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An ‛Aukward’ tale
(2017)
One hundred and seventy-three years ago, the last two Great Auks, Pinguinus impennis, ever reliably seen were killed. Their internal organs can be found in the collections of the Natural History Museum of Denmark, but the location of their skins has remained a mystery. In 1999, Great Auk expert Errol Fuller proposed a list of five potential candidate skins in museums around the world. Here we take a palaeogenomic approach to test which—if any—of Fuller’s candidate skins likely belong to either of the two birds. Using mitochondrial genomes from the five candidate birds (housed in museums in Bremen, Brussels, Kiel, Los Angeles, and Oldenburg) and the organs of the last two known individuals, we partially solve the mystery that has been on Great Auk scholars’ minds for generations and make new suggestions as to the whereabouts of the still-missing skin from these two birds.
Analyse der Funktion der dualen Lokalisation der 3-Mercaptopyruvat Sulfurtransferase im Menschen
(2017)
Changes in body height throughout extended historic periods are very complex and dynamic processes. Thispilot study aimed to investigate the pattern of longitudinal height z-scores changes in children before and after entering kindergarten. In summer 2016, we measured height and weight of 32 children from 4 groups of two kindergartens aged 3–6 years. All ages were centered according to the age of entry into the kindergarten. For each child we determined mean z-scores for height before and after entering the kindergarten, and assessed the variances for each kindergarten group. Twenty-two children targeted in height z-scores towards average height of their respective kindergarten group, 10 children did not. Due to the small numbers, the convergence in height variance however, remained insignificant (chi-squared independence test, p = 0.127). Additional studies with larger sample sizes are needed to confirm this pilot study.
Rubisco catalyses the first step of CO2 assimilation into plant biomass. Despite its crucial role, it is notorious for its low catalytic rate and its tendency to fix O2 instead of CO2, giving rise to a toxic product that needs to be recycled in a process known as photorespiration. Since almost all our food supply relies on Rubisco, even small improvements in its specificity for CO2 could lead to an improvement of photosynthesis and ultimately, crop yield. In this work, we attempted to improve photosynthesis by decreasing photorespiration with an artificial CCM based on a fusion between Rubisco and a carbonic anhydrase (CA).
A preliminary set of plants contained fusions between one of two CAs, bCA1 and CAH3, and the N- or C-terminus of RbcL connected by a small flexible linker of 5 amino acids. Subsequently, further fusion proteins were created between RbcL C-terminus and bCA1/CAH3 with linkers of 14, 23, 32, and 41 amino acids. The transplastomic tobacco plants carrying fusions with bCA1 were able to grow autotrophically even with the shortest linkers, albeit at a low rate, and accumulated very low levels of the fusion protein. On the other hand, plants carrying fusions with CAH3 were autotrophic only with the longer linkers. The longest linker permitted nearly wild-type like growth of the plants carrying fusions with CAH3 and increased the levels of fusion protein, but also of smaller degradation products.
The fusion of catalytically inactive CAs to RbcL did not cause a different phenotype from the fusions with catalytically active CAs, suggesting that the selected CAs were not active in the fusion with RbcL or their activity did not have an effect on CO2 assimilation. However, fusions to RbcL did not abolish RbcL catalytic activity, as shown by the autotrophic growth, gas exchange and in vitro activity measurements. Furthermore, Rubisco carboxylation rate and specificity for CO2 was not altered in some of the fusion proteins, suggesting that despite the defect in RbcL folding or assembly caused by the fusions, the addition of 60-150 amino acids to RbcL does not affect its catalytic properties. On the contrary, most growth defects of the plants carrying RbcL-CA fusions are related to their reduced Rubisco content, likely caused by impaired RbcL folding or assembly. Finally, we found that fusions with RbcL C-terminus were better tolerated than with the N-terminus, and increasing the length of the linker relieved the growth impairment imposed by the fusion to RbcL. Together, the results of this work constitute considerable relevant findings for future Rubisco engineering.
The increasing prevalence of methicillin-resistant Staphylococcus aureus has become a major public health threat. While lactobacilli were recently found useful in combating various pathogens, limited data exist on their therapeutic potential for S. aureus infections. The aim of this study was to determine whether Lactobacillus salivarius was able to produce bactericidal activities against S. aureus and to determine whether the inhibition was due to a generalized reduction in pH or due to secreted Lactobacillus product(s). We found an 8.6-log10 reduction of planktonic and a 6.3-log10 reduction of biofilm S. aureus. In contrast, the previously described anti-staphylococcal effects of L. fermentum only caused a 4.0-log10 reduction in planktonic S. aureus cells, with no effect on biofilm S. aureus cells. Killing of S. aureus was partially pH dependent, but independent of nutrient depletion. Cell-free supernatant that was pH neutralized and heat inactivated or proteinase K treated had significantly reduced killing of L. salivarius than with pH-neutralized supernatant alone. Proteomic analysis of the L. salivarius secretome identified a total of five secreted proteins including a LysM-containing peptidoglycan binding protein and a protein peptidase M23B. These proteins may represent potential novel anti-staphylococcal agents that could be effective against S. aureus biofilms.
The Arabidopsis thaliana NAC transcription factor JUNGBRUNNEN1 (AtJUB1) regulates growth by directly repressing GA3ox1 and DWF4, two key genes involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis, respectively, leading to GA and BR deficiency phenotypes. AtJUB1 also reduces the expression of PIF4, a bHLH transcription factor that positively controls cell elongation, while it stimulates the expression of DELLA genes, which are important repressors of growth. Here, we extend our previous findings by demonstrating that AtJUB1 induces similar GA and BR deficiency phenotypes and changes in gene expression when overexpressed in tomato (Solanum lycopersicum). Importantly, and in accordance with the growth phenotypes observed, AtJUB1 inhibits the expression of growth-supporting genes, namely the tomato orthologs of GA3ox1, DWF4 and PIF4, but activates the expression of DELLA orthologs, by directly binding to their promoters. Overexpression of AtJUB1 in tomato delays fruit ripening, which is accompanied by reduced expression of several ripeningrelated genes, and leads to an increase in the levels of various amino acids (mostly proline, beta-alanine, and phenylalanine), gamma-aminobutyric acid (GABA), and major organic acids including glutamic acid and aspartic acid. The fact that AtJUB1 exerts an inhibitory effect on the GA/BR biosynthesis and PIF4 genes but acts as a direct activator of DELLA genes in both, Arabidopsis and tomato, strongly supports the model that the molecular constituents of the JUNGBRUNNEN1 growth control module are considerably conserved across species.
Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis (Arabidopsis thaliana). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells.
Bacterial Molybdoenzymes
(2017)
The biogenesis of molybdoenzymes is a cytoplasmic event requiring both the folded apoenzymes and the matured molybdenum cofactor. The structure and the complexity of the molybdenum cofactor varies in each molybdoenzyme family and consequently different accessory proteins are required for the maturation of the respective enzymes. Thus, for enzymes of both the DMSO reductase and xanthine oxidase families, specific chaperones exist which are dedicated to increase the stability and the folding of specific members of each family. In this review, we describe the role of these chaperones for molybdoenzyme maturation. We present a model which describes step by step the mechanism of the maturation of representative molybdoenzymes from each family.
Background
Non-typhoid Salmonella Typhimurium (S. Typhimurium) accounts for a high number of registered salmonellosis cases, and O-serotyping is one important tool for monitoring epidemiology and spread of the disease. Moreover, variations in glucosylated O-antigens are related to immunogenicity and spread in the host. However, classical autoagglutination tests combined with the analysis of specific genetic markers cannot always reliably register phase variable glucose modifications expressed on Salmonella O-antigens and additional tools to monitor O-antigen glucosylation phenotypes of S. Typhimurium would be desirable.
Results
We developed a test for the phase variable O-antigen glucosylation state of S. Typhimurium using the tailspike proteins (TSP) of Salmonella phages 9NA and P22. We used this ELISA like tailspike adsorption (ELITA) assay to analyze a library of 44 Salmonella strains. ELITA was successful in discriminating strains that carried glucose 1-6 linked to the galactose of O-polysaccharide backbone (serotype O1) from non-glucosylated strains. This was shown by O-antigen compositional analyses of the respective strains with mass spectrometry and capillary electrophoresis. The ELITA test worked rapidly in a microtiter plate format and was highly O-antigen specific. Moreover, TSP as probes could also detect glucosylated strains in flow cytometry and distinguish multiphasic cultures differing in their glucosylation state.
Conclusions
Tailspike proteins contain large binding sites with precisely defined specificities and are therefore promising tools to be included in serotyping procedures as rapid serotyping agents in addition to antibodies. In this study, 9NA and P22TSP as probes could specifically distinguish glucosylation phenotypes of Salmonella on microtiter plate assays and in flow cytometry. This opens the possibility for flow sorting of cell populations for subsequent genetic analyses or for monitoring phase variations during large scale O-antigen preparations necessary for vaccine production.
Twenty two species of ectoparasites (Family Nycteribiidae: Nycteribia (Listropoda) schmidlii schmidlii, Nycteribia (Nycteribia) latreillii, Nycteribia (Nycteribia) pedicularia, Penicillidia (Penicillidia) dufourii, and Phthiridium biarticulatum; Family Streblidae: Brachytarsina (Brachytarsina) flavipennis and Raymondia huberi; Order Siphonaptera: Rhinolophopsylla unipectinata arabs, Nycteridopsylla longiceps, Araeopsylla gestroi, Ischnopsyllus intermedius, and Ischnopsyllus octactenus; Order Heteroptera: Cimex pipistrelli, Cimex lectularius, and Cacodmus vicinus; Class Arachnida: Order Mesostigmata: Spinturnix myoti and Eyndhovenia euryalis; Order Ixodida: Family Argasidae: Argas transgariepinus and Argas vespertilionis; Family Ixodidae: Hyalomma dromedarii, Ixodes ricinus, and Ixodes vespertilionis) were recovered from 19 bat species in Algeria. New host records for bats are recorded for the first time: N. schmidlii from Rh. clivosus and R. cystops; N. latreillii from Rh. blasii and P. gaisleri; R. huberi from Rh. clivosus; C. pipistrelli from E. isabellinus and H. savii; C. vicinus from E. isabellinus; S. myoti from P. gaisleri; E. euryalis from P. gaisleri and Rh. blasii; A. vespertilionis from P. gaisleri; I. ricinus from T. teniotis and Rh. hipposideros and H. dromedarii from P. kuhlii. Raymondia huberi is recorded for the first time from Algeria.
Biodiversity-multifunctionality relationships depend on identity and number of measured functions
(2017)
Biodiversity ensures ecosystem functioning and provisioning of ecosystem services, but it remains unclear how biodiversity-ecosystem multifunctionality relationships depend on the identity and number of functions considered. Here, we demonstrate that ecosystem multifunctionality, based on 82 indicator variables of ecosystem functions in a grassland biodiversity experiment, increases strongly with increasing biodiversity. Analysing subsets of functions showed that the effects of biodiversity on multifunctionality were stronger when more functions were included and that the strength of the biodiversity effects depended on the identity of the functions included. Limits to multifunctionality arose from negative correlations among functions and functions that were not correlated with biodiversity. Our findings underline that the management of ecosystems for the protection of biodiversity cannot be replaced by managing for particular ecosystem functions or services and emphasize the need for specific management to protect biodiversity. More plant species from the experimental pool of 60 species contributed to functioning when more functions were considered. An individual contribution to multifunctionality could be demonstrated for only a fraction of the species.
The heme-undecapeptide microperoxidase-11 (MP-11) was immobilized on mesoporous antimony-doped tin oxide (ATO) thin-film electrodes modified with the positively charged binding promotor polydiallyldimethylammonium chloride. Surface concentrations of MP-11 of 1.5 nmol cm(-2) were sufficiently high to enable spectroelectrochemical analyses. UV/Vis spectroscopy and resonance Raman spectroscopy revealed that immobilized MP-11 adopts a six-coordinated low-spin conformation, as in solution in the presence of a polycation. Cathodic reduction of hydrogen peroxide at potentials close to +500mV versus Ag/AgCl indicates that the reaction proceeds via a Compound I-type like intermediate, analogous to natural peroxidases, and confirms mesoporous ATO as a suitable host material for adsorbing the heme-peptide in its native state. A hydrogen peroxide sensor is proposed by using the bioelectrocatalytic properties of the MP-11-modified ATO.
Background: Rodents of the genus Rattus are among the most pervasive and successful invasive species, causing major vicissitudes in native ecological communities. A broad and flexible generalist diet has been suggested as key to the invasion success of Rattus spp. Here, we use an indirect approach to better understand foraging niche width, plasticity, and overlap within and between introduced Rattus spp. in anthropogenic habitats and natural humid forests of Madagascar.
Results: Based on stable carbon and nitrogen isotope values measured in hair samples of 589 individual rodents, we found that Rattus rattus had an extremely wide foraging niche, encompassing the isotopic space covered by a complete endemic forest-dwelling Malagasy small mammal community. Comparisons of Bayesian standard ellipses, as well as (multivariate) mixed-modeling analyses, revealed that the stable isotope niche of R. rattus tended to change seasonally and differed between natural forests and anthropogenic habitats, indicating plasticity in feeding niches. In co-occurrence, R. rattus and Rattus norvegicus partitioned feeding niches. Isotopic mismatch of signatures of individual R. rattus and the habitat in which they were captured, indicate frequent dispersal movements for this species between natural forest and anthropogenic habitats.
Conclusions: Since R. rattus are known to transmit a number of zoonoses, potentially affecting communities of endemic small mammals, as well as humans, these movements presumably increase transmission potential. Our results suggest that due to their generalist diet and potential movement between natural forest and anthropogenic habitats, Rattus spp. might affect native forest-dependent Malagasy rodents as competitors, predators, and disease vectors. The combination of these effects helps explain the invasion success of Rattus spp. and the detrimental effects of this genus on the endemic Malagasy rodent fauna.
Mosses are a major component of the arctic vegetation, particularly in wetlands. We present C / N atomic ratio, delta C-13 and delta N-15 data of 400 brown-moss samples belonging to 10 species that were collected along hydrological gradients within polygonal mires located on the southern Taymyr Peninsula and the Lena River delta in northern Siberia. Additionally, n-alkane patterns of six of these species (16 samples) were investigated. The aim of the study is to see whether the inter-and intraspecific differences in C / N, isotopic compositions and n-alkanes are indicative of habitat, particularly with respect to water level. Overall, we find high variability in all investigated parameters for two different moisture-related groups of moss species. The C / N ratios range between 11 and 53 (median: 32) and show large variations at the intraspecific level. However, species preferring a dry habitat (xero-mesophilic mosses) show higher C / N ratios than those preferring a wet habitat (meso-hygrophilic mosses). The delta C-13 values range between 37.0 and 22.5% (median D 27.8 %). The delta N-15 values range between 6.6 and C 1.7%(median D 2.2 %). We find differences in delta C-13 and delta N-15 compositions between both habitat types. For some species of the meso-hygrophilic group, we suggest that a relationship between the individ-ual habitat water level and isotopic composition can be inferred as a function of microbial symbiosis. The n-alkane distribution also shows differences primarily between xeromesophilic and meso-hygrophilic mosses, i. e. having a dominance of n-alkanes with long (n-C29, n-C31 /and intermediate (n-C25 /chain lengths, respectively. Overall, our results reveal that C / N ratios, isotopic signals and n-alkanes of studied brown-moss taxa from polygonal wetlands are characteristic of their habitat.
Mosses are a major component of the arctic vegetation, particularly in wetlands. We present C / N atomic ratio, delta C-13 and delta N-15 data of 400 brown-moss samples belonging to 10 species that were collected along hydrological gradients within polygonal mires located on the southern Taymyr Peninsula and the Lena River delta in northern Siberia. Additionally, n-alkane patterns of six of these species (16 samples) were investigated. The aim of the study is to see whether the inter-and intraspecific differences in C / N, isotopic compositions and n-alkanes are indicative of habitat, particularly with respect to water level. Overall, we find high variability in all investigated parameters for two different moisture-related groups of moss species. The C / N ratios range between 11 and 53 (median: 32) and show large variations at the intraspecific level. However, species preferring a dry habitat (xero-mesophilic mosses) show higher C / N ratios than those preferring a wet habitat (meso-hygrophilic mosses). The delta C-13 values range between 37.0 and 22.5% (median D 27.8 %). The delta N-15 values range between 6.6 and C 1.7%(median D 2.2 %). We find differences in delta C-13 and delta N-15 compositions between both habitat types. For some species of the meso-hygrophilic group, we suggest that a relationship between the individ-ual habitat water level and isotopic composition can be inferred as a function of microbial symbiosis. The n-alkane distribution also shows differences primarily between xeromesophilic and meso-hygrophilic mosses, i. e. having a dominance of n-alkanes with long (n-C29, n-C31 /and intermediate (n-C25 /chain lengths, respectively. Overall, our results reveal that C / N ratios, isotopic signals and n-alkanes of studied brown-moss taxa from polygonal wetlands are characteristic of their habitat.
Can't remember to forget you
(2017)
In nature plants are exposed to frequent changes in their abiotic and biotic environment. While some environmental cues are used to gauge the environment and align growth and development, others are beyond the regularly encountered spectrum of a species and trigger stress responses. Such stressful conditions provide a potential threat to survival and integrity. Plants adapt to extreme environmental conditions through physiological adaptations that are usually transient and are maintained until stressful environments subside. It is increasingly appreciated that in some cases environmental cues activate a stress memory that persists for some time after the extreme condition has subsided. Recent research has shown that this stress-induced environmental memory is mediated by epigenetic and chromatin-based mechanisms and both histone methylation and nucleosome occupancy are associated with it.
Mathematical models of bacterial growth have been successfully applied to study the relationship between antibiotic drug exposure and the antibacterial effect. Since these models typically lack a representation of cellular processes and cell physiology, the mechanistic integration of drug action is not possible on the cellular level. The cellular mechanisms of drug action, however, are particularly relevant for the prediction, analysis and understanding of interactions between antibiotics. Interactions are also studied experimentally, however, a lacking consent on the experimental protocol hinders direct comparison of results. As a consequence, contradictory classifications as additive, synergistic or antagonistic are reported in literature.
In the present thesis we developed a novel mathematical model for bacterial growth that integrates cell-level processes into the population growth level. The scope of the model is to predict bacterial growth under antimicrobial perturbation by multiple antibiotics in vitro.
To this end, we combined cell-level data from literature with population growth data for Bacillus subtilis, Escherichia coli and Staphylococcus aureus. The cell-level data described growth-determining characteristics of a reference cell, including the ribosomal concentration and efficiency. The population growth data comprised extensive time-kill curves for clinically relevant antibiotics (tetracycline, chloramphenicol, vancomycin, meropenem, linezolid, including dual combinations).
The new cell-level approach allowed for the first time to simultaneously describe single and combined effects of the aforementioned antibiotics for different experimental protocols, in particular different growth phases (lag and exponential phase). Consideration of ribosomal dynamics and persisting sub-populations explained the decreased potency of linezolid on cultures in the lag phase compared to exponential phase cultures. The model captured growth rate dependent killing and auto-inhibition of meropenem and - also for vancomycin exposure - regrowth of the bacterial cultures due to adaptive resistance development. Stochastic interaction surface analysis demonstrated the pronounced antagonism between meropenem and linezolid to be robust against variation in the growth phase and pharmacodynamic endpoint definition, but sensitive to a change in the experimental duration.
Furthermore, the developed approach included a detailed representation of the bacterial cell-cycle. We used this representation to describe septation dynamics during the transition of a bacterial culture from the exponential to stationary growth phase. Resulting from a new mechanistic understanding of transition processes, we explained the lag time between the increase in cell number and bacterial biomass during the transition from the lag to exponential growth phase. Furthermore, our model reproduces the increased intracellular RNA mass fraction during long term exposure of bacteria to chloramphenicol.
In summary, we contribute a new approach to disentangle the impact of drug effects, assay readout and experimental protocol on antibiotic interactions. In the absence of a consensus on the corresponding experimental protocols, this disentanglement is key to translate information between heterogeneous experiments and also ultimately to the clinical setting.
Single-cell quantification of transcription kinetics and variability promotes a mechanistic understanding of gene regulation. Here, using single-molecule RNA fluorescence in situ hybridization and mathematical modeling, we dissect cellular RNA dynamics for Arabidopsis FLOWERING LOCUS C (FLC). FLC expression quantitatively determines flowering time and is regulated by antisense (COOLAIR) transcription. In cells without observable COOLAIR expression, we quantify FLC transcription initiation, elongation, intron processing, and lariat degradation, as well as mRNA release from the locus and degradation. In these heterogeneously sized cells, FLC mRNA number increases linearly with cell size, resulting in a large cell-to-cell variability in transcript level. This variation is accounted for by cell-sizedependent, Poissonian FLC mRNA production, but not by large transcriptional bursts. In COOLAIRexpressing cells, however, antisense transcription increases with cell size and contributes to FLC transcription decreasing with cell size. Our analysis therefore reveals an unexpected role for antisense transcription in modulating the scaling of transcription with cell size.
Head and neck squamous cell carcinomas (HNSCC) exhibiting resistance to the EGFR-targeting drug cetuximab poses a challenge to their effective clinical management. Here, we report a specific mechanism of resistance in this setting based upon the presence of a single nucleotide polymorphism encoding EGFR-K-521 (K-allele), which is expressed in > 40% of HNSCC cases. Patients expressing the K-allele showed significantly shorter progressionfree survival upon palliative treatment with cetuximab plus chemotherapy or radiation. In several EGFR-mediated cancer models, cetuximab failed to inhibit downstream signaling or to kill cells harboring a high K-allele frequency. Cetuximab affinity for EGFR-K-521 was reduced slightly, but ligand-mediated EGFR acti-vation was intact. We found a lack of glycan sialyation on EGFR-K-521 that associated with reduced protein stability, suggesting a structural basis for reduced cetuximab efficacy. CetuGEX, an antibody with optimized Fc glycosylation targeting the same epitope as cetuximab, restored HNSCC sensitivity in a manner associated with antibody-dependent cellular cytotoxicity rather than EGFR pathway inhibition. Overall, our results highlight EGFR-K-521 expression as a key mechanism of cetuximab resistance to evaluate prospectively as a predictive biomarker in HNSCC patients. Further, they offer a preclinical rationale for the use of ADCC-optimized antibodies to treat tumors harboring this EGFR isoform.
Chloroplast membranes have a unique composition characterized by very high contents of the galactolipids, MGDG and DGDG. Many studies on constitutive, galactolipid-deficient mutants revealed conflicting results about potential functions of galactolipids in photosynthetic membranes. Likely, this was caused by pleiotropic effects such as starvation artefacts because of impaired photosynthesis from early developmental stages of the plants onward. Therefore, an ethanol inducible RNAi-approach has been taken to suppress two key enzymes of galactolipid biosynthesis in the chloroplast, MGD1 and DGD1. Plants were allowed to develop fully functional source leaves prior to induction, which then could support plant growth. Then, after the ethanol induction, both young and mature leaves were investigated over time.
Our studies revealed similar changes in both MGDG- and DGDG-deficient lines, however young and mature leaves of transgenic lines showed a different response to galactolipid deficiency. While no changes of photosynthetic parameters and minor changes in lipid content were observed in mature leaves of transgenic lines, strong reductions in total chlorophyll content and in the accumulation of all photosynthetic complexes and significant changes in contents of various lipid groups occurred in young leaves. Microscopy studies revealed an appearance of lipid droplets in the cytosol of young leaves in all transgenic lines which correlates with significantly higher levels of TAGs. Since in young leaves the production of membrane lipids is lowered, the excess of fatty acids is used for storage lipids production, resulting in the accumulation of TAGs.
Our data indicate that both investigated galactolipids serve as structural lipids since changes in photosynthetic parameters were mainly the result of reduced amounts of all photosynthetic constituents. In response to restricted galactolipid synthesis, thylakoid biogenesis is precisely readjusted to keep the proper stoichiometry and functionality of the photosynthetic apparatus. Ultimately, the data revealed that downregulation of one galactolipid triggers changes not only in chloroplasts but also in the nucleus as shown by downregulation of nuclear encoded subunits of the photosynthetic complexes.
In this work the human AOX1 was characterized and detailed aspects regarding the expression, the enzyme kinetics and the production of reactive oxygen species (ROS) were investigated. The hAOX1 is a cytosolic enzyme belonging to the molybdenum hydroxylase family. Its catalytically active form is a homodimer with a molecular weight of 300 kDa. Each monomer (150 kDa) consists of three domains: a N-terminal domain (20 kDa) containing two [2Fe-2S] clusters, a 40 kDa intermediate domain containing a flavin adenine dinucleotide (FAD), and a C-terminal domain (85 kDa) containing the substrate binding pocket and the molybdenum cofactor (Moco). The hAOX1 has an emerging role in the metabolism and pharmacokinetics of many drugs, especially aldehydes and N- heterocyclic compounds.
In this study, the hAOX1 was hetereogously expressed in E. coli TP1000 cells, using a new codon optimized gene sequence which improved the expressed protein yield of around 10-fold compared to the previous expression systems for this enzyme. To increase the catalytic activity of hAOX1, an in vitro chemical sulfuration was performed to favor the insertion of the equatorial sulfido ligand at the Moco with consequent increased enzymatic activity of around 10-fold. Steady-state kinetics and inhibition studies were performed using several substrates, electron acceptors and inhibitors. The recombinant hAOX1 showed higher catalytic activity when molecular oxygen was used as electron acceptor. The highest turn over values were obtained with phenanthridine as substrate. Inhibition studies using thioridazine (phenothiazine family), in combination with structural studies performed in the group of Prof. M.J. Romão, Nova Universidade de Lisboa, showed a new inhibition site located in proximity of the dimerization site of hAOX1. The inhibition mode of thioridazine resulted in a noncompetitive inhibition type. Further inhibition studies with loxapine, a thioridazine-related molecule, showed the same type of inhibition. Additional inhibition studies using DCPIP and raloxifene were carried out.
Extensive studies on the FAD active site of the hAOX1 were performed. Twenty new hAOX1 variants were produced and characterized. The hAOX1 variants generated in this work were divided in three groups: I) hAOX1 single nucleotide polymorphisms (SNP) variants; II) XOR- FAD loop hAOX1 variants; III) additional single point hAOX1 variants. The hAOX1 SNP variants G46E, G50D, G346R, R433P, A439E, K1231N showed clear alterations in their catalytic activity, indicating a crucial role of these residues into the FAD active site and in relation to the overall reactivity of hAOX1.
Furthermore, residues of the bovine XOR FAD flexible loop (Q423ASRREDDIAK433) were introduced in the hAOX1. FAD loop hAOX1 variants were produced and characterized for their stability and catalytic activity. Especially the variants hAOX1 N436D/A437D/L438I, N436D/A437D/L438I/I440K and Q434R/N436D/A437D/L438I/I440K showed decreased catalytic activity and stability. hAOX1 wild type and variants were tested for reactivity toward NADH but no reaction was observed.
Additionally, the hAOX1 wild type and variants were tested for the generation of reactive oxygen species (ROS). Interestingly, one of the SNP variants, hAOX1 L438V, showed a high ratio of superoxide prodction. This result showed a critical role for the residue Leu438 in the mechanism of oxygen radicals formation by hAOX1. Subsequently, further hAOX1 variants having the mutated Leu438 residue were produced. The variants hAOX1 L438A, L438F and L438K showed superoxide overproduction of around 85%, 65% and 35% of the total reducing equivalent obtained from the substrate oxidation.
The results of this work show for the first time a characterization of the FAD active site of the hAOX1, revealing the importance of specific residues involved in the generation of ROS and effecting the overall enzymatic activity of hAOX1. The hAOX1 SNP variants presented here indicate that those allelic variations in humans might cause alterations ROS balancing and clearance of drugs in humans.
Im Sinne des Refinements von Tierversuchen sollen alle Bedingungen während der Zucht, der Haltung und des Transports von zu Versuchszwecken gehaltenen Tieren und alle Methoden während des Versuchs so verbessert werden, dass die verwendeten Tiere ein minimales Maß an potentiellem Distress, Schmerzen oder Leiden erfahren. Zudem soll ihr Wohlbefinden durch die Möglichkeit des Auslebens speziesspezifischer Verhaltensweisen und die Anwendung tierschonender Verfahren maximal gefördert werden. Zur Etablierung von Grundsätzen des Refinements sind grundlegende Kenntnisse über die physiologischen Bedürfnisse und Verhaltensansprüche der jeweiligen Spezies unabdingbar. Die Experimentatoren sollten das Normalverhalten der Tiere kennen, um potentielle Verhaltensabweichungen, wie Stereotypien, zu verstehen und interpretieren zu können. Standardisierte Haltungsbedingungen von zu Versuchszwecken gehaltenen Mäusen weichen in diversen Aspekten von der natürlichen Umgebung ab und erfordern eine gewisse Adaptation. Ist ein Tier über einen längeren Zeitraum unfähig, sich an die gegebenen Umstände anzupassen, können abnormale Verhaltensweisen, wie Stereotypien auftreten. Stereotypien werden definiert als Abweichungen vom Normalverhalten, die repetitiv und ohne Abweichungen im Ablauf ausgeführt werden, scheinbar keiner Funktion dienen und der konkreten Umweltsituation nicht immer entsprechen.
Bisher war unklar, in welchem Ausmaß stereotypes Verhalten den metabolischen Phänotyp eines Individuums beeinflusst. Ziel dieser Arbeit war es daher, das stereotype Verhalten der FVB/NJ-Maus erstmals detailliert zu charakterisieren, systematisch zusammenzutragen, welche metabolischen Konsequenzen dieses Verhalten bedingt und wie sich diese auf das Wohlbefinden der Tiere und die Verwendung stereotyper Tiere in Studien mit tierexperimentellem Schwerpunkt auswirken.
Der Versuch begann mit der Charakterisierung der mütterlichen Fürsorge in der Parentalgeneration. Insgesamt wurden 35 Jungtiere der F1-Generation vom Absatz an, über einen Zeitraum von 11 Wochen einzeln gehalten, kontinuierlich beobachtet, bis zum Versuchsende wöchentlich Kotproben gesammelt und das Körpergewicht bestimmt. Zusätzlich erfolgten begleitende Untersuchungen wie Verhaltenstests und die Erfassung der physischen Aktivität und metabolischer Parameter. Anschließend wurden u.a. die zerebralen Serotonin- und Dopamingehalte, fäkale Glucocorticoidlevels, hepatisches Glykogen und muskuläre Glykogen- und Triglyceridlevels bestimmt.
Nahezu unabhängig von der mütterlichen Herkunft entwickelte sich bei mehr als der Hälfte der 35 Jungtiere in der F1-Generation stereotypes Verhalten. Diese Daten deuten darauf hin, dass es keine Anzeichen für das Erlernen oder eine direkte genetische Transmission stereotypen Verhaltens bei der FVB/NJ-Maus gibt. Über den gesamten Beobachtungszeitraum zeichneten sich die stereotypen FVB/NJ-Mäuse durch ein eingeschränktes Verhaltensrepertoire aus. Zu Gunsten der erhöhten Aktivität und des Ausübens stereotypen Verhaltens lebten sie insgesamt weniger andere Verhaltensweisen (Klettern, Graben, Nagen) aus. Darüber hinaus waren Stereotypien sowohl im 24-Stunden Open Field Test als auch in der Messeinrichtung der indirekten Tierkalorimetrie mit einer erhöhten Aktivität und Motilität assoziiert, während die circadiane Rhythmik nicht divergierte. Diese erhöhte körperliche Betätigung spiegelte sich in den niedrigeren Körpergewichtsentwicklungen der stereotypen Tiere wieder. Außerdem unterschieden sich die Körperfett- und Körpermuskelanteile.
Zusammenfassend lässt sich sagen, dass das Ausüben stereotypen Verhaltens zu Differenzen im metabolischen Phänotyp nicht-stereotyper und stereotyper FVB/NJ-Mäuse führt. Im Sinne der „Guten Wissenschaftlichen Praxis“ sollte das zentrale Ziel jedes Wissenschaftlers sein, aussagekräftige und reproduzierbare Daten hervorzubringen. Jedoch können keine validen Resultate von Tieren erzeugt werden, die in Aspekten variieren, die für den vorgesehenen Zweck der Studie nicht berücksichtigt wurden. Deshalb sollten nicht-stereotype und stereotype Individuen nicht innerhalb einer Versuchsgruppe randomisiert werden. Stereotype Tiere demzufolge von geplanten Studien auszuschließen, würde allerdings dem Gebot des zweiten R’s – der Reduction – widersprechen. Um Refinement zu garantieren, sollte der Fokus auf der maximal erreichbaren Prävention stereotypen Verhaltens liegen. Diverse Studien haben bereits gezeigt, dass die Anreicherung der Haltungsumwelt (environmental enrichment) zu einer Senkung der Prävalenz von Stereotypien bei Mäusen führt, dennoch kommen sie weiterhin vor. Daher sollte environmental enrichment zukünftig weniger ein „Kann“, sondern ein „Muss“ sein – oder vielmehr: der Goldstandard. Zudem würde eine profunde phänotypische Charakterisierung dazu beitragen, Mausstämme zu erkennen, die zu Stereotypien neigen und den für den spezifischen Zweck am besten geeigneten Mausstamm zu identifizieren, bevor ein Experiment geplant wird.
Citizen science projects have a long history in ecological studies. The research usefulness of such projects is dependent on applying simple and standardized methods. Here, we conducted a citizen science project that involved more than 3500 Swedish high school students to examine the temperature difference between surface water and the overlying air (T-w-T-a) as a proxy for sensible heat flux (Q(H)). If Q(H) is directed upward, corresponding to positive T-w-T-a, it can enhance CO2 and CH4 emissions from inland waters, thereby contributing to increased greenhouse gas concentrations in the atmosphere. The students found mostly negative T-w-T-a across small ponds, lakes, streams/rivers and the sea shore (i.e. downward Q(H)), with T-w-T-a becoming increasingly negative with increasing T-a. Further examination of T-w-T-a using high-frequency temperature data from inland waters across the globe confirmed that T-w-T-a is linearly related to T-a. Using the longest available high-frequency temperature time series from Lake Erken, Sweden, we found a rapid increase in the occasions of negative T-w-T-a with increasing annual mean T-a since 1989. From these results, we can expect that ongoing and projected global warming will result in increasingly negative T-w-T-a, thereby reducing CO2 and CH4 transfer velocities from inland waters into the atmosphere.
We investigated 4.84-m-long sediment record spanning over the Late Glacial and Holocene from Lake Donggi Cona to be able to reconstruct circulation pattern on the Tibetan Plateau (TP). Presently, Lake Donggi Cona is located at the boundaries of Westerlies and Asian monsoon circulations in the northeastern TP. However, the exact timing and stimulating mechanisms for climatic changes and monsoon shifts in this region are still debated. We used a 19-ka-long stable isotope record of sedimentary n-alkanes to address this discrepancy by providing insights into paleohydrological conditions. The SD of nC(23) is influenced by lake water evaporation; the BD. values of sedimentary nC(29) are mainly controlled by moisture source and temperature changes. Long-chain n-alkanes dominate over the core whereas three mean clusters (i.e. microbial, aquatic and terrestrial) can be inferred. Multi-proxies suggest five major episodes in the history of Lake Donggi Cona. The Lake Donggi Cona record indicates that the Late Glacial(18.4-14.8 cal ka BP) was dominated by low productivity of mainly microbial and aquatic organisms. Relatively low delta D values suggest low temperatures and moist conditions eventually caused by stronger Westerlies, winter monsoon and melt-water influence. Likely, the shift (similar to 17.9 cal ka BP) from microbial to enhanced aquatic input suggests either a change from deep to shallow water lake or a break in local stratification. Between 14.8 and 13.0 cal ka BP, variable climatic conditions prevailed. Although the Westerlies weekend, the increase in temperature enhanced the permafrost and snow melting (displayed by a high sedimentary accumulation rate). Higher delta D values indicate increasingly arid conditions with higher temperatures which eventually lead to high evaporative conditions and lowest lake levels. Low vegetation cover and high erosion rates led to high sediment accumulation resulting in stratification followed by anoxia in the terminal lake. From 13.0 to 9.2 cal ka BP, lowered values of 813 along with high contents of terrestrial organic matter marked the early-Holocene warming indicating a further strengthening of summer precipitation and higher lake levels. A cooling trend was observed in the mid-Holocene between 9.2 and 3.0 cal ka BP accompanied by higher moisture availability (displayed by lowered SD values) caused by reduced evaporative conditions due to a drop in temperature and recovering Westerlies. After 3.0 cal ka BP, a decrease in lake productivity and cold and semi-arid conditions prevailed suggesting lower lake levels and reduced moisture from recycled air masses and Westerlies. We propose that the summer monsoon was the predominant moisture source during the Belling-Allered warm complex and early -Holocene followed by Westerlies in mid-to-late Holocene period. Stable carbon isotope values-32%o indicate the absence of C-4 -type vegetation in the region contradicting with their presence in the Lake Qinghai record. The 81) record from lake Donggi Cona highlights the importance of the interplay between Westerlies and summer monsoon circulation at this location, which is highly dynamic in northeastern plateau compared to the North Atlantic circulation and insolation changes. Consequently lake Donggi Cona might be an important anchor point for environmental reconstructions on the Tibetan Plateau. (C) 2017 The Authors. Published by Elsevier Ltd.
The performance of hybridization capture combined with next-generation sequencing (NGS) has seen limited investigation with samples from hot and arid regions until now. We applied hybridization capture and shotgun sequencing to recover DNA sequences from bone specimens of ancient-domestic dromedary (Camelus dromedarius) and its extinct ancestor, the wild dromedary from Jordan, Syria, Turkey and the Arabian Peninsula, respectively. Our results show that hybridization capture increased the percentage of mitochondrial DNA (mtDNA) recovery by an average 187-fold and in some cases yielded virtually complete mitochondrial (mt) genomes at multifold coverage in a single capture experiment. Furthermore, we tested the effect of hybridization temperature and time by using a touchdown approach on a limited number of samples. We observed no significant difference in the number of unique dromedary mtDNA reads retrieved with the standard capture compared to the touchdown method. In total, we obtained 14 partial mitochondrial genomes from ancient-domestic dromedaries with 17-95% length coverage and 1.27-47.1-fold read depths for the covered regions. Using whole-genome shotgun sequencing, we successfully recovered endogenous dromedary nuclear DNA (nuDNA) from domestic and wild dromedary specimens with 1-1.06-fold read depths for covered regions. Our results highlight that despite recent methodological advances, obtaining ancient DNA (aDNA) from specimens recovered from hot, arid environments is still problematic. Hybridization protocols require specific optimization, and samples at the limit of DNA preservation need multiple replications of DNA extraction and hybridization capture as has been shown previously for Middle Pleistocene specimens.
The performance of hybridization capture combined with next-generation sequencing (NGS) has seen limited investigation with samples from hot and arid regions until now. We applied hybridization capture and shotgun sequencing to recover DNA sequences from bone specimens of ancient-domestic dromedary (Camelus dromedarius) and its extinct ancestor, the wild dromedary from Jordan, Syria, Turkey and the Arabian Peninsula, respectively. Our results show that hybridization capture increased the percentage of mitochondrial DNA (mtDNA) recovery by an average 187-fold and in some cases yielded virtually complete mitochondrial (mt) genomes at multifold coverage in a single capture experiment. Furthermore, we tested the effect of hybridization temperature and time by using a touchdown approach on a limited number of samples. We observed no significant difference in the number of unique dromedary mtDNA reads retrieved with the standard capture compared to the touchdown method. In total, we obtained 14 partial mitochondrial genomes from ancient-domestic dromedaries with 17-95% length coverage and 1.27-47.1-fold read depths for the covered regions. Using whole-genome shotgun sequencing, we successfully recovered endogenous dromedary nuclear DNA (nuDNA) from domestic and wild dromedary specimens with 1-1.06-fold read depths for covered regions. Our results highlight that despite recent methodological advances, obtaining ancient DNA (aDNA) from specimens recovered from hot, arid environments is still problematic. Hybridization protocols require specific optimization, and samples at the limit of DNA preservation need multiple replications of DNA extraction and hybridization capture as has been shown previously for Middle Pleistocene specimens.
Comparing mitogenomic timetrees for two African savannah primate genera (Chlorocebus and Papio)
(2017)
Complete mitochondrial (mtDNA) genomes have proved to be useful in reconstructing primate phylogenies with higher resolution and confidence compared to reconstructions based on partial mtDNA sequences. Here, we analyse complete mtDNA genomes of African green monkeys (genus Chlorocebus), a widely distributed primate genus in Africa representing an interesting phylogeographical model for the evolution of savannah species. Previous studies on partial mtDNA sequences revealed nine major clades, suggesting several cases of para- and polyphyly among Chlorocebus species. However, in these studies, phylogenetic relationships among several clades were not resolved, and divergence times were not estimated. We analysed complete mtDNA genomes for ten Chlorocebus samples representing major mtDNA clades to find stronger statistical support in the phylogenetic reconstruction than in the previous studies and to estimate divergence times. Our results confirmed para- and polyphyletic relationships of most Chlorocebus species, while the support for the phylogenetic relationships between the mtDNA clades increased compared to the previous studies. Our results indicate an initial west-east division in the northern part of the Chlorocebus range with subsequent divergence into north-eastern and southern clades. This phylogeographic scenario contrasts with that for another widespread African savannah primate genus, the baboons (Papio), for which a dispersal from southern Africa into East and West Africa was suggested.
The bat-eared fox, Otocyon megalotis, is the only member of its genus and is thought to occupy a basal position within the dog family. These factors can lead to challenges in complete mitochondrial reconstructions and accurate phylogenetic positioning. Here, we present the first complete mitochondrial genome of the bat-eared fox recovered using shotgun sequencing and iterative mapping to three distantly related species. Phylogenetic analyses placed the bat-eared fox basal in the Canidae family within the clade including true foxes (Vulpes) and the raccoon dog (Nyctereutes) with high support values. This position is in good agreement with previously published results based on short fragments of mitochondrial and nuclear genes, therefore adding more support to the basal positioning of the bat-eared fox within Canidae.
The combination of the biocatalytic features of enzymes with the unique physical properties of nanoparticles in a biohybrid system provides a promising approach for the development of advanced bioelectrocatalytic devices. This study describes the construction of photoelectrochemical signal chains based on CdSe/ZnS quantum dot (QD) modified gold electrodes as light switchable elements, and low molecular weight redox molecules for the combination with different biocatalysts. Photoelectrochemical and photoluminescence experiments verify that electron transfer can be achieved between the redox molecules hexacyanoferrate and ferrocene, and the QDs under illumination. Since for both redox mediators a concentration dependent photocurrent change has been found, light switchable enzymatic signal chains are built up with fructose dehydrogenase (FDH) and pyrroloquinoline quinone-dependent glucose dehydrogenase ((PQQ) GDH) for the detection of sugars. After immobilization of the enzymes at the QD electrode the biocatalytic oxidation of the substrates can be followed by conversion of the redox mediator in solution and subsequent detection at the QD electrode. Furthermore, (PQQ) GDH has been assembled together with ferrocenecarboxylic acid on top of the QD electrode for the construction of a funtional biohybrid architecture, showing that electron transfer can be realized from the enzyme over the redox mediator to the QDs and subsequently to the electrode in a completely immobilized fashion. The results obtained here do not only provide the basis for light-switchable biosensing and bioelectrocatalytic applications, but may also open the way for self-driven point-of-care systems by combination with solar cell approaches (power generation at the QD electrode by enzymatic substrate consumption).
Background: Animals show consistent individual behavioural patterns over time and over situations. This phenomenon has been referred to as animal personality or behavioural syndromes. Little is known about consistency of animal personalities over entire life times. We investigated the repeatability of behaviour in common voles (Microtus arvalis) at different life stages, with different time intervals, and in different situations. Animals were tested using four behavioural tests in three experimental groups: 1. before and after maturation over three months, 2. twice as adults during one week, and 3. twice as adult animals over three months, which resembles a substantial part of their entire adult life span of several months.
Results: Different behaviours were correlated within and between tests and a cluster analysis showed three possible behavioural syndrome-axes, which we name boldness, exploration and activity. Activity and exploration behaviour in all tests was highly repeatable in adult animals tested over one week. In animals tested over maturation, exploration behaviour was consistent whereas activity was not. Voles that were tested as adults with a three-month interval showed the opposite pattern with stable activity but unstable exploration behaviour.
Conclusions: The consistency in behaviour over time suggests that common voles do express stable personality over short time. Over longer periods however, behaviour is more flexible and depending on life stage (i.e. tested before/after maturation or as adults) of the tested individual. Level of boldness or activity does not differ between tested groups and maintenance of variation in behavioural traits can therefore not be explained by expected future assets as reported in other studies.
All life-sustaining processes are ultimately driven by thousands of biochemical reactions occurring in the cells: the metabolism. These reactions form an intricate network which produces all required chemical compounds, i.e., metabolites, from a set of input molecules. Cells regulate the activity through metabolic reactions in a context-specific way; only reactions that are required in a cellular context, e.g., cell type, developmental stage or environmental condition, are usually active, while the rest remain inactive. The context-specificity of metabolism can be captured by several kinds of experimental data, such as by gene and protein expression or metabolite profiles. In addition, these context-specific data can be assimilated into computational models of metabolism, which then provide context-specific metabolic predictions.
This thesis is composed of three individual studies focussing on context-specific experimental data integration into computational models of metabolism. The first study presents an optimization-based method to obtain context-specific metabolic predictions, and offers the advantage of being fully automated, i.e., free of user defined parameters. The second study explores the effects of alternative optimal solutions arising during the generation of context-specific metabolic predictions. These alternative optimal solutions are metabolic model predictions that represent equally well the integrated data, but that can markedly differ. This study proposes algorithms to analyze the space of alternative solutions, as well as some ways to cope with their impact in the predictions.
Finally, the third study investigates the metabolic specialization of the guard cells of the plant Arabidopsis thaliana, and compares it with that of a different cell type, the mesophyll cells. To this end, the computational methods developed in this thesis are applied to obtain metabolic predictions specific to guard cell and mesophyll cells. These cell-specific predictions are then compared to explore the differences in metabolic activity between the two cell types. In addition, the effects of alternative optima are taken into consideration when comparing the two cell types. The computational results indicate a major reorganization of the primary metabolism in guard cells. These results are supported by an independent 13C labelling experiment.
Intensive land use is a major cause of biodiversity loss, but most studies comparing the response of multiple taxa rely on simple diversity measures while analyses of other community attributes are only recently gaining attention. Species-abundance distributions (SADs) are a community attribute that can be used to study changes in the overall abundance structure of species groups, and whether these changes are driven by abundant or rare species. We evaluated the effect of grassland management intensity for three land-use modes (fertilization, mowing, grazing) and their combination on species richness and SADs for three belowground (arbuscular mycorrhizal fungi, prokaryotes and insect larvae) and seven aboveground groups (vascular plants, bryophytes and lichens; arthropod herbivores; arthropod pollinators; bats and birds). Three descriptors of SADs were evaluated: general shape (abundance decay rate), proportion of rare species (rarity) and proportional abundance of the commonest species (dominance). Across groups, taxonomic richness was largely unaffected by land-use intensity and only decreased with increasing mowing intensity. Of the three SAD descriptors, abundance decay rate became steeper with increasing combined land-use intensity across groups. This reflected a decrease in rarity among plants, herbivores and vertebrates. Effects of fertilization on the three descriptors were similar to the combined land-use intensity effects. Mowing intensity only affected the SAD descriptors of insect larvae and vertebrates, while grazing intensity produced a range of effects on different descriptors in distinct groups. Overall, belowground groups had more even abundance distribtitions than aboveground groups. Strong differences among aboveground groups and between above- and belowground groups indicate that no single taxonomic group can serve as an indicator for effects in other groups. In the past, the use of SADs has been hampered by concerns over theoretical models underlying specific forms of SADs. Our study shows that SAD descriptors that are not connected to a particular model are suitable to assess the effect of land use on community structure.
The acentriolar Dictyostelium centrosome is a nucleus-associated body consisting of a core structure with three plaque-like layers, which are surrounded by a microtubule-nucleating corona. The core duplicates once per cell cycle at the G2/M transition, whereby its central layer disappears and the two outer layers form the mitotic spindle poles. Through proteomic analysis of isolated centrosomes, we have identified CP39 and CP75, two essential components of the core structure. Both proteins can be assigned to the central core layer as their centrosomal presence is correlated to the disappearance and reappearance of the central core layer in the course of centrosome duplication. Both proteins contain domains with centrosome-binding activity in their N- and C-terminal halves, whereby the respective N-terminal half is required for cell cycle-dependent regulation. CP39 is capable of self-interaction and GFP-CP39 overexpression elicited supernumerary microtubule-organizing centers and pre-centrosomal cytosolic clusters. Underexpression stopped cell growth and reversed the MTOC amplification phenotype. In contrast, in case of CP75 underexpression of the protein by RNAi treatment elicited supernumerary MTOCs. In addition, CP75RNAi affects correct chromosome segregation and causes co-depletion of CP39 and CP91, another central core layer component. CP39 and CP75 interact with each other directly in a yeast two-hybrid assay. Furthermore, CP39, CP75 and CP91 mutually interact in a proximity-dependent biotin identification (BioID) assay. Our data indicate that these three proteins are all required for proper centrosome biogenesis and make up the major structural components of core structure's central layer.
Inhibition of MAP kinase pathways by selective BRAF inhibitors, such as vemurafenib and dabrafenib, have evolved as key therapies of BRAF-mutated melanoma. However, tumor relapse and therapy resistance have remained as major problems, which may be addressed by combination with other pathway inhibitors. Here we identified the potassium channel inhibitor TRAM-34 as highly effective in combination with vemurafenib. Thus apoptosis was significantly enhanced and cell viability was decreased. The combination vemurafenib/TRAM-34 was also effective in vemurafenib-resistant cells, suggesting that acquired resistance may be overcome. Vemurafenib decreased ERK phosphorylation, suppressed antiapoptotic Mcl-1 and enhanced proapoptotic Puma and Bim. The combination resulted in enhancement of proapoptotic pathways as caspase-3 and loss of mitochondrial membrane potential. Indicating a special mechanism of vemurafenib-induced apoptosis, we found strong enhancement of intracellular ROS levels already at 1 h of treatment. The critical role of ROS was demonstrated by the antioxidant vitamin E (alpha-tocopherol), which decreased intracellular ROS as well as apoptosis. Also caspase activation and loss of mitochondrial membrane potential were suppressed, proving ROS as an upstream effect. Thus ROS represents an initial and independent apoptosis pathway in melanoma cells that is of particular importance for vemurafenib and its combination with TRAM-34.
Background
Protected areas are the most common and important instrument for the conservation of biological diversity and are called for under the United Nations' Convention on Biological Diversity. Growing human population densities, intensified land-use, invasive species and increasing habitat fragmentation threaten ecosystems worldwide and protected areas are often the only refuge for endangered species. Climate change is posing an additional threat that may also impact ecosystems currently under protection. Therefore, it is of crucial importance to include the potential impact of climate change when designing future nature conservation strategies and implementing protected area management. This approach would go beyond reactive crisis management and, by necessity, would include anticipatory risk assessments. One avenue for doing so is being provided by simulation models that take advantage of the increase in computing capacity and performance that has occurred over the last two decades.
Here we review the literature to determine the state-of-the-art in modeling terrestrial protected areas under climate change, with the aim of evaluating and detecting trends and gaps in the current approaches being employed, as well as to provide a useful overview and guidelines for future research.
Results
Most studies apply statistical, bioclimatic envelope models and focus primarily on plant species as compared to other taxa. Very few studies utilize a mechanistic, process-based approach and none examine biotic interactions like predation and competition. Important factors like land-use, habitat fragmentation, invasion and dispersal are rarely incorporated, restricting the informative value of the resulting predictions considerably.
Conclusion
The general impression that emerges is that biodiversity conservation in protected areas could benefit from the application of modern modeling approaches to a greater extent than is currently reflected in the scientific literature. It is particularly true that existing models have been underutilized in testing different management options under climate change. Based on these findings we suggest a strategic framework for more effectively incorporating the impact of climate change in models exploring the effectiveness of protected areas.
Carbon (C) sequestration in soils plays a key role in the global C cycle. It is therefore crucial to adequately monitor dynamics in soil organic carbon (Delta SOC) stocks when aiming to reveal underlying processes and potential drivers. However, small-scale spatial (10-30 m) and temporal changes in SOC stocks, particularly pronounced in arable lands, are hard to assess. The main reasons for this are limitations of the well-established methods. On the one hand, repeated soil inventories, often used in long-term field trials, reveal spatial patterns and trends in Delta SOC but require a longer observation period and a sufficient number of repetitions. On the other hand, eddy covariance measurements of C fluxes towards a complete C budget of the soil-plant-atmosphere system may help to obtain temporal Delta SOC patterns but lack small-scale spatial resolution.
To overcome these limitations, this study presents a reliable method to detect both short-term temporal dynamics as well as small-scale spatial differences of Delta SOC using measurements of the net ecosystem carbon balance (NECB) as a proxy. To estimate the NECB, a combination of automatic chamber (AC) measurements of CO2 exchange and empirically modeled aboveground biomass development (NPPshoot / were used. To verify our method, results were compared with Delta SOC observed by soil resampling.
Soil resampling and AC measurements were performed from 2010 to 2014 at a colluvial depression located in the hummocky ground moraine landscape of northeastern Germany. The measurement site is characterized by a variable groundwater level (GWL) and pronounced small-scale spatial heterogeneity regarding SOC and nitrogen (Nt) stocks. Tendencies and magnitude of Delta SOC values derived by AC measurements and repeated soil inventories corresponded well. The period of maximum plant growth was identified as being most important for the development of spatial differences in annual Delta SOC. Hence, we were able to confirm that AC-based C budgets are able to reveal small-scale spatial differences and short-term temporal dynamics of Delta SOC.
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.
With Saccharomyces cerevisiae being a commonly used host organism for synthetic biology and biotechnology approaches, the work presented here aims at the development of novel tools to improve and facilitate pathway engineering and heterologous protein production in yeast. Initially, the multi-part assembly strategy AssemblX was established, which allows the fast, user-friendly and highly efficient construction of up to 25 units, e.g. genes, into a single DNA construct. To speed up complex assembly projects, starting from sub-gene fragments and resulting in mini-chromosome sized constructs, AssemblX follows a level-based approach: Level 0 stands for the assembly of genes from multiple sub-gene fragments; Level 1 for the combination of up to five Level 0 units into one Level 1 module; Level 2 for linkages of up to five Level 1 modules into one Level 2 module. This way, all Level 0 and subsequently all Level 1 assemblies can be carried out simultaneously. Individually planned, overlap-based Level 0 assemblies enable scar-free and sequence-independent assemblies of transcriptional units, without limitations in fragment number, size or content. Level 1 and Level 2 assemblies, which are carried out via predefined, computationally optimized homology regions, follow a standardized, highly efficient and PCR-free scheme. AssemblX follows a virtually sequence-independent scheme with no need for time-consuming domestication of assembly parts. To minimize the risk of human error and to facilitate the planning of assembly projects, especially for individually designed Level 0 constructs, the whole AssemblX process is accompanied by a user-friendly webtool. This webtool provides the user with an easy-to-use operating surface and returns a bench-protocol including all cloning steps. The efficiency of the assembly process is further boosted through the implementation of different features, e.g. ccdB counter selection and marker switching/reconstitution. Due to the design of homology regions and vector backbones the user can flexibly choose between various overlap-based cloning methods, enabling cost-efficient assemblies which can be carried out either in E. coli or yeast. Protein production in yeast is additionally supported by a characterized library of 40 constitutive promoters, fully integrated into the AssemblX toolbox. This provides the user with a starting point for protein balancing and pathway engineering. Furthermore, the final assembly cassette can be subcloned into any vector, giving the user the flexibility to transfer the individual construct into any host organism different from yeast.
As successful production of heterologous compounds generally requires a precise adjustment of protein levels or even manipulation of the host genome to e.g. inhibit unwanted feedback regulations, the optogenetic transcriptional regulation tool PhiReX was designed. In recent years, light induction was reported to enable easy, reversible, fast, non-toxic and nearly gratuitous regulation, thereby providing manifold advantages compared to conventional chemical inducers. The optogenetic interface established in this study is based on the photoreceptor PhyB and its interacting protein PIF3. Both proteins, derived from Arabidopsis thaliana, dimerize in a red/far-red light-responsive manner. This interaction depends on a chromophore, naturally not available in yeast. By fusing split proteins to both components of the optical dimerizer, active enzymes can be reconstituted in a light-dependent manner. For the construction of the red/far-red light sensing gene expression system PhiReX, a customizable synTALE-DNA binding domain was fused to PhyB, and a VP64 activation domain to PIF3. The synTALE-based transcription factor allows programmable targeting of any desired promoter region. The first, plasmid-based PhiReX version mediates chromophore- and light-dependent expression of the reporter gene, but required further optimization regarding its robustness, basal expression and maximum output. This was achieved by genome-integration of the optical regulator pair, by cloning the reporter cassette on a high-copy plasmid and by additional molecular modifications of the fusion proteins regarding their cellular localization. In combination, this results in a robust and efficient activation of cells over an incubation time of at least 48 h. Finally, to boost the potential of PhiReX for biotechnological applications, yeast was engineered to produce the chromophore. This overcomes the need to supply the expensive and photo-labile compound exogenously. The expression output mediated through PhiReX is comparable to the strong constitutive yeast TDH3 promoter and - in the experiments described here - clearly exceeds the commonly used galactose inducible GAL1 promoter.
The fast-developing field of synthetic biology enables the construction of complete synthetic genomes. The upcoming Synthetic Yeast Sc2.0 Project is currently underway to redesign and synthesize the S. cerevisiae genome. As a prerequisite for the so-called “SCRaMbLE” system, all Sc2.0 chromosomes incorporate symmetrical target sites for Cre recombinase (loxPsym sites), enabling rearrangement of the yeast genome after induction of Cre with the toxic hormonal substance beta-estradiol. To overcome the safety concern linked to the use of beta-estradiol, a red light-inducible Cre recombinase, dubbed L-SCRaMbLE, was established in this study. L-SCRaMbLE was demonstrated to allow a time- and chromophore-dependent recombination with reliable off-states when applied to a plasmid containing four genes of the beta-carotene pathway, each flanked with loxPsym sites. When directly compared to the original induction system, L-SCRaMbLE generates a larger variety of recombination events and lower basal activity. In conclusion, L-SCRaMbLE provides a promising and powerful tool for genome rearrangement.
The three tools developed in this study provide so far unmatched possibilities to tackle complex synthetic biology projects in yeast by addressing three different stages: fast and reliable biosynthetic pathway assembly; highly specific, orthogonal gene regulation; and tightly controlled synthetic evolution of loxPsym-containing DNA constructs.
Plant cells host two important organelles: mitochondria, known as the cell’s ‘powerhouse’, which act by converting oxygen and nutrients into ATP, and plastids, which perform photosynthesis. These organelles contain their own genomes that encode proteins required for gene expression and energy metabolism. Transformation technologies offer great potential for investigating all aspects of the physiology and gene expression of these organelles in vivo. In addition, organelle transformation can be a valuable tool for biotechnology and molecular plant breeding. Plastid transformation systems are well-developed for a few higher plants, however, mitochondrial transformation has so far only been reported for Saccharomyces cerevisiae and the unicellular alga Chlamydomonas reinhardtii.
Development of an efficient new selection marker for plastid transformation is important for several reasons, including facilitating supertransformation of the plastid genome for metabolic engineering purposes and for producing multiple knock-outs or site-directed mutagenesis of two unlinked genes. In this work, we developed a novel selection system for Nicotiana tabacum (tobacco) chloroplast transformation with an alternative marker. The marker gene, aac(6′)-Ie/aph(2′′)-Ia, was cloned into different plastid transformation vectors and several candidate aminoglycoside antibiotics were investigated as selection agents. Generally, the efficiency of selection and the transformation efficiency with aac(6′)-Ie/aph(2′′)-Ia as selectable marker in combination with the aminoglycoside antibiotic tobramycin was similarly high as that with the standard marker gene aadA and spectinomycin selection. Furthermore, our new selection system may be useful for the development of plastid transformation for new species, including cereals, the world’s most important food crops, and could also be helpful for the establishment of a selection system for mitochondrial transformation.
To date, all attempts to achieve mitochondrial transformation for higher plants have been unsuccessful. A mitochondrial transformation system for higher plants would not only provide a potential for studying mitochondrial physiology but could also provide a method to introduce cytoplasmic male sterility into crops to produce hybrid seeds. Establishing a stable mitochondrial transformation system in higher plants requires several steps including delivery of foreign DNA, stable integration of the foreign sequences into the mitochondrial genome, efficient expression of the transgene, a highly regenerable tissue culture system that allows regeneration of the transformed cells into plants, and finally, a suitable selection system to identify cells with transformed mitochondrial genomes. Among all these requirements, finding a good selection is perhaps the most important obstacle towards the development of a mitochondrial transformation system for higher plants. In this work, two selection systems were tested for mitochondrial transformation: kanamycin as a selection system in combination with the antibiotic-inactivating marker gene nptII, and sulfadiazine as a selection agent that inhibits the folic acid biosynthesis pathway residing in plant mitochondria in combination with the sul gene encoding an enzyme that is insensitive to inhibition by sulfadiazine. Nuclear transformation experiments were considered as proof of the specificity of the sulfadiazine selection system for mitochondria. We showed that an optimized sulfadiazine selection system, with the Sul protein targeted to mitochondria, is much more efficient than the previous sulfadiazine selection system, in which the Sul protein was targeted to the chloroplast. We also showed by systematic experiments that the efficiency of selection and nuclear transformation of the optimized sulfadiazine selection was higher compared to the standard kanamycin selection system. Finally, we also investigated the suitability of this selection system for nuclear transformation of the model alga Chlamydomonas reinhardtii, obtaining promising results. Although we designed several mitochondrial transformation vectors with different expression elements and integration sites in the mitochondrial genome based on the sulfadiazine system, and different tissue culture condition were also considered, we were not able to obtain mitochondrial transformation with this system. Nonetheless, establishing the sul gene as an efficient and specific selection marker for mitochondria addresses one of the major bottlenecks and may pave the way to achieve mitochondrial transformation in higher plants.
We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments.
We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments.
We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D-1/2 = 1.7 +/- 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods.
Die Hybridomtechnik zur Produktion von monoklonalen Antikörpern ermöglichte einen großen Schritt in der Entwicklung von Immunoassays für die biochemische Forschung und klinische Diagnostik. Auch die Produktion von Antikörpern gegen niedermolekulare Analyten, Haptene, typische Targets in der Lebensmittel- und Umweltanalytik, erlangte in den letzten Jahren eine immer größere Bedeutung. Im Zuge der Durchführung der Hybridomtechnik werden tausende Antikörper-sezernierende und nicht-sezernierende Zellen generiert. Die Selektion der wenigen antigenselektiven Hybridomzellen zählt dabei zu den herausforderndsten Schritten für die Antikörpergewinnung. Bisherige Selektionsverfahren, wie die Limiting-Dilution-Klonierung in Verbindung mit Enzyme-linked Immunosorbent Assays (ELISAs), garantieren keine Monoklonalität und erlauben nur das Screening von einigen wenigen Zellklonen. Hingegen ermöglichen Hochdurchsatz-Selektionsmethoden, wie die Fluoreszenz-aktivierte Zellsortierung (FACS), einen sehr hohen Probendurchsatz. Eine Einzelzellablage garantiert hierbei Monoklonalität. Jedoch sind die dafür erforderlichen Zellmarkierungen oftmals zellschädigend oder aufwendig zu generieren. Auch ist bisher noch keine Markierungsmethode bekannt, die es ermöglicht, Hapten-selektive Hybridomzellen durchflusszytometrisch zu analysieren und eine FACS-Selektion durchzuführen.
Aus diesem Grund wurden in dieser Arbeit zwei Zellmarkierungsmethoden entwickelt, die dies ermöglichen sollten. Die membranständigen Antikörper von Hybridomzellen sollten entweder direkt oder indirekt immunfluoreszenz-markiert und dadurch für die Durchflusszytometrie und FACS-Selektion zugänglich gemacht werden. Die direkte Markierung wurde mittels eines Hapten-Fluorophor-Konjugats durchgeführt. Sie ermöglichte erstmalig den Anteil an Haptenselektiven Hybridomzellen in einer Hybridomzelllinie zu überprüfen. Dies konnte für zwei Hapten-selektive Hybridomzelllinien, die Antikörper gegen das Hormon 17β-Estradiol und das Cardenolid Digoxigenin bilden, gezeigt werden. Durchflusszytometrie und ELISAs lieferten vergleichbare Ergebnisse. Zellen, die Hapten-selektiv markiert werden konnten, sezernierten ebenfalls Hapten-selektive Antikörper. Des Weiteren konnte die direkte Markierung dazu genutzt werden, zwei Mykotoxin-selektive Hybridomzelllinien, welche Antikörper gegen Aflatoxin und Zearalenon bilden, auf Monoklonalität zu testen. Dies ist mittels ELISA nicht möglich. Die Markierungsmethode eignete sich jedoch nur für fixierte Hybridomzellen. Eine Markierung von lebenden Zellen konnte weder durchflusszytometrisch noch mittels konfokaler Laser-Scanning-Mikroskopie gezeigt werden.
Dies gelang erst mit einer neu entwickelten indirekten Immunfluoreszenzmarkierung. Dabei wurden die Zellen zunächst mit einem Hapten-Peroxidase-Konjugat inkubiert, gefolgt von einem Fluorophor-markierten anti-HRP-Antikörper-Konjugat. Dies wurde für zwei Analyten, das Hormon Estron und das Antiepileptikum Carbamazepin, gezeigt. Die indirekte Markierung wurde erfolgreich dazu verwendet, Carbamazepin-selektive Hybridomzellen aus einem Fusionsansatz für die monoklonale Antikörperproduktion auszusortieren. Damit wurde erstmalig eine Zellmarkierungsmethode entwickelt, die eine Hochdurchsatz-Selektion lebender Hybridomzellen aus einem Fusionsansatz ermöglicht. Sie ist nicht zellschädigend und kann zusätzlich zur Selektion Hapten-selektiver Plasmazellen verwendet werden.
Epigenetic maintenance of gene repression is essential for development. Polycomb complexes are central to this memory, but many aspects of the underlying mechanism remain unclear. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binds Polycomb-deposited H3K27me3 and is required for repression of many Polycomb target genes in Arabidopsis. Here we show that LHP1 binds RNA in vitro through the intrinsically disordered hinge region. By independently perturbing the RNA-binding hinge region and H3K27me3 (trimethylation of histone H3 at Lys27) recognition, we found that both facilitate LHP1 localization and H3K27me3 maintenance. Disruption of the RNAbinding hinge region also prevented formation of subnuclear foci, structures potentially important for epigenetic repression.
Reproductive development of grapevine and berry composition are both strongly influenced by temperature. To date, the molecular mechanisms involved in grapevine berries response to high temperatures are poorly understood. Unlike recent data that addressed the effects on berry development of elevated temperatures applied at the whole plant level, the present work particularly focuses on the fruit responses triggered by direct exposure to heat treatment (HT). In the context of climate change, this work focusing on temperature effect at the microclimate level is of particular interest as it can help to better understand the consequences of leaf removal (a common viticultural practice) on berry development. HT (+8 degrees C) was locally applied to clusters from Cabernet Sauvignon fruiting cuttings at three different developmental stages (middle green, veraison and middle ripening). Samples were collected 1, 7, and 14 days after treatment and used for metabolic and transcriptomic analyses. The results showed dramatic and specific biochemical and transcriptomic changes in heat exposed berries, depending on the developmental stage and the stress duration. When applied at the herbaceous stage, HT delayed the onset of veraison. Heating also strongly altered the berry concentration of amino acids and organic acids (e.g., phenylalanine, raminobutyric acid and malate) and decreased the anthocyanin content at maturity. These physiological alterations could be partly explained by the deep remodeling of transcriptome in heated berries. More than 7000 genes were deregulated in at least one of the nine experimental conditions. The most affected processes belong to the categories "stress responses," protein metabolism" and "secondary metabolism," highlighting the intrinsic capacity of grape berries to perceive HT and to build adaptive responses. Additionally, important changes in processes related to "transport," "hormone" and "cell wall" might contribute to the postponing of veraison. Finally, opposite effects depending on heating duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HI induced decrease in anthocyanin content may result from a combination of transcript abundance and product degradation.
Aerobic anoxygenic phototrophs (AAPs) have been shown to exist in numerous marine and brackish environments where they are hypothesized to play important ecological roles. Despite their potential significance, the study of freshwater AAPs is in its infancy and limited to local investigations. Here, we explore the occurrence, diversity and distribution of AAPs in lakes covering a wide latitudinal gradient: Mongolian and German lakes located in temperate regions of Eurasia, tropical Great East African lakes, and polar permanently ice-covered Antarctic lakes. Our results show a widespread distribution of AAPs in lakes with contrasting environmental conditions and confirm that this group is composed of different members of the Alpha- and Betaproteobacteria. While latitude does not seem to strongly influence AAP abundance, clear patterns of community structure and composition along geographic regions were observed as indicated by a strong macro-geographical signal in the taxonomical composition of AAPs. Overall, our results suggest that the distribution patterns of freshwater AAPs are likely driven by a combination of small-scale environmental conditions (specific of each lake and region) and large-scale geographic factors (climatic regions across a latitudinal gradient).
The effects of biodiversity on ecosystem functioning generally increase over time, but the underlying processes remain unclear. Using 26 long-term grassland and forest experimental ecosystems, we demonstrate that biodiversity-ecosystem functioning relationships strengthen mainly by greater increases in functioning in high-diversity communities in grasslands and forests. In grasslands, biodiversity effects also strengthen due to decreases in functioning in low-diversity communities. Contrasting trends across grasslands are associated with differences in soil characteristics.
Do cities represent sources, sinks or isolated islands for urban wild boar population structure?
(2017)
ecoAO
(2017)
Although aldehyde oxidase (AO) is an important hepatic drug-metabolizing enzyme, it remains understudied and is consequently often overlooked in preclinical studies, an oversight that has resulted in the failure of multiple clinical trials. AO’s preclusion to investigation stems from the following: (1) difficulties synthesizing metabolic standards due to the chemospecificity and regiospecificity of the enzyme and (2) significant inherent variability across existing in vitro systems including liver cytosol, S9 fractions, and primary hepatocytes, which lack specificity and generate discordant expression and activity profiles. Here, we describe a practical bacterial biotransformation system, ecoAO, addressing both issues simultaneously. ecoAO is a cell paste of MoCo-producing Escherichia coli strain TP1017 expressing human AO. It exhibits specific activity toward known substrates, zoniporide, 4-trans-(N,N-dimethylamino)cinnamaldehyde, O6-benzylguanine, and zaleplon; it also has utility as a biocatalyst, yielding milligram quantities of synthetically challenging metabolite standards such as 2-oxo-zoniporide. Moreover, ecoAO enables routine determination of kcat and V/K, which are essential parameters for accurate in vivo clearance predictions. Furthermore, ecoAO has potential as a preclinical in vitro screening tool for AO activity, as demonstrated by its metabolism of 3-aminoquinoline, a previously uncharacterized substrate. ecoAO promises to provide easy access to metabolites with the potential to improve pharmacokinetic clearance predictions and guide drug development.
Mixing events in stratified lakes result in microalgae being exposed to varying conditions in light and organic carbon concentrations. Stratified lakes consist of an upper illuminated strata and a lower, darker strata where organic carbon accumulates. Therefore, in this contribution we explore the importance of dissolved organic carbon for growth under various light intensities by measuring some ecophysiological adaptations in two green microalgae. We compared the non-motile Chlorella vulgaris with the flagellated Chlamydomonas acidophila under auto-, mixo-, and heterotrophic growth conditions. In both algae the maximum photosynthetic and growth rates were highest under mixotrophy, and both algae appeared inhibited in their phosphorus acquisition under heterotrophy. Heterotrophic conditions provoked the largest differences as C. vulgaris produced chlorophyll a in darkness and grew as well as in autotrophic conditions, whereas Chl. acidophila bleached and could not grow heterotrophically. Although the fatty acid composition of both phytoplankton species differed, both species reacted in a similar way to changes in their growth conditions, mainly by a decrease of C18:3n-3 and an increase of C18:1n-9 from auto- to heterotrophic conditions. The two contrasting responses within the group of green microalgae suggest that dissolved organic carbon has a high deterministic potential to explain the survival and behaviour of green algae in the deeper strata of lakes.
EDTA and NTA effectively tune the mineralization of calcium phosphate from bulk aqueous solution
(2017)
This study describes the effects of nitrilotriacetic acid (NTA) and ethylenediaminotetraacetic acid (EDTA) on themineralization of calciumphosphate from bulk aqueous solution. Mineralization was performed between pH 6 and 9 and with NTA or EDTA concentrations of 0, 5, 10, and 15 mM. X-ray diffraction and infrared spectroscopy show that at low pH, mainly brushite precipitates and at higher pH, mostly hydroxyapatite forms. Both additives alter the morphology of the precipitates. Without additive, brushite precipitates as large plates. With NTA, the morphology changes to an unusual rod-like shape. With EDTA, the edges of the particles are rounded and disk-like particles form. Conductivity and pH measurements suggest that the final products form through several intermediate steps.
Core-specific sensorimotor exercises are proven to enhance neuromuscular activity of the trunk. However, the influence of high-intensity perturbations on training efficiency is unclear within this context. Sixteen participants (29 +/- 2 yrs; 175 +/- 8 cm; 69 +/- 13 kg) were prepared with a 12-lead bilateral trunk EMG. Warm-up on a dynamometer was followed by maximum voluntary isometric trunk (flex/ext) contraction (MVC). Next, participants performed four conditions for a one-legged stance with hip abduction on a stable surface (HA) repeated randomly on an unstable surface (HAP), on a stable surface with perturbation (HA + P), and on an unstable surface with perturbation (HAP + P). Afterwards, bird dog (BD) was performed under the same conditions (BD, BDP, BD + P, BDP + P). A foam pad under the foot (HA) or the knee (BD) was used as an unstable surface. Exercises were conducted on a moveable platform. Perturbations (ACC 50 m/sec(2);100 ms duration;10rep.) were randomly applied in the anterior-posterior direction. The root mean square (RMS) normalized to MVC (%) was calculated (whole movement cycle). Muscles were grouped into ventral right and left (VR;VL), and dorsal right and left (DR;DL). Ventral Dorsal and right-left ratios were calculated (two way repeated-measures ANOVA;alpha = 0,05). Amplitudes of all muscle groups in bird dog were higher compared to hip abduction (p <= 0.0001; Range: BD: 14 +/- 3% (BD;VR) to 53 +/- 4%; HA: 7 +/- 2% (HA;DR) to 16 +/- 4% (HA;DR)). EMG-RMS showed significant differences (p < 0.001) between conditions and muscle groups per exercise. Interaction effects were only significant for HA (p = 0.02). No significant differences were present in EMG ratios (p > 0.05). Additional high-intensity perturbations during core-specific sensorimotor exercises lead to increased neuromuscular activity and therefore higher exercise intensities. However, the beneficial effects on trunk function remain unclear. Nevertheless, BD is more suitable to address trunk muscles.
This is a cumulative dissertation comprising three original studies (one published, one in revision, one submitted; Effective December 2017) investigating how reptile species in arid Australia respond to various climatic parameters at different spatial scales and analysing the two potential main underlying mechanisms: thermoregulatory behaviour and species interactions. This dissertation combines extensive individual-based field data across trophic levels, selected field experiments, statistical analyses, and predictive modelling techniques. Mechanisms and processes detected in this dissertation can now be used to predict potential future changes in the community of arid-zone lizards. This knowledge will help improving our fundamental understanding of the consequences of global change and thereby prevent biodiversity loss in a vulnerable ecosystem.
In mormyrid weakly electric fish, the electric organ discharge (EOD) is used for species recognition, orientation and prey localization. Produced in the muscle-derived adult electric organ, the EOD exhibits a wide diversity across species in both waveform and duration. While certain defining EOD characteristics can be linked to anatomical features of the electric organ, many factors underlying EOD differentiation are yet unknown. Here, we report the differential expression of 13 Kv1 voltage-gated potassium channel genes, two inwardly rectifying potassium channel genes, two previously studied sodium channel genes and an ATPase pump in two sympatric species of the genus Campylomormyrus in both the adult electric organ and skeletal muscle. Campylomormyrus compressirostris displays a basal EOD, largely unchanged during development, while C. tshokwe has an elongated, putatively derived discharge. We report an upregulation in all Kv1 genes in the electric organ of Campylomormyrus tshokwe when compared to both skeletal muscle and C. compressirostris electric organ. This pattern of upregulation in a species with a derived EOD form suggests that voltage-gated potassium channels are potentially involved in the diversification of the EOD signal among mormyrid weakly electric fish.
Lying is an everyday moral phenomenon about which philosophers have written a lot. Not only the moral status of lying has been intensively discussed but also what it means to lie in the first place. Perhaps the most important criterion for an adequate definition of lying is that it fits with people’s understanding and use of this concept. In this light, it comes as a surprise that researchers only recently started to empirically investigate the folk concept of lying. In this paper, we describe three experimental studies which address the following questions: Does a statement need to be objectively false in order to constitute lying? Does lying necessarily include the intention to deceive? Can one lie by omitting relevant facts?
Natural products and their derivatives have always been a source of drug leads. In particular, bacterial compounds have played an important role in drug development, for example in the field of antibiotics. A decrease in the discovery of novel leads from natural sources and the hope of finding new leads through the generation of large libraries of drug-like compounds by combinatorial chemistry aimed at specific molecular targets drove the pharmaceutical companies away from research on natural products. However, recent technological advances in genetics, bioinformatics and analytical chemistry have revived the interest in natural products. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of natural products generated by the action of post-translationally modifying enzymes on precursor peptides translated from mRNA by ribosomes. The great substrate promiscuity exhibited by many of the enzymes from RiPP biosynthetic pathways have led to the generation of hundreds of novel synthetic and semisynthetic variants, including variants carrying non-canonical amino acids (ncAAs). The microviridins are a family of RiPPs characterized by their atypical tricyclic structure composed of lactone and lactam rings, and their activity as serine protease inhibitors. The generalities of their biosynthetic pathway have already been described, however, the lack of information on details such as the protease responsible for cleaving off the leader peptide from the cyclic core peptide has impeded the fast and cheap production of novel microviridin variants. In the present work, knowledge on leader peptide activation of enzymes from other RiPP families has been extrapolated to the microviridin family, making it possible to bypass the need of a leader peptide. This feature allowed for the exploitation of the microviridin biosynthetic machinery for the production of novel variants through the establishment of an efficient one-pot in vitro platform. The relevance of this chemoenzymatic approach has been exemplified by the synthesis of novel potent serine protease inhibitors from both rationally-designed peptide libraries and bioinformatically predicted microviridins. Additionally, new structure-activity relationships (SARs) could be inferred by screening microviridin intermediates. The significance of this technique was further demonstrated by the simple incorporation of ncAAs into the microviridin scaffold.
Recent advances in gene function prediction rely on ensemble approaches that integrate results from multiple inference methods to produce superior predictions. Yet, these developments remain largely unexplored in plants. We have explored and compared two methods to integrate 10 gene co-function networks for Arabidopsis thaliana and demonstrate how the integration of these networks produces more accurate gene function predictions for a larger fraction of genes with unknown function. These predictions were used to identify genes involved in mitochondrial complex I formation, and for five of them, we confirmed the predictions experimentally. The ensemble predictions are provided as a user-friendly online database, EnsembleNet. The methods presented here demonstrate that ensemble gene function prediction is a powerful method to boost prediction performance, whereas the EnsembleNet database provides a cutting-edge community tool to guide experimentalists.
Spatial environmental heterogeneity is considered a fundamental factor for the maintenance of plant species richness. However, it still remains unclear whether heterogeneity may also facilitate coexistence at fine grain sizes or whether other processes, like mass effects and source sink dynamics due to dispersal, control species composition and diversity at these scales. In this study, we used two complimentary analyses to identify the role of heterogeneity within 15 m x 15 m plots for the coexistence of species-rich annual communities in a semi-arid environment along a steep precipitation gradient. Specifically, we: (a) analyzed the effect of environmental heterogeneity on species, functional and phylogenetic diversity within microsites (alpha diversity, 0.06 m(2) and 1 m(2)), across microsites (beta diversity), and diversity at the entire plot (gamma diversity); (b) further we used two null models to detect non-random trait and phylogenetic patterns in order to infer assembly processes, i.e. whether co-occurring species tend to share similar traits (trait convergence) or dissimilar traits (trait divergence). In general, our results showed that heterogeneity had a positive effect on community diversity. Specifically, for alpha diversity, the effect was significant for functional diversity, and not significant for either species or phylogenetic diversities. For beta diversity, all three measures of community diversity (species, functional, and phylogenetic) increased significantly, as they also did for gamma diversity, where functional measures were again stronger than for species or phylogenetic measures. In addition, the null model approach consistently detected trait convergence, indicating that species with similar traits tended to co-occur and had high abundances in a given microsite. While null model analysis across the phylogeny partly supported these trait findings, showing phylogenetic underdispersion at the 1m(2) grain size, surprisingly when species abundances in microsites were analyzed they were more evenly distributed across the phylogenetic tress than expected (phylogenetic overdispersion). In conclusion, our results provide compelling support that environmental heterogeneity at a relatively fine scale is an important factor for species co-existence as it positively affects diversity as well as influences species assembly. Our study underlines the need for trait-based approaches conducted at fine grain sizes in order to better understand species coexistence and community assembly. (C) 2017 Elsevier GmbH. All rights reserved.
Enzymes from the xanthine oxidase (XO) family of molybdenum enzymes are generally, with some exceptions, molybdenum iron–sulfur flavin hydroxylases. Mammalian xanthine oxidoreductase and aldehyde oxidase were among the first enzymes to be studied in detail more than 100 years ago and, surprisingly, they continue to be thoroughly studied in molecular detail with many open and unresolved questions remaining. Enzymes of the XO family are characterized by a molybdenum cofactor (Moco) active site with a MoVIOS(OH) ligand sphere where substrate hydroxylation of either aromatic or aliphatic carbon centers is catalyzed. During the reaction, electrons are transferred to the oxidizing substrate, most commonly O2 or NAD+, which react at the FAD site.
Plants frequently have to weather both biotic and abiotic stressors, and have evolved sophisticated adaptation and defense mechanisms. In recent years, chromatin modifications, nucleosome positioning, and DNA methylation have been recognized as important components in these adaptations. Given their potential epigenetic nature, such modifications may provide a mechanistic basis for a stress memory, enabling plants to respond more efficiently to recurring stress or even to prepare their offspring for potential future assaults. In this review, we discuss both the involvement of chromatin in stress responses and the current evidence on somatic, intergenerational, and transgenerational stress memory.
Exploring the Arabidopsis metabolic landscape by genetic mapping integrated with network analysis
(2017)
Motivated by conflicting evidence in the literature, we re-assessed the role of facial feedback when detecting quantitative or qualitative changes in others’ emotional expressions. Fifty-three healthy adults observed self-paced morph sequences where the emotional facial expression either changed quantitatively (i.e., sad-to-neutral, neutral-to-sad, happy-to-neutral, neutral-to-happy) or qualitatively (i.e. from sad to happy, or from happy to sad). Observers held a pen in their own mouth to induce smiling or frowning during the detection task. When morph sequences started or ended with neutral expressions we replicated a congruency effect: Happiness was perceived longer and sooner while smiling; sadness was perceived longer and sooner while frowning. Interestingly, no such congruency effects occurred for transitions between emotional expressions. These results suggest that facial feedback is especially useful when evaluating the intensity of a facial expression, but less so when we have to recognize which emotion our counterpart is expressing.
To study the role of the TTR-RBP4-ROH complex components (transthyretin, serum retinol binding protein, retinol) and of angiogenic factors PlGF (placental growth factor) and sFlt-1 (soluble fms-like tyrosine kinase-1) in pregnancies complicated by small for gestational age infants (SGA). Case control study conducted on maternal serum collected between 11 + 0 to 13 + 6 weeks of gestation. TTR, RBP4, ROH, PlGF and sFlt-1 were measured in SGA patients (birth weight < 10%) who delivered at term (n = 37) and before 37 weeks of gestation (n = 17) and in a matched control group with uneventful pregnancies (n = 37). We found decreased RBP4 in SGA patients that delivered fetuses < 3% and in fetuses delivered after the 37 weeks of gestation compared to controls [1.50 (95% CI 1.40-1.75) vs 1.62 (95% CI 1.47-1.98), p < 0.05]. Further, we found lower PlGF and sFlt-1 concentrations in SGA that delivered before 37 weeks of gestation compared to controls (respectively, PIGF and sFlt-1: 39.7 pg/ml (95% CI 32.3-66.3) vs 62.9 pg/ml (95% CI 45.2-78.4) and 906 pg/ml (95% CI 727-1626) vs 1610 pg/ml (95% CI 1088-212), p < 0.05). First trimester maternal serum RBP4 and angiogenic factors PlGF and sFlt-1 can differently predict the timing of delivery of pregnancies complicated by SGA fetuses.
Fat loads were quantified for 2125 Yellow-browed Warblers Phylloscopus inornatus trapped at a stop-over site in Far East Russia during autumn migration. Flight ranges of 660-820km were estimated for the fattest individuals, suggesting that they would need to stop for refuelling at least six times to reach their wintering areas in South East Asia.
Late embryogenesis abundant (LEA) proteins are related to cellular dehydration tolerance. Most LEA proteins are predicted to have no stable secondary structure in solution, i.e., to be intrinsically disordered proteins (IDPs), but they may acquire alpha-helical structure upon drying. In the model plant Arabidopsis thaliana, the LEA proteins COR15A and COR15B are highly induced upon cold treatment and are necessary for the plants to attain full freezing tolerance. Freezing leads to increased intracellular crowding due to dehydration by extracellular ice crystals. In vitro, crowding by high glycerol concentrations induced partial folding of COR15 proteins. Here, we have extended these investigations to two related proteins, LEA11 and LEA25. LEA25 is much longer than LEA11 and COR15A, but shares a conserved central sequence domain with the other two proteins. We have created two truncated versions of LEA25 (2H and 4H) to elucidate the structural and functional significance of this domain. Light scattering and CD spectroscopy showed that all five proteins were largely unstructured and monomeric in dilute solution. They folded in the presence of increasing concentrations of trifluoroethanol and glycerol. Additional folding was observed in the presence of glycerol and membranes. Fourier transform infra red spectroscopy revealed an interaction of the LEA proteins with membranes in the dry state leading to a depression in the gel to liquid-crystalline phase transition temperature. Liposome stability assays revealed a cryoprotective function of the proteins. The C- and N-terminal extensions of LEA25 were important in cryoprotection, as the central domain itself (2H, 4H) only provided a low level of protection.
Background: Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA) may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle".
Results: Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni) and 22,737 bp (P. panini), they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i. e., in every generation.
Conclusions: The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB), which has been described from the chicken mitochondrial genome. As this RFB is supposed to halt replication, it offers a potential mechanistic explanation for frequent recombination in mitochondrial genomes.
Single molecule RNA fluorescent in situ hybridization (smFISH) enables gene transcription to be assessed at the cellular level. In this point of view article, we describe our recent smFISH research in the model plant Arabidopsis thaliana and discuss how this technique could further knowledge of plant gene transcription in the future.
Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.
The Leopard cat Prionailurus bengalensis is a habitat generalist that is widely distributed across Southeast Asia. Based on morphological traits, this species has been subdivided into 12 subspecies. Thus far, there have been few molecular studies investigating intraspecific variation, and those had been limited in geographic scope. For this reason, we aimed to study the genetic structure and evolutionary history of this species across its very large distribution range in Asia. We employed both PCR-based (short mtDNA fragments, 94 samples) and high throughput sequencing based methods (whole mitochondrial genomes, 52 samples) on archival, noninvasively collected and fresh samples to investigate the distribution of intraspecific genetic variation. Our comprehensive sampling coupled with the improved resolution of a mitochondrial genome analyses provided strong support for a deep split between Mainland and Sundaic Leopard cats. Although we identified multiple haplogroups within the species’ distribution, we found no matrilineal evidence for the distinction of 12 subspecies. In the context of Leopard cat biogeography, we cautiously recommend a revision of the Prionailurus bengalensis subspecific taxonomy: namely, a reduction to 4 subspecies (2 mainland and 2 Sundaic forms).
Genome-wide association studies of birth weight have focused on fetal genetics, whereas relatively little is known about the role of maternal genetic variation. We aimed to identify maternal genetic variants associated with birth weight that could highlight potentially relevant maternal determinants of fetal growth. We meta-analysed data on up to 8.7 million SNPs in up to 86 577 women of European descent from the Early Growth Genetics (EGG) Consortium and the UK Biobank. We used structural equation modelling (SEM) and analyses of mother–child pairs to quantify the separate maternal and fetal genetic effects. Maternal SNPs at 10 loci (MTNR1B, HMGA2, SH2B3, KCNAB1, L3MBTL3, GCK, EBF1, TCF7L2, ACTL9, CYP3A7) were associated with offspring birth weight at P < 5 Â 10 À8 . In SEM analyses, at least 7 of the 10 associations were consistent with effects of the maternal genotype acting via the intrauterine environment, rather than via effects of shared alleles with the fetus. Variants, or correlated proxies, at many of the loci had been previously associated with adult traits, including fasting glucose (MTNR1B, GCK and TCF7L2) and sex hormone levels (CYP3A7), and one (EBF1) with gestational duration. The identified associations indicate that genetic effects on maternal glucose, cytochrome P450 activity and gestational duration, and potentially on maternal blood pressure and immune function, are relevant for fetal growth. Further characterization of these associations in mechanistic and causal analyses will enhance understanding of the potentially modifiable maternal determinants of fetal growth, with the goal of reducing the morbidity and mortality associated with low and high birth weights.
Global effects of income and income inequality on adult height and sexual dimorphism in height
(2017)
Objectives: Average adult height of a population is considered a biomarker of the quality of the health environment and economic conditions. The causal relationships between height and income inequality are not well understood. We analyze data from 169 countries for national average heights of men and women and national-level economic factors to test two hypotheses: (1) income inequality has a greater association with average adult height than does absolute income; and (2) neither income nor income inequality has an effect on sexual dimorphism in height. Methods: Average height data come from the NCD-RisC health risk factor collaboration. Economic indicators are derived from the World Bank data archive and include gross domestic product (GDP), Gross National Income per capita adjusted for personal purchasing power (GNI_ PPP), and income equality assessed by the Gini coefficient calculated by the Wagstaff method. Results: Hypothesis 1 is supported. Greater income equality is most predictive of average height for both sexes. GNI_ PPP explains a significant, but smaller, amount of the variation. National GDP has no association with height. Hypothesis 2 is rejected. With greater average adult height there is greater sexual dimorphism. Conclusions: Findings support a growing literature on the pernicious effects of inequality on growth in height and, by extension, on health. Gradients in height reflect gradients in social disadvantage. Inequality should be considered a pollutant that disempowers people from the resources needed for their own healthy growth and development and for the health and good growth of their children.
A synthetic protocol for the fabrication of ultrathin polymeric films containing the enzyme 2-deoxy-D-ribose-5-phosphate aldolase from Escherichia coli (DERA(EC)) is presented. Ultrathin enzymatically active films are useful for applications in which only small quantities of active material are needed and at the same time quick response and contact times without diffusion limitation are wanted. We show how DERA as an exemplary enzyme can be immobilized in a thin polymer layer at the air-water interface and transferred to a suitable support by the Langmuir-Schaefer technique under full conservation of enzymatic activity. The polymer in use is a poly(N-isopropylacrylamide-co-N-2-thiolactone acrylamide) (P(NIPAAm-co-TlaAm)) statistical copolymer in which the thiolactone units serve a multitude of purposes including hydrophobization of the polymer, covalent binding of the enzyme and the support and finally cross-linking of the polymer matrix. The application of this type of polymer keeps the whole approach simple as additional cocomponents such as cross-linkers are avoided.
Impact of normal weight obesity on fundamental motor skills in pre-school children aged 3 to 6 years
(2017)
Normal weight obesity is defined as having excessive body fat, but normal BMI. Even though previous research revealed that excessive body fat in children inhibited their physical activity and decreased motor performance, there has been only little evidence about motor performance of normal weight obese children. This study aims to establish whether normal weight obese pre-school children aged 3-6 years will have a significantly worse level of fundamental motor skills compared to normal weight non-obese counterparts. The research sample consisted of 152 pre-schoolers selected from a specific district of Prague, the Czech Republic. According to values from four skinfolds: triceps, subscapula, suprailiaca, calf, and BMI three categories of children aged 3-6 years were determined: A) normal weight obese n = 51; B) normal weight non-obese n = 52; C) overweight and obese n = 49. The Movement Assessment Battery for Children (MABC-2) was used for the assessment of fundamental motor skills. Normal weight obese children had significantly higher amount of adipose tissue p < 0.001 than normal weight non-obese children but the same average BMI. Moreover, normal weight obese children did not have significantly less amount of subcutaneous fat on triceps and calf compared to their overweight and obese peers. In majority of MABC-2 tests, normal weight obese pre-schoolers showed the poorest performance. Moreover, normal weight obese children had significantly worse total standard score = 38.82 compared to normal weight non-obese peers = 52.27; p < 0.05. In addition, normal weight obese children had a more than three times higher frequency OR = 3.69 CI95% (1.10; 12.35) of severe motor deficit performance <= 5th centile of the MABC-2 norm. These findings are strongly alarming since indices like BMI are not able to identify normal weight obese individual. We recommend verifying real portion of normal weight obese children as they are probably in higher risk of health and motor problems than overweight and obese population due to their low lean mass.
Import and decomposition of dissolved organic carbon in pre-dams of drinking water reservoirs
(2017)
Dissolved organic carbon (DOC) depicts a key component in the aquatic carbon cycle as well as for drinking water production from surface waters. DOC concentrations increased in water bodies of the northern hemisphere in the last decades, posing ecological consequences and water quality problems. Within the pelagic zone of lakes and reservoirs, the DOC pool is greatly affected by biological activity as DOC is simultaneously produced and decomposed. This thesis aimed for a conceptual understanding of organic carbon cycling and DOC quality changes under differing hydrological and trophic conditions. Further, the occurrence of aquatic priming was investigated, which has been proposed as a potential process facilitating the microbial decomposition of stable allochthonous DOC within the pelagic zone.
To study organic carbon cycling under different hydrological conditions, quantitative and qualitative investigations were carried out in three pre-dams of drinking water reservoirs exhibiting a gradient in DOC concentrations and trophic states. All pre-dams were mainly autotrophic in their epilimnia. Discharge and temperature were identified as the key factors regulating net production and respiration in the upper water layers of the pre-dams. Considerable high autochthonous production was observed during the summer season under higher trophic status and base flow conditions. Up to 30% of the total gained organic carbon was produced within the epilimnia. Consequently, this affected the DOC quality within the pre-dams over the year and enhanced characteristics of algae-derived DOC were observed during base flow in summer. Allochthonous derived DOC dominated at high discharges and oligotrophic conditions when production and respiration were low. These results underline that also small impoundments with typically low water residence times are hotspots of carbon cycling, significantly altering water quality in dependence of discharge conditions, temperature and trophic status. Further, it highlights that these factors need to be considered in future water management as increasing temperatures and altered precipitation patterns are predicted in the context of climate change.
Under base flow conditions, heterotrophic bacteria preferentially utilized older DOC components with a conventional radiocarbon age of 195-395 years before present (i.e. before 1950). In contrast, younger carbon components (modern, i.e. produced after 1950) were mineralized following a storm flow event. This highlights that age and recalcitrance of DOC are independent from each other. To assess the ages of the microbially consumed DOC, a simplified method was developed to recover the respired CO2 from heterotrophic bacterioplankton for carbon isotope analyses (13C, 14C). The advantages of the method comprise the operation of replicate incubations at in-situ temperatures using standard laboratory equipment and thus enabling an application in a broad range of conditions.
Aquatic priming was investigated in laboratory experiments during the microbial decomposition of two terrestrial DOC substrates (peat water and soil leachate). Thereby, natural phytoplankton served as a source of labile organic matter and the total DOC pool increased throughout the experiments due to exudation and cell lysis of the growing phytoplankton. A priming effect for both terrestrial DOC substrates was revealed via carbon isotope analysis and mixing models. Thereby, priming was more pronounced for the peat water than for the soil leachate. This indicates that the DOC source and the amount of the added labile organic matter might influence the magnitude of a priming effect. Additional analysis via high-resolution mass spectrometry revealed that oxidized, unsaturated compounds were more strongly decomposed under priming (i.e. in phytoplankton presence). Given the observed increase in DOC concentrations during the experiments, it can be concluded that aquatic priming is not easily detectable via net concentration changes alone and could be considered as a qualitative effect.
The knowledge gained from this thesis contributes to the understanding of aquatic carbon cycling and demonstrated how DOC dynamics in freshwaters vary with hydrological, seasonal and trophic conditions. It further demonstrated that aquatic priming contributes to the microbial transformation of organic carbon and the observed decay of allochthonous DOC during transport in inland waters.
A recent full species-level phylogeny of tits, titmice and chickadees (Paridae) has placed the Chinese endemic black-bibbed tit (Poecile hypermelaenus) as the sister to the Palearctic willow tit (P. montanus). Because this sister-group relationship is in striking disagreement with the traditional affiliation of P. hypermelaenus close to the marsh tit (P. palustris) we tested this phylogenetic hypothesis in a multi locus analysis with an extended taxon sampling including sixteen subspecies of willow tits and marsh tits. As a taxonomic reference we included type specimens in our analysis. The molecular genetic study was complemented with an analysis of biometric data obtained from museum specimens. Our phylogenetic reconstructions, including a comparison of all GenBank data available for our target species, clearly show that the genetic lineage previously identified as P. hypermelaenus actually refers to P. weigoldicus because sequences were identical to that of a syntype of this taxon. The close relationship of P. weigoldicus and P. montanus - despite large genetic distances between the two taxa - is in accordance with current taxonomy and systematics. In disagreement with the previous phylogenetic hypothesis but in accordance with most taxonomic authorities, all our P. hypermelaenus specimens fell in the sister Glade of all western and eastern Palearctic P. palustris. Though shared haplotypes among the Chinese populations of the two latter species might indicate mitochondrial introgression in this part of the breeding range, further research is needed here due to the limitations of our own sampling.
BACKGROUND: Reduced left ventricular ejection fraction (LVEF) ≤30% is the most powerful prognostic indicator for sudden cardiac death (SCD) in patients after myocardial infarction (MI), but there are little data about long-term changes of LVEF after revascularization and the following implantation of a cardioverter defibrillator (ICD).
METHODS: We performed a retrospective analysis of 277 patients with reduced LVEF at least 1month after MI and complete revascularization. Patients (median time post-MI 23.4months; 74.3% after PCI, 25.7% after CABG were assigned either to group 1 (LVEF<30%) or group 2 (LVEF 30-40%). Biplane echocardiography was redone after a mean follow-up of 441±220days.
RESULTS: LVEF increased significantly in both two groups (group 1: 26.2±4.8% to 32.4±8.5%; p<0.001; group 2: 38.2±2.5% to 44.4±9.6%; p<0.001). However, statistical analysis of first and second LVEF measurement by means of a LOWESS regression and with an appropriate correction of the regression towards the mean effect revealed only a moderate increase of the mean LVEF from 35 to 37% (p<0.001) with a large interindividual variation.
CONCLUSIONS: The impact of early revascularization on LVEF appears to be low in the majority of post-MI heart failure patients. Owing to the high variability, a single measurement may not be reliable enough to justify a decision on ICD indication.
Poly(ether imide) (PEI), which can be chemically functionalized with biologically active ligands, has emerged as a potential biomaterial for medical implants. Electrospun PEI scaffolds have shown advantageous properties, such as enhanced endothelial cell adherence, proliferation and low platelet adhesion in in vitro experiments. In this study, the in vivo behaviour of electrospun PEI scaffolds and PEI films was examined in a murine subcutaneous implantation model. Electrospun PEI scaffolds and films were surgically implanted subcutaneously in the dorsae of mice. The surrounding subcutaneous tissue response was examined via histopathological examination at 7 and 28days after implantation. No serious adverse events were observed for both types of PEI implants. The presence of macrophages or foreign body giant cells in the vicinity of the implants and the formation of a fibrous capsule indicated a normal foreign body reaction towards PEI films and scaffolds. Capsule thickness and inflammatory infiltration cells significantly decreased for PEI scaffolds during days 7-28 while remaining unchanged for PEI films. The infiltration of cells into the implant was observed for PEI scaffolds 7days after implantation and remained stable until 28days of implantation. Additionally some, but not all, PEI scaffold implants induced the formation of functional blood vessels in the vicinity of the implants. Conclusively, this study demonstrates the in vivo biocompatibility of PEI implants, with favourable properties of electrospun PEI scaffolds regarding tissue integration and wound healing.
Dam construction on the Zeya River, which is an important tributary of the Amur River in Far East Russia, has caused significant declines in water levels and frequency of floods in the adjacent floodplains since 1980. However, an extreme flood event occurred in 2013. Populations of six crane species were monitored before and after these drastic water level changes at Muraviovka Park in Far East Russia, an important breeding and stop-over site. Individuals were counted by territory mapping during the breeding season (2000-2015) and by roosting site counts during autumn migration (2006-2015). The objective of this study was to evaluate whether changes in water levels had a significant impact on local and migratory crane populations. We found a positive effect of flooding on numbers of breeding Red-crowned Cranes (Grus japonensis) and White-naped Cranes (Antigone vipio), as well as on numbers of roosting Hooded Cranes (Grus monacha) in autumn. Siberian Cranes (Leucogeranus leucogeranus) were only observed after the wetlands were flooded. The results of this study highlight the importance of elevated Amur River water levels for crane populations of global importance.
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g., high-fat diets) or overweight and insulin resistance (e.g., methionine-choline-deficient diets), or they are based on monogenetic defects (e.g., ob/ob mice). In the current study, a Western-type diet containing soybean oil with high n-6-PUFA and 0.75% cholesterol (SOD + Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice, which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast, a soybean oil-containing Western-type diet without cholesterol (SOD) induced only mild steatosis but not hepatic inflammation, fibrosis, weight gain or insulin resistance. Another high-fat diet, mainly consisting of lard and supplemented with fructose in drinking water (LAD + Fru), resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD + Cho, but livers were devoid of inflammation and fibrosis. Although both LAD + Fru-and SOD + Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD + Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. In summary, dietary cholesterol in the SOD + Cho diet may trigger hepatic inflammation and fibrosis. SOD + Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH.
Background: Abdominal aortic aneurysm (AAA) is a deadly irreversible weakening and distension of the abdominal aortic wall. The pathogenesis of AAA remains poorly understood. Investigation into the physical and molecular characteristics of perivascular adipose tissue (PVAT) adjacent to AAA has not been done before and is the purpose of this study.
Methods and Results: Human aortae, periaortic PVAT, and fat surrounding peripheral arteries were collected from patients undergoing elective surgical repair of AAA. Control aortas were obtained from recently deceased healthy organ donors with no known arterial disease. Aorta and PVAT was found in AAA to larger extent compared with control aortas. Immunohistochemistry revealed neutrophils, macrophages, mast cells, and T-cells surrounding necrotic adipocytes. Gene expression analysis showed that neutrophils, mast cells, and T-cells were found to be increased in PVAT compared with AAA as well as cathepsin K and S. The concentration of ceramides in PVAT was determined using mass spectrometry and correlated with content of T-cells in the PVAT.
Conclusions: Our results suggest a role for abnormal necrotic, inflamed, proteolytic adipose tissue to the adjacent aneurysmal aortic wall in ongoing vascular damage.
Plastid protein biosynthesis occurs on bacterial-type 70S ribosomes consisting of a large (50S) and a small (30S) subunit. However, since many steps of ribosome biogenesis are not thermodynamically favorable at biological conditions, it requires many assembly factors. One group of assembly factors, circularly permuted GTPases, was implicated in 30S subunit maturation in E. coli, by a protein RsgA. RsgA orthologues are present in bacteria and plastid-containing species and in silico analysis revealed presence of a RsgA-like protein in Arabidopsis thaliana. To functionally characterize the Arabidopsis orthologue, two AtRsgA T-DNA insertion lines were analyzed in this study. The exon line (rsgA-e) led to embryo lethality, while the intron line (rsgA-i) caused severe dwarf, pale green phenotype. Further investigation of rsgA-i mutant line revealed defects in chloroplast biogenesis which led to increased number of chloroplasts, decreased chloroplast size, decreased air space between mesophyll cells and smaller shoot apical meristems, which showed unusual proplastid accumulation. Moreover, rsgA-i plants showed reduction in chlorophyll A and B content, decreased electron transport rate and photosynthetic efficiency. Further analyses revealed that the protein is involved in chloroplast 30S subunit maturation. Interestingly, we observed that while chloroplast-targeted and chloroplast-encoded proteins are generally downregulated in the mutant, a contrasting upregulation of the corresponding transcripts is observed, indicating an elaborate compensatory mechanism. To conclude, the study presented here reveals a ribosome assembly factor and a compensatory mechanism activated during impaired chloroplast function.
Ionogels (IGs) based on poly(methyl methacrylate) (PMMA) and the metal-containing ionic liquids (ILs) bis-1-butyl-3-methlimidazolium tetrachloridocuprate(II), tetrachloride cobaltate(II), and tetrachlorido manganate(II) have been synthesized and their mechanical and electrical properties have been correlated with their microstructure. Unlike many previous examples, the current IGs show a decreasing stability in stress-strain experiments on increasing IL fractions. The conductivities of the current IGs are lower than those observed in similar examples in the literature. Both effects are caused by a two-phase structure with micrometer-sized IL-rich domains homogeneously dispersed an IL-deficient continuous PMMA phase. This study demonstrates that the IL-polymer miscibility and the morphology of the IGs are key parameters to control the (macroscopic) properties of IGs.
Hintergrund: Etablierte Protein- und Nukleinsäure-basierte Methoden für den spezifischen Pathogennachweis sind nur unter standardisierten Laborbedingungen von geschultem Personal durchführbar und daher mit einem hohen Zeit- und Kostenaufwand verbunden. In der Nukleinsäure-basierten Diagnostik kann durch die Einführung der isothermalen Amplifikation eine schnelle und kostengünstige Alternative zur Polymerase-Kettenreaktion (PCR) verwendet werden. Die Loop-mediated isothermal amplification (LAMP) bietet aufgrund der hohen Amplifikationseffizienz vielfältige Detektionsmöglichkeiten, die sowohl für Schnelltest- als auch für Monitoring-Anwendungen geeignet sind.
Ein wesentliches Ziel dieser Arbeit war die Verbesserung der Anwendbarkeit der LAMP und die Entwicklung einer neuen Methode für den einfachen, schnellen und günstigen Nachweis von Pathogenen mittels alternativer DNA- oder Pyrophosphat-abhängiger Detektionsverfahren. Hier wurden zunächst direkte und indirekte Detektionsmethoden untersucht und darauf aufbauend ein Verfahren entwickelt, mit dem neue Metallionen-abhängige Fluoreszenzfarbstoffe für die selektive Detektion von Pyrophosphat in der LAMP und anderen enzymatischen Reaktionen identifiziert werden können. Als Alternative für die DNA-basierte Detektion in der digitalen LAMP sollten die zuvor etablierten Farbstoffe für den Pyrophosphatnachweis in einer Emulsion getestet werden. Abschließend wurde ein neuer Reaktionsmechanismus für die effiziente Generierung hochmolekularer DNA unter isothermalen Bedingungen als Alternative zur LAMP entwickelt.
Ergebnisse: Für den Nachweis RNA- und DNA-basierter Phythopathogene konnte die Echtzeit- und Endpunktdetektion mit verschiedenen Farbstoffen in einem geschlossenen System etabliert werden. Hier wurde Berberin als DNA-interkalierender Fluoreszenzfarbstoff mit vergleichbarer Sensitivität zu SYBR Green und EvaGreen erfolgreich in der LAMP mit Echtzeitdetektion eingesetzt. Ein Vorteil von Berberin gegenüber den anderen Farbstoffen ist die Toleranz der DNA-Polymerase auch bei hohen Farbstoffkonzentrationen. Berberin kann daher auch in der geschlossenen LAMP-Reaktion ohne zusätzliche Anpassung der Reaktionsbedingungen für die Endpunktdetektion verwendet werden. Darüber hinaus konnte Hydroxynaphtholblau (HNB), das für den kolorimetrischen Endpunktnachweis bekannt ist, erstmals auch für die fluorimetrische Detektion der LAMP in Echtzeit eingesetzt werden. Zusätzlich konnten in der Arbeit weitere Metallionen-abhängige Farbstoffe zur indirekten Detektion der LAMP über das Pyrophosphat identifiziert werden. Dafür wurde eine iterative Methode entwickelt, mit der potenzielle Farbstoffe hinsichtlich ihrer Enzymkompatibilität und ihrer spektralen Eigenschaften bei An- oder Abwesenheit von Manganionen selektiert werden können. Mithilfe eines kombinatorischen Screenings im Mikrotiterplattenformat konnte die komplexe Konzentrationsabhängigkeit zwischen den einzelnen Komponenten für einen fluorimetrischen Verdrängungsnachweis untersucht werden. Durch die Visualisierung des Signal-Rausch-Verhältnis’ als Intensitätsmatrix (heatmap) konnten zunächst Alizarinrot S und Tetrazyklin unter simulierten Reaktionsbedingungen selektiert werden. In der anschließenden enzymatischen LAMP-Reaktion konnte insbesondere Alizarinrot S als günstiger, nicht-toxischer und robuster Fluoreszenzfarbstoff identifiziert werden und zeigte eine Pyrophosphat-abhängige Zunahme der Fluoreszenzintensität. Die zuvor etablierten Farbstoffe (HNB, Calcein und Alizarinrot S) konnten anschließend erfolgreich für die indirekte, fluorimetrische Detektion von Pyrophosphat in einer LAMP-optimierten Emulsion eingesetzt werden. Die Stabilität und Homogenität der generierten Emulsion wurde durch den Zusatz des Emulgators Poloxamer 188 verbessert. Durch die fluoreszenzmikroskopische Analyse der Emulsion war eine eindeutige Diskriminierung der positiven und negativen Tröpfchen vor allem bei Einsatz von Calcein und Alizarinrot S möglich. Aufgrund des komplexen Primer-Designs und der hohen Wahrscheinlichkeit unspezifischer Amplifikation in der LAMP wurde eine neue Bst DNA-Polymerase-abhängige isothermale Amplifikationsreaktion entwickelt. Durch die Integration einer spezifischen Linkerstruktur (abasische Stelle oder Hexaethylenglykol) zwischen zwei Primersequenzen konnte ein bifunktioneller Primer die effiziente Regenerierung der Primerbindungsstellen gewährleisten. Der neue Primer induziert nach der spezifischen Hybridisierung auf dem Templat die Rückfaltung zu einer Haarnadelstruktur und blockiert gleichzeitig die Polymeraseaktivität am Gegenstrang, wodurch eine autozyklische Amplifikation trotz konstanter Reaktionstemperatur möglich ist. Die Effizienz der „Hinge-initiated Primer dependent Amplification“ (HIP) konnte abschließend durch die Verkürzung der Distanz zwischen einem modifizierten Hinge-Primer und einem PCR-ähnlichen Primer verbessert werden.
Schlussfolgerung: Die LAMP hat sich aufgrund der hohen Robustheit und Effizienz zu einer leistungsfähigen Alternative für die klassische PCR in der molekularbiologischen Diagnostik entwickelt. Unterschiedliche Detektionsverfahren verbessern die Leistungsfähigkeit der qualitativen und quantitativen LAMP für die Feldanwendungen und für die Diagnostik, da die neuen DNA- und Pyrophosphat-abhängigen Nachweismethoden in einer geschlossenen Reaktion eingesetzt werden können und so eine einfache Pathogendiagnostik ermöglichen. Die gezeigten Methoden können darüber hinaus zu einer Kostensenkung und Zeitersparnis gegenüber den herkömmlichen Methoden beitragen. Ein attraktives Ziel stellt die Weiterentwicklung der HIP für den Pathogennachweis als Alternative zur LAMP dar. Hierbei können die neuen LAMP-Detektionsverfahren ebenfalls Anwendung finden. Die Verwendung von Bst DNA-Polymerase-abhängigen Reaktionen ermöglicht darüber hinaus die Integration einer robusten isothermalen Amplifikation in mikrofluidische Systeme. Durch die Kombination der Probenvorbereitung, Amplifikation und Detektion sind zukünftige Anwendungen mit kurzer Analysezeit und geringem apparativen Aufwand insbesondere in der Pathogendiagnostik möglich.
Cellular membranes constantly experience remodeling, as exemplified by morphological changes during endo- and exocytosis. Regulation of membrane morphology is essential for these processes. In this work, we attempt to establish a regulation path based on the use of photoswitches exhibiting conformational changes in model membranes, namely, giant unilamellar vesicles (GUVs). The mechanism of the changes in the GUVs’ morphology caused by isomerization of the photosensitive molecules has been previously explored but still remains elusive. We examine the morphological reshaping of GUVs in the presence of the photoswitch o-tetrafluoroazobenzene (F-azo) and show that the mechanism behind the resulting morphological changes involves both an increase in the membrane area and generation of a positive spontaneous curvature. First, we characterize the partitioning of F-azo in a single-component membrane using both experimental and computational approaches. The partition coefficient calculated from molecular dynamic simulations agrees with experimental data obtained with size-exclusion chromatography. Then, we implement the approach of vesicle electrodeformation in order to assess the increase in the membrane area, which is observed as a result of the conformational change of F-azo. Finally, the local and the effective membrane spontaneous curvatures were estimated from the observed shapes of vesicles exhibiting outward budding. We then extend the application of the F-azo to multicomponent lipid membranes, which exhibit a coexistence of domains in different liquid phases due to a miscibility gap between the lipids. We perform initial experiments to investigate whether F-azo can be employed to modulate the lateral lipid packing and organization. We observe either complete mixing of the domains or the appearing of disordered domains within the domains of more ordered phase. The type of behavior observed in response to the photoisomerization of F-azo was dependent on the used lipid composition. We believe that the findings introduced here will have an impact in understanding and controlling both lipid phase modulation and regulation of the membrane morphology in membrane systems.