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Ecophysiological studies on Antarctic cryptophytes to assess whether climatic changes such as ocean acidification and enhanced stratification affect their growth in Antarctic coastal waters in the future are lacking so far. This is the first study that investigated the combined effects of increasing availability of pCO2 (400 and 1000 µatm) and irradiance (20, 200 and 500 μmol photons m−2 s −1) on growth, elemental composition and photophysiology of the Antarctic cryptophyte Geminigera cryophila. Under ambient pCO2, this species was characterized by a pronounced sensitivity to increasing irradiance with complete growth inhibition at the highest light intensity. Interestingly, when grown under high pCO2 this negative light effect vanished and it reached highest rates of growth and particulate organic carbon production at the highest irradiance compared to the other tested experimental conditions. Our results for G. cryophila reveal beneficial effects of ocean acidification in conjunction with enhanced irradiance on growth and photosynthesis. Hence, cryptophytes such as G. cryophila may be potential winners of climate change, potentially thriving better in more stratified and acidic coastal waters and contributing in higher abundance to future phytoplankton assemblages of coastal Antarctic waters.
1. Plant-fungal interactions are important for plant community assembly, but quantifying these relationships remains challenging. High throughput sequencing of fungal communities allows us to identify plant-fungal associations at a high level of resolution, but often fails to provide information on taxonomic and functional assignment of fungi. 2. We transplanted seeds of Pinus cembra across an elevational gradient (1850–2250 m a.s.l.) and identified environmental factors and known fungal associates important for seedling establishment and survival. We then applied null model tests to identify taxonomically unassigned fungi associated with pine recruitment. 3. Early seedling establishment was determined by abiotic environmental factors, while seedling survival was predominantly affected by biotic environmental factors (i.e., the abundance of a fungal pathogen known from literature and the distance to adult trees). Null model tests identified known mycorrhizal partners and a large number of unknown operational taxonomic units (OTUs) associated with seedling survival, including saprotrophic and pathogenic species. These results highlight that unknown fungal OTUs, which are usually discarded from analyses, could play a crucial role for plant survival. 4. Synthesis. We conclude that high throughput metabarcoding paired with null model tests, is a valuable approach for identifying hidden plant-fungal associations within large and complex DNA metabarcoding datasets. Such an approach can be an important tool in illuminating the black box of plant-microbe interactions, and thus understanding ecosystem dynamics.
A1AO ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg−1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na+)-binding site. Indeed, ATP hydrolysis was strictly Na+-dependent. N,N′-dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+. Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD-4 for a common binding site. After incorporation of the A1AO ATP synthase into liposomes, ATP-dependent primary transport of 22Na+ as well as ΔµNa+-driven ATP synthesis could be demonstrated. The Na+ A1AO ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases.
Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
Acetogenic bacteria are a group of strictly anaerobic bacteria that may have been first life forms on Earth since they employ an ancient pathway for CO2 fixation into acetyl-CoA that is coupled to the synthesis of ATP, the Wood–Ljungdahl pathway. Electrons for CO2 reduction are derived from oxidation of H2 or CO and thus, these bacteria can grow lithotrophically on gases present on early Earth. Among the organic molecules present on early Earth is acetaldehyde, a highly volatile C2 compound. Here, we demonstrate that the acetogenic model bacterium Acetobacterium woodii grows on acetaldehyde. Acetaldehyde is dismutated to ethanol and acetyl-CoA, most likely by the bifunctional alcohol dehydrogenase AdhE. Acetyl-CoA is converted to acetate by two subsequent enzymes, phosphotransacetylase and acetate kinase, accompanied by the synthesis of ATP by substrate-level phosphorylation. Apparently, growth on acetaldehyde does not employ the Wood–Ljungdahl pathway. Our finding opens the possibility of a simple and ancient metabolic pathway with only three enzymes that allows for biomass (acetyl-CoA) and ATP formation on early Earth.
Frontal areas of the mammalian cortex are thought to be important for cognitive control and complex behaviour. These areas have been studied mostly in humans, non-human primates and rodents. In this article, we present a quantitative characterization of response properties of a frontal auditory area responsive to sound in the brain of Carollia perspicillata, the frontal auditory field (FAF). Bats are highly vocal animals, and they constitute an important experimental model for studying the auditory system. We combined electrophysiology experiments and computational simulations to compare the response properties of auditory neurons found in the bat FAF and auditory cortex (AC) to simple sounds (pure tones). Anatomical studies have shown that the latter provides feedforward inputs to the former. Our results show that bat FAF neurons are responsive to sounds, and however, when compared to AC neurons, they presented sparser, less precise spiking and longer-lasting responses. Based on the results of an integrate-and-fire neuronal model, we suggest that slow, subthreshold, synaptic dynamics can account for the activity pattern of neurons in the FAF. These properties reflect the general function of the frontal cortex and likely result from its connections with multiple brain regions, including cortico-cortical projections from the AC to the FAF.
The UN 2030 Agenda for Sustainable Development stresses the fundamental role science should play in implementing the 17 Sustainable Development Goals endorsed by the global community. But how can and should researchers respond to this societal demand on science? We argue that answering this question requires systematic engagement with the fundamental normative dimensions of the 2030 Agenda and those of the scientific community—and with the implications these dimensions have for research and practice. We suggest that the production of knowledge relevant to sustainable development entails analytic engagement with norms and values through four tasks. First, to unravel and critically reflect on the ethical values involved in sustainability, values should increasingly become an empirical and theoretical object of sustainability research. Second, to ensure that research on social–ecological systems is related to sustainability values, researchers should reflect on and spell out what sustainability values guide their research, taking into account possible interdependencies, synergies, and trade-offs. Third, to find common ground on what sustainability means for specific situations, scientists should engage in deliberative learning processes with societal actors, with a view to jointly reflecting on existing development visions and creating new, contextualized ones. Fourth, this implies that researchers and scientific disciplines must clarify their own ethical and epistemic values, as this defines accountability and shapes identification of problems, research questions, and results. We believe that ignoring these tasks, whether one is in favor or critical of the 2030 Agenda, will undermine the credibility and relevance of scientific contributions for sustainable development.
A new cyclic lipopeptide, phototemtide A (1), was isolated from Escherichia coli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR‐ESI‐MS and NMR experiments. The absolute configurations of amino acids and 3‐hydroxyoctanoic acid in 1 were determined by using the advanced Marfey's method and comparison after total synthesis of 1, respectively. Additionally, three new minor derivatives, phototemtides B–D (2–4), were identified by detailed HPLC–MS analysis. Phototemtide A (1) showed weak antiprotozoal activity against Plasmodium falciparum, with an IC50 value of 9.8 μm. The biosynthesis of phototemtides A–D (1–4) was also proposed.
Atelopus is a species-rich group of Neotropical bufonids. Present knowledge on bioacoustics in this genus is relatively poor, as vocalisations have been described in only about one fifth of the ca. 100 species known. All studied members of the genus produce vocalisations although, with a few exceptions, most species lack a middle ear. Nonetheless, hearing has been demonstrated even in earless Atelopus making bioacoustics in these toads an inspiring research field. So far, three structural call types have been identified in the genus. As sympatry is uncommon in Atelopus, calls of the same type often vary little between species. Based on recordings from the 1980s, we describe vocalisations of three Venezuelan species (A. carbonerensis, A. mucubajiensis, A. tamaense) from the Cordillera de Mérida, commonly known as the Andes of Venezuela and the Tamá Massif, a Venezuelan spur of the Colombian Cordillera Oriental. Vocalisations correspond, in part, to the previously identified call types in Atelopus. Evaluation of the vocalisations of the three species presented in this study leads us to recognise a fourth structural call type for the genus. With this new addition, the Atelopus acoustic repertoire now includes (1) pulsed calls, (2) pure tone calls, (3) pulsed short calls and (4) pure tone short calls. The call descriptions provided here are valuable contributions to the bioacoustics of these Venezuelan Atelopus species, since all of them have experienced dramatic population declines that limit possibilities of further studies.
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.
Climate change forces many species to move their ranges to higher latitudes or elevations. Resulting immigration or emigration of species might lead to functional changes, e.g., in the trait distribution and composition of ecological assemblages. Here, we combined approaches from biogeography (species distribution models; SDMs) and community ecology (functional diversity) to investigate potential effects of climate-driven range changes on frugivorous bird assemblages along a 3000 m elevational gradient in the tropical Andes. We used SDMs to model current and projected future occurrence probabilities of frugivorous bird species from the lowlands to the tree line. SDM-derived probabilities of occurrence were combined with traits relevant for seed dispersal of fleshy-fruited plants to calculate functional dispersion (FDis; a measure of functional diversity) for current and future bird assemblages. Comparisons of FDis between current and projected future assemblages showed consistent results across four dispersal scenarios, five climate models and two representative concentration pathways. Projections indicated a decrease of FDis in the lowlands, an increase of FDis at lower mid-elevations and little changes at high elevations. This suggests that functional dispersion responds differently to global warming at different elevational levels, likely modifying avian seed dispersal functions and plant regeneration in forest ecosystems along tropical mountains.
Active species reintroduction is an important conservation tool when aiming for the restoration of biological communities and ecosystems. The effective monitoring of reintroduction success is a crucial factor in this process. Here, we used a combination of environmental DNA (eDNA) techniques and species distribution models (SDMs) to evaluate the success of recent reintroductions of the freshwater fish Alburnoides bipunctatus in central Germany. We built SDMs without and with eDNA presence data to locate further suitable reintroduction sites and potentially overlooked populations of the species. We successfully detected eDNA of A. bipunctatus at all reintroduction sites, as well as several adjacent sites mostly in downstream direction, which supports the success of reintroduction efforts. eDNA‐based species detection considerably improved SDMs for A. bipunctatus, which allowed to identify species presence in previously unknown localities. Our results confirm the usefulness of eDNA techniques as standard tool to monitor reintroduced fish populations. We propose that combining eDNA with SDMs is a highly effective approach for long‐term monitoring of reintroduction success in aquatic species.
Aim: To provide distribution information and preliminary conservation assessments for all species of the pineapple family (Bromeliaceae), one of the most diverse and ecologically important plant groups of the American tropics—a global biodiversity hotspot. Furthermore, we aim to analyse patterns of diversity, endemism and the conservation status of the Bromeliaceae on the continental level in the light of their evolutionary history.
Location: The Americas.
Methods: We compiled a dataset of occurrence records for 3,272 bromeliad species (93.4% of the family) and modelled their geographic distribution using either climate‐based species distribution models, convex hulls or geographic buffers dependent on the number of occurrences available. We then combined this data with information on taxonomy and used the ConR software for a preliminary assessment of the conservation status of all species following Criterion B of the International Union for the Conservation of Nature (IUCN).
Results: Our results stress the Atlantic Forest in eastern Brazil, the Andean slopes, Central America and the Guiana Highlands as centres of bromeliad diversity and endemism. Phylogenetically ancient subfamilies of bromeliads are centred in the Guiana highlands whereas the large radiations of the group spread across different habitats and large geographic area. A total of 81% of the evaluated bromeliad species are Possibly Threatened with extinction. We provide range polygons for 3,272 species, as well as newly georeferenced point localities for 911 species in the novel “bromeliad” r package, together with functions to generate diversity maps for individual taxonomic or functional groups.
Main conclusions: Diversity centres of the Bromeliaceae agreed with macroecological patterns of other plant and animal groups, but show some particular patterns related to the evolutionary origin of the family, especially ancient dispersal corridors. A staggering 2/3rds of Bromeliaceae species might be threatened with extinction, especially so in tropical rain forests, raising concerns about the conservation of the family and bromeliad‐dependent animal species.
Summary
Wild relatives of crops thrive in habitats where environmental conditions can be restrictive for productivity and survival of cultivated species. The genetic basis of this variability, particularly for tolerance to high temperatures, is not well understood. We examined the capacity of wild and cultivated accessions to acclimate to rapid temperature elevations that cause heat stress (HS).
We investigated genotypic variation in thermotolerance of seedlings of wild and cultivated accessions. The contribution of polymorphisms associated with thermotolerance variation was examined regarding alterations in function of the identified gene.
We show that tomato germplasm underwent a progressive loss of acclimation to strong temperature elevations. Sensitivity is associated with intronic polymorphisms in the HS transcription factor HsfA2 which affect the splicing efficiency of its pre‐mRNA. Intron splicing in wild species results in increased synthesis of isoform HsfA2‐II, implicated in the early stress response, at the expense of HsfA2‐I which is involved in establishing short‐term acclimation and thermotolerance.
We propose that the selection for modern HsfA2 haplotypes reduced the ability of cultivated tomatoes to rapidly acclimate to temperature elevations, but enhanced their short‐term acclimation capacity. Hence, we provide evidence that alternative splicing has a central role in the definition of plant fitness plasticity to stressful conditions.
Aim: Predicting future changes in species richness in response to climate change is one of the key challenges in biogeography and conservation ecology. Stacked species distribution models (S‐SDMs) are a commonly used tool to predict current and future species richness. Macroecological models (MEMs), regression models with species richness as response variable, are a less computationally intensive alternative to S‐SDMs. Here, we aim to compare the results of two model types (S‐SDMS and MEMs), for the first time for more than 14,000 species across multiple taxa globally, and to trace the uncertainty in future predictions back to the input data and modelling approach used.
Location: Global land, excluding Antarctica.
Taxon: Amphibians, birds and mammals.
Methods: We fitted S‐SDMs and MEMs using a consistent set of bioclimatic variables and model algorithms and conducted species richness predictions under current and future conditions. For the latter, we used four general circulation models (GCMs) under two representative concentration pathways (RCP2.6 and RCP6.0). Predicted species richness was compared between S‐SDMs and MEMs and for current conditions also to extent‐of‐occurrence (EOO) species richness patterns. For future predictions, we quantified the variance in predicted species richness patterns explained by the choice of model type, model algorithm and GCM using hierarchical cluster analysis and variance partitioning.
Results: Under current conditions, species richness predictions from MEMs and S‐SDMs were strongly correlated with EOO‐based species richness. However, both model types over‐predicted areas with low and under‐predicted areas with high species richness. Outputs from MEMs and S‐SDMs were also highly correlated among each other under current and future conditions. The variance between future predictions was mostly explained by model type.
Main conclusions: Both model types were able to reproduce EOO‐based patterns in global terrestrial vertebrate richness, but produce less collinear predictions of future species richness. Model type by far contributes to most of the variation in the different future species richness predictions, indicating that the two model types should not be used interchangeably. Nevertheless, both model types have their justification, as MEMs can also include species with a restricted range, whereas S‐SDMs are useful for looking at potential species‐specific responses.
In 2010, the Conference of the Parties of the Convention on Biological Diversity agreedon the Strategic Plan for Biodiversity 2011–2020 in Aichi Prefecture, Japan. As this planapproaches its end, we discussed whether marine biodiversity and prediction studieswere nearing the Aichi Targets during the 4th World Conference on Marine Biodiversityheld in Montreal, Canada in June 2018. This article summarises the outcome of a five-day group discussion on how global marine biodiversity studies should be focusedfurther to better understand the patterns of biodiversity. We discussed and reviewedseven fundamental biodiversity priorities related to nine Aichi Targets focusing onglobal biodiversity discovery and predictions to improve and enhance biodiversitydata standards (quantity and quality), tools and techniques, spatial and temporal scaleframing, and stewardship and dissemination. We discuss how identifying biodiversityknowledge gaps and promoting efforts have and will reduce such gaps, including via theuse of new databases, tools and technology, and how these resources could be improvedin the future. The group recognised significant progress toward Target 19 in relationto scientific knowledge, but negligible progress with regard to Targets 6 to 13 whichaimed to safeguard and reduce human impacts on biodiversity.
Soil degradation can have an impact on the soil microbiota, but its specific effects on soil fungal communities are poorly understood. In this work, we studied the impact of soil degradation on the richness and diversity of communities of soil fungi, including three different degrees of degradation in Germany and Panama. Soil fungi were isolated monthly using the soil-sprinkling method for 8 months in Germany and 3 months in Panama, and characterized by morphological and molecular data. Soil physico-chemical properties were measured and correlated with the observed values of fungal diversity. We isolated a total of 71 fungal species, 47 from Germany, and 32 from Panama. Soil properties were not associated with fungal richness, diversity, or composition in soils, with the exception of soil compaction in Germany. The geographic location was a strong determinant of the soil fungal species composition although in both countries there was dominance by members of the orders Eurotiales and Hypocreales. In conclusion, the results of this work do not show any evident influence of soil degradation on communities of soil fungi in Germany or Panama.
Diatoms are thought to provide about 40% of total global photosynthesis and diatoms of the genus Coscinodiscus are an important, sometimes dominant, cosmopolitan component of the marine diatom community. The oomycete parasitoid Lagenisma coscinodisci is widespread in the northern hemisphere on its hosts in the genus Coscinodiscus. Because of its potential ecological importance, it would be a suitable pathogen model to investigate plankton/parasite interactions, but the species cannot be cultivated on media without its host, so far. Thus, it was the aim of this study to explore the potential of dual culture of host and pathogen in the laboratory and to optimise cultivation to ensure a long-term cultivation of the pathogen. Here, we report successful cultivation of a single spore strain of L. coscinodisci (Isla), on several Coscinodiscus species and strains, as well as the establishment of a cultivation routine with Coscinodiscus granii (CGS1 and CG36), which enabled us to maintain the single spore strain for more than 3 years in 6 cm Petri dishes and 10 ml tissue culture flasks. This opens up the opportunity to study the processes and mechanism in plankton/parasitoid interactions under controlled conditions.
Holocarpic oomycetes are poorly known but widespread parasites in freshwater and marine ecosystems. Most of the holocarpic species seem to belong to clades that diverge before the two crown lineages of the oomycetes, the Saprolegniomycetes and the Peronosporomycetes. Recently, the genus Miracula was described to accommodate Miracula helgolandica, a holocarpic parasitoid of Pseudo-nitzschia diatoms, which received varying support for its placement as the earliest-diverging oomycete lineage. In the same phylogenetic reconstruction, Miracula helgolandica was grouped with some somewhat divergent sequences derived from environmental sequencing, indicating that Miracula would not remain monotypic. Here, a second species of Miracula is reported, which was found as a parasitoid in the limnic centric diatom Pleurosira leavis. Its life-cycle stages are described and depicted in this study and its phylogenetic placement in the genus Miracula revealed. As a consequence, the newly discovered species is introduced as Miracula moenusica.
Olpidiopsis is a genus of obligate holocarpic endobiotic oomycetes. Most of the species classified in the genus are known only from their morphology and life cycle, and a few have been examined for their ultrastructure or molecular phylogeny. However, the taxonomic placement of all sequenced species is provisional, as no sequence data are available for the type species, O. saprolegniae, to consolidate the taxonomy of species currently placed in the genus. Thus, efforts were undertaken to isolate O. saprolegniae from its type host, Saprolegnia parasitica and to infer its phylogenetic placement based on 18S rDNA sequences. As most species of Olpidiopsis for which sequence data are available are from rhodophyte hosts, we have also isolated the type species of the rhodophyte-parasitic genus Pontisma, P. lagenidioides and obtained partial 18S rDNA sequences. Phylogenetic reconstructions in the current study revealed that O. saprolegniae from Saprolegnia parasitica forms a monophyletic group with a morphologically similar isolate from S. ferax, and a morphologically and phylogenetically more divergent species from S. terrestris. However, they were widely separated from a monophyletic, yet unsupported clade containing P. lagenidioides and red algal parasites previously classified in Olpidiopsis. Consequently, all holocarpic parasites in red algae should be considered to be members of the genus Pontisma as previously suggested by some researchers. In addition, a new species of Olpidiopsis, O. parthenogenetica is introduced to accommodate the pathogen of S. terrestris.
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to reliably detect RBP binding sites from iCLIP data. The workflow covers all steps from the initial quality control of the sequencing reads up to peak calling and quantification of RBP binding. For each tool, we explain the specific requirements for iCLIP data analysis and suggest optimised parameter settings.
Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.
Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.
Wolves (Canis lupus) are currently showing a remarkable comeback in the highly frag-mented cultural landscapes of Germany. We here show that wolf numbers increasedexponentially between 2000 and 2015 with an annual increase of about 36%. Wedemonstrate that the first territories in each newly colonized region were establishedover long distances from the nearest known reproducing pack on active militarytraining areas (MTAs). We show that MTAs, rather than protected areas, served asstepping-stones for the recolonization of Germany facilitating subsequent spreadingof wolf territories in the surrounding landscape. We did not find any significant differ-ence between MTAs and protected areas with regard to habitat. One possible reasonfor the importance of MTAs may be their lower anthropogenic mortality rates com-pared to protected and other areas. To our knowledge, this is the first documented casewhere MTAs facilitate the recolonization of an endangered species across large areas.
In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10–12 µm. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.
Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide–sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.
The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tcey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.
Petroleum products including crude oils and refined distillates are unique environmental pollutants consisting of thousands of compounds with varying physical-chemical properties and resulting toxicity for aquatic biota. Hence, for a reliable risk assessment individual petroleum product toxicity profiles are needed. Furthermore, the influence of oil spill response strategies like the application of chemical dispersants has to be implemented. The present study addressed the toxicity of water-accommodated fractions (WAFs) of two different oil types on fish early life stages on different biological organization levels in the laboratory model species Danio rerio. Experiments with a 3rd generation dispersant used in loading rated resembling the exposure in experiments with chemically dispersed oils were included, enabling a direct comparability of results. This approach is of high importance as especially the investigation of dispersant toxicity in relevant exposure concentrations is rather scarce. Zebrafish embryos were exposed to different WAFs shortly after and up to 120 hour post fertilization (hpf). Besides phenotypic effects including edema and spine deformations, reduced responses to dark stimuli, increased CYP1A activity and marginal AChE inhibition were observed in sublethal effect concentrations. Both oil types had varying strength of toxicity, which did not correlate with corresponding chemical analysis of target PAHs. Chemically dispersed oils induced stronger acute toxicity in zebrafish embryos compared to native (initial) oil exposure, which was further reflected by very low exposure concentrations for biomarker endpoints. Based on a comparison to the dispersant alone, a higher toxicity of dispersed oils was related to a combination of dispersant toxicity and an elevated crude oil compound bioavailability, due to dispersion-related partitioning kinetics. In contrast to LEWAF and CEWAF neither typical morphological effects nor mechanism-specific toxicity were observed for the dispersant alone, indicating narcosis as the responsible cause of effects.
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.
The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.
Brain metastases are the most common intracranial tumor in adults and are associated with poor patient prognosis and median survival of only a few months. Treatment options for brain metastasis patients remain limited and largely depend on surgical resection, radio- and/or chemotherapy. The development and pre-clinical testing of novel therapeutic strategies require reliable experimental models and diagnostic tools that closely mimic technologies that are used in the clinic and reflect histopathological and biochemical changes that distinguish tumor progression from therapeutic response. In this study, we sought to test the applicability of magnetic resonance (MR) spectroscopy in combination with MR imaging to closely monitor therapeutic efficacy in a breast-to-brain metastasis model. Given the importance of radiotherapy as the standard of care for the majority of brain metastases patients, we chose to monitor the post-irradiation response by magnetic resonance spectroscopy (MRS) in combination with MR imaging (MRI) using a 7 Tesla small animal scanner. Radiation was applied as whole brain radiotherapy (WBRT) using the image-guided Small Animal Radiation Research Platform (SARRP). Here we describe alterations in different metabolites, including creatine and N-acetylaspartate, that are characteristic for brain metastases progression and lactate, which indicates hypoxia, while choline levels remained stable. Radiotherapy resulted in normalization of metabolite levels indicating tumor stasis or regression in response to treatment. Our data indicate that the use of MR spectroscopy in addition to MRI represents a valuable tool to closely monitor not only volumetrical but also metabolic changes during tumor progression and to evaluate therapeutic efficacy of intervention strategies. Adapting the analytical technology in brain metastasis models to those used in clinical settings will increase the translational significance of experimental evaluation and thus contribute to the advancement of pre-clinical assessment of novel therapeutic strategies to improve treatment options for brain metastases patients.
Wie herzig!
(2019)
Doch Vorsicht – dies ist kein Einblick in die Hirnwindungen eines verliebten Teenagers. Vielmehr handelt es sich hier um einen wissenschaftlichen Blick in die Großhirnrinde einer Maus. Die Forscherinnen und Forscher um Prof. Amparo Acker-Palmer vom Buchmann Institut für Molekulare Lebenswissenschaften und dem Institut für Zellbiologie und Neurowissenschaften der Goethe-Universität haben 2018 in der Zeitschrift "Science" darüber berichtet, dass Blutgefäße bei der Entwicklung neuronaler Zellnetzwerke im Gehirn eine bislang unbekannte Rolle spielen ...
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.
Background: Developmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be easily observed and imaged using fluorescent proteins have become very popular. One limiting factor however is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. One way to improve this is by developing mounting methods to increase the number of embryos that can be imaged simultaneously in near-to-identical orientation.
Results: Here we present an improved mounting method allowing semi-automated and high-content imaging of zebrafish embryos. It is based on a 3D-printed stamp which is used to create a 2D coordinate system of multiple μ-wells in an agarose cast. Each μ-well models a negative of the average zebrafish embryo morphology between 22 and 96 h-post-fertilization. Due to this standardized and reproducible arrangement, it is possible to define a custom well plate in the respective imaging software that allows for a semi-automated imaging process. Furthermore, the improvement in Z-orientation significantly reduces post-processing and improves comparability of volumetric data while reducing light exposure and thus photo-bleaching and photo-toxicity, and improving signal-to-noise ratio (SNR).
Conclusions: We present here a new method that allows to standardize and improve mounting and imaging of embryos. The 3D-printed stamp creates a 2D coordinate system of μ-wells in an agarose cast thus standardizing specimen mounting and allowing high-content imaging of up to 44 live or mounted zebrafish embryos simultaneously in a semi-automated, well-plate like manner on inverted confocal microscopes. In summary, image data quality and acquisition efficiency (amount of data per time) are significantly improved. The latter might also be crucial when using the services of a microscopy facility.
Aims: The examination of histological sections is still the gold standard in diagnostic pathology. Important histopathological diagnostic criteria are nuclear shapes and chromatin distribution as well as nucleus-cytoplasm relation and immunohistochemical properties of surface and intracellular proteins. The aim of this investigation was to evaluate the benefits and drawbacks of three-dimensional imaging of CD30+ cells in classical Hodgkin Lymphoma (cHL) in comparison to CD30+ lymphoid cells in reactive lymphoid tissues.
Materials and results: Using immunoflourescence confocal microscopy and computer-based analysis, we compared CD30+ neoplastic cells in Nodular Sclerosis cHL (NScCHL), Mixed Cellularity cHL (MCcHL), with reactive CD30+ cells in Adenoids (AD) and Lymphadenitis (LAD). We confirmed that the percentage of CD30+ cell volume can be calculated. The amount in lymphadenitis was approx. 1.5%, in adenoids around 2%, in MCcHL up to 4,5% whereas the values for NScHL rose to more than 8% of the total cell cytoplasm. In addition, CD30+ tumour cells (HRS-cells) in cHL had larger volumes, and more protrusions compared to CD30+ reactive cells. Furthermore, the formation of large cell networks turned out to be a typical characteristic of NScHL.
Conclusion: In contrast to 2D histology, 3D laser scanning offers a visualisation of complete cells, their network interaction and spatial distribution in the tissue. The possibility to differentiate cells in regards to volume, surface, shape, and cluster formation enables a new view on further diagnostic and biological questions. 3D includes an increased amount of information as a basis of bioinformatical calculations.
Visual aesthetic experiences unfold over time, yet most of our understanding of such experiences comes from experiments using static visual stimuli and measuring static responses. Here, we investigated the temporal dynamics of subjective aesthetic experience using temporally extended stimuli (movie clips) in combination with continuous behavioral ratings. Two groups of participants, a rate group (n = 25) and a view group (n = 25), watched 30-second video clips of landscapes and dance performances in test and retest blocks. The rate group reported continuous ratings while watching the videos, with an overall aesthetic judgment at the end of each video, in both test and retest blocks. The view group, however, passively watched the videos in the test block, reporting only an overall aesthetic judgment at the end of each clip. In the retest block, the view group reported both continuous and overall judgments. When comparing the two groups, we found that the task of making continuous ratings did not influence overall ratings or agreement across participants. In addition, the degree of temporal variation in continuous ratings over time differed substantially by observer (from slower "integrators" to "fast responders"), but less so by video. Reliability of continuous ratings across repeated exposures was in general high, but also showed notable variance across participants. Together, these results show that temporally extended stimuli produce aesthetic experiences that are not the same from person to person, and that continuous behavioral ratings provide a reliable window into the temporal dynamics of such aesthetic experiences while not materially altering the experiences themselves.
Circular RNAs (circRNAs), an important class of regulatory RNAs, have been shown to be the most prevalent in the brain compared with other tissues. However the processes governing their biogenesis in neurons are still elusive. Moreover, little is known about whether and how different biogenesis factors work in synchrony to generate neuronal circRNAs. To address this question, we pharmacologically inhibited the spliceosome and profiled rat neuronal circRNAs using RNA sequencing. We identified over 100 circRNAs that were up-regulated and a few circRNAs that were down-regulated upon spliceosome inhibition. Bioinformatic analysis revealed that up-regulated circRNAs possess significantly longer flanking introns compared with the un-changed circRNA population. Moreover, the flanking introns of up-regulated circRNAs harbor a higher number of distinct repeat sequences and more reverse complementary motifs compared with the unchanged circRNAs. Taken together, our data demonstrate that the biogenesis of circRNAs containing distinct intronic features becomes favored under conditions of limited spliceosome activity.
The identification of heat stress (HS)-resilient germplasm is important to ensure food security under less favorable environmental conditions. For that, germplasm with an altered activity of factors regulating the HS response is an important genetic tool for crop improvement. Heat shock binding protein (HSBP) is one of the main negative regulators of HS response, acting as a repressor of the activity of HS transcription factors. We identified a TILLING allele of Solanum lycopersicum (tomato) HSBP1. We examined the effects of the mutation on the functionality of the protein in tomato protoplasts, and compared the thermotolerance capacity of lines carrying the wild-type and mutant alleles of HSBP1. The methionine-to-isoleucine mutation in the central heptad repeats of HSBP1 leads to a partial loss of protein function, thereby reducing the inhibitory effect on Hsf activity. Mutant seedlings show enhanced basal thermotolerance, while mature plants exhibit increased resilience in repeated HS treatments, as shown by several physiological parameters. Importantly, plants that are homozygous for the wild-type or mutant HSBP1 alleles showed no significant differences under non-stressed conditions. Altogether, these results indicate that the identified mutant HSBP1 allele can be used as a genetic tool in breeding, aiming to improve the thermotolerance of tomato varieties.
Truffle fungi are well known for their enticing aromas partially emitted by microbes colonizing truffle fruiting bodies. The identity and diversity of these microbes remain poorly investigated, because few studies have determined truffle-associated bacterial communities while considering only a small number of fruiting bodies. Hence, the factors driving the assembly of truffle microbiomes are yet to be elucidated. Here we investigated the bacterial community structure of more than 50 fruiting bodies of the black truffle Tuber aestivum in one French and one Swiss orchard using 16S rRNA gene amplicon high-throughput sequencing. Bacterial communities from truffles collected in both orchards shared their main dominant taxa: while 60% of fruiting bodies were dominated by α-Proteobacteria, in some cases the β-Proteobacteria or the Sphingobacteriia classes were the most abundant, suggesting that specific factors (i.e., truffle maturation and soil properties) shape differently truffle-associated microbiomes. We further attempted to assess the influence in truffle microbiome variation of factors related to collection season, truffle mating type, degree of maturation, and location within the truffle orchards. These factors had differential effects between the two truffle orchards, with season being the strongest predictor of community variation in the French orchard, and spatial location in the Swiss one. Surprisingly, genotype and fruiting body maturation did not have a significant effect on microbial community composition. In summary, our results show, regardless of the geographical location considered, the existence of heterogeneous bacterial communities within T. aestivum fruiting bodies that are dominated by three bacterial classes. They also indicate that factors shaping microbial communities within truffle fruiting bodies differ across local conditions.
This review summarizes studies of protection against singlet oxygen and radical damage by carotenoids. The main focus is on how substitutions of the carotenoid molecules determine high antioxidant activities such as singlet oxygen quenching and radical scavenging. Applied assays were carried out either in vitro in solvents or with liposomes, and in a few cases with living organisms. In the latter, protection by carotenoids especially of photosynthesis against light- and UV-stress is of major importance, but also heterotrophic organisms suffer from high light and UV exposure which can be alleviated by carotenoids. Carotenoids to be compared include C30, C40 and C50 molecules either acyclic, monocyclic or bicyclic with different substitutions including sugar and fatty acid moieties. Although some studies are difficult to compare, there is a tendency towards mono and bicyclic carotenoids with keto groups at C-4/C-4’ and the longest possible polyene structure functions to act best in singlet oxygen quenching and radical scavenging. Size of the carotenoid and lipophilic substituents such as fatty acids seem to be of minor importance for their activity but hydroxyl groups at an acyclic end and especially glycosylation of these hydroxyl groups enhance carotenoid activity.
Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As an exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension, and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was also detected for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86, and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429, and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in Drosophila melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.
The haloarchaeon Haloferax volcanii contains nearly 2800 small non-coding RNAs (sRNAs). One intergenic sRNA, sRNA132, was chosen for a detailed characterization. A deletion mutant had a growth defect and thus underscored the importance of sRNA132. A microarray analysis identified the transcript of an operon for a phosphate-specific ABC transporter as a putative target of sRNA132. Both the sRNA132 and the operon transcript accumulated under low phosphate concentrations, indicating a positive regulatory role of sRNA132. A kinetic analysis revealed that sRNA132 is essential shortly after the onset of phosphate starvation, while other regulatory processes take over after several hours. Comparison of the transcriptomes of wild-type and the sRNA132 gene deletion mutant 30 min after the onset of phosphate starvation revealed that sRNA132 controls a regulon of about 40 genes. Remarkably, the regulon included a second operon for a phosphate-specific ABC transporter, which also depended on sRNA132 for rapid induction in the absence of phosphate. Competitive growth experiments of the wild-type and ABC transporter operon deletion mutants underscored the importance of both transporters for growth at low phosphate concentrations. Northern blot analyses of four additional members of the sRNA132 regulon verified that all four transcripts depended on sRNA132 for rapid regulation after the onset of phosphate starvation. Importantly, this is the first example for the transient importance of a sRNA for any archaeal and bacterial species. In addition, this study unraveled the first sRNA regulon for haloarchaea.
As a flavor and platform chemical, m-cresol (3-methylphenol) is a valuable industrial compound that currently is mainly synthesized by chemical methods from fossil resources. In this study, we present the first biotechnological de novo production of m-cresol from sugar in complex yeast extract-peptone medium with the yeast Saccharomyces cerevisiae. A heterologous pathway based on the decarboxylation of the polyketide 6-methylsalicylic acid (6-MSA) was introduced into a CEN.PK yeast strain. For synthesis of 6-MSA, expression of different variants of 6-MSA synthases (MSASs) were compared. Overexpression of codon-optimized MSAS from Penicillium patulum together with activating phosphopantetheinyl transferase npgA from Aspergillus nidulans resulted in up to 367 mg/L 6-MSA production. Additional genomic integration of the genes had a strongly promoting effect and 6-MSA titers reached more than 2 g/L. Simultaneous expression of 6-MSA decarboxylase patG from A. clavatus led to the complete conversion of 6-MSA and production of up to 589 mg/L m-cresol. As addition of 450–750 mg/L m-cresol to yeast cultures nearly completely inhibited growth our data suggest that the toxicity of m-cresol might be the limiting factor for higher production titers.
Mapping biodiversity is the marathon of the 21st Century as an answer to the present extinction crisis. A century in which science is also characterised by large scientific datasets collected through new technologies aiming to fill gaps in our knowledge of species distributions. However, most species records rely on observations that are not linked to specimens, which does not allow verification of species hypotheses by other scientists. Natural history museums form a verifiable source of biodiversity records which were made by taxonomists. Nonetheless, these museums seem to be forgotten by biologists in scientific fields other than taxonomy or systematics. Naturalis Biodiversity Center (NBC) in Leiden is care keeper of large collections of marine organisms, which were sampled in the Northeast Atlantic during the CANCAP and Tyro Mauritania II expeditions (1976–1988). Many octocorals were sampled and deposited in the NBC collection, where they became available for study and were partially identified by the senior author (M.G.) in the 1980s. Nonetheless, no checklist or taxonomic revision was published so far with the complete results. In 2016 the first author visited NBC to examine NE Atlantic Plexauridae octocorals. Plexauridae octocoral-vouchered records were listed and mapped to reveal high standard primary biodiversity records unreported so far for the NE Atlantic Ocean. Twenty-four Plexauridae species with ~ six putative new species to science were discovered and eleven new biogeographical records were made from distinct Macaronesian archipelagos. Finally, new depth range records were found for three species at sea basin level and for eight species at a regional scale.
The nuclear exosome and its essential co-factor, the RNA helicase MTR4, play crucial roles in several RNA degradation pathways. Besides unwinding RNA substrates for exosome-mediated degradation, MTR4 associates with RNA-binding proteins that function as adaptors in different RNA processing and decay pathways. Here, we identify and characterize the interactions of human MTR4 with a ribosome processing adaptor, NVL, and with ZCCHC8, an adaptor involved in the decay of small nuclear RNAs. We show that the unstructured regions of NVL and ZCCHC8 contain short linear motifs that bind the MTR4 arch domain in a mutually exclusive manner. These short sequences diverged from the arch-interacting motif (AIM) of yeast rRNA processing factors. Our results suggest that nuclear exosome adaptors have evolved canonical and non-canonical AIM sequences to target human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA-binding proteins.
Nanoplastics (NP) and microplastics (MP) accumulate in our environment as a consequence of the massive consumption of plastics. Huge knowledge-gaps exist regarding uptake and fate of plastic particles in micro- and nano-dimensions in humans as well as on their impact on human health.
This study investigated the transport and effects of 50 nm and 0.5 μm COOH-modified polystyrene (PS) particles, as representatives for NP and MP, in different biological models in vitro. Acute toxicity and potential translocation of the particles were studied at the human intestinal and placental barrier using advanced in vitro co-culture models. Furthermore, embryotoxicity and genotoxicity were investigated as highly sensitive endpoints.
Polystyrene was not acutely toxic in both sizes (nano- and microparticles). No transport across the intestinal and placental barrier but a cellular uptake and intracellular accumulation of PS nano- and microparticles were determined. The particles were identified as weak embryotoxic and non-genotoxic.
In contrast to single-organ studies, this multi-endpoint study is providing a data-set with the exact same type of particles to compare organ-specific outcomes. Our study clearly shows the need to investigate other types of plastics as well as towards long-term or chronic effects of plastic particles in different biological models in vitro.