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- Arabidopsis thaliana (38)
- climate change (28)
- Arabidopsis (25)
- ancient DNA (19)
- biodiversity (19)
- Dictyostelium (16)
- functional traits (16)
- Climate change (14)
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- Institut für Biochemie und Biologie (3366) (remove)
Activity patterns and time budgets of bushbuck (Tragelaphus scriptus) were studied in a free-ranging population in Queen Elizabeth National Park, Uganda from August 2000 to January 2002. We investigated differences in activity patterns in relation to daytime, season, sun radiation, moonlight, age and sex. Bushbuck were found to show peak activities around sunrise and at dawn. No difference in the mean activity rates was found between the dry and wet season. Daytime activity was not predicted by differences in sun radiation, nor was nighttime activity predicted by the presence or absence of moonlight. We found the activity of adult territorial males to be strongly positively correlated with that of females, whereas the activity of young-adult non-territorial males was not significantly correlated with the activity of females. This suggests that young-adult males shift their peak activity to phases when adult territorial males are less active.
Grouping patterns within the genus Tragelaphus suggest that species inhabiting open areas tend to live in larger groups, while species preferring dense habitats live solitarily or in small family groups. We asked if similar variation would be concealed in the within-species variation of bushbuck (Tragelaphus scriptus). Recent molecular phylogeographic analyses revealed several locally adapted forms of bushbuck in different ecoregions on the African continent. We compared group sizes of south-eastern bushbuck (Tragelaphus sylvaticus) among six different populations ("ecotypes"). To date, most data on the social organization of bushbuck have been collected from only one population in Queen Elizabeth National Park (QENP) in Uganda. This particular population, however, inhabits an unusual-comparatively open-habitat type, while bushbuck otherwise inhabit dense habitats, leaving doubt whether data collected in QENP are representative of the entire species. We, therefore, compared grouping patterns between ecotypes inhabiting rather open habitats (e.g., dianae and haywoodi) and ecotypes occupying rather dense habitats (e.g., massaicus and ornatus). In bachelor groups and in all-female (spinster) groups, single sightings were the most frequent "group type" in all populations examined. We detected no significant difference among ecotypes in relative frequencies of group size categories in the case of bachelor groups. Spinster group sizes were slightly (albeit significantly) smaller in QENP than in all other areas. Moreover, a comparison of two areas inside (low human pursuit) and outside Lake Mburo National Park (high hunting pressure) in Uganda revealed no significant difference in grouping patterns in response to human pursuit (as reported for impala [Aepyceros melampus] inhabiting the same area). Altogether, our results suggest that group sizes in bushbuck are not dependent on the habitat type they inhabit; neither does human nuisance have an impact on grouping patterns. Hence, an "almost solitary" lifestyle appears to be a characteristic of the entire taxon.
A free-ranging bushbuck (Tragelaphus scriptus) population was observed over a period of 3 years, thereby enabling a detailed description of the behavioural repertoire of this widespread but barely investigated solitary African antelope species. Agonistic and submissive behaviour patterns are described, among them several hitherto un- described behaviour patterns - such as "escorting", where territorial males guide intruders to the periphery of their territory - and "push-up position", an extreme form of submissive behaviour. Furthermore, we report on behaviour patterns of males and females during mating as well as on behaviour patterns of parents directed towards their offspring. Again, we describe a hitherto unknown behaviour: the protection of calves by adult males, which may be a socio-positive behaviour directed towards their offspring led by kin selection
We examined factors predicting female densities within the common home ranges of related females ("clans") in bushbuck Tragelaphus scriptus Pallas, 1776. In this species, each female forms an individual home range, but the home ranges of matrilineal clan members strongly overlap. We found female densities to increase in areas with high canopy cover. Moreover, individual home range sizes tended to decrease with increasing cover. Food plant availability and intruder pressure by two heterospecific competitors did not significantly affect female densities. Apparently, canopy cover is the major limited resource in this species. A possible explanation is that both adult bushbuck and - even more markedly - fawns hide from predators in dense vegetation, in particular in thicket clumps and coalescences. The study shows an effect of habitat properties (eg sufficient canopy cover) on a within-population level in bushbuck, where female densities differ even among proximate clan areas
Wronski, Torsten ; Wacher, Timothy ; Hammond, Robert L. ; Winney, Bruce ; Hundertmark, Kris J. ; Blacket, Mark J. ; Mohammed, Osama B. ; Flores, Benito ; Omer, Sawsan A. ; Macasero, William ; Plath, Martin ; Tiedemann, Ralph ; Bleidorn, Christoph
Mountain gazelles (Gazella gazella) rank among the most critically endangered mammals on the Arabian Peninsula. Past conservation efforts have been plagued by confusion about the phylogenetic relationship among various 'phenotypically discernable' populations, and even the question of species boundaries was far from being certain. This lack of knowledge has had a direct impact on conservation measures, especially ex situ breeding programmes, hampering the assignment of captive stocks to potential conservation units. Here, we provide a phylogenetic framework, based on the analysis of mtDNA sequences (360 bp cytochrome b and 213 bp Control Region) of 126 individuals collected from the wild throughout the Arabian Peninsula and from captive stocks. Our analyses revealed two reciprocally monophyletic genetic lineages within the presumed species Gazella gazella: one 'northern clade' on the Golan Heights (Israel/Syrian border) and one genetically diverse larger clade from the rest of the Arabian Peninsula including the Arava Valley (Negev, Israel). Applying the Strict Phylogenetic Species Concept (sensu Mishler & Theriot, 2000) allows assigning species status to these two major clades.
Wu, Anhui ; Allu, Annapurna Devi ; Garapati, Prashanth ; Siddiqui, Hamad ; Dortay, Hakan ; Zanor, Maria-Ines ; Asensi-Fabado, Maria Amparo ; Munne-Bosch, Sergi ; Antonio, Carla ; Tohge, Takayuki ; Fernie, Alisdair R. ; Kaufmann, Kerstin ; Xue, Gang-Ping ; Müller-Röber, Bernd ; Balazadeh, Salma
The transition from juvenility through maturation to senescence is a complex process that involves the regulation of longevity. Here, we identify JUNGBRUNNEN1 (JUB1), a hydrogen peroxide (H2O2)-induced NAC transcription factor, as a central longevity regulator in Arabidopsis thaliana. JUB1 overexpression strongly delays senescence, dampens intracellular H2O2 levels, and enhances tolerance to various abiotic stresses, whereas in jub1-1 knockdown plants, precocious senescence and lowered abiotic stress tolerance are observed. A JUB1 binding site containing a RRYGCCGT core sequence is present in the promoter of DREB2A, which plays an important role in abiotic stress responses. JUB1 transactivates DREB2A expression in mesophyll cell protoplasts and transgenic plants and binds directly to the DREB2A promoter. Transcriptome profiling of JUB1 overexpressors revealed elevated expression of several reactive oxygen species-responsive genes, including heat shock protein and glutathione S-transferase genes, whose expression is further induced by H2O2 treatment. Metabolite profiling identified elevated Pro and trehalose levels in JUB1 overexpressors, in accordance with their enhanced abiotic stress tolerance. We suggest that JUB1 constitutes a central regulator of a finely tuned control system that modulates cellular H2O2 level and primes the plants for upcoming stress through a gene regulatory network that involves DREB2A.
Wu, Hao ; Han, Yijie ; Rodriguez Sillke, Yasmina ; Deng, Hongzhang ; Siddiqui, Sophiya ; Treese, Christoph ; Schmidt, Franziska ; Friedrich, Marie ; Keye, Jacqueline ; Wan, Jiajia ; Qin, Yue ; Kühl, Anja A. ; Qin, Zhihai ; Siegmund, Britta ; Glauben, Rainer
Tumor-associated macrophages (TAMs) promote tumor growth and metastasis by suppressing tumor immune surveillance. Herein, we provide evidence that the immunosuppressive phenotype of TAMs is controlled by long-chain fatty acid metabolism, specifically unsaturated fatty acids, here exemplified by oleate. Consequently, en-route enriched lipid droplets were identified as essential organelles, which represent effective targets for chemical inhibitors to block in vitro polarization of TAMs and tumor growth in vivo. In line, analysis of human tumors revealed that myeloid cells infiltrating colon cancer but not gastric cancer tissue indeed accumulate lipid droplets. Mechanistically, our data indicate that oleate-induced polarization of myeloid cells depends on the mammalian target of the rapamycin pathway. Thus, our findings reveal an alternative therapeutic strategy by targeting the pro-tumoral myeloid cells on a metabolic level.
Wu, Yabin ; Stoddart, Martin J. ; Wuertz-Kozak, Karin ; Grad, Sibylle ; Alini, Mauro ; Ferguson, Stephen J.
Wu, Yunhua ; Wollenberger, Ursula ; Hofrichter, Martin ; Ullrich, Rene ; Scheibner, Katrin ; Scheller, Frieder W.
Three different sizes of chitosan-capped Au nanoparticles were synthesized and were used to incorporate Agrocybe aegerita peroxygenase (AaeAPO) onto the surface of glassy carbon electrode. The direct electron transfer of AaeAPO was achieved in all films. The highest amount of electroactive enzyme and highest electron transfer rate constant k(s) of AaeAPO were obtained in the film with the smallest size of chitosan-capped Au nanoparticles.
In anaerobic solutions, quasi-reversible oxidation and reduction are obtained with a formal potential of -0.280V vs. Ag/AgCl 1 M KCl in 100 mM (pH 7.0) PBS at scan rate of 1 V s(-1). Bioelectrocatalytic reduction currents can be obtained with the AaeAPO-modified electrode on addition of hydrogen peroxide. This reaction was suppressed when sodium azide, an inhibitor of AaeAPO, was present. Furthermore, the peroxide-dependent conversion of aniline was characterized and it was found that a polymer product via p-aminophenol is formed. And the AaeAPO biosensor was applied to determine aniline and p-aminophenol.
Our goal was to reconstruct the late eighteenth century forest vegetation of the Prignitz region (NE Germany) at a scale of 1:50,000. We also wanted to relate the historical forest vegetation to the actual and potential natural vegetation. For these purposes, we selected 15 woody species and transferred relevant data found in historical records from various sources together with the recent localities of (very) old individuals belonging to these woody species into ArcView GIS. Following multi-step data processing including the generation of a point density layer using a moving window with kernel estimation and derivation of vegetation units applying Boolean algebra rules together with information on site conditions, we derived 17 forest communities corresponding to the potential natural vegetation. We were able to reconstruct the historical forest vegetation for 90% of the forest area ca. 1780. Only two of the 17 forest communities covered large parts of the forested area. The oak forest with Agrostis capillaris covered about 44% of the total forest area, and alder forests on fenland made up about 37%. Oak-hornbeam forests with Stellaria holostea comprised slightly less than 6% of the forest area, while all other forest communities comprised less than 1%. The historical forest vegetation is more similar to the potential forest vegetation and quite different from the actual forest vegetation because coniferous tree species currently cover approximately two-thirds of the actual forest area. The most beneficial result of this study is the map of high-resolution historical vegetation units that may serve as the basis for various further studies, e.g., modelling long-term changes in biodiversity at the landscape scale.
Wurzbacher, Christian ; Attermeyer, Katrin ; Kettner, Marie Therese ; Flintrop, Clara ; Warthmann, Norman ; Hilt, Sabine ; Grossart, Hans-Peter ; Monaghan, Michael T.
Most studies of aquatic plankton focus on either macroscopic or microbial communities, and on either eukaryotes or prokaryotes. This separation is primarily for methodological reasons, but can overlook potential interactions among groups. Here we tested whether DNA metabarcoding of unfractionated water samples with universal primers could be used to qualitatively and quantitatively study the temporal dynamics of the total plankton community in a shallow temperate lake. Significant changes in the relative proportions of normalized sequence reads of eukaryotic and prokaryotic plankton communities over a 3-month period in spring were found. Patterns followed the same trend as plankton estimates measured using traditional microscopic methods. The bloom of a conditionally rare bacterial taxon belonging to Arcicella was characterized, which rapidly came to dominate the whole lake ecosystem and would have remained unnoticed without metabarcoding. The data demonstrate the potential of universal DNA metabarcoding applied to unfractionated samples for providing a more holistic view of plankton communities.
Shifts among Eukaryota, Bacteria, and Archaea define the vertical organization of a lake sediment
(2017)
Wurzbacher, Christian ; Fuchs, Andrea ; Attermeyer, Katrin ; Frindte, Katharina ; Grossart, Hans-Peter ; Hupfer, Michael ; Casper, Peter ; Monaghan, Michael T.
Background: Lake sediments harbor diverse microbial communities that cycle carbon and nutrients while being constantly colonized and potentially buried by organic matter sinking from the water column. The interaction of activity and burial remained largely unexplored in aquatic sediments. We aimed to relate taxonomic composition to sediment biogeochemical parameters, test whether community turnover with depth resulted from taxonomic replacement or from richness effects, and to provide a basic model for the vertical community structure in sediments. Methods: We analyzed four replicate sediment cores taken from 30-m depth in oligo-mesotrophic Lake Stechlin in northern Germany. Each 30-cm core spanned ca. 170 years of sediment accumulation according to Cs-137 dating and was sectioned into layers 1-4 cm thick. We examined a full suite of biogeochemical parameters and used DNA metabarcoding to examine community composition of microbial Archaea, Bacteria, and Eukaryota. Results: Community beta-diversity indicated nearly complete turnover within the uppermost 30 cm. We observed a pronounced shift from Eukaryota- and Bacteria-dominated upper layers (<5 cm) to Bacteria-dominated intermediate layers (5-14 cm) and to deep layers (>14 cm) dominated by enigmatic Archaea that typically occur in deep-sea sediments. Taxonomic replacement was the prevalent mechanism in structuring the community composition and was linked to parameters indicative of microbial activity (e.g., CO2 and CH4 concentration, bacterial protein production). Richness loss played a lesser role but was linked to conservative parameters (e.g., C, N, P) indicative of past conditions. Conclusions: By including all three domains, we were able to directly link the exponential decay of eukaryotes with the active sediment microbial community. The dominance of Archaea in deeper layers confirms earlier findings from marine systems and establishes freshwater sediments as a potential low-energy environment, similar to deep sea sediments. We propose a general model of sediment structure and function based on microbial characteristics and burial processes. An upper "replacement horizon" is dominated by rapid taxonomic turnover with depth, high microbial activity, and biotic interactions. A lower "depauperate horizon" is characterized by low taxonomic richness, more stable "low-energy" conditions, and a dominance of enigmatic Archaea.
Fungi and bacteria are the major organic matter (OM) decomposers in aquatic ecosystems. While bacteria are regarded as primary mineralizers in the pelagic zone of lakes and oceans, fungi dominate OM decomposition in streams and wetlands. Recent findings indicate that fungal communities are also active in lakes, but little is known about their diversity and interactions with bacteria. Therefore, the decomposer niche overlap of saprotrophic fungi and bacteria was studied on pollen (as a seasonally recurring source of fine particulate OM) by performing microcosm experiments with three different lake types. Special emphasis was placed on analysis of fungal community composition and diversity. We hypothesized that (I) pollen select for small saprotrophic fungi and at the same time for typical particle-associated bacteria; (II) fungal communities form specific free-living and attached sub-communities in each lake type; (III) the ratio between fungi or bacteria on pollen is controlled by the lake's chemistry. Bacteria-to-fungi ratios were determined by quantitative PCR (qPCR), and bacterial and fungal diversity were studied by clone libraries and denaturing gradient gel electrophoresis (DGGE) fingerprints. A protease assay was used to identify functional differences between treatments. For generalization, systematic differences in bacteria-to-fungi ratios were analyzed with a dataset from the nearby Baltic Sea rivers. High abundances of Chytridiomycota as well as occurrences of Cryptomycota and yeast-like fungi confirm the decomposer niche overlap of saprotrophic fungi and bacteria on pollen. As hypothesized, microbial communities consistently differed between the lake types and exhibited functional differences. Bacteria-to-fungi ratios correlated well with parameters such as organic carbon and pH. The importance of dissolved organic carbon and nitrogen for bacteria-to-fungi ratios was supported by the Baltic Sea river dataset. Our findings highlight the fact that carbon-to-nitrogen ratios may also control fungal contributions to OM decomposition in aquatic ecosystems.
Environmental actinorhodopsin expression revealed by a new in situ filtration and fixation sampler
(2012)
Freshwater Actinobacteria are an important and dominant group of bacterioplankton in most temperate freshwater systems. Recently, metagenomic studies discovered rhodopsin-like protein-coding sequences present in Actinobacteria which could be a decisive hint for their success in freshwater ecosystems. We analysed the diversity of actinorhodopsin (ActR) in Lake Stechlin (northern Germany) and assessed the actR expression profile during a diurnal cycle. We obtained 85 positive actR clones which could be subsequently grouped to 17 operational taxonomic units assuming a 90% sequence similarity. The phylogenetic analysis points to a close relationship of all obtained sequences to the acI lineage of Actinobacteria, forming six independent clusters. For the first time, we followed in situ transcription of actR in Lake Stechlin revealing a rather constitutive circadian gene expression. For analysing in situ expression patterns of functional genes in aquatic ecosystems, such as actR, we invented a new in situ filtration and fixation sampler (IFFS). The IFFS enables the representative investigation of microbial transcriptomes in any aquatic ecosystem at all water depths. The IFFS sampler is simple and inexpensive, and we provide all engineering plans for an easy rebuild. Consequently, our IFFS is suitable to reliably study expression of any known functional gene of any aquatic microorganism.
Wurzbacher, Christian ; Warthmann, Norman ; Bourne, Elizabeth Charlotte ; Attermeyer, Katrin ; Allgaier, Martin ; Powell, Jeff R. ; Detering, Harald ; Mbedi, Susan ; Großart, Hans-Peter ; Monaghan, Michael T.
Freshwater fungi are a poorly studied ecological group that includes a high taxonomic diversity. Most studies on aquatic fungal diversity have focused on single habitats, thus the linkage between habitat heterogeneity and fungal diversity remains largely unexplored. We took 216 samples from 54 locations representing eight different habitats in the meso-oligotrophic, temperate Lake Stechlin in North-East Germany. These included the pelagic and littoral water column, sediments, and biotic substrates. We performed high throughput sequencing using the Roche 454 platform, employing a universal eukaryotic marker region within the large ribosomal subunit (LSU) to compare fungal diversity, community structure, and species turnover among habitats. Our analysis recovered 1027 fungal OTUs (97% sequence similarity). Richness estimates were highest in the sediment, biofilms, and benthic samples (189-231 OTUs), intermediate in water samples (42-85 OTUs), and lowest in plankton samples (8 OTUs). NMDS grouped the eight studied habitats into six clusters, indicating that community composition was strongly influenced by turnover among habitats. Fungal communities exhibited changes at the phylum and order levels along three different substrate categories from littoral to pelagic habitats. The large majority of OTUs (> 75%) could not be classified below the order level due to the lack of aquatic fungal entries in public sequence databases. Our study provides a first estimate of lake-wide fungal diversity and highlights the important contribution of habitat heterogeneity to overall diversity and community composition. Habitat diversity should be considered in any sampling strategy aiming to assess the fungal diversity of a water body.
Wuster, Wolfgang ; Chirio, Laurent ; Trape, Jean-Francois ; Ineich, Ivan ; Jackson, Kate ; Greenbaum, Eli ; Barron, Cesar ; Kusamba, Chifundera ; Nagy, Zoltan T. ; Storey, Richard ; Hall, Cara ; Wuster, Catharine E. ; Barlow, Axel ; Broadley, Donald G.
Cobras are among the most widely known venomous snakes, and yet their taxonomy remains incompletely understood, particularly in Africa. Here, we use a combination of mitochondrial and nuclear gene sequences and morphological data to diagnose species limits within the African forest cobra, Naja (Boulengerina) melanoleuca. Mitochondrial DNA sequences reveal deep divergences within this taxon. Congruent patterns of variation in mtDNA, nuclear genes and morphology support the recognition of five separate species, confirming the species status of N. subfulva and N. peroescobari, and revealing two previously unnamed West African species, which are described as new: Naja (Boulengerina) guineensis sp. nov. Broadley, Trape, Chirio, Ineich & Wuster, from the Upper Guinea forest of West Africa, and Naja (Boulengerina) savannula sp. nov. Broadley, Trape, Chirio & Wuster, a banded form from the savanna-forest mosaic of the Guinea and Sudanian savannas of West Africa. The discovery of cryptic diversity in this iconic group highlights our limited understanding of tropical African biodiversity, hindering our ability to conserve it effectively.
Wutke, Saskia ; Benecke, Norbert ; Sandoval-Castellanos, Edson ; Döhle, Hans-Jürgen ; Friederich, Susanne ; Gonzalez Soto, Javier Esteban ; Hallsson, Jon Hallsteinn ; Hofreiter, Michael ; Lougas, Lembi ; Magnell, Ola ; Morales-Muniz, Arturo ; Orlando, Ludovic ; Palsdottir, Albina Hulda ; Reissmann, Monika ; Ruttkay, Matej ; Trinks, Alexandra ; Ludwig, Arne
Horses have been valued for their diversity of coat colour since prehistoric times; this is especially the case since their domestication in the Caspian steppe in similar to 3,500 BC. Although we can assume that human preferences were not constant, we have only anecdotal information about how domestic horses were influenced by humans. Our results from genotype analyses show a significant increase in spotted coats in early domestic horses (Copper Age to Iron Age). In contrast, medieval horses carried significantly fewer alleles for these phenotypes, whereas solid phenotypes (i.e., chestnut) became dominant. This shift may have been supported because of (i) pleiotropic disadvantages, (ii) a reduced need to separate domestic horses from their wild counterparts, (iii) a lower religious prestige, or (iv) novel developments in weaponry. These scenarios may have acted alone or in combination. However, the dominance of chestnut is a remarkable feature of the medieval horse population.
Wutke, Saskia ; Sandoval-Castellanos, Edson ; Benecke, Norbert ; Döhle, Hans-Jürgen ; Friederich, Susanne ; Gonzalez, Javier ; Hofreiter, Michael ; Lougas, Lembi ; Magnell, Ola ; Malaspinas, Anna-Sapfo ; Morales-Muniz, Arturo ; Orlando, Ludovic ; Reissmann, Monika ; Trinks, Alexandra ; Ludwig, Arne
Present-day domestic horses are immensely diverse in their maternally inherited mitochondrial DNA, yet they show very little variation on their paternally inherited Y chromosome. Although it has recently been shown that Y chromosomal diversity in domestic horses was higher at least until the Iron Age, when and why this diversity disappeared remain controversial questions. We genotyped 16 recently discovered Y chromosomal single-nucleotide polymorphisms in 96 ancient Eurasian stallions spanning the early domestication stages (Copper and Bronze Age) to the Middle Ages. Using this Y chromosomal time series, which covers nearly the entire history of horse domestication, we reveal how Y chromosomal diversity changed over time. Our results also show that the lack of multiple stallion lineages in the extant domestic population is caused by neither a founder effect nor random demographic effects but instead is the result of artificial selection-initially during the Iron Age by nomadic people from the Eurasian steppes and later during the Roman period. Moreover, the modern domestic haplotype probably derived from another, already advantageous, haplotype, most likely after the beginning of the domestication. In line with recent findings indicating that the Przewalski and domestic horse lineages remained connected by gene flow after they diverged about 45,000 years ago, we present evidence for Y chromosomal introgression of Przewalski horses into the gene pool of European domestic horses at least until medieval times.
Wüstenhagen, Doreen Anja ; Lukas, Phil ; Müller, Christian ; Aubele, Simone A. ; Hildebrandt, Jan-Peter ; Kubick, Stefan
Synthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized hirudin showed a considerably higher anti-thrombin activity compared to recombinant hirudin produced in bacterial cells.
Xiang, Hai ; Gao, Jianqiang ; Yu, Baoquan ; Zhou, Hui ; Cai, Dawei ; Zhang, Youwen ; Chen, Xiaoyong ; Wang, Xi ; Hofreiter, Michael ; Zhao, Xingbo
Chickens represent by far the most important poultry species, yet the number, locations, and timings of their domestication have remained controversial for more than a century. Here we report ancient mitochondrial DNA sequences from the earliest archaeological chicken bones from China, dating back to similar to 10,000 B.P. The results clearly show that all investigated bones, including the oldest from the Nanzhuangtou site, are derived from the genus Gallus, rather than any other related genus, such as Phasianus. Our analyses also suggest that northern China represents one region of the earliest chicken domestication, possibly dating as early as 10,000 y B.P. Similar to the evidence from pig domestication, our results suggest that these early domesticated chickens contributed to the gene pool of modern chicken populations. Moreover, our results support the idea that multiple members of the genus Gallus, specifically Gallus gallus and Gallus sonneratii contributed to the gene pool of the modern domestic chicken. Our results provide further support for the growing evidence of an early mixed agricultural complex in northern China.
Xiao, Shangbin ; Liu, Liu ; Wang, Wei ; Lorke, Andreas ; Woodhouse, Jason Nicholas ; Grossart, Hans-Peter
Biogenic greenhouse gas emissions, e.g., of methane (CH4) and carbon dioxide (CO2) from inland waters, contribute substantially to global warming. In aquatic systems, dissolved greenhouse gases are highly heterogeneous in both space and time. To better understand the biological and physical processes that affect sources and sinks of both CH4 and CO2, their dissolved concentrations need to be measured with high spatial and temporal resolution. To achieve this goal, we developed the Fast-Response Automated Gas Equilibrator (FaRAGE) for real-time in situ measurement of dissolved CH4 and CO2 concentrations at the water surface and in the water column. FaRAGE can achieve an exceptionally short response time (t(95%) = 12 s when including the response time of the gas analyzer) while retaining an equilibration ratio of 62.6% and a measurement accuracy of 0.5% for CH4. A similar performance was observed for dissolved CO2 (t(95%) = 10 s, equilibration ratio 67.1 %). An equilibration ratio as high as 91.8% can be reached at the cost of a slightly increased response time (16 s). The FaRAGE is capable of continuously measuring dissolved CO2 and CH4 concentrations in the nM-to-submM (10(-9)-10(-3) mol L-1) range with a detection limit of subnM (10(-10) mol L-1), when coupling with a cavity ring-down greenhouse gas analyzer (Picarro GasScouter). FaRAGE allows for the possibility of mapping dissolved concentration in a "quasi" three-dimensional manner in lakes and provides an inexpensive alternative to other commercial gas equilibrators. It is simple to operate and suitable for continuous monitoring with a strong tolerance for suspended particles. While the FaRAGE is developed for inland waters, it can be also applied to ocean waters by tuning the gas-water mixing ratio. The FaRAGE is easily adapted to suit other gas analyzers expanding the range of potential applications, including nitrous oxide and isotopic composition of the gases.
Xie, B. ; Tang, X. ; Wollenberger, Ursula ; Johansson, G. ; Gorton, Lo ; Scheller, Frieder W. ; Danielsson, B.
Mitochondrial diseases may be caused by numerous mutations that alter proteins of the respiratory chain and of other metabolic pathways in the mitochondrium. For clinicians this disease group poses a considerable diagnostic challenge due to ambiguous genotype-phenotype relationships. Until now, only 30 % of the mitochondriopathies can be diagnosed at the molecular level. We therefore need a new diagnostic tool that offers a wide view on the mitochondrial proteins. Here, we present a method to generate a high-resolution, large-gel two-dimensional gel electrophoretic (2-DE) map of a purified fraction of mitochondrial proteins from Epstein-Barr virus-immortalized lymphoblastoid cell line (LCL). LCLs can be easily obtained from patients and control subjects in a routine clinical setting. They often express the biochemical phenotype and can be cultured to high cell numbers, sufficient to gain enough purified material for 2- DE. In total we identified 166 mitochondrial proteins. Thirteen proteins were earlier not known to be of mitochondrial origin. Thirty-nine proteins were associated with human diseases ranging from respiratory chain enzyme deficiencies to disorders of P-oxidation and amino acid metabolism. This 2-DE map is intended to be the first step to diagnose mitochondrial diseases at the proteomic level
Xu, Huizhen ; Giannetti, Alessandro ; Sugiyama, Yuki ; Zheng, Wenna ; Schneider, René ; Watanabe, Yoichiro ; Oda, Yoshihisa ; Persson, Staffan
All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose-the load-bearing structure in cell walls-at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing's reaction-diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.
Xu, J. ; Brearley, C. A. ; Lin, W. H. ; Wang, Y. ; Ye, R. ; Müller-Röber, Bernd ; Xu, Z. H. ; Xue, H. W.
Inositol polyphosphates, such as inositol trisphosphate, are pivotal intracellular signaling molecules in eukaryotic cells. In higher plants the mechanism for the regulation of the type and the level of these signaling molecules is poorly understood. In this study we investigate the physiological function of an Arabidopsis (Arabidopsis thaliana) gene encoding inositol polyphosphate kinase (AtIPK2alpha), which phosphorylates inositol 1,4,5-trisphosphate successively at the D-6 and D-3 positions, and inositol 1,3,4,5-tetrakisphosphate at D-6, resulting in the generation of inositol 1,3,4,5,6-pentakisphosphate. Semiquantitative reverse transcription-PCR and promoter-beta-glucuronidase reporter gene analyses showed that AtIPK2alpha is expressed in various tissues, including roots and root hairs, stem, leaf, pollen grains, pollen tubes, the flower stigma, and siliques. Transgenic Arabidopsis plants expressing the AtIPK2alpha antisense gene under its own promoter were generated. Analysis of several independent transformants exhibiting strong reduction in AtIPK2alpha transcript levels showed that both pollen germination and pollen tube growth were enhanced in the antisense lines compared to wild-type plants, especially in the presence of nonoptimal low Ca2+ concentrations in the culture medium. Furthermore, root growth and root hair development were also stimulated in the antisense lines, in the presence of elevated external Ca2+ concentration or upon the addition of EGTA. In addition, seed germination and early seedling growth was stimulated in the antisense lines. These observations suggest a general and important role of AtIPK2alpha, and hence inositol polyphosphate metabolism, in the regulation of plant growth most likely through the regulation of calcium signaling, consistent with the well-known function of inositol trisphosphate in the mobilization of intracellular calcium stores
Motivation:
Constraint-based modeling approaches allow the estimation of maximal in vivo enzyme catalytic rates that can serve as proxies for enzyme turnover numbers. Yet, genome-scale flux profiling remains a challenge in deploying these approaches to catalogue proxies for enzyme catalytic rates across organisms.
Results:
Here, we formulate a constraint-based approach, termed NIDLE-flux, to estimate fluxes at a genome-scale level by using the principle of efficient usage of expressed enzymes. Using proteomics data from Escherichia coli, we show that the fluxes estimated by NIDLE-flux and the existing approaches are in excellent qualitative agreement (Pearson correlation > 0.9). We also find that the maximal in vivo catalytic rates estimated by NIDLE-flux exhibits a Pearson correlation of 0.74 with in vitro enzyme turnover numbers. However, NIDLE-flux results in a 1.4-fold increase in the size of the estimated maximal in vivo catalytic rates in comparison to the contenders. Integration of the maximum in vivo catalytic rates with publically available proteomics and metabolomics data provide a better match to fluxes estimated by NIDLE-flux. Therefore, NIDLE-flux facilitates more effective usage of proteomics data to estimate proxies for kcatomes.
Nitric oxide synthase (NOS) plays a critical role in a number of key physiological and pathological processes. Investigation of electron-transfer reactions in NOS would contribute to a better understanding of the nitric oxide (NO) synthesis mechanism. Herein, we describe an electrochemically driven catalytic strategy, using a nanocomposite that consisted of the oxygenase domain of neuronal NOS (D290nNOSoxy), indium tin oxide (ITO) nanopartides and polyvinyl alcohol (PVA). Fast direct electron transfer between electrodes and D290nNOSoxy was observed with the heterogeneous electron transfer rate constant (k(er)) of 154.8 +/- 0.1 s(-1) at the scan rate of 5 V s(-1). Moreover, the substrate IV-hydroxy-L-arginine (NHA) was used to prove the concept of electrochemically driven biocatalysis of D290nNOSoxy. In the presence of the oxygen cosubstrate and tetrahydrobiopterin (BH4) cofactor, the addition of NHA caused the decreases of both oxidation current at + 0.1 V and reduction current at potentials ranging from -0.149 V to -0.549 V vs Ag/AgCl. Thereafter, a series of control experiments such as in the absence of BH4 or D290nNOSoxy were performed. All the results demonstrated that D290nNOSoxy biocatalysis was successfully driven by electrodes in the presence of BH4 and oxygen. This novel bioelectronic system showed potential for further investigation of NOS and biosensor applications. (C) 2013 Elsevier B.V. All rights reserved.
Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability.
Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution.
The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy.
It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70% confluence (SC 70% hom) or a clump seeding group with heterogeneously distributed cells at 90% confluence (CL 90% het), can be observed as early as 9 days post induction.
These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine.
Yan, Jiawei ; Frøkjær, Emil Egede ; Engelbrekt, Christian ; Leimkühler, Silke ; Ulstrup, Jens ; Wollenberger, Ulla ; Xiao, Xinxin ; Zhang, Jingdong
Human sulfite oxidase (hSO) is a homodimeric two-domain enzyme central in the biological sulfur cycle. A pyranopterin molybdenum cofactor (Moco) is the catalytic site and a heme b(5) group located in the N-terminal domain. The two domains are connected by a flexible linker region. Electrons produced at the Moco in sulfite oxidation, are relayed via heme b(5) to electron acceptors or an electrode surface. Inter-domain conformational changes between an open and a closed enzyme conformation, allowing "gated" electron transfer has been suggested. We first recorded cyclic voltammetry (CV) of hSO on single-crystal Au(111)-electrode surfaces modified by self-assembled monolayers (SAMs) both of a short rigid thiol, cysteamine and of a longer structurally flexible thiol, omega-amino-octanethiol (AOT). hSO on cysteamine SAMs displays a well-defined pair of voltammetric peaks around -0.207 V vs. SCE in the absence of sulfite substrate, but no electrocatalysis. hSO on AOT SAMs displays well-defined electrocatalysis, but only "fair" quality voltammetry in the absence of sulfite. We recorded next in situ scanning tunnelling spectroscopy (STS) of hSO on AOT modified Au(111)-electrodes, disclosing, a 2-5 % surface coverage of strong molecular scale contrasts, assigned to single hSO molecules, notably with no contrast difference in the absence and presence of sulfite. In situ STS corroborated this observation with a sigmoidal tunnelling current/overpotential correlation.
The Eukaryotic-Specific ISD11 Is a Complex-Orphan Protein with Ability to Bind the Prokaryotic IscS
(2016)
Yan, Robert ; Friemel, Martin ; Aloisi, Claudia ; Huynen, Martijn ; Taylor, Ian A. ; Leimkühler, Silke ; Pastore, Annalisa
The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction.
Yan, Wan ; Fang, Liang ; Nöchel, Ulrich ; Gould, Oliver E. C. ; Behl, Marc ; Kratz, Karl ; Lendlein, Andreas
The variation of the molecular architecture of multiblock copolymers has enabled the introduction of functional behaviour and the control of key mechanical properties. In the current study, we explore the synergistic relationship of two structural components in a shape-memory material formed of a multiblock copolymer with crystallizable poly(epsilon-caprolactone) and crystallizable polyfoligo(3S-iso-butylmorpholine-2,5-dione) segments (PCL-PIBMD). The thermal and structural properties of PCL-PIBMD films were compared with PCI.-PU and PMMD-PU investigated by means of DSC, SAXS and WARS measurements. The shape-memory properties were quantified by cyclic, thermomechanical tensile tests, where deformation strains up to 900% were applied for programming PCL-PIBMD films at 50 degrees C. Toluene vapor treatment experiments demonstrated that the temporary shape was fixed mainly by glassy PIBMD domains at strains lower than 600% with the PCL contribution to fixation increasing to 42 +/- 2% at programming strains of 900% This study into the shape-memory mechanism of PCL-PIBMD provides insight into the structure function relation in multiblock copolymers with both crystallizable and glassy switching segments.
Background
The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment.
Results
Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression.
Conclusions
Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
Results: Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression. Conclusions: Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
Yan, Wenhao ; Chen, Dijun ; Schumacher, Julia ; Durantini, Diego ; Engelhorn, Julia ; Chen, Ming ; Carles, Cristel C. ; Kaufmann, Kerstin
Enhancers are critical for developmental stage-specific gene expression, but their dynamic regulation in plants remains poorly understood. Here we compare genome-wide localization of H3K27ac, chromatin accessibility and transcriptomic changes during flower development in Arabidopsis. H3K27ac prevalently marks promoter-proximal regions, suggesting that H3K27ac is not a hallmark for enhancers in Arabidopsis. We provide computational and experimental evidence to confirm that distal DNase. hypersensitive sites are predictive of enhancers. The predicted enhancers are highly stage-specific across flower development, significantly associated with SNPs for flowering-related phenotypes, and conserved across crucifer species. Through the integration of genome-wide transcription factor (TF) binding datasets, we find that floral master regulators and stage-specific TFs are largely enriched at developmentally dynamic enhancers. Finally, we show that enhancer clusters and intronic enhancers significantly associate with stage-specific gene regulation by floral master TFs. Our study provides insights into the functional flexibility of enhancers during plant development, as well as hints to annotate plant enhancers.
Yang, Lei ; Perrera, Valentina ; Saplaoura, Eleftheria ; Apelt, Federico ; Bahin, Mathieu ; Kramdi, Amira ; Olas, Justyna Jadwiga ; Müller-Röber, Bernd ; Sokolowska, Ewelina ; Zhang, Wenna ; Li, Runsheng ; Pitzalis, Nicolas ; Heinlein, Manfred ; Zhang, Shoudong ; Genovesio, Auguste ; Colot, Vincent ; Kragler, Friedrich
In plants, transcripts move to distant body parts to potentially act as systemic signals regulating development and growth. Thousands of messenger RNAs (mRNAs) are transported across graft junctions via the phloem to distinct plant parts. Little is known regarding features, structural motifs, and potential base modifications of transported transcripts and how these may affect their mobility. We identified Arabidopsis thalianam RNAs harboring the modified base 5-methylcytosine (m(5)C) and found that these are significantly enriched in mRNAs previously described as mobile, moving over graft junctions to distinct plant parts. We confirm this finding with graft-mobile methylated mRNAs TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1 (TCTP1) and HEAT SHOCK COGNATE PROTEIN 70.1 (HSC70.1), whose mRNA transport is diminished in mutants deficient in m(5)C mRNA methylation. Together, our results point toward an essential role of cytosine methylation in systemic mRNA mobility in plants and that TCTP1 mRNA mobility is required for its signaling function.
QuestionDisturbed areas offer great opportunities for restoring native biodiversity, but they are also prone to invasion by alien plants. Following the limiting similarity hypothesis, we address the question of whether or not similarity of plant functional traits helps developing seed mixtures of native communities with high resistance to invasive species at an early stage of restoration. LocationCentre of Greenhouses and Laboratories Durnast, Technische Universitat Munchen, Freising, Germany. MethodsUsing a system of linear equations, we designed native communities maximizing the similarity between the native and two invasive species according to ten functional traits. We used native grassland plants, two invasive alien species that are often problematic in disturbed areas (i.e., Ambrosia artemisiifolia and Solidago gigantea) and trait information obtained from databases. The two communities were then tested for resistance against establishment of the two invaders separately in a greenhouse experiment. We measured height of the invasive species and above-ground biomass, along with leaf area index, 4 and 8months after sowing respectively. ResultsBoth invasive species were successfully reduced by the native community designed to suppress S. gigantea dominated by small-seeded species. These results could be considered as partial support for the limiting similarity hypothesis. However, given the success of this mixture against both invasive species, suppression was better explained by a seed density effect resulting from the smaller seed mass of the native species included in this mixture. Further, the dominance of a fast-developing competitive species could also contribute to its success. ConclusionsThere was no unequivocal support for the limiting similarity hypothesis in terms of the traits selected. Instead we found that increasing seeding density of native species and selecting species with a fast vegetative development is an effective way to suppress invasive plants during early stages of restoration. If limiting similarity is used to design communities for restoration, early life-history traits should be taken into account.
The first molecularly imprinted polymer (MIP) for the recognition of the copper-enzyme laccase was successfully prepared by electropolymerizing scopoletin in the presence of alkaline-inactivated enzyme. Laccase-MIP and the control polymer without laccase (nonimprinted polymer, NIP) were characterized by voltammetry using the redox marker ferricyanide. After electropolymerization, the signals for ferricyanide for both the MIP and the NIP were almost completely suppressed and increased after removal of the target from the polymer layer. Rebinding of both inactivated and active laccase decreased the ferricyanide peak currents to almost equal extent. The relative decrease of signal suppression approached saturation above 10 nM. Furthermore, the surface activity of rebound laccase toward the oxidation of catechol was investigated. The surface activity approached saturation above 10 nM, a value close to the value of the measurements with ferricyanide. Interaction of NIP with laccase brought about a six times smaller signal of catechol oxidation.
For the first time a molecularly imprinted polymer (MIP)-based sensor for tyrosinase is described. This sensor is based on the electropolymerization of scopoletin or o-phenylenediamine in the presence of tyrosinase from mushrooms, which has a high homology to the human enzyme. The template was removed either by treatment with proteinase Kor by alkaline treatment. The measuring signal was generated either by measuring the formation of a product by the target enzyme or by evaluation of the permeability of the redox marker ferricyanide. The o-phenylenediamine-based MIP sensor has a linear measuring range up to 50 nM of tyrosinase with a limit of detection of 3.97 nM (R 2 = 0.994) and shows good discrimination towards other proteins, e.g., bovine serum albumin and cytochrome c.
Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11
(2011)
Yarman, Aysu ; Badalyan, Artavazd ; Gajovic-Eichelmann, Nenad ; Wollenberger, Ursula ; Scheller, Frieder W.
Microperoxidase-11 (MR-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline. 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-II and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.
Yarman, Aysu ; Gröbe, Glenn ; Neumann, Bettina ; Kinne, Mathias ; Gajovic-Eichelmann, Nenad ; Wollenberger, Ursula ; Hofrichter, Martin ; Ullrich, Rene ; Scheibner, Katrin ; Scheller, Frieder W.
The aromatic peroxygenase (APO; EC 1.11.2.1) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of -278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe(2+)/Fe(3+) redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed.
Simple and robust
(2021)
A spectrum of 7562 publications on Molecularly Imprinted Polymers (MIPs) has been presented in literature within the last ten years (Scopus, September 7, 2020). Around 10 % of the papers published on MIPs describe the recognition of proteins. The straightforward synthesis of MIPs is a significant advantage as compared with the preparation of enzymes or antibodies. MIPs have been synthesized from only one up to six functional monomers while proteins are made up of 20 natural amino acids. Furthermore, they can be synthesized against structures of low immunogenicity and allow multi-analyte measurements via multi-target synthesis. Electrochemical methods allow simple polymer synthesis, removal of the template and readout. Among the different sensor configurations electrochemical MIP-sensors provide the broadest spectrum of protein analytes. The sensitivity of MIP-sensors is sufficiently high for biomarkers in the sub-nanomolar region, nevertheless the cross-reactivity of highly abundant proteins in human serum is still a challenge. MIPs for proteins offer innovative tools not only for clinical and environmental analysis, but also for bioimaging, therapy and protein engineering.
We present an electrochemical MIP sensor for tamoxifen (TAM)-a nonsteroidal anti-estrogen-which is based on the electropolymerisation of an O-phenylenediamine. resorcinol mixture directly on the electrode surface in the presence of the template molecule. Up to now only. bulk. MIPs for TAM have been described in literature, which are applied for separation in chromatography columns. Electro-polymerisation of the monomers in the presence of TAM generated a film which completely suppressed the reduction of ferricyanide. Removal of the template gave a markedly increased ferricyanide signal, which was again suppressed after rebinding as expected for filling of the cavities by target binding. The decrease of the ferricyanide peak of the MIP electrode depended linearly on the TAM concentration between 1 and 100 nM. The TAM-imprinted electrode showed a 2.3 times higher recognition of the template molecule itself as compared to its metabolite 4-hydroxytamoxifen and no cross-reactivity with the anticancer drug doxorubucin was found. Measurements at + 1.1 V caused a fouling of the electrode surface, whilst pretreatment of TAM with peroxide in presence of HRP generated an oxidation product which was reducible at 0 mV, thus circumventing the polymer formation and electrochemical interferences.
A new electrochemical MIP sensor for the most frequently used drug paracetamol (PAR) was prepared by electropolymerization of mixtures containing the template molecule and the functional monomers ophenylenediamine, resorcinol and aniline. The imprinting factor of 12 reflects the effective target binding to the MIP as compared with the non-imprinted electropolymer. Combination of the MIP with a nonspecific esterase allows the measurement of phenacetin - another analgesic drug. In the second approach the PAR containing sample solution was pretreated with tyrosinase in order to prevent electrochemical interferences by ascorbic acid and uric acid. Interference-free indication at a very low electrode potential without fouling of the electrode surface was achieved with the o-phenylenediamine: resorcinol-based MIP.
Yarman, Aysu ; Schulz, Christopher ; Sygmund, Cristoph ; Ludwig, Roland ; Gorton, Lo ; Wollenberger, Ursula ; Scheller, Frieder W.
For the first time the direct electron transfer of an enzyme - cellobiose dehydrogenase, CDH - has been coupled with the hexokinase catalyzed competition for glucose in a sensor for ATP. To enhance the signal output for ATP, pyruvate kinase was coimmobilized to recycle ADP by the phosphoenolpyruvate driven reaction. The new sensor overcomes the limit of 1:1 stoichiometry of the sequential or competitive conversion of ATP by effective enzymatic recycling of the analyte. The anodic oxidation of the glucose converting CDH proceeds at electrode potentials below 0 mV vs. Ag vertical bar AgCl thus potentially interfering substances like ascorbic acid or catecholamines do not influence the measuring signal. The combination of direct electron transfer of CDH with the enzymatic recycling results in an interference-free and oxygen-independent measurement of ATP in the lower mu molar concentration range with a lower limit of detection of 63.3 nM (S/N=3).
Drosophila melanogaster photoreceptors are highly polarized cells and their plasma membrane is organized into distinct domains. Zonula adherens junctions separate a smooth peripheral surface, the equivalent of the basolateral surface in other epithelial cells, from the central surface (cong apical surface). The latter consists of the microvillar rhabdomere and the juxtarhabdomeric domain, a nonmicrovillar area between the rhabdomere and the zonulae adherens. The distribution of Na/K-ATPase over these domains was examined by immunocytochemical, developmental, and genetic approaches. Immunofluorescence and immunogold labeling of adult compound eyes reveal that the distribution of Na/ K-ATPase is concentrated at the peripheral surface in the photoreceptors R1-R6, but extends over the juxtarhabdomeric domain to the rhabdomere in the photoreceptors R7/R8. Developmental analysis demonstrates further that Na/K-ATPase is localized over the entire plasma membrane in all photoreceptors in early pupal eyes. Redistribution of Na/K-ATPase in R1- R6 occurs at about 78% of pupal life, coinciding with the onset of Rh1-rhodopsin expression on the central surface of these cells. Despite the essential role of Rh1 in structural development and intracellular trafficking, Rh1 mutations do not affect the distribution of Na/K-ATPase. These results suggest that Na/K-ATPase and rhodopsin are involved in distinct intracellular localization mechanisms, which are maintained independent of each other.
Yates, Colin J. ; Elith, Jane ; Latimer, Andrew M. ; Le Maitre, David ; Midgley, Guy F. ; Schurr, Frank Martin ; West, Adam G.
Increasing evidence shows that anthropogenic climate change is affecting biodiversity. Reducing or stabilizing greenhouse gas emissions may slow global warming, but past emissions will continue to contribute to further unavoidable warming for more than a century. With obvious signs of difficulties in achieving effective mitigation worldwide in the short term at least, sound scientific predictions of future impacts on biodiversity will be required to guide conservation planning and adaptation. This is especially true in Mediterranean type ecosystems that are projected to be among the most significantly affected by anthropogenic climate change, and show the highest levels of confidence in rainfall projections. Multiple methods are available for projecting the consequences of climate change on the main unit of interest - the species - with each method having strengths and weaknesses. Species distribution models (SDMs) are increasingly applied for forecasting climate change impacts on species geographic ranges. Aggregation of models for different species allows inferences of impacts on biodiversity, though excluding the effects of species interactions. The modelling approach is based on several further assumptions and projections and should be treated cautiously. In the absence of comparable approaches that address large numbers of species, SDMs remain valuable in estimating the vulnerability of species. In this review we discuss the application of SDMs in predicting the impacts of climate change on biodiversity with special reference to the species-rich South West Australian Floristic Region and South African Cape Floristic Region. We discuss the advantages and challenges in applying SDMs in biodiverse regions with high levels of endemicity, and how a similar biogeographical history in both regions may assist us in understanding their vulnerability to climate change. We suggest how the process of predicting the impacts of climate change on biodiversity with SDMs can be improved and emphasize the role of field monitoring and experiments in validating the predictions of SDMs.
An electrochemical assay for the indication of the activity of the cell bound differentiation marker alkaline phosphatase (ALP) is proposed using voltammetry on an in-vitro cell culture. The basis of the assay is cultivation of cells on gold microelectrodes in wells of a microplate, catalytic hydrolysis of p-aminophenyl phosphate by ALP and indication of p-aminophenol oxidation by square wave voltammetry (SWV) with the sensors onto which the cells attached. The morphology of the bone marrow stromal cell line (MBA-15) on the electrode surface was investigated and it exhibited in vitro osteogenic characteristics. Since ALP is expressed on the cell surface in early differentiation stage of osteoblastic cells, its activity was followed after different culture times over a period of 144 h by recording repetitive voltammograms at different time points upon addition of the substrate p-aminophenyl phosphate. The ALP activity was estimated from the signal increase related to formation rate of p-aminophenol and the number of cells. The highest value was measured at 120 h, when the cells reached confluence. The results of the electrochemical activity assay are consistent with the colorimetric acquired value from p-nitrophenol formation rate.