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Enzyme cascade reactions
(2015)
The rise of evolutionary novelties is one of the major drivers of evolutionary diversification. African weakly-electric fishes (Teleostei, Mormyridae) have undergone an outstanding adaptive radiation, putatively owing to their ability to communicate through species-specific Electric Organ Discharges (EODs) produced by a novel, muscle-derived electric organ. Indeed, such EODs might have acted as effective pre-zygotic isolation mechanisms, hence favoring ecological speciation in this group of fishes. Despite the evolutionary importance of this organ, genetic investigations regarding its origin and function have remained limited.
The ultimate aim of this study is to better understand the genetic basis of EOD production by exploring the transcriptomic profiles of the electric organ and of its ancestral counterpart, the skeletal muscle, in the genus Campylomormyrus. After having established a set of reference transcriptomes using “Next-Generation Sequencing” (NGS) technologies, I performed in silico analyses of differential expression, in order to identify sets of genes that might be responsible for the functional differences observed between these two kinds of tissues. The results of such analyses indicate that: i) the loss of contractile activity and the decoupling of the excitation-contraction processes are reflected by the down-regulation of the corresponding genes in the electric organ; ii) the metabolic activity of the electric organ might be specialized towards the production and turnover of membrane structures; iii) several ion channels are highly expressed in the electric organ in order to increase excitability, and iv) several myogenic factors might be down-regulated by transcription repressors in the EO.
A secondary task of this study is to improve the genus level phylogeny of Campylomormyrus by applying new methods of inference based on the multispecies coalescent model, in order to reduce the conflict among gene trees and to reconstruct a phylogenetic tree as closest as possible to the actual species-tree. By using 1 mitochondrial and 4 nuclear markers, I was able to resolve the phylogenetic relationships among most of the currently described Campylomormyrus species. Additionally, I applied several coalescent-based species delimitation methods, in order to test the hypothesis that putatively cryptic species, which are distinguishable only from their EOD, belong to independently evolving lineages. The results of this analysis were additionally validated by investigating patterns of diversification at 16 microsatellite loci. The results suggest the presence of a new, yet undescribed species of Campylomormyrus.
Background African weakly-electric fishes of the family Mormyridae are able to produce and perceive weak electric signals (typically less than one volt in amplitude) owing to the presence of a specialized, muscle-derived electric organ (EO) in their tail region. Such electric signals, also known as Electric Organ Discharges (EODs), are used for objects/prey localization, for the identification of conspecifics, and in social and reproductive behaviour. This feature might have promoted the adaptive radiation of this family by acting as an effective pre-zygotic isolation mechanism. Despite the physiological and evolutionary importance of this trait, the investigation of the genetic basis of its function and modification has so far remained limited. In this study, we aim at: i) identifying constitutive differences in terms of gene expression between electric organ and skeletal muscle (SM) in two mormyrid species of the genus Campylomormyrus: C. compressirostris and C. tshokwe, and ii) exploring cross-specific patterns of gene expression within the two tissues among C. compressirostris, C. tshokwe, and the outgroup species Gnathonemus petersii. Results Twelve paired-end (100 bp) strand-specific RNA-seq Illumina libraries were sequenced, producing circa 330 M quality-filtered short read pairs. The obtained reads were assembled de novo into four reference transcriptomes. In silico cross-tissue DE-analysis allowed us to identify 271 shared differentially expressed genes between EO and SM in C. compressirostris and C.tshokwe. Many of these genes correspond to myogenic factors, ion channels and pumps, and genes involved in several metabolic pathways. Cross-species analysis has revealed that the electric organ transcriptome is more variable in terms of gene expression levels across species than the skeletal muscle transcriptome. Conclusions The data obtained indicate that: i) the loss of contractile activity and the decoupling of the excitation-contraction processes are reflected by the down-regulation of the corresponding genes in the electric organ’s transcriptome; ii) the metabolic activity of the EO might be specialized towards the production and turn-over of membrane structures; iii) several ion channels are highly expressed in the EO in order to increase excitability; iv) several myogenic factors might be down-regulated by transcription repressors in the EO.
Background: African weakly-electric fishes of the family Mormyridae are able to produce and perceive weak electric signals (typically less than one volt in amplitude) owing to the presence of a specialized, muscle-derived electric organ (EO) in their tail region. Such electric signals, also known as Electric Organ Discharges (EODs), are used for objects/prey localization, for the identification of conspecifics, and in social and reproductive behaviour. This feature might have promoted the adaptive radiation of this family by acting as an effective pre-zygotic isolation mechanism. Despite the physiological and evolutionary importance of this trait, the investigation of the genetic basis of its function and modification has so far remained limited. In this study, we aim at: i) identifying constitutive differences in terms of gene expression between electric organ and skeletal muscle (SM) in two mormyrid species of the genus Campylomormyrus: C. compressirostris and C. tshokwe, and ii) exploring cross-specific patterns of gene expression within the two tissues among C. compressirostris, C. tshokwe, and the outgroup species Gnathonemus petersii.
Results: Twelve paired-end (100 bp) strand-specific RNA-seq Illumina libraries were sequenced, producing circa 330 M quality-filtered short read pairs. The obtained reads were assembled de novo into four reference transcriptomes. In silico cross-tissue DE-analysis allowed us to identify 271 shared differentially expressed genes between EO and SM in C. compressirostris and C. tshokwe. Many of these genes correspond to myogenic factors, ion channels and pumps, and genes involved in several metabolic pathways. Cross-species analysis has revealed that the electric organ transcriptome is more variable in terms of gene expression levels across species than the skeletal muscle transcriptome.
Conclusions: The data obtained indicate that: i) the loss of contractile activity and the decoupling of the excitation-contraction processes are reflected by the down-regulation of the corresponding genes in the electric organ's transcriptome; ii) the metabolic activity of the EO might be specialized towards the production and turn-over of membrane structures; iii) several ion channels are highly expressed in the EO in order to increase excitability; iv) several myogenic factors might be down-regulated by transcription repressors in the EO.
The electric organ (EO) of weakly electric mormyrids consists of flat, disk-shaped electrocytes with distinct anterior and posterior faces. There are multiple species-characteristic patterns in the geometry of the electrocytes and their innervation. To further correlate electric organ discharge (EOD) with EO anatomy, we examined four species of the mormyrid genus Campylomormyrus possessing clearly distinct EODs. In C. compressirostris, C. numenius, and C. tshokwe, all of which display biphasic EODs, the posterior face of the electrocytes forms evaginations merging to a stalk system receiving the innervation. In C. tamandua that emits a triphasic EOD, the small stalks of the electrocyte penetrate the electrocyte anteriorly before merging on the anterior side to receive the innervation. Additional differences in electrocyte anatomy among the former three species with the same EO geometry could be associated with further characteristics of their EODs. Furthermore, in C. numenius, ontogenetic changes in EO anatomy correlate with profound changes in the EOD. In the juvenile the anterior face of the electrocyte is smooth, whereas in the adult it exhibits pronounced surface foldings. This anatomical difference, together with disparities in the degree of stalk furcation, probably contributes to the about 12 times longer EOD in the adult.
Audition in bats serves passive orientation, alerting functions and communication as it does in other vertebrates. In addition, bats have evolved echolocation for orientation and prey detection and capture. This put a selective pressure on the auditory system in regard to echolocation-relevant temporal computation and frequency analysis. The present review attempts to evaluate in which respect the processing modules of bat auditory cortex (AC) are a model for typical mammalian AC function or are designed for echolocation-unique purposes. We conclude that, while cortical area arrangement and cortical frequency processing does not deviate greatly from that of other mammals, the echo delay time-sensitive dorsal cortex regions contain special designs for very powerful time perception. Different bat species have either a unique chronotopic cortex topography or a distributed salt-and-pepper representation of echo delay. The two designs seem to enable similar behavioural performance.
Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.
The current eukaryotic tree of life groups most eukaryotes into one of five supergroups, the Opisthokonta, Amoebozoa, Archaeplastida, Excavata and SAR (Stramenopile, Alveolata, Rhizaria). Molecular and comparative morphological analyses revealed that the last eukaryotic common ancestor (LECA) already contained a rather sophisticated equipment of organelles including a mitochondrion, an endomembrane system, a nucleus with a lamina, a microtubule-organizing center (MTOC), and a flagellar apparatus. Recent studies of MTOCs, basal bodies/centrioles, and nuclear envelope organization of organisms in different supergroups have clarified our picture of how the nucleus and MTOCs co-evolved from LECA to extant eukaryotes. In this review we summarize these findings with special emphasis on valuable contributions of research on a lamin-like protein, nuclear envelope proteins, and the MTOC in the amoebozoan model organism Dictyostelium discoideum. (C) 2015 Elsevier GmbH. All rights reserved.
Der Na⁺-K⁺-2Cl⁻-Kotransporter (NKCC2) wird im distalen Nephron der Niere exprimiert. Seine Verteilung umfasst die Epithelien der medullären und kortikalen Teile der dicken aufsteigenden Henle-Schleife (Thick ascending limb, TAL) und die Macula densa. Resorptiver NaCl-Transport über den NKCC2 dient dem renalen Konzentrierungsmechanismus und reguliert systemisch auch Volumenstatus und Blutdruck. Die Aktivität des NKCC2 ist mit der Phosphorylierung seiner N-terminalen Aminosäurereste Serin 126 und Threonin 96/101 verbunden. Vermittelt wird diese durch die homologen Kinasen SPAK (SPS-related proline/alanine-rich kinase) und OSR1 (Oxidative stress responsive kinase 1), die hierzu ihrerseits phosphoryliert werden müssen. Der regulatorische Kontext dieser Kinasen ist mittlerweile gut charakterisiert. Über Mechanismen und Produkte, die den NKCC2 deaktivieren, war hingegen weniger bekannt. Ziel der Arbeit war daher zu untersuchen, welche Wege zur Deaktivierung des Transporters führen. Der intrazelluläre Sortierungsrezeptor SORLA (Sorting-protein-related receptor with A-type repeats) war zuvor in seiner Bedeutung für das Nephron charakterisiert worden. Ein SORLA-defizientes Mausmodell weist unter anderem eine stark verringerte NKCC2-Phosphorylierung auf. Unter osmotischem Stress können SORLA-defiziente Mäuse ihren Urin weniger effizient konzentrieren. Meine Resultate zeigen mit hochauflösender Technik, dass SORLA apikal im TAL lokalisiert ist und dass mit NKCC2 eine anteilige Kolokalisation besteht. Unter SORLA Defizienz war die für die NKCC2 Aktivität maßgebliche SPAK/OSR1-Phosphorylierung gegenüber dem Wildtyp nicht verändert. Jedoch war die ebenfalls im TAL exprimierte Phosphatase Calcineurin Aβ (CnAβ) per Western blot um das zweifache gesteigert. Parallel hierzu wurde immunhistochemisch die Kolokalisation von verstärktem CnAβ-Signal und NKCC2 bestätigt. Beide Befunde geben zusammen den Hinweis auf einen Bezug zwischen der reduzierten NKCC2-Phosphorylierung und der gesteigerten Präsenz von CnAβ bei SORLA Defizienz. Die parallel induzierte Überexpression von SORLA in HEK-Zellen zeigte entsprechend eine Halbierung der CnAβ Proteinmenge. SORLA steuert demzufolge sowohl die Abundanz als auch die zelluläre Verteilung der Phosphatase. Weiterhin ließ sich die Interaktion zwischen CnAβ und SORLA (intrazelluläre Domäne) mittels Co-Immunpräzipitation bzw. GST-pulldown assay nachweisen. Auch die Interaktion zwischen CnAβ und NKCC2 wurde auf diesem Weg belegt. Da allerdings weder SORLA noch NKCC2 ein spezifisches Bindungsmuster für CnAβ aufweisen, sind vermutlich intermediäre Adapterproteine bei ihrer Bindung involviert. Die pharmakologische Inhibition von CnAβ mittels Cyclosporin A (CsA; 1 h) führte bei SORLA Defizienz zur Normalisierung der NKCC2-Phosphorylierung. Entsprechend führte in vitro die Gabe von CsA bei TAL Zellen zu einer 7-fach gesteigerten NKCC2-Phosphorylierung. Zusammenfassend zeigen die Ergebnisse, dass die Phosphatase CnAβ über ihre Assoziation mit NKCC2 diesen im adluminalen Zellkompartiment deaktivieren kann. Gesteuert wird dieser Vorgang durch die Eigenschaft von SORLA, CnAβ apikal zu reduzieren und damit die adluminale Phosphorylierung und Aktivität von NKCC2 zu unterstützen. Da Calcineurin-Inhibitoren derzeit die Grundlage der immunsupprimierenden Therapie darstellen, haben die Ergebnisse eine klinische Relevanz. Angesichts der Co-Expression von SORLA und CnAβ in verschiedenen anderen Organen können die Ergebnisse auch über die Niere hinaus Bedeutung erlangen.
Trade plays a key role in the spread of alien species and has arguably contributed to the recent enormous acceleration of biological invasions, thus homogenizing biotas worldwide. Combining data on 60-year trends of bilateral trade, as well as on biodiversity and climate, we modeled the global spread of plant species among 147 countries. The model results were compared with a recently compiled unique global data set on numbers of naturalized alien vascular plant species representing the most comprehensive collection of naturalized plant distributions currently available. The model identifies major source regions, introduction routes, and hot spots of plant invasions that agree well with observed naturalized plant numbers. In contrast to common knowledge, we show that the 'imperialist dogma,' stating that Europe has been a net exporter of naturalized plants since colonial times, does not hold for the past 60 years, when more naturalized plants were being imported to than exported from Europe. Our results highlight that the current distribution of naturalized plants is best predicted by socioeconomic activities 20 years ago. We took advantage of the observed time lag and used trade developments until recent times to predict naturalized plant trajectories for the next two decades. This shows that particularly strong increases in naturalized plant numbers are expected in the next 20 years for emerging economies in megadiverse regions. The interaction with predicted future climate change will increase invasions in northern temperate countries and reduce them in tropical and (sub) tropical regions, yet not by enough to cancel out the trade-related increase.
Introduction
The transition from cross-fertilisation (outcrossing) to self-fertilisation (selfing) frequently coincides with changes towards a floral morphology that optimises self-pollination, the selfing syndrome. Population genetic studies have reported the existence of both outcrossing and selfing populations in Arabis alpina (Brassicaceae), which is an emerging model species for studying the molecular basis of perenniality and local adaptation. It is unknown whether its selfing populations have evolved a selfing syndrome.
Methods
Using macro-photography, microscopy and automated cell counting, we compared floral syndromes (size, herkogamy, pollen and ovule numbers) between three outcrossing populations from the Apuan Alps and three selfing populations from the Western and Central Alps (Maritime Alps and Dolomites). In addition, we genotyped the plants for 12 microsatellite loci to confirm previous measures of diversity and inbreeding coefficients based on allozymes, and performed Bayesian clustering.
Results and Discussion
Plants from the three selfing populations had markedly smaller flowers, less herkogamy and lower pollen production than plants from the three outcrossing populations, whereas pistil length and ovule number have remained constant. Compared to allozymes, microsatellite variation was higher, but revealed similar patterns of low diversity and high Fis in selfing populations. Bayesian clustering revealed two clusters. The first cluster contained the three outcrossing populations from the Apuan Alps, the second contained the three selfing populations from the Maritime Alps and Dolomites.
Conclusion
We conclude that in comparison to three outcrossing populations, three populations with high selfing rates are characterised by a flower morphology that is closer to the selfing syndrome. The presence of outcrossing and selfing floral syndromes within a single species will facilitate unravelling the genetic basis of the selfing syndrome, and addressing which selective forces drive its evolution.
Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations. Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.
Cancer cachexia, of which the most notable symptom is severe and rapid weight loss, is present in the majority of patients with advanced cancer. Inflammatory mediators play an important role in the development of cachexia, envisaged as a chronic inflammatory syndrome. The white adipose tissue (WAT) is one of the first compartments affected in cancer cachexia and suffers a high rate of lipolysis. It secretes several cytokines capable of directly regulating intermediate metabolism. A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-B). We have examined the gene expression of the subunits NF-Bp65 and NF-Bp50, as well as NF-Bp65 and NF-Bp50 binding, the gene expression of pro-inflammatory mediators under NF-B control (IL-1, IL-6, INF-, TNF-, MCP-1), and its inhibitory protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IB-). The observational study involved 35 patients (control group, n = 12 and cancer group, n = 23, further divided into cachectic and non-cachectic). NF-Bp65 and its target genes expression (TNF-, IL-1, MCP-1 and IB-) were significantly higher in cachectic cancer patients. Moreover, NF-Bp65 gene expression correlated positively with the expression of its target genes. The results strongly suggest that the NF-B pathway plays a role in the promotion of WAT inflammation during cachexia.
Non-alcoholic fatty liver disease is a growing problem in industrialized and developing countries. Hepatic lipid accumulation is the result of an imbalance between fatty acid uptake, fatty acid de novo synthesis, beta-oxidation and secretion of triglyceride-rich lipoproteins from the hepatocyte. A central regulator of hepatic lipid metabolism is cytosolic citrate that can either be derived from the mitochondrium or be taken up from the blood via the plasma membrane sodium citrate transporter NaCT, the product of the mammalian INDY gene (SLC13A5). mINDY ablation protects against diet-induced steatosis whereas mINDY expression is increased in patients with hepatic steatosis. Diet-induced hepatic steatosis is also enhanced by activation of the arylhyrocarbon receptor (AhR) both in humans and animal models. Therefore, the hypothesis was tested whether the mINDY gene might be a target of the AhR. In accordance with such a hypothesis, the AhR activator benzo[a]pyrene induced the mINDY expression in primary cultures of rat hepatocytes in an AhR-dependent manner. This induction resulted in an increased citrate uptake and citrate incorporation into lipids which probably was further enhanced by the benzo[a]pyrene-dependent induction of key enzymes of fatty acid synthesis. A potential AhR binding site was identified in the mINDY promoter that appears to be conserved in the human promoter. Elimination or mutation of this site largely abolished the activation of the mINDY promoter by benzo[a]pyrene. This study thus identified the mINDY as an AhR target gene. AhR-dependent induction of the mINDY gene might contribute to the development of hepatic steatosis. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
Plant community assembly at small scales: Spatial vs. environmental factors in a European grassland
(2015)
Dispersal limitation and environmental conditions are crucial drivers of plant species distribution and establishment. As these factors operate at different spatial scales, we asked: Do the environmental factors known to determine community assembly at broad scales operate at fine scales (few meters)? How much do these factors account for community variation at fine scales? In which way do biotic and abiotic interactions drive changes in species composition?
We surveyed the plant community within a dry grassland along a very steep gradient of soil characteristics like pH and nutrients. We used a spatially explicit sampling design, based on three replicated macroplots of 15 x 15, 12 x 12 and 12 x 12 m in extent. Soil samples were taken to quantify several soil properties (carbon, nitrogen, plant available phosphorus, pH, water content and dehydrogenase activity as a proxy for overall microbial activity). We performed variance partitioning to assess the effect of these variables on plant composition and statistically controlled for spatial autocorrelation via eigenvector mapping. We also applied null model analysis to test for non-random patterns in species co-occurrence using randomization schemes that account for patterns expected under species interactions.
At a fine spatial scale, environmental factors explained 18% of variation when controlling for spatial autocorrelation in the distribution of plant species, whereas purely spatial processes accounted for 14% variation. Null model analysis showed that species spatially segregated in a non-random way and these spatial patterns could be due to a combination of environmental filtering and biotic interactions. Our grassland study suggests that environmental factors found to be directly relevant in broad scale studies are present also at small scales, but are supplemented by spatial processes and more direct interactions like competition. (C) 2015 Elsevier Masson SAS. All rights reserved.
Magnetite nanoparticles and their assembly comprise a new area of development for new technologies. The magnetic particles can interact and assemble in chains or networks. Magnetotactic bacteria are one of the most interesting microorganisms, in which the assembly of nanoparticles occurs. These microorganisms are a heterogeneous group of gram negative prokaryotes, which all show the production of special magnetic organelles called magnetosomes, consisting of a magnetic nanoparticle, either magnetite (Fe3O4) or greigite (Fe3S4), embedded in a membrane. The chain is assembled along an actin-like scaffold made of MamK protein, which makes the magnetosomes to arrange in mechanically stable chains. The chains work as a compass needle in order to allow cells to orient and swim along the magnetic field of the Earth.
The formation of magnetosomes is known to be controlled at the molecular level. The physico–chemical conditions of the surrounding environment also influence biomineralization. The work presented in this manuscript aims to understand how such external conditions, in particular the extracellular oxidation reduction potential (ORP) influence magnetite formation in the strain Magnetospirillum magneticum AMB-1. A controlled cultivation of the microorganism was developed in a bioreactor and the formation of magnetosomes was characterized.
Different techniques have been applied in order to characterize the amount of iron taken up by the bacteria and in consequence the size of magnetosomes produced at different ORP conditions. By comparison of iron uptake, morphology of bacteria, size and amount of magnetosomes per cell at different ORP, the formation of magnetosomes was inhibited at ORP 0 mV, whereas reduced conditions, ORP – 500 mV facilitate biomineralization process.
Self-assembly of magnetosomes occurring in magnetotactic bacteria became an inspiration to learn from nature and to construct nanoparticles assemblies by using the bacteriophage M13 as a template. The M13 bacteriophage is an 800 nm long filament with encapsulated single-stranded DNA that has been recently used as a scaffold for nanoparticle assembly. I constructed two types of assemblies based on bacteriophages and magnetic nanoparticles. A chain – like assembly was first formed where magnetite nanoparticles are attached along the phage filament. A sperm – like construct was also built with a magnetic head and a tail formed by phage filament.
The controlled assembly of magnetite nanoparticles on the phage template was possible due to two different mechanism of nanoparticle assembly. The first one was based on the electrostatic interactions between positively charged polyethylenimine coated magnetite nanoparticles and negatively charged phages. The second phage –nanoparticle assembly was achieved by bioengineered recognition sites. A mCherry protein is displayed on the phage and is was used as a linker to a red binding nanobody (RBP) that is fused to the one of the proteins surrounding the magnetite crystal of a magnetosome.
Both assemblies were actuated in water by an external magnetic field showing their swimming behavior and potentially enabling further usage of such structures for medical applications. The speed of the phage - nanoparticles assemblies are relatively slow when compared to those of microswimmers previously published. However, only the largest phage-magnetite assemblies could be imaged and it is therefore still unclear how fast these structures can be in their smaller version.
Transitory starch metabolism is a nonlinear and highly regulated process. It originated very early in the evolution of chloroplast-containing cells and is largely based on a mosaic of genes derived from either the eukaryotic host cell or the prokaryotic endosymbiont. Initially located in the cytoplasm, starch metabolism was rewired into plastids in Chloroplastida. Relocation was accompanied by gene duplications that occurred in most starch-related gene families and resulted in subfunctionalization of the respective gene products. Starch-related isozymes were then evolutionary conserved by constraints such as internal starch structure, posttranslational protein import into plastids and interactions with other starch-related proteins. 25 starch-related genes in 26 accessions of Arabidopsis thaliana were sequenced to assess intraspecific diversity, phylogenetic relationships, and modes of selection. Furthermore, sequences derived from additional 80 accessions that are publicly available were analyzed. Diversity varies significantly among the starch-related genes. Starch synthases and phosphorylases exhibit highest nucleotide diversities, while pyrophosphatases and debranching enzymes are most conserved. The gene trees are most compatible with a scenario of extensive recombination, perhaps in a Pleistocene refugium. Most genes are under purifying selection, but disruptive selection was inferred for a few genes/substitutiones. To study transcript levels, leaves were harvested throughout the light period. By quantifying the transcript levels and by analyzing the sequence of the respective accessions, we were able to estimate whether transcript levels are mainly determined by genetic (i.e., accession dependent) or physiological (i.e., time dependent) parameters. We also identified polymorphic sites that putatively affect pattern or the level of transcripts.
Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'(SHG)) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'(SHG) values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices. The maximum SHG intensity occurs at the highest moisture and amylopectin content. These results support the hypothesis that SHG is caused by ordered hydrogen and hydroxyl bond networks which increase with hydration of starch granules. (C) 2015 Optical Society of America
Photosynthetic acclimation of phytoplankton to lower irradiation can be met by several strategies such as increasing the affinity for light or increasing antenna size and stacking of the thylakoids. The latter is reflected by a higher proportion of polyunsaturated fatty acids (PUFAs). Additionally, photosynthetic capacity (P-max), respiratory losses, and proton leakage can be reduced under low light. Here we consider the effect of light intensity and phosphorus availability simultaneously on the photosynthetic acclimation and fatty acid composition of four phytoplankters. We studied representatives of the Chlorophyceae, Cryptophyceae and Mediophyceae, all of which are important components of plankton communities in temperate lakes. In our analysis, excluding fatty acid composition, we found different acclimation strategies in the chlorophytes Scenedesmus quadricauda, Chlamydomonas globosa, cryptophyte Cryptomonas ovata and ochrophyte Cyclotella meneghiniana. We observed interactive effects of light and phosphorus conditions on photosynthetic capacity in S. quadricauda and Cry. ovata. Cry. ovata can be characterized as a low light-acclimated species, whereas S. quadricauda and Cyc. meneghiniana can cope best with a combination of high light intensities and low phosphorus supply. Principal component analyses (PCA), including fatty acid composition, showed further species-specific patterns in their regulation of P-max with PUFAs and light. In S. quadricauda and Cyc. meneghiniana, PUFAs negatively affected the relationship between P-max and light. In Chl. globosa, lower light coincided with higher PUFAs and lower P-max, but PCA also indicated that PUFAs had no direct influence on P-max. PUFAs and P-max were unaffected by light in Cry. ovata. We did not observe a general trend in the four species tested and concluded that, in particular, the interactive effects highlight the importance of taking into account more than one environmental factor when assessing photosynthetic acclimation to lower irradiation.
Long-chain polyunsaturated fatty acids (LC-PUFA) are critical for the health of aquatic and terrestrial organisms; therefore, understanding the production, distribution, and abundance of these compounds is imperative. Although the dynamics of LC-PUFA production and distribution in aquatic environments has been well documented, a systematic and comprehensive comparison to LC-PUFA in terrestrial environments has not been rigorously investigated. Here we use a data synthesis approach to compare and contrast fatty acid profiles of 369 aquatic and terrestrial organisms. Habitat and trophic level were interacting factors that determined the proportion of individual omega-3 (n-3) or omega-6 (n-6) PUFA in aquatic and terrestrial organisms. Higher total n-3 content compared with n-6 PUFA and a strong prevalence of the n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) characterized aquatic versus terrestrial organisms. Conversely, terrestrial organisms had higher linoleic acid (LNA) and alpha-linolenic acid (ALA) contents than aquatic organisms; however, the ratio of ALA: LNA was higher in aquatic organisms. The EPA + DHA content was higher in aquatic animals than terrestrial organisms, and increased from algae to invertebrates to vertebrates in the aquatic environment. An analysis of covariance (ANCOVA) revealed that fatty acid composition was highly dependent on the interaction between habitat and trophic level. We conclude that freshwater ecosystems provide an essential service through the production of n-3 LC-PUFA that are required to maintain the health of terrestrial organisms including humans.
Previous studies examining the effects of food quality on zooplankton often controlled for maternal effects of resource provisioning using standardized maternal diets. However, varying nutritional history of mothers may change resource provisioning to their progeny, especially regarding polyunsaturated fatty acids (PUFAs), which may change the interpretation of previously observed fitness responses of offspring. To assess PUFA-mediated maternal provisioning effects on offspring, we raised females of the cladoceran Daphnia magna on diets differing considerably in PUFA composition and raised their offspring on a PUFA-lacking diet supplemented with the omega 3 PUFAs alpha-linolenic acid (ALA) and/or eicosapentaenoic acid (EPA). The mass-specific growth responses of offspring to their own diets were affected by the maternal diet regime, probably due to varying maternal PUFA provisioning. A low maternal provisioning of EPA or ALA was sufficient to prevent growth limitation of offspring by these PUFAs until reaching maturity. A comparison with results of published ALA and EPA supplementation experiments suggests that the previously observed limitation effects depended on the usage of a single algae genus as maternal diet. Therefore, we suggest that maternal diets should be deliberately varied in future studies assessing ecological relevant food quality effects on zooplankton, especially regarding PUFAs.
The underlying mechanisms and consequences of competition and diversity are central themes in ecology. A higher diversity of primary producers often results in higher resource use efficiency in aquatic and terrestrial ecosystems. This may result in more food for consumers on one hand, while, on the other hand, it can also result in a decreased food quality for consumers; higher biomass combined with the same availability of the limiting compound directly reduces the dietary proportion of the limiting compound. Here we tested whether and how interspecific competition in phytoplankton communities leads to changes in resource use efficiency and cellular concentrations of nutrients and fatty acids. The measured particulate carbon : phosphorus ratios (C:P) and fatty acid concentrations in the communities were compared to the theoretically expected ratios and concentrations of measurements on simultaneously running monocultures. With interspecific competition, phytoplankton communities had higher concentrations of the monounsaturated fatty acid oleic acid and also much higher concentrations of the ecologically and physiologically relevant long-chain polyunsaturated fatty acid eicosapentaenoic acid than expected concentrations based on monocultures. Such higher availability of essential fatty acids may contribute to the positive relationship between phytoplankton diversity and zooplankton growth, and may compensate limitations by mineral nutrients in higher trophic levels.
In freshwater systems, Daphnia has been demonstrated to show adaptive responses following the light-dark cycle. The adjustment of these responses to the change of day and night is probably transmitted via the hormone melatonin. The rate-limiting enzyme in melatonin synthesis is the arylalkylamine N-transferase (AANAT). We identified three genes coding for insect-like AANATs in Daphnia, of which we measured the gene expression in an ecologically relevant light-dark cycle. We demonstrated that Daphnia's insect-like AANAT gene expression oscillated in a daily manner, and that the highest peak of expression after the onset of darkness was followed by a peak of melatonin production at midnight. Moreover, we could show an oscillation of endogenous melatonin synthesis in Daphnia. In most organisms, melatonin synthesis is due to rhythmic expression of genes of the circadian clock, since transcription of aanats is directly linked to a circadian transcription factor. We could demonstrate that putative clock genes and insect-like AANAT genes of Daphnia were equally expressed. Therefore, we propose that melatonin synthesis is coupled to the expression of Daphnia clock genes, and that insect-like AANATs of crustaceans have a similar function as AANATs of vertebrates: The initiation of melatonin synthesis. In future studies with Daphnia, it will be necessary to take the time of day into account since melatonin concentrations might influence stress responses.
Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor beta 1 integrin and occurs in the absence of blood flow. Downregulation of beta 1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a beta 1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.
The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.
Application of hybridisation capture to investigate complete mitogenomes from ancient samples
(2015)
We extend the scope of European palaeogenomics by sequencing the genomes of Late Upper Palaeolithic (13,300 years old, 1.4-fold coverage) and Mesolithic (9,700 years old, 15.4-fold) males from western Georgia in the Caucasus and a Late Upper Palaeolithic (13,700 years old, 9.5-fold) male from Switzerland. While we detect Late Palaeolithic–Mesolithic genomic continuity in both regions, we find that Caucasus hunter-gatherers (CHG) belong to a distinct ancient clade that split from western hunter-gatherers ∼45 kya, shortly after the expansion of anatomically modern humans into Europe and from the ancestors of Neolithic farmers ∼25 kya, around the Last Glacial Maximum. CHG genomes significantly contributed to the Yamnaya steppe herders who migrated into Europe ∼3,000 BC, supporting a formative Caucasus influence on this important Early Bronze age culture. CHG left their imprint on modern populations from the Caucasus and also central and south Asia possibly marking the arrival of Indo-Aryan languages.
We extend the scope of European palaeogenomics by sequencing the genomes of Late Upper Palaeolithic (13,300 years old, 1.4-fold coverage) and Mesolithic (9,700 years old, 15.4-fold) males from western Georgia in the Caucasus and a Late Upper Palaeolithic (13,700 years old, 9.5-fold) male from Switzerland. While we detect Late Palaeolithic-Mesolithic genomic continuity in both regions, we find that Caucasus hunter-gatherers (CHG) belong to a distinct ancient clade that split from western hunter-gatherers similar to 45 kya, shortly after the expansion of anatomically modern humans into Europe and from the ancestors of Neolithic farmers similar to 25 kya, around the Last Glacial Maximum. CHG genomes significantly contributed to the Yamnaya steppe herders who migrated into Europe similar to 3,000 BC, supporting a formative Caucasus influence on this important Early Bronze age culture. CHG left their imprint on modern populations from the Caucasus and also central and south Asia possibly marking the arrival of Indo-Aryan languages.
Cardiovascular metallic stents established in clinical application are typically coated by a thin polymeric layer on the stent struts to improve hemocompatibility, whereby often a drug is added to the coating to inhibit neointimal hyperplasia. Besides such thin film coatings recently nano/microfiber coated stents are investigated, whereby the fibrous coating was applied circumferential on stents. Here, we explored whether a thin fibrous encasement of metallic stents with preferentially longitudinal aligned fibers and different local fiber densities can be achieved by electrospinning. An elastic degradable copolyetheresterurethane, which is reported to selectively enhance the adhesion of endothelial cells, while simultaneously rejecting smooth muscle cells, was utilized for stent coating. The fibrous stent encasements were microscopically assessed regarding their single fiber diameters, fiber covered area and fiber alignment at three characteristic stent regions before and after stent expansion. Stent coatings with thicknesses in the range from 30 to 50 mu m were achieved via electrospinning with 1,1,1,3,3,3-hexafluoro-2-propanol (HFP)-based polymer solution, while a mixture of HFP and formic acid as solvent resulted in encasements with a thickness below 5 mu m comprising submicron sized single fibers. All polymeric encasements were mechanically stable during expansion, whereby the fibers deposited on the struts remained their position. The observed changes in fiber density and diameter indicated diverse local deformation mechanisms of the microfibers at the different regions between the struts. Based on these results it can be anticipated that the presented fibrous encasement of stents might be a promising alternative to stents with polymeric strut coatings releasing anti-proliferative drugs. Copyright (c) 2015 John Wiley & Sons, Ltd.
The standing stock and production of organismal biomass depends strongly on the organisms’ biotic environment, which arises from trophic and non-trophic interactions among them. The trophic interactions between the different groups of organisms form the food web of an ecosystem, with the autotrophic and bacterial production at the basis and potentially several levels of consumers on top of the producers. Feeding interactions can regulate communities either by severe grazing pressure or by shortage of resources or prey production, termed top-down and bottom-up control, respectively. The limitations of all communities conglomerate in the food web regulation, which is subject to abiotic and biotic forcing regimes arising from external and internal constraints. This dissertation presents the effects of alterations in two abiotic, external forcing regimes, terrestrial matter input and long-lasting low temperatures in winter. Diverse methodological approaches, a complex ecosystem model study and the analysis of two whole-lake measurements, were performed to investigate effects for the food web regulation and the resulting consequences at the species, community and ecosystem scale. Thus, all types of organisms, autotrophs and heterotrophs, at all trophic levels were investigated to gain a comprehensive overview of the effects of the two mentioned altered forcing regimes. In addition, an extensive evaluation of the trophic interactions and resulting carbon fluxes along the pelagic and benthic food web was performed to display the efficiencies of the trophic energy transfer within the food webs. All studies were conducted in shallow lakes, which is worldwide the most abundant type of lakes. The specific morphology of shallow lakes allows that the benthic production contributes substantially to the whole-lake production. Further, as shallow lakes are often small they are especially sensitive to both, changes in the input of terrestrial organic matter and the atmospheric temperature. Another characteristic of shallow lakes is their appearance in alternative stable states. They are either in a clear-water or turbid state, where macrophytes and phytoplankton dominate, respectively. Both states can stabilize themselves through various mechanisms.
These two alternative states and stabilizing mechanisms are integrated in the complex ecosystem model PCLake, which was used to investigate the effects of the enhanced terrestrial particulate organic matter (t-POM) input to lakes. The food web regulation was altered by three distinct pathways: (1) Zoobenthos received more food, increased in biomass which favored benthivorous fish and those reduced the available light due to bioturbation. (2) Zooplankton substituted autochthonous organic matter in their diet by suspended t-POM, thus the autochthonous organic matter remaining in the water reduced its transparency. (3) T-POM suspended into the water and reduced directly the available light. As macrophytes are more light-sensitive than phytoplankton they suffered the most from the lower transparency. Consequently, the resilience of the clear-water state was reduced by enhanced t-POM inputs, which makes the turbid state more likely at a given nutrient concentration. In two subsequent winters long-lasting low temperatures and a concurrent long duration of ice coverage was observed which resulted in low overall adult fish biomasses in the two study lakes – Schulzensee and Gollinsee, characterized by having and not having submerged macrophytes, respectively. Before the partial winterkill of fish Schulzensee allowed for a higher proportion of piscivorous fish than Gollinsee. However, the partial winterkill of fish aligned both communities as piscivorous fish are more sensitive to low oxygen concentrations. Young of the year fish benefitted extremely from the absence of adult fish due to lower predation pressure. Therefore, they could exert a strong top-down control on crustaceans, which restructured the entire zooplankton community leading to low crustacean biomasses and a community composition characterized by copepodites and nauplii. As a result, ciliates were released from top-down control, increased to high biomasses compared to lakes of various trophic states and depths and dominated the zooplankton community. While being very abundant in the study lakes and having the highest weight specific grazing rates among the zooplankton, ciliates exerted potentially a strong top-down control on small phytoplankton and particle-attached bacteria. This resulted in a higher proportion of large phytoplankton compared to other lakes. Additionally, the phytoplankton community was evenly distributed presumably due to the numerous fast growing and highly specific ciliate grazers. Although, the pelagic food web was completely restructured after the subsequent partial winterkills of fish, both lakes were resistant to effects of this forcing regime at the ecosystem scale. The consistently high predation pressure on phytoplankton prevented that Schulzensee switched from the clear-water to the turbid state. Further mechanisms, which potentially stabilized the clear-water state, were allelopathic effects by macrophytes and nutrient limitation in summer. The pelagic autotrophic and bacterial production was an order of magnitude more efficient transferred to animal consumers than the respective benthic production, despite the alterations of the food web structure after the partial winterkill of fish. Thus, the compiled mass-balanced whole-lake food webs suggested that the benthic bacterial and autotrophic production, which exceeded those of the pelagic habitat, was not used by animal consumers. This holds even true if the food quality, additional consumers such as ciliates, benthic protozoa and meiobenthos, the pelagic-benthic link and the potential oxygen limitation of macrobenthos were considered. Therefore, low benthic efficiencies suggest that lakes are primarily pelagic systems at least at the animal consumer level.
Overall, this dissertation gives insights into the regulation of organism groups in the pelagic and benthic habitat at each trophic level under two different forcing regimes and displays the efficiency of the carbon transfer in both habitats. The results underline that the alterations of external forcing regimes affect all hierarchical level including the ecosystem.
A straightforward synthesis strategy to multimerize a peptide mimotopes for antibody B13-DE1 recognition is described based on lysine dendrons as multivalent scaffolds. Lysine dendrons that possess N-terminal alkyne residues at the periphery were quantitative functionalized with azido peptides using click chemistry. The solid-phase peptide synthesis (SPPS) allows preparing the peptide dendron in high purity and establishing the possibility of automation. The presented peptide dendron is a promising candidate as multivalent ligand and was used for antibody B13-DE1 recognition. The binding affinity increases with higher dendron generation without loss of specificity. The analysis of biospecific interaction between the synthesized peptide dendron and the antibody was done via surface plasmon resonance (SPR) technique. The presented results show a promising tool for investigations of antigen-antibody reactions.
The recently extinct (ca. 1768) Steller's sea cow (Hydrodamalis gigas) was a large, edentulous North Pacific sirenian. The phylogenetic affinities of this taxon to other members of this clade, living and extinct, are uncertain based on previous morphological and molecular studies. We employed hybridization capture methods and second generation sequencing technology to obtain >30 kb of exon sequences from 26 nuclear genes for both H. gigas and Dugong dugon. We also obtained complete coding sequences for the tooth-related enamelin (ENAM) gene. Hybridization probes designed using dugong and manatee sequences were both highly effective in retrieving sequences from H. gigas (mean = 98.8% coverage), as were more divergent probes for regions of ENAM (99.0% coverage) that were designed exclusively from a proboscidean (African elephant) and a hyracoid (Cape hyrax). New sequences were combined with available sequences for representatives of all other afrotherian orders. We also expanded a previously published morphological matrix for living and fossil Sirenia by adding both new taxa and nine new postcranial characters. Maximum likelihood and parsimony analyses of the molecular data provide robust support for an association of H. gigas and D. dugon to the exclusion of living trichechids (manatees). Parsimony analyses of the morphological data also support the inclusion of H. gigas in Dugongidae with D. dugon and fossil dugongids. Timetree analyses based on calibration density approaches with hard- and soft-bounded constraints suggest that H. gigas and D. dugon diverged in the Oligocene and that crown sirenians last shared a common ancestor in the Eocene. The coding sequence for the ENAM gene in H. gigas does not contain frameshift mutations or stop codons, but there is a transversion mutation (AG to CG) in the acceptor splice site of intron 2. This disruption in the edentulous Steller's sea cow is consistent with previous studies that have documented inactivating mutations in tooth-specific loci of a variety of edentulous and enamelless vertebrates including birds, turtles, aardvarks, pangolins, xenarthrans, and baleen whales. Further, branch-site dN/dS analyses provide evidence for positive selection in ENAM on the stem dugongid branch where extensive tooth reduction occurred, followed by neutral evolution on the Hydrodamalis branch. Finally, we present a synthetic evolutionary tree for living and fossil sirenians showing several key innovations in the history of this clade including character state changes that parallel those that occurred in the evolutionary history of cetaceans. (C) 2015 Elsevier Inc. All rights reserved.
Technological innovations such as next generation sequencing and DNA hybridisation enrichment have resulted in multi-fold increases in both the quantity of ancient DNA sequence data and the time depth for DNA retrieval. To date, over 30 ancient genomes have been sequenced, moving from 0.7x coverage (mammoth) in 2008 to more than 50x coverage (Neanderthal) in 2014. Studies of rapid evolutionary changes, such as the evolution and spread of pathogens and the genetic responses of hosts, or the genetics of domestication and climatic adaptation, are developing swiftly and the importance of palaeogenomics for investigating evolutionary processes during the last million years is likely to increase considerably. However, these new datasets require new methods of data processing and analysis, as well as conceptual changes in interpreting the results. In this review we highlight important areas of future technical and conceptual progress and discuss research topics in the rapidly growing field of palaeogenomics.
Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.
We selected the immunogenic cell wall beta-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells.