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Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.
Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'(SHG)) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'(SHG) values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices. The maximum SHG intensity occurs at the highest moisture and amylopectin content. These results support the hypothesis that SHG is caused by ordered hydrogen and hydroxyl bond networks which increase with hydration of starch granules. (C) 2015 Optical Society of America
A nanohybrid consisting of poly(3-aminobenzenesulfonic acid-co-aniline) and multiwalled carbon nanotubes [MWCNT-P(ABS-A)]) on a gold electrode was used to immobilize the hexameric tyrosine-coordinated heme protein (HTHP). The enzyme showed direct electron transfer between the heme group of the protein and the nanostructured surface. Desorption of the noncovalently bound heme from the protein could be excluded by control measurements with adsorbed hemin on aminohexanthiol-modified electrodes. The nanostructuring and the optimised charge characteristics resulted in a higher protein coverage as compared with MUA/MU modified electrodes. The adsorbed enzyme shows catalytic activity for the cathodic H2O2 reduction and oxidation of NADH.
Cardiovascular metallic stents established in clinical application are typically coated by a thin polymeric layer on the stent struts to improve hemocompatibility, whereby often a drug is added to the coating to inhibit neointimal hyperplasia. Besides such thin film coatings recently nano/microfiber coated stents are investigated, whereby the fibrous coating was applied circumferential on stents. Here, we explored whether a thin fibrous encasement of metallic stents with preferentially longitudinal aligned fibers and different local fiber densities can be achieved by electrospinning. An elastic degradable copolyetheresterurethane, which is reported to selectively enhance the adhesion of endothelial cells, while simultaneously rejecting smooth muscle cells, was utilized for stent coating. The fibrous stent encasements were microscopically assessed regarding their single fiber diameters, fiber covered area and fiber alignment at three characteristic stent regions before and after stent expansion. Stent coatings with thicknesses in the range from 30 to 50 mu m were achieved via electrospinning with 1,1,1,3,3,3-hexafluoro-2-propanol (HFP)-based polymer solution, while a mixture of HFP and formic acid as solvent resulted in encasements with a thickness below 5 mu m comprising submicron sized single fibers. All polymeric encasements were mechanically stable during expansion, whereby the fibers deposited on the struts remained their position. The observed changes in fiber density and diameter indicated diverse local deformation mechanisms of the microfibers at the different regions between the struts. Based on these results it can be anticipated that the presented fibrous encasement of stents might be a promising alternative to stents with polymeric strut coatings releasing anti-proliferative drugs. Copyright (c) 2015 John Wiley & Sons, Ltd.
Polyelectrolyte multilayer films are nowadays very attractive for bioapplications due to their tunable properties and ability to control cellular response. Here we demonstrate that multilayers made of hyaluronic acid and poly-l-lysine act as high-capacity reservoirs for small charged molecules. Strong accumulation within the film is explained by electrostatically driven binding to free charges of polyelectrolytes. Binding and release mechanisms are discussed based on charge balance and polymer dynamics in the film. Our results show that transport of molecules through the film-solution interface limits the release rate. The multilayers might serve as an effective platform for drug delivery and tissue engineering due to high potential for drug loading and controlled release.
Marine and limnic particles are hotspots of organic matter mineralization significantly affecting biogeochemical element cycling. Fluorescence in-situ hybridization and pyrosequencing of 16S rRNA genes were combined to investigate bacterial diversity and community composition on limnic and coastal marine particles >5 and >10m respectively. Limnic particles were more abundant (average: 1x10(7)l(-1)), smaller in size (average areas: 471 versus 2050m(2)) and more densely colonized (average densities: 7.3 versus 3.6 cells 100m(-2)) than marine ones. Limnic particle-associated (PA) bacteria harboured Alphaproteobacteria and Betaproteobacteria, and unlike previously suggested sizeable populations of Gammaproteobacteria, Actinobacteria and Bacteroidetes. Marine particles were colonized by Planctomycetes and Betaproteobacteria additionally to Alphaproteobacteria, Bacteroidetes and Gammaproteobacteria. Large differences in individual particle colonization could be detected. High-throughput sequencing revealed a significant overlap of PA and free-living (FL) bacteria highlighting an underestimated connectivity between both fractions. PA bacteria were in 14/21 cases more diverse than FL bacteria, reflecting a high heterogeneity in the particle microenvironment. We propose that a ratio of Chao 1 indices of PA/FL<1 indicates the presence of rather homogeneously colonized particles. The identification of different bacterial families enriched on either limnic or marine particles demonstrates that, despite the seemingly similar ecological niches, PA communities of both environments differ substantially.
Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.
In roots of Arabidopsis (Arabidopsis thaliana), L-lactate is generated by the reduction of pyruvate via L-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative L-lactate-metabolizing enzymes based on their homology to CYB2, the L-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses L-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than L-lactate. The key factor making GOX3 more efficient with L-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize L-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-of-function and overexpressor plants indicate that L-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on L-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes L-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of L-lactate after its formation under normoxia.
Abiotic stresses, such as salinity, cause global yield loss of all major crop plants. Factors and mechanisms that can aid in plant breeding for salt stress tolerance are therefore of great importance for food and feed production. Here, we identified a MYB-like transcription factor, Salt-Related MYB1 (SRM1), that negatively affects Arabidopsis (Arabidopsis thaliana) seed germination under saline conditions by regulating the levels of the stress hormone abscisic acid (ABA). Accordingly, several ABA biosynthesis and signaling genes act directly downstream of SRM1, including SALT TOLERANT1/NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, RESPONSIVE TO DESICCATION26, and Arabidopsis NAC DOMAIN CONTAINING PROTEIN19. Furthermore, SRM1 impacts vegetative growth and leaf shape. We show that SRM1 is an important transcriptional regulator that directly targets ABA biosynthesis and signaling-related genes and therefore may be regarded as an important regulator of ABA-mediated salt stress tolerance.
Downward fluxes of particulate organic matter (POM) are the major process for sequestering atmospheric CO2 into aquatic sediments for thousands of years. Budget calculations of the biological carbon pump are heavily based on the ratio between carbon export (sedimentation) and remineralization (release to the atmosphere). Current methodologies determine microbial dynamics on POM using closed vessels, which are strongly biased towards heterotrophy due to rapidly changing water chemistry (Bottle Effect). We developed a flow-through rolling tank for long term studies that continuously maintains POM at near in-situ conditions. There, bacterial communities resembled in-situ communities and greatly differed from those in the closed systems. The active particle-associated community in the flow-through system was stable for days, contrary to hours previously reported for closed incubations. In contrast to enhanced respiration rates, the decrease in photosynthetic rates on particles throughout the incubation was much slower in our system than in traditional ones. These results call for reevaluating experimentally-derived carbon fluxes estimated using traditional methods.
For over a hundred years, the "river sharks" of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks.
Small scale distribution of insect root herbivores may promote plant species diversity by creating patches of different herbivore pressure. However, determinants of small scale distribution of insect root herbivores, and impact of land use intensity on their small scale distribution are largely unknown. We sampled insect root herbivores and measured vegetation parameters and soil water content along transects in grasslands of different management intensity in three regions in Germany. We calculated community-weighted mean plant traits to test whether the functional plant community composition determines the small scale distribution of insect root herbivores. To analyze spatial patterns in plant species and trait composition and insect root herbivore abundance we computed Mantel correlograms. Insect root herbivores mainly comprised click beetle (Coleoptera, Elateridae) larvae (43%) in the investigated grasslands. Total insect root herbivore numbers were positively related to community-weighted mean traits indicating high plant growth rates and biomass (specific leaf area, reproductive-and vegetative plant height), and negatively related to plant traits indicating poor tissue quality (leaf C/N ratio). Generalist Elaterid larvae, when analyzed independently, were also positively related to high plant growth rates and furthermore to root dry mass, but were not related to tissue quality. Insect root herbivore numbers were not related to plant cover, plant species richness and soil water content. Plant species composition and to a lesser extent plant trait composition displayed spatial autocorrelation, which was not influenced by land use intensity. Insect root herbivore abundance was not spatially autocorrelated. We conclude that in semi-natural grasslands with a high share of generalist insect root herbivores, insect root herbivores affiliate with large, fast growing plants, presumably because of availability of high quantities of food. Affiliation of insect root herbivores with large, fast growing plants may counteract dominance of those species, thus promoting plant diversity.
Fertilization causes species loss and species dominance changes in plant communities worldwide. However, it still remains unclear how fertilization acts upon species functional traits, e.g. seed mass. Seed mass is a key trait of the regeneration strategy of plants, which influences a range of processes during the seedling establishment phase. Fertilization may select upon seed mass, either directly by increased nutrient availability or indirectly by increased competition. Since previous research has mainly analyzed the indirect effects of fertilization, we disentangled the direct and indirect effects to examine how nutrient availability and competition influence the seed mass relationships on four key components during seedling establishment: seedling emergence, time of seedling emergence, seedling survival and seedling growth. We conducted a common garden experiment with 22 dry grassland species with a two-way full factorial design that simulated additional nutrient supply and increased competition. While we found no evidence that fertilization either directly by additional nutrient supply or indirectly by increased competition alters the relationship between seed mass and (time of) seedling emergence, we revealed that large seed mass is beneficial under nutrient-poor conditions (seedlings have greater chances of survival, particularly in nutrient-poor soils) as well as under competition (large-seeded species produced larger seedlings, which suffered less from competition than small-seeded species). Based on these findings, we argue that both factors, i.e. nutrient availability and competition intensity, ought to be considered to understand how fertilization influences seedling establishment and species composition with respect to seed mass in natural communities. We propose a simple conceptual model, in which seed mass in natural communities is determined by competition intensity and nutrient availability. Here, we hypothesize that seed mass shows a U-shaped pattern along gradients of soil fertility, which may explain the contrasting soil fertility-seed mass relationships found in the recent literature.
Copepods are exposed to a high non-predatory mortality and their decomposing carcasses act as microniches with intensified microbial activity. Sinking carcasses could thereby represent anoxic microenvironment sustaining anaerobic microbial pathways in otherwise oxic water columns. Using non-invasive O-2 imaging, we document that carcasses of Calanus finmarchicus had an anoxic interior even at fully air-saturated ambient O-2 level. The extent of anoxia gradually expanded with decreasing ambient O-2 levels. Concurrent microbial sampling showed the expression of nitrite reductase genes (nirS) in all investigated carcass samples and thereby documented the potential for microbial denitrification in carcasses. The nirS gene was occasionally expressed in live copepods, but not as consistently as in carcasses. Incubations of sinking carcasses in (15)NO3-amended seawater demonstrated denitrification, of which on average 34%+/- 17% (n=28) was sustained by nitrification. However, the activity was highly variable and was strongly dependent on the ambient O-2 levels. While denitrification was present even at air-saturation (302 mol L-1), the average carcass specific activity increased several orders of magnitude to approximate to 1 nmol d(-1) at 20% air-saturation (55 mol O-2 L-1) at an ambient temperature of 7 degrees C. Sinking carcasses of C. finmarchicus therefore represent hotspots of pelagic denitrification, but the quantitative importance as a sink for bioavailable nitrogen is strongly dependent on the ambient O-2 level. The importance of carcass associated denitrification could be highly significant in O-2 depleted environments such as Oxygen Minimum Zones (OMZ).
Population models in ecology are often not good at predictions, even if they are complex and seem to be realistic enough. The reason for this might be that Occam's razor, which is key for minimal models exploring ideas and concepts, has been too uncritically adopted for more realistic models of systems. This can tic models too closely to certain situations, thereby preventing them from predicting the response to new conditions. We therefore advocate a new kind of parsimony to improve the application of Occam's razor. This new parsimony balances two contrasting strategies for avoiding errors in modeling: avoiding inclusion of nonessential factors (false inclusions) and avoiding exclusion of sometimes-important factors (false exclusions). It involves a synthesis of traditional modeling and analysis, used to describe the essentials of mechanistic relationships, with elements that arc included in a model because they have been reported to be or can arguably be assumed to be important under certain conditions. The resulting models should be able to reflect how the internal organization of populations change and thereby generate representations of the novel behavior necessary for complex predictions, including regime shifts.
Trade plays a key role in the spread of alien species and has arguably contributed to the recent enormous acceleration of biological invasions, thus homogenizing biotas worldwide. Combining data on 60-year trends of bilateral trade, as well as on biodiversity and climate, we modeled the global spread of plant species among 147 countries. The model results were compared with a recently compiled unique global data set on numbers of naturalized alien vascular plant species representing the most comprehensive collection of naturalized plant distributions currently available. The model identifies major source regions, introduction routes, and hot spots of plant invasions that agree well with observed naturalized plant numbers. In contrast to common knowledge, we show that the 'imperialist dogma,' stating that Europe has been a net exporter of naturalized plants since colonial times, does not hold for the past 60 years, when more naturalized plants were being imported to than exported from Europe. Our results highlight that the current distribution of naturalized plants is best predicted by socioeconomic activities 20 years ago. We took advantage of the observed time lag and used trade developments until recent times to predict naturalized plant trajectories for the next two decades. This shows that particularly strong increases in naturalized plant numbers are expected in the next 20 years for emerging economies in megadiverse regions. The interaction with predicted future climate change will increase invasions in northern temperate countries and reduce them in tropical and (sub) tropical regions, yet not by enough to cancel out the trade-related increase.
Patterns of phenotypic trait variation in two temperate forest herbs along a broad climatic gradient
(2015)
Phenotypic trait variation plays a major role in the response of plants to global environmental change, particularly in species with low migration capabilities and recruitment success. However, little is known about the variation of functional traits within populations and about differences in this variation on larger spatial scales. In a first approach, we therefore related trait expression to climate and local environmental conditions, studying two temperate forest herbs, Milium effusum and Stachys sylvatica, along a similar to 1800-2500 km latitudinal gradient. Within each of 9-10 regions in six European countries, we collected data from six populations of each species and recorded several variables in each region (temperature, precipitation) and population (light availability, soil parameters). For each plant, we measured height, leaf area, specific leaf area, seed mass and the number of seeds and examined environmental effects on within-population trait variation as well as on trait means. Most importantly, trait variation differed both between and within populations. Species, however, differed in their response. Intrapopulation variation in Milium was consistently positively affected by higher mean temperatures and precipitation as well as by more fertile local soil conditions, suggesting that more productive conditions may select for larger phenotypic variation. In Stachys, particularly light availability positively influenced trait variation, whereas local soil conditions had no consistent effects. Generally, our study emphasises that intra-population variation may differ considerably across larger scales-due to phenotypic plasticity and/or underlying genetic diversity-possibly affecting species response to global environmental change.
We extend the scope of European palaeogenomics by sequencing the genomes of Late Upper Palaeolithic (13,300 years old, 1.4-fold coverage) and Mesolithic (9,700 years old, 15.4-fold) males from western Georgia in the Caucasus and a Late Upper Palaeolithic (13,700 years old, 9.5-fold) male from Switzerland. While we detect Late Palaeolithic-Mesolithic genomic continuity in both regions, we find that Caucasus hunter-gatherers (CHG) belong to a distinct ancient clade that split from western hunter-gatherers similar to 45 kya, shortly after the expansion of anatomically modern humans into Europe and from the ancestors of Neolithic farmers similar to 25 kya, around the Last Glacial Maximum. CHG genomes significantly contributed to the Yamnaya steppe herders who migrated into Europe similar to 3,000 BC, supporting a formative Caucasus influence on this important Early Bronze age culture. CHG left their imprint on modern populations from the Caucasus and also central and south Asia possibly marking the arrival of Indo-Aryan languages.
MYB transcription factors (TFs) are important regulators of flavonoid biosynthesis in plants. Here, we report MYB112 as a formerly unknown regulator of anthocyanin accumulation in Arabidopsis (Arabidopsis thaliana). Expression profiling after chemically induced overexpression of MYB112 identified 28 up-and 28 down-regulated genes 5 h after inducer treatment, including MYB7 and MYB32, which are both induced. In addition, upon extended induction, MYB112 also positively affects the expression of PRODUCTION OF ANTHOCYANIN PIGMENT1, a key TF of anthocyanin biosynthesis, but acts negatively toward MYB12 and MYB111, which both control flavonol biosynthesis. MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C) CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative polymerase chain reaction, we show that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters, revealing them as direct downstream target genes. We further show that MYB112 expression is up-regulated by salinity and high light stress, environmental parameters that both require the MYB112 TF for anthocyanin accumulation under these stresses. In contrast to several other MYB TFs affecting anthocyanin biosynthesis, MYB112 expression is not controlled by nitrogen limitation or an excess of carbon. Thus, MYB112 constitutes a regulator that promotes anthocyanin accumulation under abiotic stress conditions.
The Norway lobster, Nephrops norvegicus, is a burrowing decapod with a rhythmic burrow emergence (24 h) governed by the circadian system. It is an important resource for European fisheries and its behavior deeply affects its availability. The current knowledge of Nephrops circadian biology is phenomenological as it is currently the case for almost all crustaceans. In attempt to elucidate the putative molecular mechanisms underlying circadian gene regulation in Nephrops, we used a transcriptomics approach on cDNA extracted from the eyestalk, a structure playing a crucial role in controlling behavior of decapods. We studied 14 male lobsters under 12-12 light-darkness blue light cycle. We used the Hiseq 2000 Illumina platform to sequence two eyestalk libraries (under light and darkness conditions) obtaining about 90 millions 100-bp paired-end reads. Trinity was used for the de novo reconstruction of transcriptomes; the size at which half of all assembled bases reside in contigs (N50) was equal to 1796 (light) and 2055 (darkness). We found a list of candidate clock genes and focused our attention on canonical ones: timeless, period, clock and bmal1. The cloning of assembled fragments validated Trinity outputs. The putative Nephrops clock genes showed high levels of identity (blastx on NCBI) with known crustacean clock gene homologs such as Eurydice pulchra (period: 47%, timeless: 59%, bmal1: 79%) and Macrobrachium rosenbergii (clock: 100%). We also found a vertebrate-like cryptochrome 2. RT-qPCR showed that only timeless had a robust diel pattern of expression. Our data are in accordance with the current knowledge of the crustacean circadian clock, reinforcing the idea that the molecular clockwork of this group shows some differences with the established model in Drosophila melanogaster.