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Amphibians of Malawi : an analysis of their richness and community diversity in a changing landscape
(2009)
This study summarizes the state of the knowledge of the amphibian diversity in Malawi highlighting the possible threats impending on this fauna correlated with human encroachment and land use change. New data about diversity, distribution and ecology have been gathered, whereas the old ones have been summarised, reviewed and commented. In order to put in context the responses of the amphibian communities to land use change, the main environmental characteristics of the country at a broad space and time scale have been explored. Furthermore, the original habitats and vegetation have been described, and their status in the present day Malawi discussed. In the same way, an overview of the actual state of the knowledge about the Malawian amphibians has been provided, and their ability to act as surrogate of environmental integrity in Sub-Saharan Africa commented on the basis of the available studies. Afterwards, the results of the study of the selected areas and samples have been analysed within this newly generated context. Different field and laboratory methods were applied for the quantitative analysis of the richness and diversity of the communities. Opportunistic search was used to detect species richness, whereas the visual encounter survey was applied to detect the relative abundance of species. Several indices of diversity and similarity, and extrapolations by means of true richness estimators were used for the analysis of the alpha and beta diversities. Additional information were gathered by means of pitfall traps with drift fence, and by the recording of the advertisement calls. Supplementary methods were applied for the analysis of the taxonomic composition of the collected material. In Malawi 84 amphibian species are recorded, two of which still undescribed (Leptopelis sp. and Phrynobatrachus sp.). Three further species need to be confirmed and might be possibly present too: Amietia viridireticulata, Hemisus guineensis, and Hyperolius minutissimus. Additionally, other unrecognised cryptic species — at least one — are present within the Hyperolius nasutus complex. Most of the species belong to the order Anura (82 species; 97.6%), whereas only two species belong to the Gymnophiona (2.4%). Anurans are divided into 12 families and 23 genera, whereas the two caecilians species into one family (Caecilidae) and two genera. The more diverse family is the Hyperoliidae (21 species, 25%) followed by the families Ptychadenidae (13 species, 15%), Arthroleptidae (11 species, 13%), Phrynobatrachidae (10 species, 12%), and Bufonidae and Pyxicephalidae (9 species, 11% respectively). The remaining high family diversity (seven families, Caecilidae included) is contrasted by a low number of species (11 species in total, 14%). Based on the available distribution data, the value of species richness of the anuran communities in Malawi is comprised between 5‒45 species. In average 16.8 ± 9.0 species (N=80) are to be found, 75% of the sites have less than 21 species, and only two sites have more than 25 species. Four hot spots of amphibian diversity were identified: the Nyika Plateau (24 species), Mangochi-Malombe (25 species), Zomba Plateau (32 species) and the Mulanje Massif (45 species). In the studied areas a mean of 14.7 ± 1.6 species was observed and extrapolations by means of the true richness estimators were in good agreement with this result. Among the studied areas the richest was Palm Forest Reserve (17 species), followed by Kaningina Forest Reserve (16 species) and Vinthukutu F. R., and Vwaza W. R (15 species). The poorest area was the Misuku Mountains with 12 species only and a slightly different ranking was generated by the true richness estimators. The mean of the species present in the samples was 4.8 ± 2.1 species, considerably less than the true species richness detected in the respective areas. Basing on the ranking generated by the K-dominance plot the most diverse samples were Palm F. R. and Misuku, whereas the less diverse were Kaningina F. R. and Fort Lister, confirmed by the values of the diversity indices. The main finding of this study was the observation of the lack of a clear match between environmental degradation and amphibian diversity, and the crucial importance of temporary water bodies for the preservation of the amphibian diversity. In fact, despite most of the original habitat formerly present in Malawi have been destroyed and replaced by cultivations, the amphibian communities of different areas showed a comparable diversity at both family and species richness level, and no evident match between environmental degradation and amphibian diversity was recognisable. Differences in species richness could mostly be explained by natural factors such the elevation gradient and the presence of temporary water bodies. However, it was not possible to exclude that the communities have changed during historical time and the shift in species composition already occurred together with the modification of their relative frequencies. Most of the species showed a remarkable ecological plasticity and several species were found in a variety of both natural and altered habitats. The classification of the Malawian amphibians on the basis of ecological guilds based on the available natural history data showed the preponderance (76%) of generalist pond breeders. As a consequence, most of these amphibians possessed a scarce capacity to act as surrogates of habitat integrity. Based on the result of this study the farm bush landscape with traditional agriculture practices bears a great potential to support amphibian diversity in terms of species richness, representing a compromise between local economic development and conservation. Furthermore, the results of this study indicate the outstanding importance of the southern-east region of Malawi for the conservation of the country’s amphibians.
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.
Amino acids can induce yeast cell adhesion but how amino acids are sensed and signal the modulation of the FLO adhesion genes is not clear. We discovered that the budding yeast Saccharomyces cerevisiae CEN.PK evolved invasive growth ability under prolonged nitrogen limitation. Such invasive mutants were used to identify amino acid transporters as regulators of FLO11 and invasive growth. One invasive mutant had elevated levels of FLO11 mRNA and a Q320STOP mutation in the SFL1 gene that encodes a protein kinase A pathway regulated repressor of FLO11. Glutamine-transporter genes DIP5 and GNP1 were essential for FLO11 expression, invasive growth and biofilm formation in this mutant. Invasive growth relied on known regulators of FLO11 and the Ssy1-Ptr3-Ssy5 complex that controls DIP5 and GNP1, suggesting that Dip5 and Gnp1 operates downstream of the Ssy1-Ptr3-Ssy5 complex for regulation of FLO11 expression in a protein kinase A dependent manner. The role of Dip5 and Gnp1 appears to be conserved in the S. cerevisiae strain ∑1278b since the dip5 gnp1 ∑1278b mutant showed no invasive phenotype.
Secondly, the amino acid transporter gene GAP1 was found to influence invasive growth through FLO11 as well as other FLO genes. Cells carrying a dominant loss-of-function PTR3647::CWNKNPLSSIN allele had increased transcription of the adhesion genes FLO1, 5, 9, 10, 11 and the amino acid transporter gene GAP1. Deletion of GAP1 caused loss of FLO11 expression and invasive growth. However, deletions of FLO11 and genes encoding components of the mitogen-activated protein kinase pathway or the protein kinase A pathway were not sufficient to abolish invasive growth, suggesting involvement of other FLO genes and alternative pathways. Increased intracellular amino acid pools in the PTR3647::CWNKNPLSSIN-containing strain opens the possibility that Gap1 regulates the FLO genes through alteration of the amino acid pool sizes.
Heat stress transcription factors (Hsfs) play essential role in heat stress response and thermotolerance by controlling the transcriptional activation of heat stress response (HSR) genes including molecular chaperones. Plant Hsf families show a striking multiplicity, with more than 20 members in the many plant species. Among Hsfs, HsfA1s act as the master regulators of heat stress (HS) response and HsfA2 becomes one of the most abundant Hsfs during HS. Using transgenic plans with suppressed expression of HsfA2 we have shown that this Hsf is involved in acquired thermotolerance of S. lycopersicum cv Moneymaker as HsfA2 is required for high expression and maintenance of increased levels of Hsps during repeated cycles of HS treatment.
Interestingly, HsfA2 undergoes temperature-dependent alternative splicing (AS) which results in the generation of seven transcript variants. Three of these transcripts (HsfA2-Iα-γ), generated due to alternative splicing of a second, newly identified intron encode for the full length protein involved in acquired thermotolerance. Another 3 transcripts (HsfA2-IIIα-γ) are generated due to alternative splicing in intron 1, leading in all cases to a premature termination codon and targeting of these transcripts for degradation via the non-sense mRNA decay mechanism (NMD).
Interestingly, excision of intron 2, results into the generation of a second previously unreported protein isoform, annotated as HsfA2-II. HsfA2-II shows similar transcriptional activity to the full-length protein HsfA2-I in the presence of HsfA1a but lacks the nuclear export signal (NES) required for nucleocytoplasmic shuttling which allows efficient nuclear retention and stimulation of transcription of HS-induced genes. Furthermore, stability assays showed that HsfA2-II exhibits lower protein stability compared to HsfA2-I.
The presence of a second intron and the generation of a second protein isoform we identified in other Solanaceae species as well. Remarkably, we observed major differences in the splicing efficiency of HsfA2 intron 2 among different tomato species. Several wild tomato accessions exhibit higher splicing efficiency that favors the generation of HsfA2-II, while in these species the splice variant HsfA2-Iγ is absent. This natural variation in splicing efficiency specifically occurring at temperatures around 37.5oC is associated with the presence of 3 intronic polymorphisms. In the case of wild species these polymorphisms seemingly restrict the binding of RS2Z36, identified as a putative splicing silencer for HsfA2 intron 2.
Tomato accessions with the polymorphic “wild” HsfA2 show enhanced thermotolerance against a direct severe heat stress incident due to the stronger increase of Hsps and other stress induced genes. Introgression of the “wild” S. pennellii HsfA2 locus into the cultivar M82, resulted in enhanced seedling thermotolerance highlighting the potential use of the polymorphic HsfA2 for breeding.
We conclude that alterations in the splicing efficiency of HsfA2 have contributed to the adaption of tomato species to different environments and these differences might be directly related to natural variation in their thermotolerance.
The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.
Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.
The Alpine Region, constituting the Alps and the Dinaric Alps, has played a major role in the formation of current patterns of biodiversity either as a contact zone of postglacial expanding lineages or as the origin of genetic diversity. In our study, we tested these hypotheses for two widespread, sympatric microgastropod taxa - Carychium minimum O.F. Müller, 1774 and Carychium tridentatum (Risso, 1826) (Gastropoda, Eupulmonata, Carychiidae) - by using COI sequence data and species potential distribution models analyzed in a statistical phylogeographical framework. Additionally, we examined disjunct transatlantic populations of those taxa from the Azores and North America. In general, both Carychium taxa demonstrate a genetic structure composed of several differentiated haplotype lineages most likely resulting from allopatric diversification in isolated refugial areas during the Pleistocene glacial periods. However, the genetic structure of Carychium minimum is more pronounced, which can be attributed to ecological constraints relating to habitat proximity to permanent bodies of water. For most of the Carychium lineages, the broader Alpine Region was identified as the likely origin of genetic diversity. Several lineages are endemic to the broader Alpine Region whereas a single lineage per species underwent a postglacial expansion to (re)colonize previously unsuitable habitats, e.g. in Northern Europe. The source populations of those expanding lineages can be traced back to the Eastern and Western Alps. Consequently, we identify the Alpine Region as a significant 'hot-spot' for the formation of genetic diversity within European Carychium lineages. Passive dispersal via anthropogenic means best explains the presence of transatlantic European Carychium populations on the Azores and in North America. We conclude that passive (anthropogenic) transport could mislead the interpretation of observed phylogeographical patterns in general.
The accumulation and distribution of characteristic secondary products in the different organs of an Aloe plant (A. succotrina Lam.) were studied by high performance liquid chromatography for the first time. In the leaves of the Aloe plant, only anthrone-C-glycosyls of the 7-hydroxyaloin type and, for the first time in plant material, the free anthraquinone 7-hydroxyaloeemodin were found. In contrast to previous reports on the distribution of secondary products in Aloe plants, anthrone-C-glycosyls were also detected in flowers, bracts and the inflorescence axis of the species examined. Aloesaponol I, a tetrahydroanthracene aglycone, was only present in the underground organs and in the stem. The 2-alkylchromone-C-glucosyl aloeresin B showed no specific occurrence as it was found in every type of organ. Based on these results and the findings of recent studies on Aloe roots and flowers, a distribution scheme of polyketide types in the Aloe plant was established. It suggests a separate and independent anthranoid metabolism for underground Aloe organs and stem on the one hand, and for leaves and inflorescence organs on the other hand. In the latter structures anthranoid metabolism seems to be additionally compartmentalized as the anthranoid pro files of inflorescence organs and leaves differ in two points relevant to anthranoid biosynthe sis: firstly, the occurrence of anthrone aglycones and secondly, the individual content of corresponding anthrone-C-glucosyl diastereomers.
The radiation-sensitive mutant pso4-1 of Saccharomyces cerevisiae shows a pleiotropic phenotype, including sensitivity to DNA cross-linking agents, nearly blocked sporulation and reduced mutability. We have cloned the putative yeast DNA repair gene PSO4 from a genomic library by complementation of the blocked UV-induced mutagenesis and of sporulation in diploids homozygous for pso4-1. Sequence analysis revealed that gene PSO4 consists of 1512 bp located upstream of UBI4 on chromosome XII and encodes a putative protein of 56.7 kDa. PSO4 is allelic to PRP19, a gene encoding a spliceosome-associated protein, but shares no significant homology with other yeast genes. Gene disruption with a destroyed reading frame of our PSO4 clone resulted in death of haploid cells, confirming the finding that PSO4/PRP19 is an essential gene. Thus, PSO4 is the third essential DNA repair gene found in the yeast S.cerevisiae.
Background: The industrial production of various alcohols from organic carbon compounds may be performed at high rates and with a low risk of contamination using thermophilic microorganisms as whole-cell catalysts. Thermoanaerobacter species that thrive around 50–75 °C not only perform fermentation of sugars to alcohols, but some also utilize different organic acids as electron acceptors, reducing them to their corresponding alcohols. Results: We purified AdhE as the major NADH- and AdhB as the major NADPH-dependent alcohol dehydrogenase (ADH) from the cell extract of the organic acid-reducing Thermoanaerobacter sp. strain X514. Both enzymes were present in high amounts during growth on glucose with and without isobutyrate, had broad substrate spectra including different aldehydes, with high affinities (< 1 mM) for acetaldehyde and for NADH (AdhE) or NADPH (AdhB). Both enzymes were highly thermostable at the physiological temperature of alcohol production. In addition to AdhE and AdhB, we identified two abundant AdhA-type ADHs based on their genes, which were recombinantly produced and biochemically characterized. The other five ADHs encoded in the genome were only expressed at low levels. Conclusions: According to their biochemical and kinetic properties, AdhE and AdhB are most important for ethanol formation from sugar and reduction of organic acids to alcohols, while the role of the two AdhA-type enzymes is less clear. AdhE is the only abundant aldehyde dehydrogenase for the acetyl-CoA reduction to aldehydes, however, acid reduction may also proceed directly by aldehyde:ferredoxin oxidoreductase. The role of the latter in bio-alcohol formation from sugar and in organic acid reduction needs to be elucidated in future studies.
Background: Otic neurons and sensory cells derive from common progenitors whose transition into mature cells requires the coordination of cell survival, proliferation and differentiation programmes. Neurotrophic support and survival of post-mitotic otic neurons have been intensively studied, but the bases underlying the regulation of programmed cell death in immature proliferative otic neuroblasts remains poorly understood. The protein kinase AKT acts as a node, playing a critical role in controlling cell survival and cell cycle progression. AKT is activated by trophic factors, including insulin-like growth factor I (IGF-I), through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by phosphatidylinositol 3-kinase (PI3K). Here we have investigated the role of IGF-dependent activation of the PI3K-AKT pathway in maintenance of otic neuroblasts.
Methodology/Principal Findings: By using a combination of organotypic cultures of chicken (Gallus gallus) otic vesicles and acoustic-vestibular ganglia, Western blotting, immunohistochemistry and in situ hybridization, we show that IGF-I-activation of AKT protects neural progenitors from programmed cell death. IGF-I maintains otic neuroblasts in an undifferentiated and proliferative state, which is characterised by the upregulation of the forkhead box M1 (FoxM1) transcription factor. By contrast, our results indicate that post-mitotic p27Kip-positive neurons become IGF-I independent as they extend their neuronal processes. Neurons gradually reduce their expression of the Igf1r, while they increase that of the neurotrophin receptor, TrkC.
Conclusions/Significance: Proliferative otic neuroblasts are dependent on the activation of the PI3K-AKT pathway by IGF-I for survival during the otic neuronal progenitor phase of early inner ear development.
Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.
The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.
In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.
Bioaerosols are considered to play a relevant role in atmospheric processes, but their sources, properties, and spatiotemporal distribution in the atmosphere are not yet well characterized. In the Amazon Basin, primary biological aerosol particles (PBAPs) account for a large fraction of coarse particulate matter, and fungal spores are among the most abundant PBAPs in this area as well as in other vegetated continental regions. Furthermore, PBAPs could also be important ice nuclei in Amazonia. Measurement data on the release of fungal spores under natural conditions, however, are sparse. Here we present an experimental approach to analyze and quantify the spore release from fungi and other spore-producing organisms under natural and laboratory conditions. For measurements under natural conditions, the samples were kept in their natural environment and a setup was developed to estimate the spore release numbers and sizes as well as the microclimatic factors temperature and air humidity in parallel to the mesoclimatic parameters net radiation, rain, and fog occurrence. For experiments in the laboratory, we developed a cuvette to assess the particle size and number of newly released fungal spores under controlled conditions, simultaneously measuring temperature and relative humidity inside the cuvette. Both approaches were combined with bioaerosol sampling techniques to characterize the released particles using microscopic methods. For fruiting bodies of the basidiomycetous species, Rigidoporus microporus, the model species for which these techniques were tested, the highest frequency of spore release occurred in the range from 62 % to 96 % relative humidity. The results obtained for this model species reveal characteristic spore release patterns linked to environmental or experimental conditions, indicating that the moisture status of the sample may be a regulating factor, whereas temperature and light seem to play a minor role for this species. The presented approach enables systematic studies aimed at the quantification and validation of spore emission rates and inventories, which can be applied to a regional mapping of cryptogamic organisms under given environmental conditions.
Bioaerosols are considered to play a relevant role in atmospheric processes, but their sources, properties and spatiotemporal distribution in the atmosphere are not yet well characterized. In the Amazon Basin, primary biological aerosol particles (PBAP) account for a large fraction of coarse particulate matter, and fungal spores are among the most abundant PBAP there as well as in other vegetated continental regions. furthermore, PBAP could also be important ice nuclei in Amazonia. Measurement data on the release of fungal spores under natural conditions, however, are sparse. Here we present an experimental approach to analyze and quantify the spore release from fungi and other spore producing organisms under natural and laboratory conditions. For measurements under natural conditions, the samples were kept in their natural environment and a setup was developed to estimate the spore release numbers and sizes together with the microclimatic factors temperature and air humidity, as well as the mesoclimatic parameters net radiation, rain, and fog occurrence. For experiments in the laboratory, we developed a cuvette to assess the particle size and number of newly released fungal spores under controlled conditions, simultaneously measuring temperature and relative humidity inside the cuvette. Both approaches were combined with bioaerosol sampling techniques to characterize the released particles by microscopic methods. For fruiting bodies of the basidiomycetous species, Rigidoporus microporus, the model species for which these techniques were tested, the highest frequency of spore release occurred in the range of 62 and 96 % relative humidity. The results obtained for this model species reveal characteristic spore release patterns linked to environmental or experimental conditions, indicating that the moisture status of the sample may be a regulating factor, while temperature and light seem to play a minor role for this species. The presented approach enables systematic studies aimed at the quantification and validation of spore emission rates and inventories, which can be applied to a regional mapping of cryptogamic organisms under given environmental conditions.
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.
Background: Aedes albopictus and Ae. japonicus are two of the most widespread invasive mosquito species that have recently become established in western Europe. Both species are associated with the transmission of a number of serious diseases and are projected to continue their spread in Europe.
Methods: In the present study, we modelled the habitat suitability for both species under current and future climatic conditions by means of an Ensemble forecasting approach. We additionally compared the modelled MAXENT niches of Ae. albopictus and Ae. japonicus regarding temperature and precipitation requirements.
Results: Both species were modelled to find suitable habitat conditions in distinct areas within Europe: Ae. albopictus within the Mediterranean regions in southern Europe, Ae. japonicus within the more temperate regions of central Europe. Only in few regions, suitable habitat conditions were projected to overlap for both species. Whereas Ae. albopictus is projected to be generally promoted by climate change in Europe, the area modelled to be climatically suitable for Ae. japonicus is projected to decrease under climate change. This projection of range reduction under climate change relies on the assumption that Ae. japonicus is not able to adapt to warmer climatic conditions. The modelled MAXENT temperature niches of Ae. japonicus were found to be narrower with an optimum at lower temperatures compared to the niches of Ae. albopictus.
Conclusions: Species distribution models identifying areas with high habitat suitability can help improving monitoring programmes for invasive species currently in place. However, as mosquito species are known to be able to adapt to new environmental conditions within the invasion range quickly, niche evolution of invasive mosquito species should be closely followed upon in future studies.
Gene families evolve by the processes of speciation (creating orthologs), gene duplication (paralogs), and horizontal gene transfer (xenologs), in addition to sequence divergence and gene loss. Orthologs in particular play an essential role in comparative genomics and phylogenomic analyses. With the continued sequencing of organisms across the tree of life, the data are available to reconstruct the unique evolutionary histories of tens of thousands of gene families. Accurate reconstruction of these histories, however, is a challenging computational problem, and the focus of the Quest for Orthologs Consortium. We review the recent advances and outstanding challenges in this field, as revealed at a symposium and meeting held at the University of Southern California in 2017. Key advances have been made both at the level of orthology algorithm development and with respect to coordination across the community of algorithm developers and orthology end-users. Applications spanned a broad range, including gene function prediction, phylostratigraphy, genome evolution, and phylogenomics. The meetings highlighted the increasing use of meta-analyses integrating results from multiple different algorithms, and discussed ongoing challenges in orthology inference as well as the next steps toward improvement and integration of orthology resources.
Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM‐CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM‐CF operations elaborated by the workgroups of the German network of ALM‐CFs, German Bio‐Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM‐CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences.
In the adult mammalian central nervous system, two defined neurogenic regions retain the capacity to generate new neurons throughout adulthood, namely the subependymal zone (SEZ) at the lateral ventricles and the subgranular layer of the hippocampus (SGL). Adult neurogenesis consists of a whole set of events including proliferation, fate specification, migration, survival and finally synaptic integration of newly born neurons. Each of these events is controlled by the interplay of numerous factors. In this study two signalling systems were analysed with regard to their functional role in adult neurogenesis in vivo, namely the purinergic system and the growth factor EGF. Neither short- nor long-term application of the P2Y receptor agonists UTP and ADPβS and the P2Y receptor antagonist suramin into the lateral ventricle of adult mice altered cell responses as compared to vehicle controls in vivo. In contrast, analysis of the expansion rates of cultured neural stem cells (NSCs) from knockout mice revealed a strong increase in the number of NSCs from NTPDase2-/- mice, whereas cell numbers of NSCs from P2Y1-/- and P2Y2-/- mice were significantly reduced in comparison to wildtype levels. Notably, in vivo proliferation rates were potently elevated in the SGL and the SEZ of NTPDase2-deficient mice. However, in vivo proliferation in both neurogenic niches of the single receptor knockout mice P2Y1-/- and P2Y2-/- and P2Y1-/- P2Y2-/-double-knockout mice did not differ significantly from the wildtype. In mice lacking the P2Y2 receptor the survival of newly born neurons in the hippocampal granule cell layer was significantly increased. These data provide the first line of evidence that purinergic signalling is involved in the control of neural stem cells behaviour not only in vitro but also in vivo. In order to further characterise the role of epidermal growth factor (EGF) in adult neurogenesis, transit amplifying precursors (TAPs) and type B astrocytes were identified as EGF-responsive cell populations following ventricular EGF injection, whereas ependymal cells, neuroblasts and NG2-positive cells did not or only to a minor extent respond to EGF injection. These EGF-responsive cell populations were found on both, the septal as well as striatal lateral ventricle walls. Long-term ventricular EGF infusion for 6d, 1. increased cell proliferation of both ventricle walls revealing a gradient along the rostro-caudal axis, 2. altered the balance between neuronal and macroglial cell fates to generate oligodendrocyte precursors and 3. lead to an entire remodelling of the classical architecture of the SEZ.
Bisphenols and phthalates, chemicals frequently used in plastic products, promote obesity in cell and animal models. However, these well-known metabolism-disrupting chemicals (MDCs) represent only a minute fraction of all compounds found in plastics. To gain a comprehensive understanding of plastics as a source of exposure to MDCs, we characterized the chemicals present in 34 everyday products using nontarget high-resolution mass spectrometry and analyzed their joint adipogenic activities by high-content imaging. We detected 55,300 chemical features and tentatively identified 629 unique compounds, including 11 known MDCs. Importantly, the chemicals extracted from one-third of the products caused murine 3T3-L1 preadipocytes to proliferate, and differentiate into adipocytes, which were larger and contained more triglycerides than those treated with the reference compound rosiglitazone. Because the majority of plastic extracts did not activate the peroxisome proliferator-activated receptor γ and the glucocorticoid receptor, the adipogenic effects are mediated via other mechanisms and, thus, likely to be caused by unknown MDCs. Our study demonstrates that daily-use plastics contain potent mixtures of MDCs and can, therefore, be a relevant yet underestimated environmental factor contributing to obesity.
Bisphenols and phthalates, chemicals frequently used in plastic products, promote obesity in cell and animal models. However, these well-known metabolism disrupting chemicals (MDCs) represent only a minute fraction of all compounds found in plastics. To gain a comprehensive understanding of plastics as a source of exposure to MDCs, we characterized all chemicals present in 34 everyday products using nontarget high-resolution mass spectrometry and analyzed their joint adipogenic activities by high-content imaging. We detected 55,300 chemical features and tentatively identified 629 unique compounds, including 11 known MDCs. Importantly, chemicals that induced proliferation, growth, and triglyceride accumulation in 3T3-L1 adipocytes were found in one third of the products. Since the majority did not target peroxisome proliferator-activated receptor γ, the effects are likely to be caused by unknown MDCs. Our study demonstrates that daily-use plastics contain potent mixtures of MDCs and can, therefore, be a relevant yet underestimated environmental factor contributing to obesity.
Teaser Plastics contain a potent mixture of chemicals promoting adipogenesis, a key process in developing obesity.
Acetobacterium woodii utilizes the Wood‐Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non‐acetogenic growth on 1,2‐propanediol (1,2‐PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2‐PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC‐encapsulated propanol‐generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH‐dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2‐PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.
Signal-dependent regulation of actin dynamics is essential for many cellular processes, including directional cell migration. In particular, cell migration is initiated by lamellipodia, actin-based protrusions of the plasma membrane. The formation of these protruding structures require incessant assembly and disassembly of actin filaments. The Arp2/3 complex and WAVE proteins are essential for both lamellipodium formation and its dynamics. WAVEs mediate the activation of the Arp2/3 complex downstream of the small GTPase Rac, thus being critical for Rac- and RTK-induced actin polymerization and cell migration. The WAVE-family proteins are always found associated with multiprotein complexes. The most abundant WAVE-based complex is referred to as the WANP (WAVE2-Abi-1-Nap1-PIR121) complex. IQGAP1 is a huge scaffolding protein with multiple protein-interacting domains. IQGAP1 participates in many fundamental activities, including regulation of the actin cytoskeleton, mitogenic, adhesive and migratory responses, as well as in cell polarity and cellular trafficking. IQGAP1 binds to N-WASP, thus raising the possibility that it might control actin nucleation by the Arp2/3 complex. In this study, IQGAP1 was found co-immunoprecipitated not only with WAVE, but also with the endogenous WANP-complex subunits. Correspondingly, IQGAP1 associated to both anti-WAVE and anti-Abi-1 immuno-complexes. Pull-down experiments proved that IQGAP1 binds directly to the WANP-complex subunits. Physical interaction between IQGAP1 and the reconstituted WANP complex could also be demonstrated. Together, these data indicate that IQGAP1 is an accessory component of the WANP complex. Interestingly, the IQGAP-WANP complex disassembled after either EGF stimulation or transfection with constitutively active Cdc42 and Rac1. HeLa cells devoid of IQGAP1 showed diminished and less persistent ruffling upon EGF, but not HGF, stimulation in comparison with the control. This phenotype was accompanied by a strong reduction in chemotaxis towards both growth factors, which was as dramatic as in WANP-complex knockdown (KD) cells. Moreover, GM130 and Giantin showed a polarized and flat ribbon-like pattern in control cells, as it is expected for cis- and cis/medial-Golgi markers. Conversely, small and dispersed vesicular structures were found in both IQGAP1 KD and WANP-complex KD cells. Importantly, Arp2/3-complex silencing resulted in the same phenotypes. Consistently, Brefeldin A-induced disassembly of the Golgi strongly inhibited the IQGAP1-WANP-complex interaction and chemotaxis towards EGF in wild-type cells. The re-expression of an RNAi-resistant wild-type IQGAP1 in IQGAP1 KD cells fully rescued both the ruffling abilities and Golgi structure. A constitutively active mutant, unable to bind to neither Rac1 /Cdc42 nor the WANP complex, could reconstitute only the former defect. Hence, this study shows that actin dynamics regulated by the IQGAP1-WANP complex controls Golgi-apparatus architecture and its contribution to cell chemotaxis. The working model here proposes that at the Golgi apparatus, recruitment of the WANP complex by IQGAP1 leads to the assembly of actin filaments required to maintain the appropriated Golgi morphology. The dissociation of the complex may be required to allow the remodeling of the Golgi membranes in order to respond following a chemoattractant gradient.
Summary statement When echolocating under demanding conditions e.g. noisy, narrow space, or cluttered environments, frugivorous bats adapt their call pattern by increasing the call rate within biosonar groups.
Abstract For orientation, echolocating bats emit biosonar calls and use echoes arising from call reflections. They often pattern their calls into groups which increases the rate of sensory feedback over time. Insectivorous bats emit call groups at a higher rate when orienting in cluttered compared to uncluttered environments. Frugivorous bats increase the rate of call group emission when they echolocate in noisy environments. Here, calls emitted by conspecifics potentially interfere with the bat’s biosonar signals and complicate the echolocation behavior. To minimize the information loss followed by signal interference, bats may profit from a temporally increased sensory acquisition rate, as it is the case for the call groups. In frugivorous bats, it remains unclear if call group emission represents an exclusive adaptation to avoid interference by signals from other bats or if it represents an adaptation that allows to orient under demanding environmental conditions. Here, we compared the emission pattern of the frugivorous bat Carollia perspicillata when the bats were flying in noisy versus silent, narrow versus wide or cluttered versus non-cluttered corridors. According to our results, the bats emitted larger call groups and they increased the call rate within the call groups when navigating in narrow, cluttered, or noisy environments. Thus, call group emission represents an adaptive behavior when the bats orient in complex environments.
In our rapidly changing world, land use has been recognized as having one of the strongest impacts on species and genetic diversity. The present state of temperate forests in Europe is a product of decisions made by former and current management and policy actions, rather than natural factors. Alterations of crown projection areas, structural complexity of the forest stand caused by thinning and cuttings, and changes in tree species composition caused by regeneration or plantings not only affect forest interior buffering against warming, but also the understorey light environment and nutrient availability. Ultimately, current silvicultural management practices have deep impact on the forest ecosystems, microenvironmental changes and forest floor understorey herbs. In response to environmental changes, plants rely on genetically heritable phenotypic variation, an important level of variation in the population, as it is prerequisite for adaptation. However, until now most studies on plant adaptation to land use focus on grassland management. Yet, studies on the adaptation of forest understorey herbs to forest management have been absent so far. This is important because understanding adaptation of understorey herbs is crucial for biodiversity conservation, forest restoration, and climate change mitigation. Studying current adaptation of understorey herbs to forest management yields insights into the evolutionary consequences of management practices, which could be employed to improve sustainable use of forest habitat.
In sum, my conducted experiments complement each other well and managed to fill in research gaps on the topic of genetically heritable phenotypic variation in understorey herbs and how it is affected by forest management and related microenvironmental variables. I showed that forest management has direct evolutionary consequences on the genetic basis of understorey herbs, but also indirectly through the microenvironment. Furthermore, I revealed that local adaptation and phenotypic plasticity of understorey herbs to forest structural attributes act along continuous gradients. And lastly, I highlighted the important role of intra-individual variation by revealing plastic responses to drought and shading, urging researchers to not ignore this important level of trait variation. Ultimately, understorey herbs in temperate forests employ phenotypic plasticity as a flexible strategy to adapt to varying environmental conditions. By adjusting their leaf characteristics, reproductive investment, and phenology, they can optimize their fitness and survival in response to changes in light availability, resource availability, and seasonal cues. The anthropogenic impact on temperate forests and understorey herbs will continue and likely increase in the future. This should urge foresters to adapt their silvicultural management decisions towards the long-term preservation of genetic diversity and, through this, the evolvability and adaptability of forest understorey herbs and associated organisms. Based on the results shown in my dissertation, variation in forest management regimes and types could be beneficial for promoting genetic diversity within several species of forest understorey herbs. Lastly, in the face of future climatic changes, the mechanisms by which plants can cope with increasing stressful environmental conditions might very well rely heavily on intra-individual variation, providing the necessary rapid plastic adjustment to changing microclimatic conditions within populations and thus increase climate change resilience.
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...
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.
ADAM15, which belongs to the family of the disintegrin and metalloproteinases, is a multi-domain transmembrane protein. A strongly upregulated expression of ADAM15 is found in inflamed synovial membranes from articular joints affected by osteoarthritis and especially rheumatoid arthritis (RA). During the chronic inflammatory process in RA the synovial membrane gets hyperplastic, resulting eventually in the formation of a pannus tissue, which can invade into the adjacent cartilage and bone thereby destroying their integrity. Previously, the expression of ADAM15 in fibroblasts of the RA synovial membrane was found to confer a significant anti-apoptotic response upon triggering of the Fas receptor, which resulted in the activation of two survival kinases, focal adhesion kinase (FAK) and Src. The Fas receptor, also named CD95, belongs to the death receptor family of the tumor necrosis factor receptors and stimulation of Fas/CD95 by its ligand FasL results in the execution of apoptotic cell death in synovial membranes of RA patients. However, the occurrence of apoptotic cell death in vivo in RA synovial tissues is considerably low despite the presence of FasL at high concentrations in the chronically inflamed joint. Accordingly, a general apoptosis resistance is a characteristic of RA-synovial fibroblasts that contributes considerably to the formation the hyperplastic aggressive pannus tissue. The objective of this study was to investigate the mechanisms underlying the capability of ADAM15 to transform FasL-mediated death- inducing signals into pro-survival activation of Src and FAK in rheumatoid arthritis fibroblasts (RASFs).
In the present study, the down-regulation of ADAM15 by RNA interference resulted in a significant increase of caspase 3/7 activity upon stimulation of the Fas receptor in RASFs. Likewise, chondrocytes expressing a deletion mutant of ADAM15 (ΔC), lacking the cytoplasmic domain, revealed increased caspase activities upon Fas ligation in comparison to cells transfected with full-length ADAM15, clearly demonstrating the importance of the cytoplasmic domain for an increased apoptosis resistance. Furthermore, activation of the Fas receptor triggered the phosphorylation of Src at Y416, which results in the active conformation of Src, as well as the phosphorylation of FAK at Y576/577 and Y861 – the target tyrosines phosphorylated by Src - in full-length ADAM15-transfected chondrocytes. However, cells transfected with ADAM15 mutant (ΔC) or with vector control did not exhibit any activation of Src and FAK upon Fas ligation. This suggested the presence of an as yet unknown protein interaction mediating the Fas triggered activation of the two kinases.
In order to identify this mechanism, the application of signal transduction inhibitors interfering with Calcium signaling either by inhibiting calmodulin with trifluoperazine (TFP) or the Calcium release-activated channel (CRAC/Orai1) with BTP-2 efficiently inhibited the phosphorylation of FAK and Src, revealing a role of calmodulin, the major Ca2+ sensor in cells, in ADAM15-dependent and Fas-elicited activation of the two survival kinases. Also, a direct Ca2+ -dependent binding of calmodulin to ADAM15 could be demonstrated by pull-down assays using calmodulin-conjugated sepharose and by protein binding assays using the recombinant cytoplasmic domain of ADAM15 and calmodulin.
Furthermore, it could be demonstrated in living synovial fibroblasts by double immunofluorescence stainings that triggering the Fas receptor by its ligand FasL or a Fas-activating antibody resulted in the recruitment of calmodulin to ADAM15 as well as to the Fas receptor in patch-like structures at the cell membrane. Simultaneously, Src associated with calmodulin was shown to become engaged in an ADAM15 complex, also containing cytoplasmic-bound FAK, by co-immunoprecipitations.
Additional studies were performed to analyze the efficacy of TFP and BTP-2 on apoptosis induction in synovial fibroblasts from 10 RA patients. Using caspase 3/7 and annexin V stainings for determining apoptosis, it could be shown that both inhibitors did not possess any apoptosis inducing capacity. However, when co-incubated with FasL both compounds synergistically enhanced apoptosis rates in the RASFs. Moreover, an additional silencing of ADAM15 revealed a further significant rise in apoptosis rates upon incubation with FasL/TFP or FasL/BTP-2, providing unequivocal evidence for an involvement of ADAM15 in facilitating apoptosis resistance in RASFs.
Taken together, these results demonstrate that ADAM15 provides a scaffold for the formation of calmodulin-dependent pro-survival signaling complexes upon CRAC/Orai1 coactivation by Fas ligation, which provides a new potential therapeutic target to break the apoptosis resistance in RASFs that critically contributes to joint destruction in RA.
Diese Dissertation befasst sich mit den Auswirkungen von nicht letalen Dosen von Neonikotinoiden auf Bienen. Neonikotinoide stellen eine Klasse von Insektiziden dar, die auf den nikotinischen Acetylcholin Rezeptor wirken. In dieser Dissertation wurden die Neonikotinoide Imidacloprid, Clothianidin und Thiacloprid benutzt. Die beiden erst genannten unterliegen zum Zeitpunkt des Verfassens dieser Arbeit einem temporären Verkaufs- und Ausbringungs-Stopp. Damit sind die Ergebnisse dieser Arbeit wichtig für die Bewertung der Gefahren von Neonikotinoiden. Neonikotinoide werden im großen Maße in der Landwirtschaft als Spritzmittel und Saatgutbeize eingesetzt. Dabei können sie in Rückständen von Bienen beim Sammeln von Nektar und Pollen aufgenommen und zum Stock gebracht werden. Um einen weiten Blick auf die Auswirkungen der Stoffe zu werfen wurden deshalb Experimente an einzelnen Sammlerinnen durchgeführt, ebenso wie an Bienenvölkern, bei denen die Substanzen verfüttert wurden. Als neuronal aktive Substanzen können sie die normale Funktion des Nervensystems von Bienen beeinflussen, was Veränderungen im Verhalten hervorrufen kann. Dies zeigt sich in Veränderungen in der Bewegung, Orientierung oder auch Interaktion mit anderen Bienen. Die Wirkung am Rezeptor variiert, trotz gleichen molekularen Ziels, stark zwischen den verwendeten Neonikotinoiden. Clothianidin wurde als Agonist beschrieben, der sogar stärkere Ströme als Acetylcholin bei gleicher Konzentration hervorrufen kann. Imidacloprid dagegen wurde bereits als partieller Agonist beschrieben, der geringere Ströme über den Rezeptor auslöst. In dieser Arbeit wurde ein erster Versuch durchgeführt um Thiacloprid ebenfalls als Agonist am nikotinischen Acetylcholin Rezeptor der Biene zu beschreiben. Hierbei wurde an einer Zelle in Kultur ein geringerer Strom ausgelöst.
Bienenvölker wurden unter kontrollierten Bedingungen gehalten, bei denen je eins der Neonikotinoide Clothianidin, Imidacloprid oder Thiacloprid in das Futter gemischt wurden. Hierfür wurden Dosen gewählt, bei denen davon ausgegangen werden konnte, dass keine akute Beeinflussung der Sammlerinnen bestand. Es konnte festgestellt werden, dass chronisches Füttern mit einer Zuckerlösung mit 8,876 mg/kg Thiacloprid zu einer verringerten Sammelleistung führte. Ebenso wurde die Entwicklung der Eier stark eingeschränkt, wobei die Königin weiterhin Eier legte. Es konnten nur vereinzelte verdeckelte Brutzellen, die ein spätes Entwicklungsstadium der Bienen darstellen, gefunden werden. Damit konnte gezeigt werden, dass geringe Dosen die Larval-Entwicklung von Bienen beeinflussen, eventuell durch Einflüsse auf die Kommunikation zwischen Ammenbienen und der Brut.
Um Auswirkungen auf einzelne Tiere zu zeigen, wurden unterschiedliche Parameter im Heimflug von Bienen nach Fütterung mit je einem der Neonikotinoide analysiert. Bienen mussten sich nach der Fütterung orientieren und von einer neuen Position den Heimweg zum Stock finden. Der Heimflug wurde per Radar verfolgt und so ein Flugprofil erstellt, das aus zwei Flugphasen bestand. Diese wurden durch die Navigation nach Vektorintegration und durch Landmarken unterteilt. Aus dem Flugprofil konnte abgelesen werden, wie lange die Bienen für die Phasen des Flugs benötigten, in welchem Hauptflugwinkel sie die erste Flugphase absolvierten, in welche Richtung sie am Ende der ersten Flugphase flogen und wie gerichtet der Flug war. Auch wurde erfasst, ob die Bienen überhaupt in der Lage waren zum Stock zurückzukehren. Hier zeigte sich, dass die Fütterung mit Zuckerwasser mit 0,6 µM und 0,9 µM Imidacloprid, ebenso wie mit 0,1 mM Thiacloprid zu einer verringerten Heimkehrwahrscheinlichkeit führte. In der ersten Flugphase konnte auch gezeigt werden, dass 0,2 µM Clothianidin im Zuckerwasser zu einem schnelleren Flug führte und dass der Flugwinkel im Vergleich zur Kontrolle in Richtung der wahren Position des Stocks verschoben war. Beide Imidacloprid-Gruppen zeigten eine ähnliche, signifikante Verschiebung des Flugwinkels, ebenso konnte im Flug selbst eine häufige Änderung der Richtung festgestellt werden. In der zweiten Flugphase zeigte sich, dass Bienen, welche mit Thiacloprid behandelt wurden häufiger eine inkorrekte Heimflugrichtung wählten, was in längeren Heimflügen resultierte. Die mit Clothianidin behandelten Bienen legten eine längere Flugstrecke zurück. Bienen, welche Imidacloprid beider Konzentrationen konsumierten, zeigten einen häufigen Wechsel ihrer Flugrichtung. Damit konnten bei allen drei gewählten Neonikotinoiden Einflüsse auf spezifische Komponenten der Navigation von Bienen gefunden und Einschränkungen im Heimkehr- und Orientierungsverhalten einzelner Sammlerinnen gezeigt werden. Somit konnten die eingehenden Fragen zumindest teilweise beantwortet werden und die Datenlage zur Frage der Schädlichkeit der, auch politisch umstrittenen, Substanzen erweitert werden.
The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881–1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
Whereas ribosome preparations of freshly sliced potato disks do not show appreciable activity in an in-vitro amino acid incorporation system, aging of the tissue leads to a greatly enhanced incorporation activity which reaches its maximum 24 hours after slicing. If ribosomes from freshly excised disks are provided with polyuridylic acid, their activity in the incorporation of phenylalanine is increased about 8 fold.
Moreover, an RNA-fraction can be dissociated by EDTA from ribosomes of aged potato tuber slices, which sediments at 15 —18S, has a base composition different from that of 16S — rRNA, 5S-and 4S —RNA, and is not present on ribosomes of fresh slices. Its appearance is inhibited by actinomycin D and therefore most probably dependent on transcription. This compound, purified from sucrose gradients, enhances in vitro leucine incorporation into peptide material by ribosomes of fresh potato slices.
The possibility is discussed that this fraction-among other factors-is responsible for the enhanced protein synthesis after slicing plant storage organs, and is indicative of a general derepression phenomenon in these tissues.
Chromatin, RNA Polymerase, Potato Tuber Tissue, Aging Phenomenon The synthesis of RNA by chromatin-bound RNA polymerase (E.C. 2.7.7.6.) from white potato tubers proceeds at a low rate, which is enhanced after slicing the tissue, however. Concomitantly DNA template availability as measured with saturating amounts of Escherichia coli polymerase is diminished drastically. Nearest neighbor frequency analysis proved that the RNA synthesized on chromatin of intact tubers is different from that synthesized on chromatin of sliced tissue.
The RNA polymerase of white potato tubers is dependent on all four ribonucleoside triphos phates and a divalent metal ion such as Mg2+ or Mn2+ and totally inhibited by the presence of pyrophosphate. Actinomycin D blocks the formation of the RNA product, which could be shown to be a heteropolymer by nearest neighbour frequency technique. The Km of the chromatin-bound enzyme with regard to ATP, GTP, CTP and UTP was 5.1 X10-5 M, 1.6X10-5 M, 0.9X10-5 M and 0.45 X 10-5M/1 respectively, α-amanitin inhibits the overall activity to about 50%, which indicates the presence of equal amounts of polymerase I and polymerase If.
Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.
Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.
Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.
Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.
Echolocation allows bats to orientate in darkness without using visual information. Bats emit spatially directed high frequency calls and infer spatial information from echoes coming from call reflections in objects (Simmons 2012; Moss and Surlykke 2001, 2010). The echoes provide momentary snapshots, which have to be integrated to create an acoustic image of the surroundings. The spatial resolution of the computed image increases with the quantity of received echoes. Thus, a high call rate is required for a detailed representation of the surroundings.
One important parameter that the bats extract from the echoes is an object’s distance. The distance is inferred from the echo delay, which represents the duration between call emission and echo arrival (Kössl et al. 2014). The echo delay decreases with decreasing distance and delay-tuned neurons have been characterized in the ascending auditory pathway, which runs from the inferior colliculus (Wenstrup et al. 2012; Macías et al. 2016; Wenstrup and Portfors 2011; Dear and Suga 1995) to the auditory cortex (Hagemann et al. 2010; Suga and O'Neill 1979; O'Neill and Suga 1982).
Electrophysiological studies usually characterize neuronal processing by using artificial and simplified versions of the echolocation signals as stimuli (Hagemann et al. 2010; Hagemann et al. 2011; Hechavarría and Kössl 2014; Hechavarría et al. 2013). The high controllability of artificial stimuli simplifies the inference of the neuronal mechanisms underlying distance processing. But, it remains largely unexplored how the neurons process delay information from echolocation sequences. The main purpose of the thesis is to investigate how natural echolocation sequences are processed in the brain of the bat Carollia perspicillata. Bats actively control the sensory information that it gathers during echolocation. This allows experimenters to easily identify and record the acoustic stimuli that are behaviorally relevant for orientation. For recording echolocation sequences, a bat was placed in the mass of a swinging pendulum (Kobler et al. 1985; Beetz et al. 2016b). During the swing the bat emitted echolocation calls that were reflected in surrounding objects. An ultrasound sensitive microphone traveling with the bat and positioned above the bat’s head recorded the echolocation sequence. The echolocation sequence carried delay information of an approach flight and was used as stimulus for neuronal recordings from the auditory cortex and inferior colliculus of the bats.
Presentation of high stimulus rates to other species, such as rats, guinea pigs, suppresses cortical neuron activity (Wehr and Zador 2005; Creutzfeldt et al. 1980). Therefore, I tested if neurons of bats are suppressed when they are stimulated with high acoustic rates represented in echolocation sequences (sequence situation). Additionally, the bats were stimulated with randomized call echo elements of the sequence and an interstimulus time interval of 400 ms (element situation). To quantify neuronal suppression induced by the sequence, I compared the response pattern to the sequence situation with the concatenated response patterns to the element situation. Surprisingly, although the bats should be adapted for processing high acoustic rates, their cortical neurons are vastly suppressed in the sequence situation (Beetz et al. 2016b). However, instead of being completely suppressed during the sequence situation, the neurons partially recover from suppression at a unit specific call echo element. Multi-electrode recordings from the cortex allow assessment of the representation of echo delays along the cortical surface. At the cortical level, delay-tuned neurons are topographically organized. Cortical suppression improves sharpness of neuronal tuning and decreases the blurriness of the topographic map. With neuronal recordings from the inferior colliculus, I tested whether the echolocation sequence also induced neuronal suppression at subcortical level. The sequence induced suppression was weaker in the inferior colliculus than in the cortex. The collicular response makes the neurons able to track the acoustic events in the echolocation sequence. Collicular suppression mainly improves the signal-to-noise ratio. In conclusion, the results demonstrate that cortical suppression is not necessarily a shortcoming for temporal processing of rapidly occurring stimuli as it has previously been interpreted.
Natural environments are usually composed of multiple objects. Thus, each echolocation call reflects off multiple objects resulting in multiple echoes following the calls. At present, it is largely unexplored how neurons process echolocation sequences containing echo information from more than one object (multi-object sequences). Therefore, I stimulated bats with a multi-object sequence which contained echo information from three objects. The objects were different distances away from each other. I tested the influence of each object on the neuronal tuning by stimulating the bats with different sequences created from filtering object specific echoes from the multi-object sequence. The cortex most reliably processes echo information from the nearest object whereas echo information from distant objects is not processed due to neuronal suppression. Collicular neurons process less selectively echo information from certain objects and respond to each echo.
For proper echolocation, bats have to distinguish between own biosonar signals and the signals coming from conspecifics. This can be quite challenging when many bats echolocate adjacent to each other. In behavioral experiments, the echolocation performance of C. perspicillata was tested in the presence of potentially interfering sounds. In the presence of acoustic noise, the bats increase the sensory acquisition rate which may increase the update rate of sensory processing. Neuronal recordings from the auditory cortex and inferior colliculus could strengthen the hypothesis. Although there were signs of acoustic interference or jamming at neuronal level, the neurons were not completely suppressed and responded to the rest of the echolocation sequence.
Vocal communication is essential to coordinate social interactions in mammals and it requires a fine discrimination of communication sounds. Auditory neurons can exhibit selectivity for specific calls, but how it is affected by preceding sounds is still debated. We tackled this using ethologically relevant vocalizations in a highly vocal mammalian species: Seba’s short-tailed bat. We show that cortical neurons present several degrees of selectivity for echolocation and distress calls. Embedding vocalizations within natural acoustic streams leads to stimulus-specific suppression of neuronal responses that changes sound selectivity in disparate manners: increases in neurons with poor discriminability in silence and decreases in neurons selective in silent settings. A computational model indicates that the observed effects arise from two forms of adaptation: presynaptic frequency specific adaptation acting in cortical inputs and stimulus unspecific postsynaptic adaptation. These results shed light into how acoustic context modulates natural sound discriminability in the mammalian cortex.
Acinetobacter baumannii virulence is mediated by the concerted action of three phospholipases D
(2015)
Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.
Acetylcholine (ACh) is the major excitatory neurotransmitter in the insect central nervous system (CNS). However, besides the neuronal expression of ACh receptors (AChR), the existence of non-neuronal AChR in honeybees is plausible. The cholinergic system is a popular target of insecticides because the pharmacology of insect nicotinic acetylcholine receptors (nAChRs) differs substantially from their vertebrate counterparts. Neonicotinoids are agonists of the nAChR and are largely used in crop protection. In contrast to their relatively high safety for humans and livestock, neonicotinoids pose a threat to pollinating insects such as bees. In addition to its effects on behavior, it becomes increasingly evident that neonicotinoids affect developmental processes in bees that appear to be independent of neuronal AChRs. Brood food (royal jelly, worker jelly, or drone jelly) produced in the hypopharyngeal glands of nurse bees contains millimolar concentrations of ACh, which is required for proper larval development. Neonicotinoids reduce the secreted ACh-content in brood food, reduce hypopharyngeal gland size, and lead to developmental impairments within the colony. We assume that potential hazards of neonicotinoids on pollinating bees occur neuronally causing behavioral impairments on adult individuals, and non-neuronally causing developmental disturbances as well as destroying gland functioning.
One of the key functions of blood vessels is to transport nutrients and oxygen to distant tissues and organs in the body. When blood supply is insufficient, new vessels form to meet the metabolic tissue demands and to re-establish cellular homeostasis. Expansion of the vascular network through sprouting angiogenesis requires the specification of ECs into leading (sprouting) tip and following (non-sprouting) stalk cells. Attracted by guidance cues tip cells dynamically extend and retract filopodia to navigate the nascent vessel sprout, whereas trailing stalk cells proliferate to form the extending vascular tube. All of these processes are under the control of environmental signals (e.g. hypoxia, metabolism) and numerous cytokines and peptide growth factors. The Dll4/Notch pathway coordinates several critical steps of angiogenic blood vessel growth. Even subtle alterations in Notch activity can profoundly influence endothelial cell behavior and blood vessel formation, yet little is known about the intrinsic regulation and dynamics of Notch signaling in endothelial cells. In addition, it remains an open question, how different growth factor signals impinging on sprouting ECs are coordinated with local environmental cues originating from nutrient-deprived, hypoxic tissue to achieve a balanced endothelial cell response. Acetylation of lysines is a critical posttranslational modification of histones, which acts as an important regulatory mechanism to control chromatin structure and gene transcription. In addition to histones, several non-histone proteins are targeted for acetylation reversible acetylation is emerging as a fundamental regulatory mechanism to control protein function, interaction and stability. Previous studies from our group identified the NAD+-dependent deacetylase SIRT1 as a key regulator of blood vessel growth controlling endothelial angiogenic responses. These studies revealed that SIRT1 is highly expressed in the vascular endothelium during blood vessel development, where it controls the angiogenic activity of endothelial cells. Moreover, in this work SIRT1 has been shown to control the activity of key regulators of cardiovascular homeostasis such as eNOS, Foxo1 and p53. The present study describes that SIRT1 antagonizes Notch signaling by deacetylating the Notch intracellular domain (NICD). We showed that loss of SIRT1 enhances DLL4-induced endothelial Notch responses as assessed by different luciferase responsive elements as well as transcriptional analysis of Notch endogenous target genes activation. Conversely, SIRT1 gain of function by overexpression of pharmacological activation decreases induction of Notch targets in response to DLL4 stimulation. We also showed that the NICD can be directly acetylated by PC AF and p300 and that SIRT1 promotes deacetylation of NICD. We have identified 14 lysines that are targeted for acetylation and their mutation abolishes the effects of SIRT1 of Notch responses. Furthermore, over-expression or activation of SIRT1 significantly reduces the levels of NICD protein. Moreover, SIRT1-mediated NICD degradation can be reversed by blockade of the proteasome suggesting a mechanism resulting from ubiquitin-mediated proteolysis. Indeed, we have shown that SIRT1 knockdown or pharmacological inhibition decreased NICD ubiquitination. We propose a novel molecular mechanism of modulation of the amplitude and duration of Notch responses in which acetylation increases NICD stability and therefore permanence at the promoters, while SIRT1, by inducing NICD degradation through its deacetylation, shortens Notch responses. In order to evaluate the physiological relevance of our findings we used different models in which the Notch functions during blood vessel formation have been extensively characterized. First, retinal angiogenesis in mice lacking SIRT1 activity shows decreased branching and reduced endothelial proliferation, similar to what happens after Notch gain of function mutations. ECs from these mice exhibit increased expression of Notch target genes. Second, these results were reproducible during intersomitic vessel growth in sirt1-deficient zebrafish. In both models, the defects could be partially rescued by inhibition of Notch activation. Third, we used an in vitro model of vessel sprouting from differentiating embryonic bodies in response to VEGF in a collagen matrix. Our results showed that Sirt1-deficient cells shows impaired sprouting which correlated with increased NICD levels. In addition, when in competition with wild-type cells in this assay, Sirt1-deficient cells are more prone to occupy the stalk cell position. Taken together, our study identifies reversible acetylation of NICD as a novel molecular mechanism to adapt the dynamics of Notch signaling and suggest that SIRT1 acts as a rheostat to fine-tune endothelial Notch responses. The NAD+-dependent feature of SIRT1 activity possibly links endothelial Notch responses to environmental cues and metabolic changes during nutrient deprivation in ischemic environments or upon other cellular stresses.
Climate change is influencing some environmental variables in the Southern Ocean (SO) and this will have an effect on the marine biodiversity. Peracarid crustaceans are one of the dominant and most species-rich groups of the SO benthos. To date, our knowledge on the influence of environmental variables in shaping abundance and species composition in the SO’s peracarid assemblages is limited, and with regard to ice coverage it is unknown. The aim of our study was to assess the influence of sea ice coverage, chlorophyll-a, and phytoplankton concentrations on abundance, distribution and assemblage structure of peracarids. In addition, the influence of other physical parameters on peracarid abundance was assessed, including depth, temperature, salinity, sediment type, current velocity, oxygen, iron, nitrate, silicate and phosphate. Peracarids were sampled with an epibenthic sledge (EBS) in different areas of the Atlantic sector of the SO and in the Weddell Sea. Sampling areas were characterized by different regimes of ice coverage (the ice free South Orkney Islands, the seasonally ice-covered Filchner Trough and the Eastern Antarctic Peninsula including the Prince Gustav Channel which was formerly covered by a perennial ice shelf). In total 64766 individuals of peracarids were collected and identified to order level including five orders: Amphipoda, Cumacea, Isopoda, Mysidacea, and Tanaidacea. Amphipoda was the most abundant taxon, representing 32% of the overall abundances, followed by Cumacea (31%), Isopoda (29%), Mysidacea (4%), and Tanaidacea (4%). The Filchner Trough had the highest abundance of peracarids, while the South Orkney Islands showed the lowest abundance compared to other areas. Ice coverage was the main environmental driver shaping the abundance pattern and assemblage structure of peracarids and the latter were positively correlated with ice coverage and chlorophyll-a concentration. We propose that the positive correlation between sea ice and peracarid abundances is likely due to phytoplankton blooms triggered by seasonal sea ice melting, which might increase the food availability for benthos. Variations in ice coverage extent and seasonality due to climate change would strongly influence the abundance and assemblage structure of benthic peracarids.
Background: Plant hormones are well known regulators which balance plant responses to abiotic and biotic stresses. We investigated the role of abscisic acid (ABA) in resistance of barley (Hordeum vulgare L.) against the plant pathogenic fungus Magnaporthe oryzae.
Results: Exogenous application of ABA prior to inoculation with M. oryzae led to more disease symptoms on barley leaves. This result contrasted the finding that ABA application enhances resistance of barley against the powdery mildew fungus. Microscopic analysis identified diminished penetration resistance as cause for enhanced susceptibility. Consistently, the barley mutant Az34, impaired in ABA biosynthesis, was less susceptible to infection by M. oryzae and displayed elevated penetration resistance as compared to the isogenic wild type cultivar Steptoe. Chemical complementation of Az34 mutant plants by exogenous application of ABA re-established disease severity to the wild type level. The role of ABA in susceptibility of barley against M. oryzae was corroborated by showing that ABA application led to increased disease severity in all barley cultivars under investigation except for the most susceptible cultivar Pallas. Interestingly, endogenous ABA concentrations did not significantly change after infection of barley with M. oryzae.
Conclusion: Our results revealed that elevated ABA levels led to a higher disease severity on barley leaves to M. oryzae. This supports earlier reports on the role of ABA in enhancing susceptibility of rice to the same pathogen and thereby demonstrates a host plant-independent function of this phytohormone in pathogenicity of monocotyledonous plants against M. oryzae.
Aim: The identification of the mechanisms determining spatial variation in biological diversity along elevational gradients is a central objective in ecology and biogeography. Here, we disentangle the direct and indirect effects of abiotic drivers (climatic conditions, and land use) and biotic drivers (vegetation structure and food resources) on functional diversity and composition of bird and bat assemblages along a tropical elevational gradient. Location: Southern slopes of Mt. Kilimanjaro, Tanzania, East Africa. Methods: We counted birds and recorded bat sonotypes on 58 plots distributed in near-natural and anthropogenically modified habitats from 700 to 4,600 m above sea level. For the recorded taxa, we compiled functional traits related to movement, foraging and body size from museum specimens and databases. Further, we recorded mean annual temperature, precipitation, vegetation complexity as well as the number of fruits, flowers, and insect biomass as measures of resource availability on each study site. Results: Using path analyses, we found similar responses of bird and bat functional diversity to the variation in abiotic and biotic drivers along the elevational gradient. In contrast, the functional composition of both taxa showed distinct responses to abiotic and biotic drivers. For both groups, direct temperature effects were most important, followed by resource availability, precipitation and vegetation complexity. Main Conclusions: Our findings indicate that physiological and metabolic constraints imposed by temperature and resource availability determine the functional diversity of bird and bat assemblages, whereas the composition of individual functional traits is driven by taxon-specific processes. Our study illustrates that distinct filtering mechanisms can result in similar patterns of functional diversity along broad environmental gradients. Such differences need to be taken into account when it comes to conserving the functional diversity of flying vertebrates on tropical mountains.
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
Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.
A tale of two seasons: The link between seasonal migration and climatic niches in passerine birds
(2020)
The question of whether migratory birds track a specific climatic niche by seasonal movements has important implications for understanding the evolution of migration, the factors affecting species' distributions, and the responses of migrants to climate change. Despite much research, previous studies of bird migration have produced mixed results. However, whether migrants track climate is only one half of the question, the other being why residents remain in the same geographic range year-round. We provide a literature overview and test the hypothesis of seasonal niche tracking by evaluating seasonal climatic niche overlap across 437 migratory and resident species from eight clades of passerine birds. Seasonal climatic niches were based on a new global dataset of breeding and nonbreeding ranges. Overlap between climatic niches was quantified using ordination methods. We compared niche overlap of migratory species to two null expectations, (a) a scenario in which they do not migrate and (b) in comparison with the overlap experienced by closely related resident species, while controlling for breeding location and range size. Partly in accordance with the hypothesis of niche tracking, we found that the overlap of breeding versus nonbreeding climatic conditions in migratory species was greater than the overlap they would experience if they did not migrate. However, this was only true for migrants breeding outside the tropics and only relative to the overlap species would experience if they stayed in the breeding range year-round. In contrast to the hypothesis of niche tracking, migratory species experienced lower seasonal climatic niche overlap than resident species, with significant differences between tropical and nontropical species. Our study suggests that in seasonal nontropical environments migration away from the breeding range may serve to avoid seasonally harsh climate; however, different factors may drive seasonal movements in the climatically more stable tropical regions.
Many cellular processes are regulated via pH, and maintaining the pH of different organelles is crucial for cell survival. A pH-sensitive GFP variant, the so-called pHluorin, has proven to be a valuable tool to study the pH of the cytosol, mitochondria and other organelles in vivo. We found that the fluorescence intensity of Endoplasmic Reticulum (ER)-targeted pHluorin in the yeast Saccharomyces cerevisiae was very low and barely showed pH sensitivity, probably due to misfolding in the oxidative environment of the ER. We therefore developed a superfolder variant of pHluorin which enabled us to monitor pH changes in the ER and the cytosol of S. cerevisiae in vivo. The superfolder pHluorin variant is likely to be functional in cells of different organisms as well as in additional compartments that originate from the secretory pathway like the Golgi apparatus and pre-vacuolar compartments, and therefore has a broad range of possible future applications.
The dependence of the Escherichia coli Na+H+ antiporter A (EcNhaA) pH sensor mutant E241C on H+ and Na+ concentrations was tested using a solid supported membrane (SSM) based electrophysiological approach. Proteoliposome preparations with right side out (RSO) oriented carriers were used to investigate the passive downhill uptake mode (physiologically the reverse transport mode) at zero membrane potential. Na+ concentration gradients established with a rapid solution exchange acted as the driving force. When a Na+ concentration gradient was established at symmetrical pH, the transport activity of the E241C EcNhaA variant was similar to that of the wildtype EcNhaA, with no shift of the bell-shaped pH dependence, an increase of the KmNa at acidic pH and a decrease of the KmNa at alkaline pH, supporting the model of a competitive binding of Na+ and H+ to a common binding site.
The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.