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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 methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.
Background: Through the rapid development in DNA sequencing methods and tools, microbiome studies on a various number of species were performed during the last decade. This advance makes it possible to analyze hundreds of samples from different species at the same time in order to obtain a general overview of the microbiota. However, there is still uncertainty on the variability of the microbiota of different animal orders and on whether certain bacteria within a species are subject to greater fluctuations than others. This is largely due to the fact that the analysis in most extensive comparative studies is based on only a few samples per species or per study site. In our study, we aim to close this knowledge gap by analyzing multiple individual samples per species including two carnivore suborders Canoidea and Feloidea as well as the orders of herbivore Perissodactyla and Artiodactyla held in different zoos. To assess microbial diversity, 621 fecal samples from 31 species were characterized by sequencing the V3–V4 region of the 16S rRNA gene using Illumina MiSeq.
Results: We found significant differences in the consistency of microbiota composition and in fecal microbial diversity between carnivore and herbivore species. Whereas the microbiota of Carnivora is highly variable and inconsistent within and between species, Perissodactyla and Ruminantia show fewer differences across species boundaries. Furthermore, low-abundance bacterial families show higher fluctuations in the fecal microbiota than high-abundance ones.
Conclusions: Our data suggest that microbial diversity is significantly higher in herbivores than in carnivores, whereas the microbiota in carnivores, unlike in herbivores, varies widely even within species. This high variability has methodological implications and underlines the need to analyze a minimum amount of about 10 samples per species. In our study, we found considerable differences in the occurrence of different bacterial families when looking at just three and six samples. However, from a sample number of 10 onwards, these within-species fluctuations balanced out in most cases and led to constant and more reliable results.
In the insect brain, the mushroom body is a higher order brain area that is key to memory formation and sensory processing. Mushroom body (MB) extrinsic neurons leaving the output region of the MB, the lobes and the peduncle, are thought to be especially important in these processes. In the honeybee brain, a distinct class of MB extrinsic neurons, A3 neurons, are implicated in playing a role in learning. Their MB arborisations are either restricted to the lobes and the peduncle, here called A3 lobe connecting neurons, or they provide feedback information from the lobes to the input region of the MB, the calyces, here called A3 feedback neurons. In this study, we analyzed the morphology of individual A3 lobe connecting and feedback neurons using confocal imaging. A3 feedback neurons were previously assumed to innervate each lip compartment homogenously. We demonstrate here that A3 feedback neurons do not innervate whole subcompartments, but rather innervate zones of varying sizes in the MB lip, collar, and basal ring. We describe for the first time the anatomical details of A3 lobe connecting neurons and show that their connection pattern in the lobes resemble those of A3 feedback cells. Previous studies showed that A3 feedback neurons mostly connect zones of the vertical lobe that receive input from Kenyon cells of distinct calycal subcompartments with the corresponding subcompartments of the calyces. We can show that this also applies to the neck of the peduncle and the medial lobe, where both types of A3 neurons arborize only in corresponding zones in the calycal subcompartments. Some A3 lobe connecting neurons however connect multiple vertical lobe areas. Contrarily, in the medial lobe, the A3 neurons only innervate one division. We found evidence for both input and output areas in the vertical lobe. Thus, A3 neurons are more diverse than previously thought. The understanding of their detailed anatomy might enable us to derive circuit models for learning and memory and test physiological data.
The constitution and regulation of effector repertoires shape host–microbe interactions. Ustilago maydis and Sporisorium reilianum are two closely related smut fungi, which both infect maize but cause distinct disease symptoms. Understanding how effector orthologs are regulated in these two pathogens can therefore provide insights into the evolution of different infection strategies. We tracked the infection progress of U. maydis and S. reilianum in maize leaves and used two distinct infection stages for cross-species RNA-sequencing analyses. We identified 207 of 335 one-to-one effector orthologs as differentially regulated during host colonization, which might reflect the distinct disease development strategies. Using CRISPR-Cas9-mediated gene conversion, we identified two differentially expressed effector orthologs with conserved function between two pathogens. Thus, differential expression of functionally conserved genes might contribute to species-specific adaptation and symptom development. Interestingly, another differentially expressed orthogroup (UMAG_05318/Sr10075) showed divergent protein function, providing a possible case for neofunctionalization. Collectively, we demonstrated that the diversification of effector genes in related pathogens can be caused both by alteration on the transcriptional level and through functional diversification of the encoded effector proteins.
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.
PaCATB : a secreted catalase protecting Podospora anserina against exogenous oxidative stress
(2011)
A differential mass spectrometry analysis of secreted proteins from juvenile and senescentPodospora anserina cultures revealed age-related differences in protein profiles. Among other proteins with decreased abundance in the secretome of senescent cultures a catalase, termed PaCATB, was identified. Genetic modulation of the abundance of PaCATB identified differential effects on the phenotype of the corresponding strains. Deletion of PaCatB resulted in decreased resistance, over-expression in increased resistance against hydrogen peroxide. While the lifespan of the genetically modified strains was found to be unaffected under standard growth conditions, increased exogenous hydrogen peroxide stress in the growth medium markedly reduced the lifespan of the PaCatB deletion strain but extended the lifespan of PaCatB over-expressors. Overall our data identify a component of the secretome of P. anserina as a new effective factor to cope with environmental stress, stress that under natural conditions is constantly applied on organisms and influences aging processes.
Highlights
• PUR, PVC and PLA microplastics affect life-history parameters of Daphnia magna.
• Natural kaolin particles are less toxic than microplastics.
• Microplastic toxicity is material-specific, e.g. PVC is most toxic on reproduction.
• In case of PVC, plastic chemicals are the main driver of microplastic toxicity.
• PLA bioplastics are similarly toxic as conventional plastics.
Abstract
Given the ubiquitous presence of microplastics in aquatic environments, an evaluation of their toxicity is essential. Microplastics are a heterogeneous set of materials that differ not only in particle properties, like size and shape, but also in chemical composition, including polymers, additives and side products. Thus far, it remains unknown whether the plastic chemicals or the particle itself are the driving factor for microplastic toxicity. To address this question, we exposed Daphnia magna for 21 days to irregular polyvinyl chloride (PVC), polyurethane (PUR) and polylactic acid (PLA) microplastics as well as to natural kaolin particles in high concentrations (10, 50, 100, 500 mg/L, ≤ 59 μm) and different exposure scenarios, including microplastics and microplastics without extractable chemicals as well as the extracted and migrating chemicals alone. All three microplastic types negatively affected the life-history of D. magna. However, this toxicity depended on the endpoint and the material. While PVC had the largest effect on reproduction, PLA reduced survival most effectively. The latter indicates that bio-based and biodegradable plastics can be as toxic as their conventional counterparts. The natural particle kaolin was less toxic than microplastics when comparing numerical concentrations. Importantly, the contribution of plastic chemicals to the toxicity was also plastic type-specific. While we can attribute effects of PVC to the chemicals used in the material, effects of PUR and PLA plastics were induced by the mere particle. Our study demonstrates that plastic chemicals can drive microplastic toxicity. This highlights the importance of considering the individual chemical composition of plastics when assessing their environmental risks. Our results suggest that less studied polymer types, like PVC and PUR, as well as bioplastics are of particular toxicological relevance and should get a higher priority in ecotoxicological studies.
Plastic products leach chemicals that induce in vitro toxicity under realistic use conditions
(2021)
Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies.
Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of intercellular communication, signaling via nucleotides. The signaling cascades involved in purinergic signaling include intracellular storage of nucleotides, nucleotide release, extracellular hydrolysis, and the effect of the released compounds or their hydrolysis products on target tissues via specific receptor systems. In this context ectonucleotidases play several roles. They inactivate released and physiologically active nucleotides, produce physiologically active hydrolysis products, and facilitate nucleoside recycling. This review briefly highlights the development of our knowledge of two types of enzymes involved in extracellular nucleotide hydrolysis and thus purinergic signaling, the ectonucleoside triphosphate diphosphohydrolases, and ecto-5′-nucleotidase.
Divergent selection between ecologically dissimilar habitats promotes local adaptation, which can lead to reproductive isolation (RI). Populations in the Poecilia mexicana species complex have independently adapted to toxic hydrogen sulfide and show varying degrees of RI. Here, we examined the variation in the mate choice component of prezygotic RI. Mate choice tests across drainages (with stimulus males from another drainage) suggest that specific features of the males coupled with a general female preference for yellow color patterns explain the observed variation. Analyses of male body coloration identified the intensity of yellow fin coloration as a strong candidate to explain this pattern, and common-garden rearing suggested heritable population differences. Male sexual ornamentation apparently evolved differently across sulfide-adapted populations, for example because of differences in natural counterselection via predation. The ubiquitous preference for yellow color ornaments in poeciliid females likely undermines the emergence of strong RI, as female discrimination in favor of own males becomes weaker when yellow fin coloration in the respective sulfide ecotype increases. Our study illustrates the complexity of the (partly non-parallel) pathways to divergence among replicated ecological gradients. We suggest that future work should identify the genomic loci involved in the pattern reported here, making use of the increasing genomic and transcriptomic datasets available for our study system.
Background Multidirectional interactions in social (or communication) networks can have a profound effect on mate choice behavior. For example, Poecilia mexicana males show weaker expression of mating preferences when being observed by an audience male. It was suggested that this behavior is an adaptation to reduce sperm competition risk, which arises because commonly preferred female phenotypes will receive attention also by surrounding males, and/or because the audience male can copy the focal male's mate choice. Do P. mexicana males indeed respond to perceived sperm competition risk? We gave males a choice between two females and repeated the tests under one of the following conditions: (1) during the 2nd part of the tests an empty transparent cylinder was presented (control); (2) an audience male inside the cylinder observed the focal male throughout the 2nd part, or (3) the audience male was presented only before the tests, but could not eavesdrop during the actual choice tests (non-specific sperm competition risk treatments); (4) the focal male could see a rival male sexually interacting with the previously preferred, or (5) with the non-preferred female before the 2nd part of the tests (specific sperm competition risk treatments). Results When comparing the strength of individual male preferences between the 1st and 2nd part of the tests (before and after presentation of an audience), male preferences declined slightly also during the control treatment (1). However, the decrease in strength of male preferences was more than two-fold stronger in audience treatment (2), i.e., with non-specific sperm competition risk including the possibility for visual eavesdropping by the audience male. No audience effect was found in treatments (3) and (5), but a weak effect was also seen when the focal male had seen the previously preferred female sexually interact with a rival male (treatment 4; specific sperm competition risk). Conclusions When comparing the two 'non-specific sperm competition risk' treatments (2 and 3), a very strong effect was found only when the audience male could actually observe the focal male during mate choice in treatment (2). This suggests that focal males indeed attempt to conceal their mating preferences in the visual presence of other males so as to avoid mate choice copying. When there is no potential for eavesdropping [treatment (3)], non-specific specific sperm competition risk seems to play a minor or no role. Congruent with studies on other poeciliid species, our results also show that P. mexicana males respond to perceived specific sperm competition risk, and tend to share their mating effort more equally among females when the resource value of their previously preferred mate decreases (after mating with a rival male). However, this effect is comparatively weak.
Importance of latrine communication in European rabbits shifts along a rural–to–urban gradient
(2016)
Background: Information transfer in mammalian communication networks is often based on the deposition of excreta in latrines. Depending on the intended receiver(s), latrines are either formed at territorial boundaries (between-group communication) or in core areas of home ranges (within-group communication). The relative importance of both types of marking behavior should depend, amongst other factors, on population densities and social group sizes, which tend to differ between urban and rural wildlife populations. Our study is the first to assess (direct and indirect) anthropogenic influences on mammalian latrine-based communication networks along a rural-to-urban gradient in European rabbits (Oryctolagus cuniculus) living in urban, suburban and rural areas in and around Frankfurt am Main (Germany).
Results: The proportion of latrines located in close proximity to the burrow was higher at rural study sites compared to urban and suburban ones. At rural sites, we found the largest latrines and highest latrine densities close to the burrow, suggesting that core marking prevailed. By contrast, latrine dimensions and densities increased with increasing distance from the burrow in urban and suburban populations, suggesting a higher importance of peripheral marking.
Conclusions: Increased population densities, but smaller social group sizes in urban rabbit populations may lead to an increased importance of between-group communication and thus, favor peripheral over core marking. Our study provides novel insights into the manifold ways by which man-made habitat alterations along a rural-to-urban gradient directly and indirectly affect wildlife populations, including latrine-based communication networks.
Extending the carotenoid pathway to astaxanthin in plants is of scientific and industrial interest. However, expression of a microbial beta-carotene ketolase (BKT) that catalyses the formation of ketocarotenoids in transgenic plants typically results in low levels of astaxanthin. The low efficiency of BKTs in ketolating zeaxanthin to astaxanthin is proposed to be the major limitation for astaxanthin accumulation in engineered plants. To verify this hypothesis, several algal BKTs were functionally characterized using an Escherichia coli system and three BKTs were identified, with high (up to 85%), moderate (~38%), and low (~1%) conversion rate from zeaxanthin to astaxanthin from Chlamydomonas reinhardtii (CrBKT), Chlorella zofingiensis (CzBKT), and Haematococcus pluvialis (HpBKT3), respectively. Transgenic Arabidopsis thaliana expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g -1 dry weight with a 1.8-fold increase in total carotenoids. In contrast, the expression of CzBKT resulted in much lower astaxanthin content (0.24 mg g -1 dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in A. thaliana. The none-native astaxanthin was found mostly in a free form integrated into the light-harvesting complexes of photosystem II in young leaves but in esterified forms in senescent leaves. The alteration of carotenoids did not affect chlorophyll content, plant growth, or development significantly. The astaxanthin-producing plants were more tolerant to high light as shown by reduced lipid peroxidation. This study advances a decisive step towards the utilization of plants for the production of high-value astaxanthin. Keywords: Arabidopsis thaliana, astaxanthin, beta-carotene ketolase, carotenoid, Haematococcus pluvialis
Background The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome – Ph+) the derivative 9+ encodes either the p40(ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR. Methods We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1α gradient. Results Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells. Conclusion Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.
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.
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.
Characterization of a dual BET/HDAC inhibitor for treatment of pancreatic ductal adenocarcinoma
(2020)
Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo‐ and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)‐JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)‐JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)‐JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1‐directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin‐targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.
The investigated haloarchaeal species, Halobacterium salinarum, Haloferax mediterranei, and H. volcanii, have all been shown to be polyploid. They contain several replicons that have independent copy number regulation, and most have a higher copy number during exponential growth phase than in stationary phase. The possible evolutionary advantages of polyploidy for haloarchaea, most of which have experimental support for at least one species, are discussed. These advantages include a low mutation rate and high resistance toward X-ray irradiation and desiccation, which depend on homologous recombination. For H. volcanii, it has been shown that gene conversion operates in the absence of selection, which leads to the equalization of genome copies. On the other hand, selective forces might lead to heterozygous cells, which have been verified in the laboratory. Additional advantages of polyploidy are survival over geological times in halite deposits as well as at extreme conditions on earth and at simulated Mars conditions. Recently, it was found that H. volcanii uses genomic DNA as genetic material and as a storage polymer for phosphate. In the absence of phosphate, H. volcanii dramatically decreases its genome copy number, thereby enabling cell multiplication, but diminishing the genetic advantages of polyploidy. Stable storage of phosphate is proposed as an alternative driving force for the emergence of DNA in early evolution. Several additional potential advantages of polyploidy are discussed that have not been addressed experimentally for haloarchaea. An outlook summarizes selected current trends and possible future developments.
Haloferax volcanii uses extracellular DNA as a source for carbon, nitrogen, and phosphorous. However, it can also grow to a limited extend in the absence of added phosphorous, indicating that it contains an intracellular phosphate storage molecule. As Hfx. volcanii is polyploid, it was investigated whether DNA might be used as storage polymer, in addition to its role as genetic material. It could be verified that during phosphate starvation cells multiply by distributing as well as by degrading their chromosomes. In contrast, the number of ribosomes stayed constant, revealing that ribosomes are distributed to descendant cells, but not degraded. These results suggest that the phosphate of phosphate-containing biomolecules (other than DNA and RNA) originates from that stored in DNA, not in rRNA. Adding phosphate to chromosome depleted cells rapidly restores polyploidy. Quantification of desiccation survival of cells with different ploidy levels showed that under phosphate starvation Hfx. volcanii diminishes genetic advantages of polyploidy in favor of cell multiplication. The consequences of the usage of genomic DNA as phosphate storage polymer are discussed as well as the hypothesis that DNA might have initially evolved in evolution as a storage polymer, and the various genetic benefits evolved later.
Acinetobacter baumannii is a nosocomial pathogen which can persist in the hospital environment not only due to the acquirement of multiple antibiotic resistances, but also because of its exceptional resistance against disinfectants and desiccation. A suitable desiccation assay was established in which A. baumannii ATCC 19606T survived for ca. 1 month. The growth medium slightly influenced survival after subsequent desiccation. A significant effect could be attributed to the growth phase in which bacteria were dried: In exponential phase, cells were much more desiccation sensitive. The main focus of the present study was the elucidation of the role of compatible solutes, which are known to protect many bacteria under low water activity conditions, in desiccation survival of A. baumannii. Exogenous trehalose was shown to efficiently protect A. baumannii on dry surfaces, in contrast to other compatible solutes tested such as mannitol or glycine betaine. To analyze the importance of intracellularly accumulated solutes, a double mutant lacking biosynthesis pathways for mannitol and trehalose was generated. This mutant accumulated glutamate as sole solute in the presence of high NaCl concentrations and showed severe growth defects under osmotic stress conditions. However, no effect on desiccation tolerance could be seen, neither when cells were dried in water nor in the presence of NaCl.
The opportunistic human pathogen Acinetobacter baumannii is one of the leading causes of nosocomial infections. The high prevalence of multidrug‐resistant strains, a high adaptability to changing environments and an overall pronounced stress resistance contribute to persistence and spread of the bacteria in hospitals and thereby promote repeated outbreaks. Altogether, the success of A. baumannii is mainly built on adaptation and stress resistance mechanisms, rather than relying on ‘true’ virulence factors. One of the stress factors that pathogens must cope with is osmolarity, which can differ between the external environment and different body parts of the human host. A. baumannii ATCC 19606T accumulates the compatible solutes glutamate, mannitol and trehalose in response to high salinities. In this work, it was found that most of the solutes vanish immediately after reaching stationary phase, a very unusual phenomenon. While glutamate can be metabolized, mannitol produced by MtlD is excreted to the medium in high amounts. First results indicate that A. baumannii ATCC 19606T undergoes a rapid switch to a dormant state (viable but non‐culturable) after disappearance of the compatible solutes. Resuscitation from this state could easily be achieved in PBS or fresh medium.
Mannitol is the major compatible solute, next to glutamate, synthesized by the opportunistic human pathogen Acinetobacter baumannii under low water activities. The key enzyme for mannitol biosynthesis, MtlD, was identified. MtlD is highly similar to the bifunctional mannitol‐1‐phosphate dehydrogenase/phosphatase from Acinetobacter baylyi. After deletion of the mtlD gene from A. baumannii ATCC 19606T cells no longer accumulated mannitol and growth was completely impaired at high salt. Addition of glycine betaine restored growth, demonstrating that mannitol is an important compatible solute in the human pathogen. MtlD was heterologously produced and purified. Enzyme activity was strictly salt dependent. Highest stimulation was reached at 600 mmol/L NaCl. Addition of different sodium as well as potassium salts restored activity, with highest stimulations up to 41 U/mg protein by sodium glutamate. In contrast, an increase in osmolarity by addition of sugars did not restore activity. Regulation of mannitol synthesis was also assayed at the transcriptional level. Reporter gene assays revealed that expression of mtlD is strongly dependent on high osmolarity, not discriminating between different salts or sugars. The presence of glycine betaine or its precursor choline repressed promoter activation. These data indicate a dual regulation of mannitol production in A. baumannii, at the transcriptional and the enzymatic level, depending on high osmolarity.
Epigenetic dysregulation contributes to the high cardiovascular disease burden in chronic kidney disease (CKD) patients. Although microRNAs (miRNAs) are central epigenetic regulators, which substantially affect the development and progression of cardiovascular disease (CVD), no data on miRNA dysregulation in CKD-associated CVD are available until now. We now performed high-throughput miRNA sequencing of peripheral blood mononuclear cells from ten clinically stable hemodialysis (HD) patients and ten healthy controls, which allowed us to identify 182 differentially expressed miRNAs (e.g., miR-21, miR-26b, miR-146b, miR-155). To test biological relevance, we aimed to connect miRNA dysregulation to differential gene expression. Genome-wide gene expression profiling by MACE (Massive Analysis of cDNA Ends) identified 80 genes to be differentially expressed between HD patients and controls, which could be linked to cardiovascular disease (e.g., KLF6, DUSP6, KLF4), to infection / immune disease (e.g., ZFP36, SOCS3, JUND), and to distinct proatherogenic pathways such as the Toll-like receptor signaling pathway (e.g., IL1B, MYD88, TICAM2), the MAPK signaling pathway (e.g., DUSP1, FOS, HSPA1A), and the chemokine signaling pathway (e.g., RHOA, PAK1, CXCL5). Formal interaction network analysis proved biological relevance of miRNA dysregulation, as 68 differentially expressed miRNAs could be connected to 47 reciprocally expressed target genes. Our study is the first comprehensive miRNA analysis in CKD that links dysregulated miRNA expression with differential expression of genes connected to inflammation and CVD. After recent animal data suggested that targeting miRNAs is beneficial in experimental CVD, our data may now spur further research in the field of CKD-associated human CVD.
Signal transduction and the regulation of gene expression are fundamental processes in every cell. RNA-binding proteins (RBPs) play a key role in the post-transcriptional modulation of gene expression in response to both internal and external stimuli. However, how signaling pathways regulate the assembly of RBPs with mRNAs remains largely unknown. Here, we summarize observations showing that the formation and composition of messenger ribonucleoprotein particles (mRNPs) is dynamically remodeled in space and time by specific signaling cascades and the resulting post-translational modifications. The integration of signaling events with gene expression is key to the rapid adaptation of cells to environmental changes and stress. Only a combined approach analyzing the signal transduction pathways and the changes in post-transcriptional gene expression they cause will unravel the mechanisms coordinating these important cellular processes.
Lieblingsbild
(2017)
Dieses Bild ist wichtig, weil wir daran verstanden haben, wie in der Zelle fehlerhaftes Spleißen verhindert wird. Dazu muss man wissen, dass unsere Gene sich aus Exons und dazwischenliegenden Introns zusammensetzen. Während des Spleißens werden die Introns entfernt und die Exons in ein reifes Transkript zusammengefügt, das dann für ein Protein kodiert. Allerdings gibt es innerhalb der Introns viele Bereiche, die einem Exon sehr ähnlich sehen. Werden diese sogenannten "PseudoExons" fälschlicherweise während des Spleißprozesses erkannt und in das reife Transkript eingebaut, kann das fatale Folgen für das kodierte Protein und oft die gesamte Zelle haben. ...
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
A method which serves to isolate the gonads from the sea cucumber (Holothuria polii) is outlined. Criteria that will secure a well determined status of maturity of the sperm are given. From this preparation a deoxyribonucleic acid is made, purified and analysed. It is concluded that the analytical data are in compliance with the theory of Crick and Watson. The ratio of Moles for this DNA while its nitrogen to phosphorus ratio on weight basis is 1,67.
Currently, the genus Polypedates comprises 26 species distributed in South, Southeast, and East Asia. Because of their relatively low dispersal capability and intolerance to seawater, this genus is ideal for the study of terrestrial range evolution that extends into the island archipelagos of southeastern Asia. In this study, based on data compiled for Polypedates from previous studies and partial mitochondrial and nuclear genes collected in this study, we performed systematic biogeographical analysis. We confirmed a Sundaland origin for the extant genus and showed northward dispersal into mainland Southeast Asia and Asia, which coincided with the timing of paleoclimatic change from the Oligocene to Middle Miocene. Climate fluctuations had a profound impact on species diversification within the genus Polypedates. Furthermore, the Red River did not mediate species exchange between Southeast Asia and mainland Asia until the end of the Miocene, with the sudden onset of northward dispersal in several clades independently at that time. Alternatively, the lineage of widespread insular P. leucomystax strongly supports the hypothesis of terrestrial connection between island archipelagos of Southeast Asia during the Mid-Pleistocene paleoclimate fluctuations. Our biogeographical analysis also supports the recent introduction of P. leucomystax to the Philippines and Ryukyus, as previously suggested.
Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.
Aim: Knowledge concerning species distribution is important for biodiversity conservation and environmental management. Fungi form a large and diverse group of species and play a key role in nutrient cycling and carbon storage. However, our understanding of fungal diversity and distribution remains limited, particularly at large spatial scales. Here, we predicted the diversity and distribution of ectomycorrhizal and saprotrophic macrofungi at relatively fine spatial resolution at a continental scale and examined the importance of variables that affect the distribution of these two functional groups. Location: Europe. Time period: 1990–2018. Major taxa studied: Macrofungi. Methods: From observations of 1,845 macrofungal species, we predicted the diversity and distribution of two functional groups of macrofungi at a resolution of 5 km across eight European countries based on 25 environmental variables using the MAXENT model. We determined the importance of variables that affect the distribution of these two functional groups of macrofungi using the built-in jackknife test in the model. Results: Analysis of the modelling results showed that eastern Denmark and southern Sweden are biodiversity hotspots for both functional groups of macrofungal species. Tree species and human disturbance (i.e., the human footprint index) were found to be the two most important predictor variables explaining the distribution of ectomycorrhizal and saprotrophic macrofungi. Main conclusions: Overall, our study demonstrates that tree species and human disturbance have played a more important role than climatic factors in determining the diversity and distribution of macrofungi at the continental scale. Our study suggests that fungal diversity and distribution might change considerably if the strongest predictors (i.e., tree species) were to be affected by climate change and/or human activity. Changes in fungal diversity might, in turn, influence other processes, because fungi are important in driving ecosystem processes, such as nutrient and carbon cycling.
One current goal in native mass spectrometry is the assignment of binding affinities to noncovalent complexes. Here we introduce a novel implementation of the existing laser-induced liquid bead ion desorption (LILBID) mass spectrometry method: this new method, LILBID laser dissociation curves, assesses binding strengths quantitatively. In all LILBID applications, aqueous sample droplets are irradiated by 3 µm laser pulses. Variation of the laser energy transferred to the droplet during desorption affects the degree of complex dissociation. In LILBID laser dissociation curves, laser energy transfer is purposely varied, and a binding affinity is calculated from the resulting complex dissociation. A series of dsDNAs with different binding affinities was assessed using LILBID laser dissociation curves. The binding affinity results from the LILBID laser dissociation curves strongly correlated with the melting temperatures from UV melting curves and with dissociation constants from isothermal titration calorimetry, standard solution phase methods. LILBID laser dissociation curve data also showed good reproducibility and successfully predicted the melting temperatures and dissociation constants of three DNA sequences. LILBID laser dissociation curves are a promising native mass spectrometry binding affinity method, with reduced time and sample consumption compared to melting curves or titrations.
Parallel multisite recordings in the visual cortex of trained monkeys revealed that the responses of spatially distributed neurons to natural scenes are ordered in sequences. The rank order of these sequences is stimulus-specific and maintained even if the absolute timing of the responses is modified by manipulating stimulus parameters. The stimulus specificity of these sequences was highest when they were evoked by natural stimuli and deteriorated for stimulus versions in which certain statistical regularities were removed. This suggests that the response sequences result from a matching operation between sensory evidence and priors stored in the cortical network. Decoders trained on sequence order performed as well as decoders trained on rate vectors but the former could decode stimulus identity from considerably shorter response intervals than the latter. A simulated recurrent network reproduced similarly structured stimulus-specific response sequences, particularly once it was familiarized with the stimuli through non-supervised Hebbian learning. We propose that recurrent processing transforms signals from stationary visual scenes into sequential responses whose rank order is the result of a Bayesian matching operation. If this temporal code were used by the visual system it would allow for ultrafast processing of visual scenes.
Background: Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which is sometimes associated with severe central nervous system disease in children. There is currently no specific medication for EV71 infection. Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to inhibit various viral infections. However, investigation of the anti-EV71 mechanism has not been reported to date.
Methods: The anti-EV71 activity of quercetin was evaluated by phenotype screening, determining the cytopathic effect (CPE) and EV71-induced cells apoptosis. The effects on EV71 replication were evaluated further by determining virus yield, viral RNA synthesis and protein expression, respectively. The mechanism of action against EV71 was determined from the effective stage and time-of-addition assays. The possible inhibitory functions of quercetin via viral 2Apro, 3Cpro or 3Dpol were tested. The interaction between EV71 3Cpro and quercetin was predicted and calculated by molecular docking.
Results: Quercetin inhibited EV71-mediated cytopathogenic effects, reduced EV71 progeny yields, and prevented EV71-induced apoptosis with low cytotoxicity. Investigation of the underlying mechanism of action revealed that quercetin exhibited a preventive effect against EV71 infection and inhibited viral adsorption. Moreover, quercetin mediated its powerful therapeutic effects primarily by blocking the early post-attachment stage of viral infection. Further experiments demonstrated that quercetin potently inhibited the activity of the EV71 protease, 3Cpro, blocking viral replication, but not the activity of the protease, 2Apro, or the RNA polymerase, 3Dpol. Modeling of the molecular binding of the 3Cpro-quercetin complex revealed that quercetin was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity.
Conclusions: Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, quercetin potently inhibits EV71 3Cpro activity, thereby blocking EV71 replication.
Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae’s 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.
Methylation of ribose sugars at the 2′-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2′-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5′ central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D′ box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications.
Kidney injury is a common complication of severe disease. Here, we report that injuries of the zebrafish embryonal kidney are rapidly repaired by a migratory response in 2-, but not in 1-day-old embryos. Gene expression profiles between these two developmental stages identify cxcl12a and myca as candidates involved in the repair process. Zebrafish embryos with cxcl12a, cxcr4b, or myca deficiency display repair abnormalities, confirming their role in response to injury. In mice with a kidney-specific knockout, Cxcl12 and Myc gene deletions suppress mitochondrial metabolism and glycolysis, and delay the recovery after ischemia/reperfusion injury. Probing these observations in zebrafish reveal that inhibition of glycolysis slows fast migrating cells and delays the repair after injury, but does not affect the slow cell movements during kidney development. Our findings demonstrate that Cxcl12 and Myc facilitate glycolysis to promote fast migratory responses during development and repair, and potentially also during tumor invasion and metastasis.
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
Fluorescense spectra of lactate dehydrogenase * (E.C. 1.1.1.27) were investigated in the presence of the coenzyme fragments dihydronicotinamide mononucleotide and dihydronicotinamide-ribose-5'-pyrophospho- (P2) -5“-ribose. The reduced mononucleotide is enzymatically less active as a hydrogen donor. However, formation of a complex with the enzyme was not observed under the conditions used. All the other substances: dihydronicotinamide-ribose-5'-pyrophospho- (P2) -5“-ribose, dihydronicotinamide- benzimidazole-dinucleotide, dihydronicotinamide-3-desazapurine-dinucleotide and dihydronicotinamide-6-mercaptopurine-dinucleotide form more or less stable complexes with lactate dehydrogenase. The complexes do not markedly differ from the complex formed with the natural cofactor. In all cases spectra indicate change in conformation of the coenzyme by forming the coenzyme-enzyme-complex which has been proposed by VELICK 1 too. The cysteine residues of the lactate dehydrogenase are not essential for binding the coenzyme to the active center; this was shown with mercury blocked enzyme.
Background: Molecular phylogenies are being published increasingly and many biologists rely on the most recent topologies. However, different phylogenetic trees often contain conflicting results and contradict significant background data. Not knowing how reliable traditional knowledge is, a crucial question concerns the quality of newly produced molecular data. The information content of DNA alignments is rarely discussed, as quality statements are mostly restricted to the statistical support of clades. Here we present a case study of a recently published mollusk phylogeny that contains surprising groupings, based on five genes and 108 species, and we apply new or rarely used tools for the analysis of the information content of alignments and for the filtering of noise (masking of random-like alignment regions, split decomposition, phylogenetic networks, quartet mapping). Results: The data are very fragmentary and contain contaminations. We show that that signal-like patterns in the data set are conflicting and partly not distinct and that the reported strong support for a "rather surprising result" (monoplacophorans and chitons form a monophylum Serialia) does not exist at the level of primary homologies. Split-decomposition, quartet mapping and neighbornet analyses reveal conflicting nucleotide patterns and lack of distinct phylogenetic signal for the deeper phylogeny of mollusks. Conclusion: Even though currently a majority of molecular phylogenies are being justified with reference to the 'statistical' support of clades in tree topologies, this confidence seems to be unfounded. Contradictions between phylogenies based on different analyses are already a strong indication of unnoticed pitfalls. The use of tree-independent tools for exploratory analyses of data quality are highly recommended. Concerning the new mollusk phylogeny more convincing evidence is needed.
Background: In general shell-less slugs are considered to be slimy animals with a rather dull appearance and a pest to garden plants. But marine slugs usually are beautifully coloured animals belonging to the less-known Opisthobranchia. They are characterized by a large array of interesting biological phenomena, usually related to foraging and/or defence. In this paper our knowledge of shell reduction, correlated with the evolution of different defensive and foraging strategies is reviewed, and new results on histology of different glandular systems are included. Results: Based on a phylogeny obtained by morphological and histological data, the parallel reduction of the shell within the different groups is outlined. Major food sources are given and glandular structures are described as possible defensive structures in the external epithelia, and as internal glands. Conclusion: According to phylogenetic analyses, the reduction of the shell correlates with the evolution of defensive strategies. Many different kinds of defence structures, like cleptocnides, mantle dermal formations (MDFs), and acid glands, are only present in shell-less slugs. In several cases, it is not clear whether the defensive devices were a prerequisite for the reduction of the shell, or reduction occurred before. Reduction of the shell and acquisition of different defensive structures had an implication on exploration of new food sources and therefore likely enhanced adaptive radiation of several groups. © 2005 Wägele and Klussmann-Kolb; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited: http://www.frontiersinzoology.com/content/2/1/3/
The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.
Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Psi in eukaryotic 18S rRNAs.
Background Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most informations on protein translocation are obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. Results To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. Conclusion The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a primitive translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has subsequently been lost in the transit sequence, but can be found at the C-terminal position of the translocating pore. Thereby, the current hypothesis of Omp85 being the prokaryotic contribution to the ancestral Toc translocon can be supported.
Network graphs have become a popular tool to represent complex systems composed of many interacting subunits; especially in neuroscience, network graphs are increasingly used to represent and analyze functional interactions between multiple neural sources. Interactions are often reconstructed using pairwise bivariate analyses, overlooking the multivariate nature of interactions: it is neglected that investigating the effect of one source on a target necessitates to take all other sources as potential nuisance variables into account; also combinations of sources may act jointly on a given target. Bivariate analyses produce networks that may contain spurious interactions, which reduce the interpretability of the network and its graph metrics. A truly multivariate reconstruction, however, is computationally intractable because of the combinatorial explosion in the number of potential interactions. Thus, we have to resort to approximative methods to handle the intractability of multivariate interaction reconstruction, and thereby enable the use of networks in neuroscience. Here, we suggest such an approximative approach in the form of an algorithm that extends fast bivariate interaction reconstruction by identifying potentially spurious interactions post-hoc: the algorithm uses interaction delays reconstructed for directed bivariate interactions to tag potentially spurious edges on the basis of their timing signatures in the context of the surrounding network. Such tagged interactions may then be pruned, which produces a statistically conservative network approximation that is guaranteed to contain non-spurious interactions only. We describe the algorithm and present a reference implementation in MATLAB to test the algorithm’s performance on simulated networks as well as networks derived from magnetoencephalographic data. We discuss the algorithm in relation to other approximative multivariate methods and highlight suitable application scenarios. Our approach is a tractable and data-efficient way of reconstructing approximative networks of multivariate interactions. It is preferable if available data are limited or if fully multivariate approaches are computationally infeasible.
The pyrrolobenzodiazepine tilivalline (1) was originally identified in the human gut pathobiont Klebsiella oxytoca, the causative agent of antibiotic-associated hemorrhagic colitis. Here we show the identification of tilivalline and analogs thereof in the entomopathogenic bacterium Xenorhabdus eapokensis as well as the identification of its biosynthesis gene cluster encoding a bimodular non-ribosomal peptide synthetase. Heterologous expression of both genes in E. coli resulted in the production of 1 and from mutasynthesis and precursor directed biosynthesis 11 new tilivalline analogs were identified in X. eapokensis. These results allowed the prediction of the tilivalline biosynthesis being similar to that in K. oxytoca.
Ohne das Eingreifen des Menschen wäre Mitteleuropa fast ein reines Waldgebiet. Noch heute beheimaten die Wälder eine große Vielfalt an Pflanzen und Tieren, die für diese Region spezifisch sind. Regionale Besonderheiten gehen aber verloren, je mehr Menschen in die Ökosysteme eingreifen: So unterscheiden sich die Pflanzenarten auf der North Charles Street in Baltimore nur wenig von denjenigen der Mainzer Landstraße in Frankfurt. Gleichzeitig verdrängen zugewanderte und eingeschleppte Arten heimische Tiere und Pflanzen. Allerdings gibt es auch im Frankfurter Stadtgebiet echte Horte der Biodiversität.
Dem Wandel rechtzeitig begegnen : Landesförderung ermöglicht richtungsweisende Klimafolgenforschung
(2008)
Die Makrophytenvegetation eines stillgelegten Kanalabschnittes ("Alte Fahrt") bei Senden in Westfalen hat sich seit Beginn der 90er Jahre drastisch verändert. Aus einem typischen Potamogetonetum lucentis sind Reinbestände von Myriophyllum spicatum geworden, denen stellenweise Ceratophyllum demersum beigemischt ist. Die Ursachen für diese gravierenden Vegetationsveränderungen sind nicht klar. Da es sich um einen der bedeutendsten westfälischen Standorte des Potamogetonetum lucentis, einer in Nordrhein-Westfalen stark gefährdeten Pflanzengesellschaft, handelte, sind weiterführende Untersuchungen und Versuche zur Wiederansiedlung zu fordern.
In spite of enormous climatic differences between Burkina Faso and Germany, 20 species belong to the spontaneous flora of both countries, i.e. 1% of the flora of Burkina Faso and 0.15 % of the German flora. All of them are either ruderal and segetal species (16) or water and reed plants (4). All of the 16 ruderals/segetals are therophytes. From a recent point of view, most of the 20 species can be classified as cosmopolitan, because they cover three and more floristic zones, and/or at least three climatic zones, and/or are represented in at least three continents. Although Burkina Faso has a semi-arid climate, none of the species can be called a sclero- or xerophyte. Therefore, in Burkina Faso, all are more or less bound to habitats at least temporarily flooded or to humid soils. In Germany, however, the concerned ruderals, with one exception, are indicators of medium dry or dry habitats.
Here we present a formal description of Biremis panamae Barka, Witkowski et Weisenborn sp. nov., which was isolated from the marine littoral environment of the Pacific Ocean coast of Panama. The description is based on morphology (light and electron microscopy) and the rbcL, psbC and SSU sequences of one clone of this species. The new species is included in Biremis due to its morphological features; i.e. two marginal rows of foramina, chambered striae, and girdle composed of numerous punctate copulae. The new species also possesses a striated valve face which is not seen in most known representatives of marine littoral Biremis species. In this study we also present the relationship of Biremis to other taxa using morphology, DNA sequence data and observations of auxosporulation. Our results based on these three sources point to an evolutionary relationship between Biremis, Neidium and Scoliopleura. The unusual silicified incunabular caps present in them are known otherwise only in Muelleria, which is probably related to the Neidiaceae and Scoliotropidaceae. We also discuss the relationship between Biremis and the recently described Labellicula and Olifantiella.
Wirtszellreaktivierung chemisch induzierter Letalschäden im DNS-haltigen Serratia-Phagen Kappa
(1965)
After treating free phage Kappa with nitrous acid, triethylenemelamine, ethylmethanesulfonate, or hydroxylamin and using these phages for infecting Serratia H Y wildtype cells, at least 20% of the lethal damage in the phage-DNA can be reactivated by the host ( = host cell reactivation). It is known that all lethal agents tested so far attack the primary structure of the D N A in different ways. Therefore, we assume that the target for the host cell reactivation consists of some damage in the secondary structure of the DNA, because there is probably some coincidence in the action of all agents. The hypothesis that in the DNA changes of thymine are a prerequisite for host cell reactivation has been disproved by the experiments with nitrous acid and ethylmethanesulfonate because both substances do not act on thymine.
UV inactivated KAPPA can be reactivated like other temperate phages by plating on uvirradiated host cells (indicator). The capacity of the indicator Serratia HY for multiplication of unirradiated KAPPA was about 0.1% survivors (colony formers). The induction of clear plaque (c·) mutants by irradiating extracellular KAPPA and plating on untreated indicator can be increased further about 2 to 4 times by using UV irradiated indicator. The increase of the number of c mutants under the latter conditions, with increasing UV dose given to the phage, was never a firstorder reaction. The highest frequency of c mutants obtained was about 4.5 per cent. Plating of unirradiated KAPPA on irradiated indicator (lowest survival fraction was 0.01%) never increased the spontaneous mutation rate to c. Two c mutants studied in detail belong to two different cistrons as shown in a complementation test (map distance about 5.3%). Only one of both was revertible to the phenotype c+ spontaneously and with a higher rate by UV. However, as shown in crossing experiments with the wild type, the backmutants do not have the original genotype but originated from mutations in at least two different intragenic suppressor loci; the map distances between them and the original c mutation were 0.64% and 0.13 per cent. Host range (h) and virulent (v) mutants could not be induced by irradiation of the free phage and plating on untreated indicator. This indicates that the UV induced high mutability of the c loci in KAPPA represents an exceptional case of behavior (UV-hot spot). Some unstable h mutants could be isolated by plating irradiated phage on irradiated indicator.
Numerous cell–cell and cell–matrix interactions within the bone marrow microenvironment enable the controlled lifelong self-renewal and progeny of hematopoietic stem and progenitor cells (HSPCs). On the cellular level, this highly mutual interaction is granted by cell adhesion molecules (CAMs) integrating differentiation, proliferation, and pro-survival signals from the surrounding microenvironment to the inner cell. However, cell–cell and cell–matrix interactions are also critically involved during malignant transformation of hematopoietic stem/progenitor cells. It has become increasingly apparent that leukemia-associated gene products, such as activated tyrosine kinases and fusion proteins resulting from chromosomal translocations, directly regulate the activation status of adhesion molecules, thereby directing the leukemic phenotype. These observations imply that interference with adhesion molecule function represents a promising treatment strategy to target pre-leukemic and leukemic lesions within the bone marrow niche. Focusing on myeloid leukemia, we provide a current overview of the mechanisms by which leukemogenic gene products hijack control of cellular adhesion to subsequently disturb normal hematopoiesis and promote leukemia development.
The avian magnetic compass was analyzed by testing migratory birds, using their orientation as an indicator. These tests revealed some remarkable properties of the avian magnetic compass: (1) It is an inclination compass’, (2) it is light-dependent, with (3) receptors located in the right eye. These characteristics are in agreement with the Radical Pair model proposed by Ritz et al. (2000). Using the same experimental set-up, we tested the model by behavioral spectroscopy’, exposing migratory birds to radiofrequency fields of different frequencies and intensities. Such fields affected the orientation only when applied at an angle to the field lines. Tests with different frequencies led to an estimate of the life time of the crucial radical pair between 2-10 μs. We also could identify an extremely sensitive resonance at the Larmor frequency, which implies specific properties of the radical pair. Cryptochromes, a blue-light absorbing photopigment, has been proposed to be the receptor-molecule; it has been found to be present in the retina of birds.
The magnetic field of the Earth provides animals with various kinds of information. Its use as a compass was discovered in the mid-1960s in birds, when it was first met with considerable skepticism, because it initially proved difficult to obtain evidence for magnetic sensitivity by conditioning experiments. Meanwhile, a magnetic compass was found to be widespread. It has now been demonstrated in members of all vertebrate classes, in mollusks and several arthropod species, in crustaceans as well as in insects. The use of the geomagnetic field as a ‘map’ for determining position, although already considered in the nineteenth century, was demonstrated by magnetically simulating displacements only after 2000, namely when animals, tested in the magnetic field of a distant site, responded as if they were physically displaced to that site and compensated for the displacement. Another use of the magnetic field is that as a ‘sign post’ or trigger: specific magnetic conditions elicit spontaneous responses that are helpful when animals reach the regions where these magnetic characteristics occur. Altogether, the geomagnetic field is a widely used valuable source of navigational information for mobile animals.
Animals use the geomagnetic field and astronomical cues to obtain compass information. The magnetic compass is not a uniform mechanism, as several functional modes have been described in different animal groups. The Sun compass requires the internal clock to interpret the position of the Sun. For star compass orientation, night-migrating birds seem to use the star pattern as a whole, without involving the internal clock. Both the astronomical compass mechanisms are based on learning processes to adapt them to the geographic latitude where the animals live and, in long-living animals, to compensate for the seasonal changes. Several mechanisms are used to determine the compass course to a goal. Using information collected during the outward journey is mostly done by path integration: recording the direction with a compass and integrating its twists and turns. Migratory animals have innate programs to guide them to their still unknown goal. Highly mobile animals with large ranges develop a so-called navigational ‘map’, a mental representation of the spatial distribution of navigational factors within their home region and their migration route. The nature of the factors involved is not yet entirely clear; magnetic intensity and inclination are the ones best supported so far.
Iron-rich structures have been described in the beak of homing pigeons, chickens and several species of migratory birds and interpreted as magnetoreceptors. Here, we will briefly review findings associated with these receptors that throw light on their nature, their function and their role in avian navigation. Electrophysiological recordings from the ophthalmic nerve, behavioral studies and a ZENK-study indicate that the trigeminal system, the nerves innervating the beak, mediate information on magnetic changes, with the electrophysiological study suggesting that these are changes in intensity. Behavioral studies support the involvement of magnetite and the trigeminal system in magnetoreception, but clearly show that the inclination compass normally used by birds represents a separate system. However, if this compass is disrupted by certain light conditions, migrating birds show 'fixed direction' responses to the magnetic field, which originate in the receptors in the beak. Together, these findings point out that there are magnetite-based magnetoreceptors located in the upper beak close to the skin. Their natural function appears to be recording magnetic intensity and thus providing one component of the multi-factorial 'navigational map' of birds.
Background The Radical Pair model proposes that magnetoreception is a light-dependent process. Under low monochromatic light from the short-wavelength part of the visual spectrum, migratory birds show orientation in their migratory direction. Under monochromatic light of higher intensity, however, they showed unusual preferences in other directions or axial preferences. To determine whether or not these responses are still controlled by the respective light regimes, European robins, Erithacus rubecula, were tested under UV, Blue, Turquoise and Green light at increasing intensities, with orientation in migratory direction serving as a criterion whether or not magnetoreception works in the normal way. Results Under low light with a quantal flux of 8 times 10 to 15 power quanta s-1 m-2, the birds were well oriented in their seasonally appropriate migratory direction under 424 nm Blue, 502 nm Turquoise and 565 nm Green light, indicating unimpaired magnetoreception. Under 373 nm UV of the same quantal flux, they were not oriented in migratory direction, showing a preference of the east-west axis instead, but they showed excellent orientation in migratory direction under UV of lower intensity. Intensities of above 36 times 10 to 15 power quanta s-1 m-2 of Blue, Turquoise and Green light elicited a variety of responses: disorientation, headings along the east-west axis, headings along the north-south axis or 'fixed' direction tendencies. These responses changed as the intensity was increased from 36 times 10 to the 15 power quanta s-1 m-2 to 54 and 72 times 10 to 15 power quanta s-1 m-2. Conclusion The specific manifestation of responses in directions other than migratory direction clearly depends on the ambient light regime. This implies that although mechanisms normally providing magnetic compass information seem disrupted, processes that are activated by light still control the behavior. It suggests complex interactions between different types of receptors, magnetic and visual. The nature of the receptors involved and details of their connections are not yet known; however, a role of the color cones in the processes mediating magnetic input is suggested.
The geomagnetic field provides directional information for birds. The avian magnetic compass is an inclination compass that uses not the polarity of the magnetic field but the axial course of the field lines and their inclination in space. It works in a flexible functional window, and it requires short-wavelength light. These characteristics result from the underlying sensory mechanism based on radical pair processes in the eyes, with cryptochrome suggested as the receptor molecule. The chromophore of cryptochrome, flavin adenine dinucleotide (FAD), undergoes a photocycle, where radical pairs are formed during photo-reduction as well as during re-oxidation; behavioral data indicate that the latter is crucial for detecting magnetic directions. Five types of cryptochromes are found in the retina of birds: cryptochrome 1a (Cry1a), cryptochrome 1b, cryptochrome 2, cryptochrome 4a, and cryptochrome 4b. Because of its location in the outer segments of the ultraviolet cones with their clear oil droplets, Cry1a appears to be the most likely receptor molecule for magnetic compass information.
Background: European robins, Erithacus rubecula, show two types of directional responses to the magnetic field: (1) compass orientation that is based on radical pair processes and lateralized in favor of the right eye and (2) so-called 'fixed direction' responses that originate in the magnetite-based receptors in the upper beak. Both responses are light-dependent. Lateralization of the 'fixed direction' responses would suggest an interaction between the two magnetoreception systems. Results: Robins were tested with either the right or the left eye covered or with both eyes uncovered for their orientation under different light conditions. With 502 nm turquoise light, the birds showed normal compass orientation, whereas they displayed an easterly 'fixed direction' response under a combination of 502 nm turquoise with 590 nm yellow light. Monocularly right-eyed birds with their left eye covered were oriented just as they were binocularly as controls: under turquoise in their northerly migratory direction, under turquoise-and-yellow towards east. The response of monocularly left-eyed birds differed: under turquoise light, they were disoriented, reflecting a lateralization of the magnetic compass system in favor of the right eye, whereas they continued to head eastward under turquoise-and-yellow light. Conclusion: 'Fixed direction' responses are not lateralized. Hence the interactions between the magnetite-receptors in the beak and the visual system do not seem to involve the magnetoreception system based on radical pair processes, but rather other, non-lateralized components of the visual system.
Forest wildflowers bloom earlier as Europe warms: lessons from herbaria and spatial modelling
(2022)
Today plants often flower earlier due to climate warming. Herbarium specimens are excellent witnesses of such long-term changes. However, the magnitude of phenological shifts may vary geographically, and the data are often clustered. Therefore, large-scale analyses of herbarium data are prone to pseudoreplication and geographical biases.
We studied over 6000 herbarium specimens of 20 spring-flowering forest understory herbs from Europe to understand how their phenology had changed during the last century. We estimated phenology trends with or without taking spatial autocorrelation into account.
On average plants now flowered over 6 d earlier than at the beginning of the last century. These changes were strongly associated with warmer spring temperatures. Flowering time advanced 3.6 d per 1°C warming. Spatial modelling showed that, in some parts of Europe, plants flowered earlier or later than expected. Without accounting for this, the estimates of phenological shifts were biased and model fits were poor.
Our study indicates that forest wildflowers in Europe strongly advanced their phenology in response to climate change. However, these phenological shifts differ geographically. This shows that it is crucial to combine the analysis of herbarium data with spatial modelling when testing for long-term phenology trends across large spatial scales.
Background: Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood–Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study.
Results: We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+–NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis.
Discussion: We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme.
In times of global warming caused by the extensive use of fossil fuels, the need to capture gaseous carbon compounds is growing bigger. Several groups of microorganisms can fix the greenhouse gas CO2. Out of these, acetogenic bacteria are role models in their ability to reduce CO2 with hydrogen to acetate, which makes acetogens prime candidates for genetic modification towards biotechnological production of value-added compounds from CO2, such as biofuels. However, growth of acetogens on gaseous substrates is strongly energy-limited, and successful metabolic engineering requires a detailed knowledge of the bioenergetics. In 1939, Clostridium aceticum was the first acetogen to be described. A recent genomic study revealed that this organism contains cytochromes and therefore may use a proton gradient in its respiratory chain. We have followed up these studies and will present data that C. aceticum does not use a H+ but a Na+ gradient for ATP synthesis, established by a Na+-Rnf. Experimental data and in silico analyses enabled us to propose the biochemistry and bioenergetics of acetogenesis from H2 + CO2 in C. aceticum.
Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H2 + CO2 or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO2 reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy."
Insects with aquatic life stages can transfer sediment and water pollutants to terrestrial ecosystems, which has been described for metals, polyaromatic hydrocarbons, and polychlorinated chemicals. However, knowledge of the transfer of aquatic micropollutants released by wastewater treatment plants is scarce despite some preliminary studies on their occurrence in riparian spiders. In our study, we address a major analytical gap focusing on the transfer of the micropollutant carbamazepine from the larvae to the adult midges of Chironomus riparius using an optimized QuEChERS extraction method and HPLC–MS/MS applicable to both life stages down to the level of about three individuals. We show that the uptake of carbamazepine by larvae is concentration-dependent and reduces the emergence rate. Importantly, the body burden remained constant in adult midges. Using this information, we estimated the daily exposure of insectivorous tree swallows as terrestrial predators to carbamazepine using the energy demand of the predator and the energy content of the prey. Assuming environmentally relevant water concentrations of about 1 μg/L, the daily dose per kilogram of body weight for tree swallows was estimated to be 0.5 μg/kg/day. At places of high water contamination of 10 μg/L, the exposure may reach 5 μg/kg/day for this micropollutant of medium polarity. Considering body burden changes upon metamorphosis, this study fills the missing link between aquatic contamination and exposure in terrestrial habitats showing that wastewater pollutants can impact birds’ life. Clearly, further analytical methods for biota analysis in both habitats are urgently required to improve risk assessment.
The taxon Syndermata comprises the biologically interesting wheel animals (“Rotifera”: Bdelloidea + Monogononta + Seisonidea) and thorny-headed worms (Acanthocephala), and is central for testing superordinate phylogenetic hypotheses (Platyzoa, Gnathifera) in the metazoan tree of life. Recent analyses of syndermatan phylogeny suggested paraphyly of Eurotatoria (free-living bdelloids and monogononts) with respect to endoparasitic acanthocephalans. Data of epizoic seisonids, however, were absent, which may have affected the branching order within the syndermatan clade. Moreover, the position of Seisonidea within Syndermata should help in understanding the evolution of acanthocephalan endoparasitism. Here, we report the first phylogenomic analysis that includes all four higher-ranked groups of Syndermata. The analyzed data sets comprise new transcriptome data for Seison spec. (Seisonidea), Brachionus manjavacas (Monogononta), Adineta vaga (Bdelloidea), and Paratenuisentis ambiguus (Acanthocephala). Maximum likelihood and Bayesian trees for a total of 19 metazoan species were reconstructed from up to 410 functionally diverse proteins. The results unanimously place Monogononta basally within Syndermata, and Bdelloidea appear as the sister group to a clade comprising epizoic Seisonidea and endoparasitic Acanthocephala. Our results support monophyly of Syndermata, Hemirotifera (Bdelloidea + Seisonidea + Acanthocephala), and Pararotatoria (Seisonidea + Acanthocephala), rejecting monophyly of traditional Rotifera and Eurotatoria. This serves as an indication that early acanthocephalans lived epizoically or as ectoparasites on arthropods, before their complex lifecycle with arthropod intermediate and vertebrate definite hosts evolved.
Identifying unexpected acoustic inputs, which allows to react appropriately to new situations, is of major importance for animals. Neural deviance detection describes a change of neural response strength to a stimulus solely caused by the stimulus' probability of occurrence. In the present study, we searched for correlates of deviance detection in auditory brainstem responses obtained in anaesthetised bats (Carollia perspicillata). In an oddball paradigm, we used two pure tone stimuli that represented the main frequencies used by the animal during echolocation (60 kHz) and communication (20 kHz). For both stimuli, we could demonstrate significant differences of response strength between deviant and standard response in slow and fast components of the auditory brainstem response. The data suggest the presence of correlates of deviance detection in brain stations below the inferior colliculus (IC), at the level of the cochlea nucleus and lateral lemniscus. Additionally, our results suggest that deviance detection is mainly driven by repetition suppression in the echolocation frequency band, while in the communication band, a deviant-related enhancement of the response plays a more important role. This finding suggests a contextual dependence of the mechanisms underlying subcortical deviance detection. The present study demonstrates the value of auditory brainstem responses for studying deviance detection and suggests that auditory specialists, such as bats, use different frequency-specific strategies to ensure an appropriate sensation of unexpected sounds.
The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh) for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4–8 mM). ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75–90%) in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM) than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb]) or thiacloprid (8,800 ppb). Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb) or clothianidin (1 and 10 ppb) reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh content in the brood food.
Fachliche Exzellenz und Bildungsnotstand – diese beiden Extreme beherrschen gegenwärtig die Diskussion um Schul- und Hochschulausbildung. Die Universität Frankfurt stellt sich der Elitediskussion und setzt auf Fokussierung und Schwerpunktbildung. Studiengänge werden modifiziert, die Art und Vielfalt möglicher Abschlüsse internationalen Standards angepasst. Die Universität will und wird wettbewerbsfähig sein, auch im internationalen Vergleich. Darüber sprach Dr. Monika Mölders mit Prof. Dr. Günther Wess, Honorarprofessor der Universität Frankfurt, Forschungsleiter Europa von Aventis und Mitglied der Geschäftsführung der Aventis Pharma Deutschland GmbH.
In search for new natural products, which may lead to the development of new drugs for all kind of applications, novel methods are needed. Here we describe the identification of electrophilic natural products in crude extracts via their reactivity against azide as a nucleophile followed by their subsequent enrichment using a cleavable azide-reactive resin (CARR). Using this approach, natural products carrying epoxides and α,β-unsaturated enones as well as several unknown compounds were identified in crude extracts from entomopathogenic Photorhabdus bacteria.
Synthesis and SAR of the antistaphylococcal natural product nematophin from Xenorhabdus nematophila
(2019)
The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure–activity relationship of the simple and antistaphylococcal amide nematophin from Xenorhabdus nematophila and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluorescent derivatives, incorporating azaindole moieties was achieved for the first time.
The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.
This report provides a brief review of the 20th annual meeting of the German Language Branch of the Society of Environmental Toxicology and Chemistry (SETAC GLB) held from September 7th to 10th 2015 at ETH (Swiss Technical University) in Zurich, Switzerland. The event was chaired by Inge Werner, Director of the Swiss Centre for Applied Ecotoxicology (Ecotox Centre) Eawag-EPFL, and organized by a team from Ecotox Centre, Eawag, Federal Office of the Environment, Federal Office of Agriculture, and Mesocosm GmbH (Germany). Over 200 delegates from academia, public agencies and private industry of Germany, Switzerland and Austria attended and discussed the current state of science and its application presented in 75 talks and 83 posters. In addition, three invited keynote speakers provided new insights into scientific knowledge ‘brokering’, and—as it was the International Year of Soil—the important role of healthy soil ecosystems. Awards were presented to young scientists for best oral and poster presentations, and for best 2014 master and doctoral theses. Program and abstracts of the meeting (mostly in German) are provided as Additional file 1.
Strong seasonal variability of hygric and thermal soil conditions are a defining environmental feature in Northern Australia. However, how such changes affect the soil–atmosphere exchange of nitrous oxide (N2O), nitric oxide (NO) and dinitrogen (N2) is still 5 not well explored. By incubating intact soil cores from four sites (3 savanna, 1 pasture) under controlled soil temperatures (ST) and soil moisture (SM) we investigated the release of the trace gas fluxes of N2O, NO and carbon dioxide (CO2). Furthermore, the release of N2 due to denitrification was measured using the helium gas flow soil core technique. Under dry pre-incubation conditions NO and N2O emission were very low (< 7.0± 5.0 μgNO-Nm−2 h−1; < 0.0± 1.4 μgN2O-Nm−2 h−1) or in case of N2O, even a net soil uptake was observed. Substantial NO (max: 306.5 μgNm−2 h−1) and relatively small N2O pulse emissions (max: 5.8±5.0 μgNm−2 h−1) were recorded following soil wetting, but these pulses were short-lived, lasting only up to 3 days. The total atmospheric loss of nitrogen was dominated by N2 emissions (82.4–99.3% of total N lost), although NO emissions contributed almost 43.2% at 50% SM and 30 °C ST. N2O emissions were systematically higher for 3 of 12 sample locations, which indicates substantial spatial variability at site level, but on average soils acted as weak N2O sources or even sinks. Emissions were controlled by SM and ST for N2O and CO2, ST and pH for NO, and SM and pH for N2.
Strong seasonal variability of hygric and thermal soil conditions are a defining environmental feature in northern Australia. However, how such changes affect the soil–atmosphere exchange of nitrous oxide (N2O), nitric oxide (NO) and dinitrogen (N2) is still not well explored. By incubating intact soil cores from four sites (three savanna, one pasture) under controlled soil temperatures (ST) and soil moisture (SM) we investigated the release of the trace gas fluxes of N2O, NO and carbon dioxide (CO2). Furthermore, the release of N2 due to denitrification was measured using the helium gas flow soil core technique. Under dry pre-incubation conditions NO and N2O emissions were very low (<7.0 ± 5.0 μg NO-N m−2 h−1; <0.0 ± 1.4 μg N2O-N m−2 h−1) or in the case of N2O, even a net soil uptake was observed. Substantial NO (max: 306.5 μg N m−2 h−1) and relatively small N2O pulse emissions (max: 5.8 ± 5.0 μg N m−2 h−1) were recorded following soil wetting, but these pulses were short lived, lasting only up to 3 days. The total atmospheric loss of nitrogen was generally dominated by N2 emissions (82.4–99.3% of total N lost), although NO emissions contributed almost 43.2% to the total atmospheric nitrogen loss at 50% SM and 30 °C ST incubation settings (the contribution of N2 at these soil conditions was only 53.2%). N2O emissions were systematically higher for 3 of 12 sample locations, which indicates substantial spatial variability at site level, but on average soils acted as weak N2O sources or even sinks. By using a conservative upscale approach we estimate total annual emissions from savanna soils to average 0.12 kg N ha−1 yr−1 (N2O), 0.68 kg N ha−1 yr−1 (NO) and 6.65 kg N ha−1 yr−1 (N2). The analysis of long-term SM and ST records makes it clear that extreme soil saturation that can lead to high N2O and N2 emissions only occurs a few days per year and thus has little impact on the annual total. The potential contribution of nitrogen released due to pulse events compared to the total annual emissions was found to be of importance for NO emissions (contribution to total: 5–22%), but not for N2O emissions. Our results indicate that the total gaseous release of nitrogen from these soils is low and clearly dominated by loss in the form of inert nitrogen. Effects of seasonally varying soil temperature and moisture were detected, but were found to be low due to the small amounts of available nitrogen in the soils (total nitrogen <0.1%).
Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species Culex pipiens and Culex torrentium. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima's D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for Cx. pipiens, widely distributed within Germany and a more homogeneous pattern for Cx. torrentium. The low genetic differences within Cx. pipiens could be a result of an infection with Wolbachia which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (FST) ranged significantly from moderate to very great between populations of Cx. pipiens and Cx. torrentium. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations (Cx. pipiens [88.38%]; Cx. torrentium [66.54%]). Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species' genetic variation. Overall, the results confirm that Cx. pipiens and Cx. torrentium underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction.
Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties. As a result, many peptides have entered the clinics for various applications. Two main routes for the biosynthesis of complex peptides have evolved in nature: ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic pathways and non-ribosomal peptide synthetases (NRPSs). Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes. These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides. In this review, we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications. In addition, the influence of the fundamentally different biosynthetic principles on past, current and future engineering approaches is illustrated. Despite the different biosynthetic principles of both peptide biosynthetic routes, the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping. The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.
Background Identification and evaluation of surface binding-pockets and occluded cavities are initial steps in protein structure-based drug design. Characterizing the active site's shape as well as the distribution of surrounding residues plays an important role for a variety of applications such as automated ligand docking or in situ modeling. Comparing the shape similarity of binding site geometries of related proteins provides further insights into the mechanisms of ligand binding. Results We present PocketPicker, an automated grid-based technique for the prediction of protein binding pockets that specifies the shape of a potential binding-site with regard to its buriedness. The method was applied to a representative set of protein-ligand complexes and their corresponding apo-protein structures to evaluate the quality of binding-site predictions. The performance of the pocket detection routine was compared to results achieved with the existing methods CAST, LIGSITE, LIGSITEcs, PASS and SURFNET. Success rates PocketPicker were comparable to those of LIGSITEcs and outperformed the other tools. We introduce a descriptor that translates the arrangement of grid points delineating a detected binding-site into a correlation vector. We show that this shape descriptor is suited for comparative analyses of similar binding-site geometry by examining induced-fit phenomena in aldose reductase. This new method uses information derived from calculations of the buriedness of potential binding-sites. Conclusions The pocket prediction routine of PocketPicker is a useful tool for identification of potential protein binding-pockets. It produces a convenient representation of binding-site shapes including an intuitive description of their accessibility. The shape-descriptor for automated classification of binding-site geometries can be used as an additional tool complementing elaborate manual inspections.
The amyloid precursor protein (APP) was discovered in the 1980s as the precursor protein of the amyloid A4 peptide. The amyloid A4 peptide, also known as A-beta (Aβ), is the main constituent of senile plaques implicated in Alzheimer’s disease (AD). In association with the amyloid deposits, increasing impairments in learning and memory as well as the degeneration of neurons especially in the hippocampus formation are hallmarks of the pathogenesis of AD. Within the last decades much effort has been expended into understanding the pathogenesis of AD. However, little is known about the physiological role of APP within the central nervous system (CNS). Allocating APP to the proteome of the highly dynamic presynaptic active zone (PAZ) identified APP as a novel player within this neuronal communication and signaling network. The analysis of the hippocampal PAZ proteome derived from APP-mutant mice demonstrates that APP is tightly embedded in the underlying protein network. Strikingly, APP deletion accounts for major dysregulation within the PAZ proteome network. Ca2+-homeostasis, neurotransmitter release and mitochondrial function are affected and resemble the outcome during the pathogenesis of AD. The observed changes in protein abundance that occur in the absence of APP as well as in AD suggest that APP is a structural and functional regulator within the hippocampal PAZ proteome. Within this review article, we intend to introduce APP as an important player within the hippocampal PAZ proteome and to outline the impact of APP deletion on individual PAZ proteome subcommunities.
The ability to vocalize is ubiquitous in vertebrates, but neural networks underlying vocal control remain poorly understood. Here, we performed simultaneous neuronal recordings in the frontal cortex and dorsal striatum (caudate nucleus, CN) during the production of echolocation pulses and communication calls in bats. This approach allowed us to assess the general aspects underlying vocal production in mammals and the unique evolutionary adaptations of bat echolocation. Our data indicate that before vocalization, a distinctive change in high-gamma and beta oscillations (50–80 Hz and 12–30 Hz, respectively) takes place in the bat frontal cortex and dorsal striatum. Such precise fine-tuning of neural oscillations could allow animals to selectively activate motor programs required for the production of either echolocation or communication vocalizations. Moreover, the functional coupling between frontal and striatal areas, occurring in the theta oscillatory band (4–8 Hz), differs markedly at the millisecond level, depending on whether the animals are in a navigational mode (that is, emitting echolocation pulses) or in a social communication mode (emitting communication calls). Overall, this study indicates that fronto-striatal oscillations could provide a neural correlate for vocal control in bats.
Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development
(2011)
Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system, and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here, we investigate the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperature-related role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures, as they characteristically occur in nature.
Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5′splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5′SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA–ligand interaction for regulation of gene expression.
While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.
This case study reports on an e-learning course on good academic practice, compulsory for doctoral students of the central graduate academy GRADE at the Goethe University in Frankfurt (Germany). The tool comprises of six closely linked web based trainings. They are designed as a virtual PhD, depicting the different phases of a doctorate and covering the various aspects of good academic practice and potential fields of academic misconduct.
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.
BACKGROUND: Current biodiversity patterns are considered largely the result of past climatic and tectonic changes. In an integrative approach, we combine taxonomic and phylogenetic hypotheses to analyze temporal and geographic diversification of epigean (Carychium) and subterranean (Zospeum) evolutionary lineages in Carychiidae (Eupulmonata, Ellobioidea). We explicitly test three hypotheses: 1) morphospecies encompass unrecognized evolutionary lineages, 2) limited dispersal results in a close genetic relationship of geographical proximally distributed taxa and 3) major climatic and tectonic events had an impact on lineage diversification within Carychiidae.
RESULTS: Initial morphospecies assignments were investigated by different molecular delimitation approaches (threshold, ABGD, GMYC and SP). Despite a conservative delimitation strategy, carychiid morphospecies comprise a great number of unrecognized evolutionary lineages. We attribute this phenomenon to historic underestimation of morphological stasis and phenotypic variability amongst lineages. The first molecular phylogenetic hypothesis for the Carychiidae (based on COI, 16S and H3) reveals Carychium and Zospeum to be reciprocally monophyletic. Geographical proximally distributed lineages are often closely related. The temporal diversification of Carychiidae is best described by a constant rate model of diversification. The evolution of Carychiidae is characterized by relatively few (long distance) colonization events. We find support for an Asian origin of Carychium. Zospeum may have arrived in Europe before extant members of Carychium. Distantly related Carychium clades inhabit a wide spectrum of the available bioclimatic niche and demonstrate considerable niche overlap.
CONCLUSIONS: Carychiid taxonomy is in dire need of revision. An inferred wide distribution and variable phenotype suggest underestimated diversity in Zospeum. Several Carychium morphospecies are results of past taxonomic lumping. By collecting populations at their type locality, molecular investigations are able to link historic morphospecies assignments to their respective evolutionary lineage. We propose that rare founder populations initially colonized a continent or cave system. Subsequent passive dispersal into adjacent areas led to in situ pan-continental or mountain range diversifications. Major environmental changes did not influence carychiid diversification. However, certain molecular delimitation methods indicated a recent decrease in diversification rate. We attribute this decrease to protracted speciation.
A new species of the eutroglobiont gastropod taxon Zospeum Bourguignat, 1856 is described. Zospeum tholussum sp. n. is characterized based on a population from the Lukina Jama–Trojama cave system (Velebit Mts., Croatia). A single living specimen occurred at 980 m depth. The species is morphologically related to Zospeum amoenum (Frauenfeld, 1856), but can be readily distinguished from the latter by the presence of a weak columellar fold and its dome-like structured 2nd whorl. DNA barcoding is capable to clearly delineate Zospeum tholussum from other Zospeum spp. as well.
Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.
Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
Mitochondrial cristae morphology is highly variable and altered under numerous pathological conditions. The protein complexes involved are largely unknown or only insufficiently characterized. Using complexome profiling we identified apolipoprotein O (APOO) and apolipoprotein O-like protein (APOOL) as putative components of the Mitofilin/MINOS protein complex which was recently implicated in determining cristae morphology. We show that APOOL is a mitochondrial membrane protein facing the intermembrane space. It specifically binds to cardiolipin in vitro but not to the precursor lipid phosphatidylglycerol. Overexpression of APOOL led to fragmentation of mitochondria, a reduced basal oxygen consumption rate, and altered cristae morphology. Downregulation of APOOL impaired mitochondrial respiration and caused major alterations in cristae morphology. We further show that APOOL physically interacts with several subunits of the MINOS complex, namely Mitofilin, MINOS1, and SAMM50. We conclude that APOOL is a cardiolipin-binding component of the Mitofilin/MINOS protein complex determining cristae morphology in mammalian mitochondria. Our findings further assign an intracellular role to a member of the apolipoprotein family in mammals.
Nanoplastics affect the inflammatory cytokine release by primary human monocytes and dendritic cells
(2022)
So far, the human health impacts of nano- and microplastics are poorly understood. Thus, we investigated whether nanoplastics exposure induces inflammatory processes in primary human monocytes and monocyte-derived dendritic cells. We exposed these cells in vitro to nanoplastics of different shapes (irregular vs. spherical), sizes (50–310 nm and polydisperse mixtures) and polymer types (polystyrene; polymethyl methacrylate; polyvinyl chloride, PVC) using concentrations of 30–300 particles cell−1. Our results show that irregular PVC particles induce the strongest cytokine release of these nanoplastics. Irregular polystyrene triggered a significantly higher pro-inflammatory response compared to spherical nanoplastics. The contribution of chemicals leaching from the particles was minor. The effects were concentration-dependent but varied markedly between cell donors. We conclude that nanoplastics exposure can provoke human immune cells to secrete cytokines as key initiators of inflammation. This response is specific to certain polymers (PVC) and particle shapes (fragments). Accordingly, nanoplastics cannot be considered one homogenous entity when assessing their health implications and the use of spherical polystyrene nanoplastics may underestimate their inflammatory effects.
The interaction of microplastics with freshwater biota and their interaction with other stressors is still not very well understood. Therefore, we investigated the ingestion, excretion and toxicity of microplastics in the freshwater gastropod Lymnaea stagnalis.
MP ingestion was analyzed as tissues levels in L. stagnalis after 6–96 h of exposure to 5–90 μm spherical polystyrene (PS) microplastics. To understand the excretion, tissue levels were determined after 24 h of exposure followed by a 12 h–7 d depuration period. To assess the toxicity, snails were exposed for 28 d to irregular PS microplastics (<63 μm, 6.4–100,000 particles mL−1), both alone and in combination with copper as additional stressor. To compare the toxicity of natural and synthetic particles, we also included diatomite particles. Microplastics ingestion and excretion significantly depended on the particle size and the exposure/depuration duration. An exposure to irregular PS had no effect on survival, reproduction, energy reserves and oxidative stress. However, we observed slight effects on immune cell phagocytosis. Exposure to microplastics did not exacerbate the reproductive toxicity of copper. In addition, there was no pronounced difference between the effects of microplastics and diatomite. The tolerance towards microplastics may originate from an adaptation of L. stagnalis to particle-rich environments or a general stress resilience. In conclusion, despite high uptake rates, PS fragments do not appear to be a relevant stressor for stress tolerant freshwater gastropods considering current environmental levels of microplastics.
he ubiquity of microplastics in aquatic ecosystems has raised concerns over their interaction with biota. However,microplastics research on freshwater species, especially mollusks, is still scarce. We, therefore, investigated the factorsaffecting microplastics ingestion in the freshwater musselDreissena polymorpha. Using polystyrene spheres (5, 10, 45,90μm), we determined the body burden of microplastics in the mussels in relation to 1) exposure and depuration time, 2)body size, 3) food abundance, and 4) microplastic concentrations.D. polymorpharapidly ingested microplastics and ex-creted most particles within 12 h. A few microplastics were retained for up to 1 wk. Smaller individuals had a higher relativebody burden of microplastics than larger individuals. The uptake of microplastics was concentration‐dependent, whereas anadditional food supply (algae) reduced it. We also compared the ingestion of microplastics byD. polymorphawith 2 otherfreshwater species (Anodonta anatina,Sinanodonta woodiana), highlighting that absolute and relative uptake depends onthe species and the size of the mussels. In addition, we determined toxicity of polystyrene fragments (≤63μm,6.4–100 000 p mL–1) and diatomite (natural particle, 100 000 p mL–1)inD. polymorphaafter 1, 3, 7, and 42 d of exposure,investigating clearance rate, energy reserves, and oxidative stress. Despite ingesting large quantities, exposure to poly-styrene fragments only affected the clearance rate ofD. polymorpha. Further, results of the microplastic and diatomiteexposure did not differ significantly. Therefore,D. polymorphais unaffected by or can compensate for polystyrene fragmenttoxicity even at concentrations above current environmental levels.Environ Toxicol Chem2021;40:2247–2260. © 2021 TheAuthors.Environmental Toxicology and Chemistrypublished by Wiley Periodicals LLC on behalf of SETAC.Keywords:Microplastics; Toxic effects; Mollusk toxicity
Non-ribosomal peptide synthetases (NRPSs) are large multienzyme machineries. They synthesize numerous important natural products starting from amino acids. For peptide synthesis functionally specialized NRPS modules interact in a defined manner. Individual modules are either located on a single or on multiple different polypeptide chains. The “peptide-antimicrobial-Xenorhabdus” (PAX) peptide producing NRPS PaxS from Xenorhabdus bacteria consists of the three proteins PaxA, PaxB and PaxC. Different docking domains (DDs) located at the N-termini of PaxB and PaxC and at the C-termini of PaxA and BaxB mediate specific non-covalent interactions between them. The N-terminal docking domains precede condensation domains while the C-terminal docking domains follow thiolation domains. The binding specificity of individual DDs is important for the correct assembly of multi-protein NRPS systems. In many multi-protein NRPS systems the docking domains are sufficient to mediate the necessary interactions between individual protein chains. However, it remains unclear if this is a general feature for all types of structurally different docking domains or if the neighboring domains in some cases support the function of the docking domains. Here, we report the 1H, 13C and 15 N NMR resonance assignments for a C-terminal di-domain construct containing a thiolation (T) domain followed by a C-terminal docking domain (CDD) from PaxA and for its binding partner – the N-terminal docking domain (NDD) from PaxB from the Gram-negative entomopathogenic bacterium Xenorhabdus cabanillasii JM26 in their free states and for a 1:1 complex formed by the two proteins. These NMR resonance assignments will facilitate further structural and dynamic studies of this protein complex.
Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains (N/CDDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of “designer” NRPS from proteins that normally do not interact. The multiprotein “peptide-antimicrobial-Xenorhabdus” (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small CDD of PaxA cooperates with its preceding thiolation (T1) domain to bind the NDD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.