoA-derived pathway in synthetic medium. Moreover, our results indicate that the NAD+/NADH redox balance and the trans-2-enoyl-CoA reductase reaction seem to be bottlenecks for n-butanol production with yeast.
Elevated tumor interstitial fluid pressure (TIFP) is a prominent feature of solid tumors and hampers the transmigration of therapeutic macromolecules, for example, large monoclonal antibodies, from tumor-supplying vessels into the tumor interstitium. TIFP values of up to 40 mm Hg have been measured in experimental solid tumors using two conventional invasive techniques: the wick-in-needle and the micropuncture technique. We propose a novel noninvasive method of determining TIFP via ultrasonic investigation with scanning acoustic microscopy at 30-MHz frequency. In our experimental setup, we observed for the impedance fluctuations in the outer tumor hull of A431-vulva carcinoma–derived tumor xenograft mice. The gain dependence of signal strength was quantified, and the relaxation of tissue was calibrated with simultaneous hydrostatic pressure measurements. Signal patterns from the acoustical images were translated into TIFP curves, and a putative saturation effect was found for tumor pressures larger than 3 mm Hg. This is the first noninvasive approach to determine TIFP values in tumors. This technique can provide a potentially promising noninvasive assessment of TIFP and, therefore, can be used to determine the TIFP before treatment approach as well to measure therapeutic efficacy highlighted by lowered TFP values.
Cryptochrome 1a, located in the UV/violet-sensitive cones in the avian retina, is discussed as receptor molecule for the magnetic compass of birds. Our previous immunohistochemical studies of chicken retinae with an antiserum that labelled only activated cryptochrome 1a had shown activation of cryptochrome 1a under 373 nm UV, 424 nm blue, 502 nm turquoise and 565 nm green light. Green light, however, does not allow the first step of photoreduction of oxidized cryptochromes to the semiquinone. As the chickens had been kept under ‘white’ light before, we suggested that there was a supply of the semiquinone present at the beginning of the exposure to green light, which could be further reduced and then re-oxidized. To test this hypothesis, we exposed chickens to various wavelengths (1) for 30 min after being kept in daylight, (2) for 30 min after a 30 min pre-exposure to total darkness, and (3) for 1 h after being kept in daylight. In the first case, we found activated cryptochrome 1a under UV, blue, turquoise and green light; in the second two cases we found activated cryptochrome 1a only under UV to turquoise light, where the complete redox cycle of cryptochrome can run, but not under green light. This observation is in agreement with the hypothesis that activated cryptochrome 1a is found as long as there is some of the semiquinone left, but not when the supply is depleted. It supports the idea that the crucial radical pair for magnetoreception is generated during re-oxidation.
Process pharmacology : a pharmacological data science approach to drug development and therapy
(2016)
A novel functional-genomics based concept of pharmacology that uses artificial intelligence techniques for mining and knowledge discovery in "big data" providing comprehensive information about the drugs’ targets and their functional genomics is proposed. In “process pharmacology”, drugs are associated with biological processes. This puts the disease, regarded as alterations in the activity in one or several cellular processes, in the focus of drug therapy. In this setting, the molecular drug targets are merely intermediates. The identification of drugs for therapeutic or repurposing is based on similarities in the high-dimensional space of the biological processes that a drug influences. Applying this principle to data associated with lymphoblastic leukemia identified a short list of candidate drugs, including one that was recently proposed as novel rescue medication for lymphocytic leukemia. The pharmacological data science approach provides successful selections of drug candidates within development and repurposing tasks.
Global climate change is one of the major driving forces for adaptive shifts in migration and breeding phenology and possibly impacts demographic changes if a species fails to adapt sufficiently. In Western Europe, pied flycatchers (Ficedula hypoleuca) have insufficiently adapted their breeding phenology to the ongoing advance of food peaks within their breeding area and consequently suffered local population declines. We address the question whether this population decline led to a loss of genetic variation, using two neutral marker sets (mitochondrial control region and microsatellites), and one potentially selectively non-neutral marker (avian Clock gene). We report temporal changes in genetic diversity in extant populations and biological archives over more than a century, using samples from sites differing in the extent of climate change. Comparing genetic differentiation over this period revealed that only the recent Dutch population, which underwent population declines, showed slightly lower genetic variation than the historic Dutch population. As that loss of variation was only moderate and not observed in all markers, current gene flow across Western and Central European populations might have compensated local loss of variation over the last decades. A comparison of genetic differentiation in neutral loci versus the Clock gene locus provided evidence for stabilizing selection. Furthermore, in all genetic markers, we found a greater genetic differentiation in space than in time. This pattern suggests that local adaptation or historic processes might have a stronger effect on the population structure and genetic variation in the pied flycatcher than recent global climate changes.
MLL-r Leukemia
(2016)
Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.
In der vorliegenden Arbeit wurde erstmals die Interaktion von A. baumannii mit humanem Plasminogen untersucht. Mit dem Translations-Elongationsfaktor TufAb, dem äußeren Membranprotein OmpW sowie dem Lipoprotein p41 konnten insgesamt drei Plasminogen-bindende Proteine von A. baumannii identifiziert werden. Außerdem wurde ein grundlegender Beitrag zur funktionellen Charakterisierung von TufAb sowie p41 von A. baumannii erbracht.
Es konnte nachgewiesen werden, dass gereinigtes TufAb humanes Plasminogen bindet und diese Interaktion teilweise durch Lysin-Reste vermittelt und von der Ionenstärke beeinflusst ist. An TufAb-gebundenes Plasminogen war für den Plasminogen-Aktivator u-PA zugänglich und konnte zu Plasmin aktiviert werden, welches das chromogene Substrat S-2251, das physiologische Substrat Fibrinogen und die zentrale Komplementkomponente C3b proteolytisch spaltete. Schließlich konnte TufAb als „Moonlighting“-Protein auf der Zelloberfläche von A. baumannii identifiziert werden.
Für das Lipoprotein p41 konnte ebenfalls gezeigt werden, dass dieses an Plasminogen bindet. Die Bindung von Plasminogen an p41 erfolgte ebenfalls über Lysin-Reste, zeigte sich allerdings von der Ionenstärke unbeeinflusst. Im Fall von p41 konnte mit Hilfe von C-terminal verkürzten p41-Konstrukten gezeigt werden, dass C-terminale Lysin-Reste an der Bindung von Plasminogen beteiligt sind. Weitere Versuche mit p41-Proteinen, bei welchen vier C-terminale Lysin-Reste durch Alanin-Reste substituiert wurden, ergaben, dass die beiden Lysin-Reste K368 und K369 essentiell für die Bindung von Plasminogen an p41 sind. Zudem konnte gezeigt werden, dass sowohl Kringle-Domäne 1 als auch Kringle-Domäne 4 von Plasminogen bei der Interaktion mit p41 involviert sind. An p41 gebundenes Plasminogen ließ sich durch u-PA zu Plasmin aktivieren, welches Fibrinogen sowie die zentrale Komplementkomponente C3b degradierte. p41 ist außerdem in der Lage, die Komplementkomponenten C3, C3b und C5 zu binden und den alternativen Weg zu inhibieren. Zudem ergaben Untersuchungen im Rahmen dieser Arbeit erste Hinweise darauf, dass zumindest die Plasminogen-bindende Region auf der Zelloberfläche von A. baumannii lokalisiert ist.
Die Inaktivierung des p41-kodierenden Gens führte zu einer signifikanten Abnahme im Überleben von A. baumannii-Zellen in der Gegenwart von NHS. Zudem zeigte die Mutante Δp41 einen Defekt in der Plasmin-abhängigen Transmigration durch einen Endothelzell-Monolayer. Beide Versuche untermauern die physiologische Relevanz für die Interaktion von A. baumannii mit Plasminogen.
The P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.
Ceraceosorus bombacis is an early-diverging lineage of smut fungi and a pathogen of cotton trees (Bombax ceiba). To study the evolutionary genomics of smut fungi in comparison with other fungal and oomycete pathogens, the genome of C. bombacis was sequenced and comparative genomic analyses were performed. The genome of 26.09 Mb encodes for 8,024 proteins, of which 576 are putative-secreted effector proteins (PSEPs). Orthology analysis revealed 30 ortholog PSEPs among six Ustilaginomycotina genomes, the largest groups of which are lytic enzymes, such as aspartic peptidase and glycoside hydrolase. Positive selection analyses revealed the highest percentage of positively selected PSEPs in C. bombacis compared with other Ustilaginomycotina genomes. Metabolic pathway analyses revealed the absence of genes encoding for nitrite and nitrate reductase in the genome of the human skin pathogen Malassezia globosa, but these enzymes are present in the sequenced plant pathogens in smut fungi. Interestingly, these genes are also absent in cultivable oomycete animal pathogens, while nitrate reductase has been lost in cultivable oomycete plant pathogens. Similar patterns were also observed for obligate biotrophic and hemi-biotrophic fungal and oomycete pathogens. Furthermore, it was found that both fungal and oomycete animal pathogen genomes are lacking cutinases and pectinesterases. Overall, these findings highlight the parallel evolution of certain genomic traits, revealing potential common evolutionary trajectories among fungal and oomycete pathogens, shaping the pathogen genomes according to their lifestyle.
During CNS development and adult neurogenesis, immature neurons travel from the germinal zones towards their final destination using cellular substrates for their migration. Classically, radial glia and neuronal axons have been shown to act as physical scaffolds to support neuroblast locomotion in processes known as gliophilic and neurophilic migration, respectively (Hatten, 1999; Marin and Rubenstein, 2003; Rakic, 2003). In adulthood, long distance neuronal migration occurs in a glial-independent manner since radial glia cells differentiate into astrocytes after birth. A series of studies highlight a novel mode of neuronal migration that uses blood vessels as scaffolds, the so-called vasophilic migration. This migration mode allows neuroblast navigation in physiological and also pathological conditions, such as neuronal precursor migration after ischemic stroke or cerebral invasion of glioma tumor cells. Here we review the current knowledge about how vessels pave the path for migrating neurons and how trophic factors derived by glio-vascular structures guide neuronal migration both during physiological as well as pathological processes
Maintenance of mitochondria is achieved by several mechanisms, including the regulation of mitochondrial proteostasis. The matrix protease CLPXP, involved in protein quality control, has been implicated in ageing and disease. However, particularly due to the lack of knowledge of CLPXP's substrate spectrum, only little is known about the pathways and mechanisms controlled by this protease. Here we report the first comprehensive identification of potential mitochondrial CLPXP in vivo interaction partners and substrates using a combination of tandem affinity purification and differential proteomics. This analysis reveals that CLPXP in the fungal ageing model Podospora anserina is mainly associated with metabolic pathways in mitochondria, e.g. components of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle as well as subunits of electron transport chain complex I. These data suggest a possible function of mitochondrial CLPXP in the control and/or maintenance of energy metabolism. Since bioenergetic alterations are a common feature of neurodegenerative diseases, cancer, and ageing, our data comprise an important resource for specific studies addressing the role of CLPXP in these adverse processes.
In the last couple of years the research on natural products concerning ecological questions has gained more and more interest. Especially natural products play an important role for the maintenance of symbiotic relationships.
Here we present the application of the “overlap extension PCR-yeast homologous recombination“(ExRec) to simplify the availability of natural products. We successfully cloned a 45 kb gene cluster and characterized two new peptides ambactin and xenolindicin from Xenorhabdus – the latter derived from a silent gene cluster. ExRec is a very efficient cloning technique and resembles a powerful method regarding the assembly of large gene clusters as well as the cloning from metagenomic libraries or RNA pools.
In addition, we discovered bacterial pyrrolizidine alkaloids from Xenorhabdus, referred to as pyrrolizixenamides. The gene cluster consisted of a NRPS and a hydroxylase encoding gene. Surprisingly, this gene cluster and its variations (type A to D) can be found throughout the bacterial kingdom which might indicate an essential function. While these substances are mainly known to play a role in the defense mechanism of plants, the function of the identified pyrrolizixenamides from Xenorhabdus yet remains unsolved.
Moreover, we firstly identified a phosphopantetheinyl transferase (PPTase) from the lichenized fungus of Evernia prunastri. The gene eppA encoding a Sfp-type PPTase was heterologously expressed in Escherichia coli and Saccharomyces cerevisiae and functional characterized by indigoidine production and complementation of lys5, respectively. All represented results contribute to the elucidation of natural products and thereby to their role in nature with special regard to symbiotic associations.
In order to investigate the diversity of the western honeybee, Apis mellifera L., in West and Central Africa, a total of 204 colonies were sampled from 44 localities in four countries – Nigeria, Niger, Cameroon and Chad. 86 of these colonies, from 23 localities, were subjected to full morphometric analysis. In a principal component analysis (PCA) of the morphometric data, the colonies formed a single cluster. It also revealed that overall size of the body was the most important source of variation between the colonies. A hierarchical structure analysis, followed by a stepwise discriminant analysis, classified the colonies into three distinct morphoclusters; however, these clusters were not geographically demarcated. In another PCA carried out with the samples under investigation and reference samples of A. m. adansonii, A. m. jemenitica and A. m. scutellata, the colonies under investigation again formed one cluster which lying over and extended beyond the clusters of the reference subspecies. This is suggestive of a wider variation in size in the bees under investigation. In a stepwise DA, 94.2% of cross-validated grouped cases were correctly classified and the distances between group centroids were highly significant (p < 0.0005) according to F-statistic. 61 and 22 of the 83 colonies under investigation were assigned to A. m. jemenitica and A. m. adansonii, respectively. Mitochondrial DNA analysis was carried out on 148 colonies from 39 localities. Four mitochondrial haplotypes, previously reported from Africa and belonging to the African mitochondrial lineage, A, were detected: A1 (n = 62), A4 (n = 70), A4' (n = 15) and A14 (n = 1). The overall haplotype diversity was low (h = 0.478 ± S. E. 0.057). A chi-square test for association was conducted between haplotypes and type of vegetation, latitude, longitude, altitude, temperature and rainfall, severally. There was a statistically significant association between haplotype and each of the six variables and the association was strong with latitude, moderate with vegetation and rainfall and weak with the remaining variables. The neighbour-joining, maximum likelihood and maximum parsimony trees, obtained from sequence variation of the cytochrome b gene of mitochondrial DNA, showed that the samples, from the current study, unambiguously clustered with the reference sequences of A. m. scutellata from Kenya, but without showing further subdivision within this sub-Saharan cluster. 133 workers (one per colony) collected from 38 localities were subjected to microsatellite analysis. A total of 292 different alleles were recorded for the 15 microsatellite loci used. All microsatellite loci were polymorphic and the number of different alleles per locus ranged between 10, in locus At163, and 31, in locus A029. Heterozygosity (or gene diversity) was high in all loci. The unbiased expected heterozygosity, which is a better expression of gene diversity, was 0.861 ± S.E. 0.017. The overall FST value, which is a good estimate of genetic differentiation of populations, was very low: 0.007 ± S.E. 0.001 (0.001 - 0.014). AMOVA and Bayesian assignment showed no differentiation of the investigated populations. Based on morphometric analysis, the results of this study present the honeybees of western Africa as a single entity with an internal variation which lacks a geographical demarcation. Consequently the results do not support the splitting of the honeybees of the region into the two subspecies, A. m. adansonii and A. m. jemenitica, as reported in the literature. More morphometric, molecular, physiological and behavioural studies are required to confirm the taxonomic status of the honeybees of the region. Meanwhile, the use of A. m. adansonii, as the sole sub-specific name for the honeybees of West and Central Africa, is recommended.
Untersuchungen an uv-bestrahlten Schwimmhäuten von Fröschen und Rückenhäuten von Mäusen ergaben, daß mit Hilfe der Nadi - Reaktion (GRÄFF) Oxydationspotentiale an der Bestrahlungsstelle nachgewiesen werden können. Kurzwelliges UV unter λ = 300 mμ hat hierbei die beste Wirkung. Die Stellen stärkster Oxydationskraft liegen bei Schwimmhäuten an den Melanophoren des Corium, bei Mäuserücken-Häuten verteilt im Corium. Bei den Froschhäuten kann zusätzlich bei unphysiologischem pH 4,2 Oxydationswirkung in der Epidermis nachgewiesen werden. Strukturelle Veränderungen an der Schwimmhaut werden polarisationsoptisch deutlich.
Wirtszellreaktivierung chemisch induzierter Letalschäden im DNS-haltigen Serratia-Phagen Kappa
(1965)
After treating free phage Kappa with nitrous acid, triethylenemelamine, ethylmethanesulfonate, or hydroxylamin and using these phages for infecting Serratia H Y wildtype cells, at least 20% of the lethal damage in the phage-DNA can be reactivated by the host ( = host cell reactivation). It is known that all lethal agents tested so far attack the primary structure of the D N A in different ways. Therefore, we assume that the target for the host cell reactivation consists of some damage in the secondary structure of the DNA, because there is probably some coincidence in the action of all agents. The hypothesis that in the DNA changes of thymine are a prerequisite for host cell reactivation has been disproved by the experiments with nitrous acid and ethylmethanesulfonate because both substances do not act on thymine.
Phenology is the study of periodic life cycle events of living organisms and how these are influenced by environmental factors. Late phenological phases such as the timing of seed release and subsequent seed dispersal considerably affect ecology and evolution in plants. Since plants are mostly sessile organisms, seed dispersal is a crucial life cycle event for the ecology and evolution of plants. In fact, long-distance seed dispersal (LDD) is a very complex process in plant biology and significantly shapes the spatial and temporal dynamics of plant populations. For example, wind dispersal in plants is influenced by a variety of factors such as plant traits, habitat type and environmental conditions (e.g. wind speed). Considering the variability of wind conditions throughout the year, the timing of seed release and dispersal is known to have considerable effects on LDD. Even though late phenologies such as ripening duration and timing of seed release and subsequent dispersal are vital in estimating ecologically highly relevant LDD, these phenologies are not appropriately addressed in ecological research. The aim of this thesis is to gain insights into the factors that shape late plant phenologies. In particular, we address the following questions: which ecologically or evolutionary parameters drive the ripening process of plant species? How does the seasonal variability of wind affect the seed release phenology of plant species? How do these factors interact for plant species in different habitat types?
In order to address these questions, we applied different methodological approaches, ranging from fieldwork and monitoring phenology to computational simulation studies and statistical modeling. To study the ripening process of species, we monitored the flowering, ripening and seed release phenology of more than 100 Central European plant species. We conducted computational simulation studies for estimating LDD by wind to study the phenology of seed release and the parameters determining LDD by wind. In conjunction with phenological data from literature, we used the obtained simulation results to investigate evidence for the existence of phenological adaptations towards LDD in 165 plant species. Further, we used the results from simulation studies of LDD by wind to disentangle the effects of species, habitat types and meteorological conditions and their interactions on the spatial spread of plant species.
The results of the relationship between plant traits, phylogeny, the ripening process and climatic factors provide insights into the basic understanding of the ripening process of plants. We identified ecological factors that shape species’ ripening phenology and seed release timing. In particular, we suggest that the species’ seed weight, life form and phylogeny shapes ripening and seed release phenology. With the statistical models on species’ temperature demands for reproduction, we introduce data that that are well suitable for parametrization and further development of plant dispersal models. The results from the simulation study based on a seasonal perspective showed that heavier seeded tree species with medium wind dispersal potential (including genera Abies, Acer, Fraxinus and Larix) have a clear synchronisation of seed abscission with periods favouring LDD. These species, which are both ecologically and economically important, showed significant synchronisation of the highest rate of seed release with high wind-speed that promoted LDD by wind in wintertime. For the tree species mentioned, we suggest strong seasonal synchronisation as evidence for phenological adaptations in order to match favourable conditions during seed release. With a closer look at the wind conditions that promote LDD by wind, our results showed considerable differences in how specific wind conditions affect LDD in different species and habitat types. We suggest that LDD by wind in species from open habitats with high wind dispersal potential is likely to be driven by thermal updrafts that are mainly driven by the sun providing energy to the ground. By contrast, LDD of heavier-seeded species from open and forested habitats is more likely to be driven by storms that produce shear-driven turbulence. The results from this thesis contribute to an increased understanding of the complete dispersal process of plants and to making more realistic projections of (future) plant distribution.
The results obtained on factors driving ripening and release phenology provide valuable insights into their ecological and phylogenetic factor constraints. The implementation of more realistic assumptions in assessing species’ dispersal potential throughout the year could help considerably in improving landscape management (e.g. timing of mowing) and in the conservation of plant populations. The evidence found for phenological adaptations towards LDD in plants is an important step in understanding the evolutionary basis of LDD in these species.
The neural crest gives rise to the neurons and glial cells of the peripheral nervous system (PNS) (Bronner-Fraser and Fraser, 1989; Frank and Sanes, 1991). Self-renewing neural crest-derived stem cells (NCSCs) are present in migratory neural crest and various postmigratory locations, including peripheral ganglia (Duff et al., 1992; Morrison et al., 1999; Kruger er al., 2002). It is demonstrated that NCSCs from embryonic mouse dorsal root ganglia (DRG) are reprogrammed in neurosphere (NS) cultures in the presence of EGF and FGF. Reprogrammed NCSCs (rNCSCs) generate exclusively central nervous system (CNS) progeny, both in vitro and upon transplantation into the mouse brain (Binder et al., 2011). In this study the timing and mechanisms underlying the reprogramming were addressed. Most of the cells acquire CNS characteristics at passage 2, reaching a stable proportion of >90% of Olig2-positive cells at passage 3, which is maintained at least up to passage 10. The PNS marker p75 is completely lacking from passage 3 onwards. Furthermore, it was shown that the reprogramming does not involve a transient pluripotency state. This suggests a direct reprogramming of NCSCs to cells with CNS identity. The reprogramming leads to a stable CNS identity as shown by delayed BMP4 application. This result is in agreement with the previous observation that rNCSCs only generate CNS progeny, in particular mature myelinating oligodendrocytes, upon transplantation into embryonic, postnatal and lesioned adult mouse brains (Binder et al., 2011). Genome wide gene expression profiles of rNCSC NS demonstrates already in culture a complete switch to a (spinal cord stem cell) SCSC CNS identity. These results demonstrate a complete reprogramming of PNS progenitors to CNS identity without genetic modification and imply PNS cells as a source for stem cell-based CNS therapy.
The reprogramming of NCSCs is completely blocked in the presence of BMP4 in NS cultures, as shown by the expression of neural crest markers p75 and Sox10. In addition, BMP4 NCSCs generate PNS neurons (Tuj1/Phox2b- and Peripherin/Tuj1-coexpressing cells) and Schwann cells (O4/p75-coexpressing cells). Genome wide gene expression profiles of BMP NCSCs demonstrate that BMP NCSCs express genes at high levels which are characteristic for neural crest/neural crest derivatives, mesenchymal derivatives of neural crest and perivascular pericytes/MSCs. On the other hand CNS marker genes are restricted to rNCSCs and are only expressed at background or undetectable levels in BMP NCSCs. These findings imply that the CNS versus PNS identity is controlled by antagonistic functions of FGF and BMP4.
The use of rNCSCs for cell therapies requires an accessible source of these cells in the adult organism. Since the DRG is not an easily approachable tissue source, the adult mouse palate, containing NCSCs, was chosen. These results suggest that pNCSCs arise from Sox10-positive neural crest-derived stem cells, that downregulate PNS marker gene expression, such as Sox10 and p75, in NS culture. Contrary to rNCSCs, CNS marker upregulation was not observed. Notably, genome wide gene expression profiles of pNCSCs demonstrate an enrichment of genes expressed by mesenchymal derivatives and perivascular pericytes/mesenchymal stem cells. Since the cranial crest gives rise, besides PNS neural progeny and melanocytes, to mesenchymal derivatives, the results demonstrate that pNCSCs have a restricted developmental potential in comparison to rNCSCs and acquire mostly normal fates of the cranial neural crest.
Taken together, the results demonstrate that rNCSCs acquire a SCSC identity in the presence of EGF and FGF and that the reprogramming can be efficiently blocked by BMP4. On the other hand, NCSCs derived from adult palate rather acquire mesenchymal fates and do not acquire a CNS identity under the conditions used.
Tulasnella species (Tulasnellaceae, Cantharellales, Basidiomycota) form inconspicuous basidiomata on rotten branches or trunks of trees, difficult to find and recognize in nature. However, according to ultrastrucural and molecular data, species of Tulasnellaceae are the most frequent mycorrhriza forming fungi (mycobionts) of green, photosynthetic orchids worldwide. Species of Tulasnellaceae were also found as prominent mycobionts of the extraordinary diverse orchids in tropical montane rainforest of Southern Ecuador. Orchids obligately depend on mycobionts during the juvenile stage when the fungi have to deliver carbon to the non-photosynthetic protocorm and thus the fungi substantially influence the establishment of orchids in the wild. Species of Tulasnellaceae can acquire carbon from decaying bark or wood by specific saprotrophic capabilities as was recently proven through comparative genomics that included data on decay enzymes from Tulasnella cf. calospora isolated from orchid mycorrhizae (Anacamptis laxiflora, Italy). Thus, species of Tulasnellaceae can be saprotrophs and symbionts simultaneously.
It is currently under discussion, whether specific species of Tulasnella are required for seed germination and establishment of distinct terrestrial and epiphytic orchids in nature or if species of Tulasnella are generalists concerning their association with orchids. The inconsistences in species concepts and taxonomy of Tulasnella spp., however, strongly impede progress in this field of research. The aim of the present study was, therefore, to revise the species concepts by combining, for the first time, morphological and molecular data from basidiomata.
Specimens were collected in tropical Andean forest in Southern Ecuador and in temperate forests in Germany. Additional specimens were loaned from fungaria. In total, 205 specimens, corresponding to 16 own samples and 189 specimens from fungaria were analyzed. The mycobiont relationships of Tulasnella spp. with orchids from the sampling area in Ecuador were studied in populations of Epidendrum rhopalostele. The basis for molecular-phylogenetic analysis was completed by data obtained from own previous investigations on mycobionts from the investigation area and Tulasnella isolates from Australia.
30 morphospecies are illustrated and delimited by a morphological key based on traditional species concepts. Tulasnella andina from Ecuador and Tulasnella kirschneri from China are presented as species new to science. Tulasnella cruciata is described from herbarium material for the first time. Tulasnella aff. eichleriana and T. violea are reported for the first time from Ecuador. Molecular sequences of two Tulasnella spp. isolated from mycobionts of Epidendrum rhopalostele cannot be related to any morphological species concept. Statistical analyses suggest that conventional diagnostic using morphological characteristics is ambiguous for delimiting morphologically similar species.
For the first time sequences of the ITS-5.8S rDNA region were obtained after cloning from fresh basidiomata. Extraction of DNA from herbarium specimens was, however, unsuccessful. Sequences from 16 fresh basidiomata, six pure cultures, and sequences of orchids mycorrhizae (e.g. from Epidendrum rhopalostele) available in the database GenBank were analyzed. Proportional
variability of ITS-5.8S rDNA sequences within and among cultures and within and among specimens were used to designate morphospecies. Results suggest an intragenomic variation of less than 2 %, an intraspecific variation of up to 4 % and an interspecific divergence of more than 9 % for Tulasnella spp.
Four percent of intraspecific divergence was defined as a minimum threshold for delimiting phylogenetic species. This threshold corroborates the so far used 3 % to 5 % divergence in delimitation of operational taxonomic units of Tulasnella mycobionts.
Quite a number of sequences of Tulasnella are available in GenBank, mostly obtained from direct PCR amplification from orchid mycorrhizae. By including closely related sequences in the phylogenetic analysis, several morphological cryptic species of Tulasnella, mostly from Ecuador, were found. Arguments are given for molecular support of the new species Tulasnella andina and the established species Tulasnella albida, T. asymmetrica, T. eichleriana, T. tomaculum, and T. violea. Thus, by combining molecular and morphological data species concepts in Tulasnella are improved. The definitions of Tulasnella calospora and T. deliquescens, however, remain phylogenetically inconsistent.
The present investigation is a first step to expand our knowledge on the intraand interspecific morphological and molecular variability of Tulasnella spp. and to delimit species relevant for studies on ecology and communities of orchids and Tulasnellaceae.
Reaktive Sauerstoffspezies lösen molekular Schäden aus und werden als möglicher Auslöser des Alterungsprozesses gesehen. Sie sind allerdings auch wichtige Signalgeber, deren Einfluss in den letzten Jahren immer mehr Beachtung findet. Im der vorliegenden Arbeit wurde die Signalwirkung von ROS auf das Alternsmodell P. anserina untersucht. Dabei konnten folgende Erkenntnisse gewonnen werden.
1. Die H2O2–Konzentration im Extrazellularraum und im Cytoplasma steigt bei PQ-Stress und während des Alterns an.
2. Bei PQ-Stress und während des Alterns treten Ähnlichkeiten der globalen Transkriptregulation auf, die vermutlich durch die angestiegene H2O2-Konzentration ausgelöst werden.
3. Die Transkriptmenge von SODs und die Gesamt-SOD-Aktivität sind bei PQ-Stress leicht herunterreguliert. Transkripte für PaCCP1 und PaCATB treten dagegen bei PQ-Stress vermehrt auf und auch die Gesamt-Katalase-Aktivität steigt an. Dies deutet darauf hin, dass der Fokus des enzymatischen Abbaus von ROS bei PQ-Stress nicht im Abbau von Superoxid-, sondern von Wasserstoffperoxid liegt.
4. Bei PQ-Stress steigt die Transkriptmenge von Schlüsselgenen der Carotinoid-Biosynthese.
5. Transkripte von mitochondrial lokalisierten Proteinen werden bei PQ-Stress stark hochreguliert, die Menge an Mitochondrien nimmt allerdings nicht zu. Dies deutet auf einen verstärkten Abbau mitochondrialer Proteine, gefolgt von einer Neusynthese hin.
6. Bei PQ-Stress sinkt die Expression von Genen, die für den Kupferimport benötig werden. Dies wird höchstwahrscheinlich durch die Inaktivierung des Transkriptionsfaktors GRISEA ausgelöst. Es kommt zu einem Kupfermangel, der eine verstärkte alternative PaAOX-abhängige Atmung auslöst und dafür sorgt, dass kupferabhängige Prozesse, wie die Melanin- und Sterigmatocystin-Synthese transkriptionell herunterreguliert werden. Durch die verringerte Transkriptmenge von Kupferimportern bei PQ-Stress kommt es zu starken Gemeinsamkeiten in der globalen Genregulation von PQ-gestressten Kulturen und der Kupfer-depletierten Mutante grisea.
7. Kupfer und PQ haben einen synergistisch negativen Effekt auf Wuchsrate und Lebensspanne von P. anserina. Bei hohen Kupfer und Superoxid-Konzentrationen kommt es vermutlich zur verstärkten Bildung von Hydroxyl-Radikalen, wodurch molekulare Schäden entstehen. Durch eine verringerte Kupferkonzentration wird der Organismus bei PQ-Stress möglicherweise vor der Bildung von Hydroxyl-Radikalen geschützt.
Insgesamt haben die Untersuchungen gezeigt, dass ROS wichtige Signalmoleküle sind, die einen starken Einfluss auf die Regulation von Transkripten haben. Viele dieser transkriptionellen Regulationen führen zu physiologischen Veränderungen. Der Fokus der Regulationen liegt unter den verwendeten Bedingungen im Schutz vor den schädlichen Effekten von ROS.
The use of parasites as biological tags for discrimination of fish stocks has become a commonly used approach in fisheries management. Metazoan parasite community analysis and anisakid nematode population genetics based on a mitochondrial cytochrome marker were applied in order to assess the usefulness of the two parasitological methods for stock discrimination of beaked redfish Sebastes mentella of three fishing grounds in the North East Atlantic. Multivariate, model-based approaches demonstrated that the metazoan parasite fauna of beaked redfish from East Greenland differed from Tampen, northern North Sea, and Bear Island, Barents Sea. A joint model (latent variable model) was used to estimate the effects of covariates on parasite species and identified four parasite species as main source of differences among fishing grounds; namely Chondracanthus nodosus, Anisakis simplex s.s., Hysterothylacium aduncum, and Bothriocephalus scorpii. Due to its high abundance and differences between fishing grounds, Anisakis simplex s.s. was considered as a major biological tag for host stock differentiation. Whilst the sole examination of Anisakis simplex s.s. on a population genetic level is only of limited use, anisakid nematodes (in particular, A. simplex s.s.) can serve as biological tags on a parasite community level. This study confirmed the use of multivariate analyses as a tool to evaluate parasite infra-communities and to identify parasite species that might serve as biological tags. The present study suggests that S. mentella in the northern North Sea and Barents Sea is not sub-structured.
The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.
The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ1 = 3.5 μs) into another red-shifted intermediate. This species decayed in a two-step process (τ2 = 84 μs, τ3 = 11 ms), to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics.
Capoeta damascina was earlier considered by many authors as one of the most common freshwater fish species found throughout the Levant, Mesopotamia, Turkey, and Iran. However, owing to a high variation in morphological characters among and within its various populations, 17 nominal species were described, several of which were regarded as valid by subsequent revising authors. Capoeta damascina proved to be a complex of closely related species, which had been poorly studied. The current study aims at defining C. damascina and the C. damascina species complex. It investigates phylogenetic relationships among the various members of the C. damascina complex, based on mitochondrial and nuclear DNA sequences. Phylogenetic relationships were projected against paleogeographical events to interpret the geographic distribution of the taxa under consideration in relation to the area’s geological history. Samples were obtained from throughout the geographic range and were subjected to genetic analyses, using two molecular markers targeting the mitochondrial cytochrome oxidase I (n = 103) and the two adjacent divergence regions (D1-D2) of the nuclear 28S rRNA genes (n = 65). Six closely related species were recognized within the C. damascina complex, constituting two main lineages: A western lineage represented by C. caelestis, C. damascina, and C. umbla and an eastern lineage represented by C. buhsei, C. coadi, and C. saadii. The results indicate that speciation of these taxa is rather a recent event. Dispersal occurred during the Pleistocene, resulting in present-day distribution patterns. A coherent picture of the phylogenetic relationships and evolutionary history of the C. damascina species complex is drawn, explaining the current patterns of distribution as a result of paleogeographic events and ecological adaptations.
The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh) for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4–8 mM). ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75–90%) in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM) than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb]) or thiacloprid (8,800 ppb). Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb) or clothianidin (1 and 10 ppb) reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh content in the brood food.
Unusual Deep Water sponge assemblage in South China - witness of the end-Ordovician mass extinction
(2015)
There are few sponges known from the end-Ordovician to early-Silurian strata all over the world, and no records of sponge fossils have been found yet in China during this interval. Here we report a unique sponge assemblage spanning the interval of the end-Ordovician mass extinction from the Kaochiapien Formation (Upper Ordovician-Lower Silurian) in South China. This assemblage contains a variety of well-preserved siliceous sponges, including both Burgess Shale-type and modern type taxa. It is clear that this assemblage developed in deep water, low energy ecosystem with less competitors and more vacant niches. Its explosion may be related to the euxinic and anoxic condition as well as the noticeable transgression during the end-Ordovician mass extinction. The excellent preservation of this assemblage is probably due to the rapid burial by mud turbidites. This unusual sponge assemblage provides a link between the Burgess Shale-type deep water sponges and the modern forms. It gives an excellent insight into the deep sea palaeoecology and the macroevolution of Phanerozoic sponges, and opens a new window to investigate the marine ecosystem before and after the end-Ordovician mass extinction. It also offers potential to search for exceptional fossil biota across the Ordovician-Silurian boundary interval in China.
Heutzutage unterliegen insbesondere aquatische Ökosysteme durch den permanenten Anstieg nicht-heimischer Arten einem folgenreichen Wandel. Dabei stellt der Biodiversitätsverlust nur den Endpunkt einer biologischen Invasion dar. Dazwischen wirken sich Faktoren wie Konkurrenz-, Prädationsdruck oder die Übertragung von Krankheitserregern wie z.B. Parasiten auf den Rückgang der Arten aus. Als integraler Bestandteil eines jeden Ökosystems spielen Parasiten bei Invasionsprozessen eine entscheidende Rolle und können durch ihren Einfluss auf heimische und nicht-heimischen Arten Invasionen begünstigen. Fische übernehmen aufgrund ihrer vielseitigen Bedeutung im Nahrungsnetz eine Schlüsselfunktion als Wirte diverser Parasitenarten und sind deshalb ausgezeichnete Untersuchungsobjekte, um durch nicht-heimische Arten verursachte Veränderungen in Nahrungsnetzstrukturen oder Parasitenfaunen aquatischer Ökosysteme aufzudecken.
Vor diesem Hintergrund wurde die vorliegende kumulative Dissertation angefertigt, welche auf drei (ISI-) Einzelpublikationen basiert. Die Arbeit beschäftigte sich unter Verwendung morphologischer und molekularbiologischer Methoden schwerpunktmäßig mit der Parasitendiversität und Nahrungsökologie zweier eingeschleppter Fischarten in stark anthropogen beeinflussten deutschen Fließgewässern. Dabei handelte es sich um die hauptsächlich durch das Ballastwasser von Schiffen verbreitete invasive Schwarzmundgrundel Neogobius melanostomus aus den Flüssen Rhein und Main sowie den von Hobby-Aquarianern in den durch Industrieabwässer thermisch belasteten Gillbach ausgesetzten Zebrabuntbarsch Amatitlania nigrofasciata. Unter Berücksichtigung nahrungsökologischer und räumlich-zeitlicher Aspekte sollten die parasitologischen Risiken und Konsequenzen, welche durch das Einschleppen nicht-heimischer Fischarten auftreten, aufgezeigt werden, um übergeordnet die Folgenabschätzung auf dem Gebiet der Invasionsbiologie zu verbessern. Das Nahrungsspektrum beider Fischarten wies sowohl räumliche (zwischen den Flüssen und Standorten), als auch zeitliche (monatlich) Variationen auf, was die Anpassungsfähigkeit bzw. die optimale Nutzung zur Verfügung stehender Ressourcen von invasiven Arten verdeutlicht. Ebenso wurden Unterschiede in der Parasitierung nachgewiesen, was die Notwendigkeit räumlicher und zeitlicher Analysen zur Erfassung der vollständigen Parasitenfauna einer invasiven Art, inklusive aller Variationen der Befallszahlen, unterstreicht. Beide Fischarten bilden zudem Zwischenwirte für heimische, als auch nicht-heimische Parasitenarten, wobei die direkte Einschleppung von nicht-heimischen Parasiten aus dem ursprünglichen Verbreitungsgebiet der Fische auszuschießen ist und somit die Enemy-Release-Hypothese (Verlust ursprünglicher Gegenspieler wie Prädatoren oder Parasiten) unterstützt. Dies könnte zusammen mit der opportunistischen Ernährungsweise ein Grund für die starke Ausbreitung sowie die anhaltend hohen Individuenzahlen dieser Arten im jeweils betrachteten Verbreitungsgebiet (Rhein, Main sowie Gillbach) sein.
Besonders hervorzuheben ist der Nachweis der nicht-heimischen Nematoden Anguillicoloides crassus und Camallanus cotti. Durch die Aufdeckung einer besonderen Form von Hyperparasitismus konnten erstmalig hohe Befallszahlen von A. crassus in N. melanostomus dokumentiert werden. Die hoch abundante Grundelart gilt somit potentiell als entscheidender Überträger des invasiven Parasiten auf den Europäischen Aal. Der durch seine hohe Virulenz in der Aquaristik bekannte C. cotti gelangte durch das Aussetzen von Zierfischen in den Gillbach und trat mit hohen Befallszahlen in A. nigrofasciata auf und wird bereits auf die heimische Fischfauna übertragen (z.B. auf den Gründling Gobio gobio und den Döbel Squalius cephalus). Ebenso ist der starke Befall der heimischen Parasiten Pomphorhynchus sp. (Acanthocephala) und Raphidascaris acus (Nematoda) bei N. melanostomus sowie A. anguillae (Acanthocephala) bei A. nigrofasciata zu nennen. Durch die zusätzliche Wirtfunktion der Neozoen und die hohen Befallsintensitäten ist mit einem verstärkten Spillback-Effekt (Rückinfizierung) auf heimische Fischarten zu rechnen.
Die stetig steigende Anzahl biologischer Invasionen führt zu immer weitreichenderen Veränderungen heimischer Ökosysteme. Für den Erhalt der Biodiversität sowie einhergehender Ökosystemfunktionen rückt auch die Forschung über Neobiota immer mehr in den Mittelpunkt. In diesem Zusammenhang gewinnen auch Pathogene wie Parasiten immer mehr an Bedeutung, welche durch gebietsfremde Arten eingeschleppt werden und die bestehende Biodiversität gefährden können. Die Ergebnisse der durchgeführten Studien haben gezeigt, dass die Verknüpfung von parasitologischen und nahrungsökologischen Untersuchungen Einblicke in die hoch dynamischen Prozesse der Invasionsbiologie geben können, was ein Vorantreiben der Forschung und Folgenabschätzungen auf diesem Gebiet ermöglicht.
Ischemic injuries of the cardiovascular system are still the leading cause of death worldwide. They are often accompanied by loss of cardiomyocytes (CM) and their replacement by non-functional heart tissue. Cardiac fibroblasts (CF) play a major role in the recovery after ischemic injury and in the scar formation. In the last few years researchers were able to reprogram fibroblasts into CM in vitro and in murine models of myocardial infarction using various protocols including a cocktail of microRNAs (miRs). These miRs can target hundreds of messenger RNAs and inhibit their translation into proteins, potentially regulating multiple cellular signaling pathways. Because of this, there has been a rising interest in the use of miRs for therapeutic purposes. However, as different miRs have different effects in different cells, there is the danger of causing serious side effects. These could be alleviated by enacting a cell-specific transport of miRs, for example by using aptamers. Aptamers are usually short strands of DNA or RNA, which can fold into a specific three-dimensional confirmation which allows them to bind specifically to target molecules. Aptamers are commonly selected from a large library for their ability to bind to target molecules using a procedure called SELEX. Aptamers have already been used to transport miRs into cancer cells.
In this thesis, we first established the transport of miRs into cells of the cardiovascular system using aptamers. MiR-126 is an important part of the signaling in endothelial cells (EC), protects from atherosclerosis and supports angiogenesis, which is why we chose it as a candidate to transport into the vasculature. We first tested two aptamers for their ability to internalize into EC and fibroblasts. Both the aptamer for the ubiquitously expressed transferrin receptor (TRA) and a general internalizing RNA motif, but not a control construct, could internalize efficiently into all cell types tested. We then designed three chimeras (Ch) using different strategies to connect TRA to miR-126. While all chimeras could internalize efficiently, only Ch3, which connects TRA to Pre-miR-126 using a sticky bridge structure, had functional effects in EC. Ch3 reduced the protein expression of VCAM-1 in EC and increased the VEGF induced sprouting of EC in a spheroid-sprouting assay. Treatment of breast cancer cells with Ch3 emulated the effects of treatment with classical miR-126-3p and miR-126-5p mimics. In the SK-BR3 cell line Ch3 and miR-126-3p reduce the viability of the cells while they reduce recruitment of EC by the MCF7 cell line. miR-126-5p had no apparent effect in the SK-BR3 line, but increased viability of MCF7 cells, as did Ch3. This implies that Ch3 can be processed to both functional miR-126-3p and miR-126-5p in treated cells.
We were unable to achieve a reprogramming of adult murine cardiac fibroblasts into cells resembling CM using the cocktail of 4 miRs. This indicates that the miR-mediated transdifferentiation is only possible in neonatal fibroblasts. The effects in mice after an AMI might possibly be caused by an enhanced plasticity of fibroblasts in and close to the infarcted area.
We also screened to find aptamers specifically binding to cells of the cardiovascular system. We used two oligonucleotide libraries in a cell-SELEX to select candidates which bind to CF, but not EC. We observed that only the library which contains two randomized regions of 26 bases showed an enrichment of species binding to fibroblasts. We then sequenced rounds 5-7 of the SELEX and analyzed the data bioinfomatically to select 10 candidate aptamers. All candidates showed a strong binding not only to CF, but also EC. This indicates that the selection pressure against species binding to EC was not high enough and would have to be increased to find true CF-aptamers. Four promising candidates were also analyzed for their potential to be internalized and we surprisingly found that all of them were internalized by EC and CF more efficiently than TRA. The similar behavior of the candidates implies that they possibly share a ligand, which is expressed both by EC and CF, but more prominently by the latter.
This work demonstrates the possibility of using aptamers to transport miRs into cells of the cardiovascular system. It also shows that it is possible to select aptamers for non-cancerous mammalian cells, which has not been done before. It is reasonable to assume that a refinement of the cell-SELEX will allow selection of cell-specific aptamers. Due to the failure of reprogramming of adult fibroblasts into induced cardiomyocytes we were unable to test whether a miR-mediated reprogramming might be inducible using aptamer transported-miRs. Ultimately, aptamer mediated transport of miRs is a feasible and promising therapeutic option for the treatment of cardiovascular diseases and other disorders like cancer.
The development of the atrioventricular (AV) canal and the cardiac valves is tightly linked and a critically regulated process. Anomalies in components of the involved pathways can lead to congenital valve malformations, a leading cause of morbidity and mortality in neonates. Myocardial Bmp as well as endocardial Notch and Wnt signaling have been identified as critical factors for the induction of EMT during the formation of the endocardial cushions and cardiac valves. Of these, canonical Wnt signaling positively regulates endocardial proliferation and EMT but negatively regulates endocardial differentiation. Further, elevated Wnt signaling leads to the ectopic expression of myocardial Bmp ligands suggesting a high level of integration of the involved pathways and crosstalk amongst the different cardiac tissues.
Here we have identified a novel role for Id4 as a mediator between Bmp and Wnt signaling. Id4 belongs to the Id family of proteins and is known to be involved in bone and nervous system development. We found that in zebrafish, id4 is expressed in the endocardium of the AV canal at embryonic stages and throughout the atrial chamber in addition to AV canal, in adults. Using transcription activator-like effector nucleases (TALENs) we established an id4 mutant allele. Our analysis shows that id4 mutant larvae are susceptible to retrograde blood flow, and show aberrant expression of developmental valvular markers. These include expanded expression domains of markers like bmp4, cspg2a and Alcam. In contrast, valve maturation as assessed by the expression of spp1 is considerably reduced in id4 mutants. Using conditional transgenic systems, along with elegant in vivo imaging of transgenic reporter lines, we further found that id4 is a transcriptional target of Bmp signaling, and it is capable of dose dependently restricting Wnt signaling in the endocardium of the Atrioventricular Canal.
Taken together, our data identifies Id4 as a novel player in Atrioventricular Canal and valve development. We show that Id4 function is important in valve development acting downstream of Bmp signaling by restricting endocardial Wnt to allow valve maturation
Deciduous plants avoid the costs of maintaining leaves in the unfavourable season, but carry the costs of constructing new leaves every year. Deciduousness is therefore expected in ecological situations with pronounced seasonality and low costs of leaf construction. In our study system, a seasonally dry tropical savanna, many trees are deciduous, suggesting that leaf construction costs must be low. Previous studies have, however, shown that nitrogen is limiting in this system, suggesting that leaf construction costs are high. Here we examine this conundrum using a time series of soil moisture availability, leaf phenology and nitrogen distribution in the tree canopy to illustrate how trees resorb nitrogen before leaf abscission and use stored reserves of nitrogen and carbon to construct new leaves at the onset of the growing season. Our results show that trees deployed leaves shortly before and in anticipation of the first rains with its associated pulse of nitrogen mineralisation. Our results also show that trees rapidly constructed a full canopy of leaves within two weeks of the first rains. We detected an increase in leaf nitrogen content that corresponded with the first rains and with the movement of nitrogen to more distal branches, suggesting that stored nitrogen reserves are used to construct leaves. Furthermore the stable carbon isotope ratios (δ13C) of these leaves suggest the use of stored carbon for leaf construction. Our findings suggest that the early deployment of leaves using stored nitrogen and carbon reserves is a strategy that is integrally linked with the onset of the first rains. This strategy may confer a competitive advantage over species that deploy leaves at or after the onset of the rains.
Die vorliegende Studie vermittelt einen epidemiologischen Überblick über das mit Haut- und Nagelläsionen assoziierte Pilzspektrum im Westen Panamas. Hierzu wurden Proben von vermutlich durch Pilzinfektionen verursachten Haut- sowie Nagelläsionen gesammelt und zum Anlegen von Kulturen verwendet. Die isolierten Pilze wurden basierend auf dem D-H-S System (Rieth), anhand morphologischer Merkmale, rDNA Sequenzdaten sowie phylogenetischen Analysen klassifiziert und mit Hilfe von Literaturdaten sowie physiologischen Eigenschaften als saprotrophe, opportunistische oder pathogene Organismen beurteilt. In Panama wurden 52 Proben von 51 Personen gesammelt, wobei das Material von 42 Haut- und Nagelläsionen der Füße, vier Läsionen der Fingernägel, zwei Chromomykosen, einer Tinea nigra und drei sonstigen Hautläsionen stammt. Bei 75 Prozent (n = 39) der Proben konnten Pilze kultiviert und insgesamt 201 Pilzstämme isoliert und subkultiviert werden. Hiervon wurden 50 Isolate (24,9 %) als Dermatophyten, 24 Stämme (11,9 %) als Hefen und 127 Isolate (63,2 %) als Schimmelpilze klassifiziert. Bei 19 Probanden (48,7 %) konnten Dermatophyten isoliert werden, wobei aus dem Probenmaterial von 12 Personen (63,2 %) ebenfalls andere Pilzarten nachgewiesen wurden. Von zwei Läsionen (5,1 %) wurden nur Hefen isoliert, wobei einmal eine Schwarze Hefe kultiviert wurde. In dem Material acht weiterer Proben (20,5 %) wurden Schimmelpilze und Hefestämme nachgewiesen und bei zehn Probanden (25,6 %) konnten aus dem Probenmaterial nur Schimmelpilze kultiviert werden. 172 Isolate wurden taxonomisch klassifiziert und 44 Arten aus 25 Gattungen, 17 Familien, 15 Ordnungen, sechs Klassen sowie den Abteilungen Ascomycota oder Basidiomycota zugeordnet. Die Ascomyceten stellen mit 164 Stämmen 40 verschiedener Arten aus 23 Gattungen, 15 Familien, 11 Ordnungen und vier Klassen die am häufigsten isolierte und vielfältigste Gruppe dar, während die Basidiomycota nur mit acht Isolaten vier verschiedener Arten zwei unterschiedlicher Gattungen, Familien, Ordnungen und Klassen nachgewiesen wurden. Im Rahmen dieser Arbeit wurden in Panama die anthropophilen Dermatophyten Trichophyton rubrum und T. interdigitale dokumentiert, wobei T. rubrum die am häufigsten isolierte Art darstellt. Kultivierte Hefen waren Candida albicans, C. duobushaemulonii, C. tropicalis, Hortaea werneckii, Sporobolomyces sp., Trichosporon asahii, T. japonicum und T. montevideense. Die Schimmelpilze stellen die größte und ökologisch diverseste Organismengruppe der kultivierten Pilze dar. So wurden von den untersuchten Läsionen sowohl humanpathogene Erreger, als auch opportunistische Arten und rein saprotrophe Pilze sowie mehrere Vertreter wahrscheinlich bisher nicht wissenschaftlich beschriebener Arten bzw. Gattungen nachgewiesen. Aus dem Probenmaterial wurden die Pilze Acremonium collariferum, Aspergillus awamori, A. clavatus, A. flavus, A. giganteus, A. heteromorphus, A. niger, A. ochraceus, A. sclerotiorum, A. versicolor, Chaetomium globosum, Chrysosporium tuberculatum, Cladosporium sphaerospermum, C. tenuissimum, Curvularia geniculata, C. lunata, Fonsecaea pedrosoi, Fusarium oxysporum, F. solani, Lophotrichus bartlettii, Microascus cinereus, Neoscytalidium dimidiatum, Penicillium commune, Scolecobasidium sp., Scopulariopsis carbonaria, S. croci, Verticillium cf. epiphytum und Wardomycopsis litoralis isoliert. Zudem wurden vier Isolate von zwei vermutlich neuen Arten der Gattung Acremonium (Bionectriaceae, Hypocreales), zwei Stämme mit einer genetischen Affinität zu der Gattung Cryptendoxyla (Cephalothecaceae, Sordariales) und jeweils ein mit den Gattungen Fusicladium (Venturiaceae, Venturiales), Knufia (Trichomeriaceae, Chaetothyriales) bzw. Rhexothecium (Eremomycetaceae, Dothideomycetidae) assoziierter Stamm kultiviert. Im Rahmen dieser Studie wurden A. giganteus, C. tenuissimum, L. bartlettii, S. carbonaria, S. croci, V. epiphytum und W. litoralis erstmalig von Mykosen des Menschen dokumentiert und die in der Literatur als Verursacher sowie Besiedler von Haut- und Nagelläsionen beschriebenen Organismen A. clavatus, A. flavus, A. niger, A. ochraceus, C. tropicalis, C. globosum, C. sphaerospermum, C. lunata, F. oxysporum, M. cinereus, P. commune, T. asahii, T. japonicum und T. montevideense wurden das erste Mal in klinischem Probenmaterial aus Panama nachgewiesen. Die Arten A. awamori, A. heteromorphus, C. globosum, C. tenuissimum, L. bartlettii, M. cinereus, P. commune, S. croci, T. asahii, T. japonicum, T. montevideense, V. epiphytum, W. litoralis und die Gattung Scolecobasidium wurden zudem erstmalig für Panama dokumentiert. Die Isolation von W. litoralis ist ebenfalls der erste Nachweis dieses Pilzes außerhalb von Spanien und auf dem amerikanischen Kontinent. Die große Anzahl im Rahmen dieser Arbeit beschriebener, bisher für die Wissenschaft unbekannter bzw. nicht in Panama dokumentierter Pilzarten lässt auf eine große mykologische Biodiversität in Panama schließen und zeigt den Bedarf weiterer Forschung.
Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of aurantiaca. Our report describing the exclusive accumulation of pelargonidin glycosides in aurantiaca petals may facilitate the modification of gentian flower color by the production of red anthocyanins.
The Asian tiger mosquito Aedes albopictus, native to South East Asia, is listed as one of the worst invasive vector species worldwide. In Europe the species is currently restricted to Southern Europe, but due to the ongoing climate change, Ae. albopictus is expected to expand its potential range further northwards. In addition to modelling the habitat suitability for Ae. albopictus under current and future climatic conditions in Europe by means of the maximum entropy approach, we here focused on the drivers of the habitat suitability prediction. We explored the most limiting factors for Aedes albopictus in Europe under current and future climatic conditions, a method which has been neglected in species distribution modelling so far. Ae. albopictus is one of the best-studied mosquito species, which allowed us to evaluate the applied Maxent approach for most limiting factor mapping. We identified three key limiting factors for Ae. albopictus in Europe under current climatic conditions: winter temperature in Eastern Europe, summer temperature in Southern Europe. Model findings were in good accordance with commonly known establishment thresholds in Europe based on climate chamber experiments and derived from the geographical distribution of the species. Under future climatic conditions low winter temperature were modelled to remain the most limiting factor in Eastern Europe, whereas in Central Europe annual mean temperature and summer temperatures were modelled to be replaced by summer precipitation, respectively, as most limiting factors. Changes in the climatic conditions in terms of the identified key limiting factors will be of great relevance regarding the invasive potential of the Ae. albopictus. Thus, our results may help to understand the key drivers of the suggested range expansion under climate change and may help to improve monitoring programmes. The applied approach of investigating limiting factors has proven to yield valuable results and may also provide valuable insights into the drivers of the prediction of current and future distribution of other species. This might be particularly interesting for other vector species that are of increasing public health concerns.
Panama, a small country between the major continents of North and South America, is one of the lesser studied regions in Central America, but is recognized for its mega-biodiversity. This is particularly true for Eastern Panama, which I am considering as the easternmost portion of the country, covering the area from the Chepo, which is also the beginning of the San Blas mountain range, towards east, up to the Darien Mountain range on the border with its neighboring country Colombia. In the lowland region I visited two physiographic areas: the Isthmian-Atlantic Moist Forests (IAMF) and the Chocó-Darién Moist Forests (CDMF). In the IAMF I worked at the localities of Río Mono, Wacuco, La Moneda, Arretí, Metetí, Filo del Tallo, and Laguna de Matusagaratí. In the CDMF I visited the localities of Cruce de Mono, Cana, Garachiné, Sambú, and Pavarandó. And I have worked in the highlands of Darién (DM), Majé (MM), Jingurudó-Sapo (JSM), Pirre (PM) and San Blas (SSM) in the highlands.
Before my research, 138 reptile and 104 amphibian species had been reported for EP. From 2008 to 2013, I collected specimens to evaluate the diversity of amphibians and reptiles for this region. I applied an integrative approach to evaluate the taxonomy, diversity, biogeography, and conservation of the herpetofauna of EP. I included analyses of morphometrics, molecular genetics (e.g. barcoding), biogeography, bioacoustics (in anurans), hemipenial morphology (in squamates), and ecology. This is the first regional evaluation of the biodiversity in EP applying integrative taxonomy. Aside from morphological and bioacoustic data, my work is based on the barcoding of 608 specimens, from which I obtained 16S mtDNA for 486 specimens and COI mtDNA for 455. In total I have got sequences for 69.2 %of the amphibian and 48.6 % of the reptile species present in EP. For the morphological analyses, I compared 1597 specimens, including my samples complemented by specimens obtained from various museums. The bioacoustic data were obtained from the analysis of 1504 calls of 27 species of frogs. Based on specimens collected in EP and according to external morphology, I could identify 65 species of amphibians and 72 reptiles, but after applying an integrative approach these numbers increased to 79 amphibians and 88 reptiles described species within my collected specimens. Additionally, I uncovered 33 taxonomic units that could not be assigned to any described species until now, 22 of them represent confirmed candidate species (CCS), and 11 were classified as Unconfirmed candidate species (UCS). Thus, increasing the known species of amphibian by 19.4 % and of reptiles by 4.8 %. Currently, there are 145 reptiles and 129 amphibians known to occur in EP. Based on my results, I have initiated several projects to solve taxonomic uncertanties, including the species of the genera Bolitoglossa, Diasporus, Dactyloa, Ecnomiohyla, Lepidoblepharis, and the taxonomic status of the species Pristimantis caryophyllaceus and Norops tropidogaster.
Out of the 22 CCS I found, I described nine species new to science with type locality in EP, six amphibians and four reptiles. Among these is a new species of Bolitoglossa described from Cerro Chucantí, Cordillera de Majé, Provincia de Darién, Panama. Additionally, I include comments on the other species of congeneric salamanders known to occur in the region. Among the tink frogs, only Diasporus quidditus was known to occur in EP. During my field work I collected six additional species of this genus, four of which are new to science, plus two species new for this region.
I also described one new species of Dactyloa (giant anole lizards) related to the former D. chocorum. I synonymized D. chocorum with D. purpurescens, and included information about the other species of the group from EP. The new species of Dactyloa resembles D. ibanezi, D. limon, and D. purpurescens in external morphology but differs from these species in dewlap coloration, dorsal color pattern, morphometrics, and scalation. I discovered one species of the genus Ecnomiohyla, which exhibits significant genetic distances (16S mtDNA gene) and morphological differences to all known Ecnomiohyla species. Along with the description of the new Ecnomiohyla species, I provide detailed comparisons of morphological and molecular characters of almost all members of the genus in Lower Central America, as well as an identification key for the entire genus. Two new species of the genus Lepidoblepharis from EP were described. In the corresponding work, I include an analysis of Lepidoblepharis spp. in the region, including phylogeography and taxonomy. One of the new species, Lepidoblepharis emberawoundule, can be differentiated from most species in the genus by its small size and its low number of lamellae under the fourth toe and finger. The other species described from EP, Lepidoblepharis rufigularis, can be differentiated from all species in the genus by its small size and the reddish throat in males.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.