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Institute
- Institut für Biochemie und Biologie (3697) (remove)
The Proteasome Acts as a Hub for Plant Immunity and Is Targeted by Pseudomonas Type III Effectors
(2016)
Recent evidence suggests that the ubiquitin-proteasome system is involved in several aspects of plant immunity and that a range of plant pathogens subvert the ubiquitin-proteasome system to enhance their virulence. Here, we show that proteasome activity is strongly induced during basal defense in Arabidopsis (Arabidopsis thaliana). Mutant lines of the proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae pv tomato DC3000 (Pst) and Pseudomonas syringae pv maculicola ES4326. Both proteasome subunits are required for pathogen-associated molecular pattern-triggered immunity responses. Analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome also plays an important role in defense priming and SAR. In addition, we show that Pst inhibits proteasome activity in a type III secretion-dependent manner. A screen for type III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1, and HopG1 as putative proteasome inhibitors. Biochemical characterization of HopM1 by mass spectrometry indicates that HopM1 interacts with several E3 ubiquitin ligases and proteasome subunits. This supports the hypothesis that HopM1 associates with the proteasome, leading to its inhibition. Thus, the proteasome is an essential component of pathogen-associated molecular pattern-triggered immunity and SAR, which is targeted by multiple bacterial effectors.
XopJ is a Xanthomonas type III effector protein that promotes bacterial virulence on susceptible pepper plants through the inhibition of the host cell proteasome and a resultant suppression of salicylic acid (SA) - dependent defense responses. We show here that Nicotiana benthamiana leaves transiently expressing XopJ display hypersensitive response (HR) -like symptoms when exogenously treated with SA. This apparent avirulence function of XopJ was further dependent on effector myristoylation as well as on an intact catalytic triad, suggesting a requirement of its enzymatic activity for HR-like symptom elicitation. The ability of XopJ to cause a HR-like symptom development upon SA treatment was lost upon silencing of SGT1 and NDR1, respectively, but was independent of EDS1 silencing, suggesting that XopJ is recognized by an R protein of the CC-NBS-LRR class. Furthermore, silencing of NPR1 abolished the elicitation of HR-like symptoms in XopJ expressing leaves after SA application. Measurement of the proteasome activity indicated that proteasome inhibition by XopJ was alleviated in the presence of SA, an effect that was not observed in NPR1 silenced plants. Our results suggest that XopJ - triggered HR-like symptoms are closely related to the virulence function of the effector and that XopJ follows a two-signal model in order to elicit a response in the non-host plant N. benthamiana.
Because of political conflicts and climate change, migration will be increased worldwide and integration in host societies is a challenge also for migrants. We hypothesize that migrants, who take up the challenge in a new social environment are taller than migrants who do not pose this challenge. We analyze by a questionnaire possible social, nutritional and ethnic influencing factors to body height (BH) of adult offspring of Turkish migrants (n = 82, 39 males) aged from 18 to 34 years (mean age 24.6 years). The results of multiple regression (downward selection) show that the more a male adult offspring of Turkish migrants feels like belonging to the Turkish culture, the smaller he is (95% CI, -3.79, -0.323). Further, the more a male adult offspring of Turkish migrants feels like belonging to the German culture, the taller he is (95% CI, -0.152, 1.738). We discussed it comparable to primates taking up their challenge in dominance, where as a result their body size increase is associated with higher IGF-1 level. IGF-1 is associated with emotional belonging and has a fundamental role in the regulation of metabolism and growth of the human body. With all pilot characteristics of our study results show that the successful challenge of integration in a new society is strongly associated with the emotional integration and identification in the sense of a personal sense of belonging to society. We discuss taller BH as a signal of social growth adjustment. In this sense, a secular trend of BH adaptation of migrants to hosts is a sign of integration.
Next-generation sequencing methods provide comprehensive data for the analysis of structural and functional analysis of the genome. The draft genomes with low contig number and high N50 value can give insight into the structure of the genome as well as provide information on the annotation of the genome. In this study, we designed a pipeline that can be used to assemble prokaryotic draft genomes with low number of contigs and high N50 value. We aimed to use combination of two de novo assembly tools (SPAdes and IDBA-Hybrid) and evaluate the impact of this approach on the quality metrics of the assemblies. The followed pipeline was tested with the raw sequence data with short reads (< 300) for a total of 10 species from four different genera. To obtain the final draft genomes, we firstly assembled the sequences using SPAdes to find closely related organism using the extracted 16 s rRNA from it. IDBA-Hybrid assembler was used to obtain the second assembly data using the closely related organism genome. SPAdes assembler tool was implemented using the second assembly, produced by IDBA-hybrid as a hint. The results were evaluated using QUAST and BUSCO. The pipeline was successful for the reduction of the contig numbers and increasing the N50 statistical values in the draft genome assemblies while preserving the coverage of the draft genomes.
Measures for interoperability of phenotypic data: minimum information requirements and formatting
(2016)
Background: Plant phenotypic data shrouds a wealth of information which, when accurately analysed and linked to other data types, brings to light the knowledge about the mechanisms of life. As phenotyping is a field of research comprising manifold, diverse and time-consuming experiments, the findings can be fostered by reusing and combining existing datasets. Their correct interpretation, and thus replicability, comparability and interoperability, is possible provided that the collected observations are equipped with an adequate set of metadata. So far there have been no common standards governing phenotypic data description, which hampered data exchange and reuse. Results: In this paper we propose the guidelines for proper handling of the information about plant phenotyping experiments, in terms of both the recommended content of the description and its formatting. We provide a document called "Minimum Information About a Plant Phenotyping Experiment", which specifies what information about each experiment should be given, and a Phenotyping Configuration for the ISA-Tab format, which allows to practically organise this information within a dataset. We provide examples of ISA-Tab-formatted phenotypic data, and a general description of a few systems where the recommendations have been implemented. Conclusions: Acceptance of the rules described in this paper by the plant phenotyping community will help to achieve findable, accessible, interoperable and reusable data.
Necrotrophic as well as saprophytic small-spored Altemaria (A.) species are annually responsible for major losses of agricultural products, such as cereal crops, associated with the contamination of food and feedstuff with potential health-endangering Altemaria toxins. Knowledge of the metabolic capabilities of different species-groups to form mycotoxins is of importance for a reliable risk assessment. 93 Altemaria strains belonging to the four species groups Alternaria tenuissima, A. arborescens, A. altemata, and A. infectoria were isolated from winter wheat kernels harvested from fields in Germany and Russia and incubated under equal conditions. Chemical analysis by means of an HPLC-MS/MS multi-Alternaria-toxin-method showed that 95% of all strains were able to form at least one of the targeted 17 non-host specific Altemaria toxins. Simultaneous production of up to 15 (modified) Altemaria toxins by members of the A. tenuissima, A. arborescens, A. altemata species-groups and up to seven toxins by A. infectoria strains was demonstrated. Overall tenuazonic acid was the most extensively formed mycotoxin followed by alternariol and alternariol mono methylether, whereas altertoxin I was the most frequently detected toxin. Sulfoconjugated modifications of alternariol, alternariol mono methylether, altenuisol and altenuene were frequently determined. Unknown perylene quinone derivatives were additionally detected. Strains of the species-group A. infectoria could be segregated from strains of the other three species-groups due to significantly lower toxin levels and the specific production of infectopyrone. Apart from infectopyrone, alterperylenol was also frequently produced by 95% of the A. infectoria strains. Neither by the concentration nor by the composition of the targeted Altemaria toxins a differentiation between the species-groups A. altemata, A. tenuissima and A. arborescens was possible.
Alternaria (A.) is a genus of widespread fungi capable of producing numerous, possibly health-endangering Alternaria toxins (ATs), which are usually not the focus of attention. The formation of ATs depends on the species and complex interactions of various environmental factors and is not fully understood. In this study the influence of temperature (7 degrees C, 25 degrees C), substrate (rice, wheat kernels) and incubation time (4, 7, and 14 days) on the production of thirteen ATs and three sulfoconjugated ATs by three different Alternaria isolates from the species groups A. tenuissima and A. infectoria was determined. High-performance liquid chromatography coupled with tandem mass spectrometry was used for quantification. Under nearly all conditions, tenuazonic acid was the most extensively produced toxin. At 25 degrees C and with increasing incubation time all toxins were formed in high amounts by the two A. tenuissima strains on both substrates with comparable mycotoxin profiles. However, for some of the toxins, stagnation or a decrease in production was observed from day 7 to 14. As opposed to the A. tenuissima strains, the A. infectoria strain only produced low amounts of ATs, but high concentrations of stemphyltoxin III. The results provide an essential insight into the quantitative in vitro AT formation under different environmental conditions, potentially transferable to different field and storage conditions.
We recently demonstrated that the sympathetic nervous system can be voluntarily activated following a training program consisting of cold exposure, breathing exercises, and meditation. This resulted in profound attenuation of the systemic inflammatory response elicited by lipopolysaccharide (LPS) administration. Herein, we assessed whether this training program affects the plasma metabolome and if these changes are linked to the immunomodulatory effects observed. A total of 224 metabolites were identified in plasma obtained from 24 healthy male volunteers at six timepoints, of which 98 were significantly altered following LPS administration. Effects of the training program were most prominent shortly after initiation of the acquired breathing exercises but prior to LPS administration, and point towards increased activation of the Cori cycle. Elevated concentrations of lactate and pyruvate in trained individuals correlated with enhanced levels of anti-inflammatory interleukin (IL)-10. In vitro validation experiments revealed that co-incubation with lactate and pyruvate enhances IL-10 production and attenuates the release of pro-inflammatory IL-1 beta and IL-6 by LPS-stimulated leukocytes. Our results demonstrate that practicing the breathing exercises acquired during the training program results in increased activity of the Cori cycle. Furthermore, this work uncovers an important role of lactate and pyruvate in the anti-inflammatory phenotype observed in trained subjects.
Home range size and resource use of breeding and non-breeding white storks along a land use gradient
(2018)
Biotelemetry is increasingly used to study animal movement at high spatial and temporal resolution and guide conservation and resource management. Yet, limited sample sizes and variation in space and habitat use across regions and life stages may compromise robustness of behavioral analyses and subsequent conservation plans. Here, we assessed variation in (i) home range sizes, (ii) home range selection, and (iii) fine-scale resource selection of white storks across breeding status and regions and test model transferability. Three study areas were chosen within the Central German breeding grounds ranging from agricultural to fluvial and marshland. We monitored GPS-locations of 62 adult white storks equipped with solar-charged GPS/3D-acceleration (ACC) transmitters in 2013-2014. Home range sizes were estimated using minimum convex polygons. Generalized linear mixed models were used to assess home range selection and fine-scale resource selection by relating the home ranges and foraging sites to Corine habitat variables and normalized difference vegetation index in a presence/pseudo-absence design. We found strong variation in home range sizes across breeding stages with significantly larger home ranges in non-breeding compared to breeding white storks, but no variation between regions. Home range selection models had high explanatory power and well predicted overall density of Central German white stork breeding pairs. Also, they showed good transferability across regions and breeding status although variable importance varied considerably. Fine-scale resource selection models showed low explanatory power. Resource preferences differed both across breeding status and across regions, and model transferability was poor. Our results indicate that habitat selection of wild animals may vary considerably within and between populations, and is highly scale dependent. Thereby, home range scale analyses show higher robustness whereas fine-scale resource selection is not easily predictable and not transferable across life stages and regions. Such variation may compromise management decisions when based on data of limited sample size or limited regional coverage. We thus recommend home range scale analyses and sampling designs that cover diverse regional landscapes and ensure robust estimates of habitat suitability to conserve wild animal populations.
Models are useful tools for understanding and predicting ecological patterns and processes. Under ongoing climate and biodiversity change, they can greatly facilitate decision-making in conservation and restoration and help designing adequate management strategies for an uncertain future. Here, we review the use of spatially explicit models for decision support and to identify key gaps in current modelling in conservation and restoration. Of 650 reviewed publications, 217 publications had a clear management application and were included in our quantitative analyses. Overall, modelling studies were biased towards static models (79%), towards the species and population level (80%) and towards conservation (rather than restoration) applications (71%). Correlative niche models were the most widely used model type. Dynamic models as well as the gene-to-individual level and the community-to-ecosystem level were underrepresented, and explicit cost optimisation approaches were only used in 10% of the studies. We present a new model typology for selecting models for animal conservation and restoration, characterising model types according to organisational levels, biological processes of interest and desired management applications. This typology will help to more closely link models to management goals. Additionally, future efforts need to overcome important challenges related to data integration, model integration and decision-making. We conclude with five key recommendations, suggesting that wider usage of spatially explicit models for decision support can be achieved by 1) developing a toolbox with multiple, easier-to-use methods, 2) improving calibration and validation of dynamic modelling approaches and 3) developing best-practise guidelines for applying these models. Further, more robust decision-making can be achieved by 4) combining multiple modelling approaches to assess uncertainty, and 5) placing models at the core of adaptive management. These efforts must be accompanied by long-term funding for modelling and monitoring, and improved communication between research and practise to ensure optimal conservation and restoration outcomes.
SDM performance varied for different range dynamics. Prediction accuracies decreased when abrupt range shifts occurred as species were outpaced by the rate of climate change, and increased again when a new equilibrium situation was realised. When ranges contracted, prediction accuracies increased as the absences were predicted well. Far- dispersing species were faster in tracking climate change, and were predicted more accurately by SDMs than short- dispersing species. BRTs mostly outperformed GLMs. The presence of a predator, and the inclusion of its incidence as an environmental predictor, made BRTs and GLMs perform similarly. Results are discussed in light of other studies dealing with effects of ecological traits and processes on SDM performance. Perspectives are given on further advancements of SDMs and for possible interfaces with more mechanistic approaches in order to improve predictions under environmental change.
Density regulation influences population dynamics through its effects on demographic rates and consequently constitutes a key mechanism explaining the response of organisms to environmental changes. Yet, it is difficult to establish the exact form of density dependence from empirical data. Here, we developed an individual-based model to explore how resource limitation and behavioural processes determine the spatial structure of white stork Ciconia ciconia populations and regulate reproductive rates. We found that the form of density dependence differed considerably between landscapes with the same overall resource availability and between home range selection strategies, highlighting the importance of fine-scale resource distribution in interaction with behaviour. In accordance with theories of density dependence, breeding output generally decreased with density but this effect was highly variable and strongly affected by optimal foraging strategy, resource detection probability and colonial behaviour. Moreover, our results uncovered an overlooked consequence of density dependence by showing that high early nestling mortality in storks, assumed to be the outcome of harsh weather, may actually result from density dependent effects on food provision. Our findings emphasize that accounting for interactive effects of individual behaviour and local environmental factors is crucial for understanding density-dependent processes within spatially structured populations. Enhanced understanding of the ways animal populations are regulated in general, and how habitat conditions and behaviour may dictate spatial population structure and demographic rates is critically needed for predicting the dynamics of populations, communities and ecosystems under changing environmental conditions.
The ability of some plant species to dominate communities in new biogeographical ranges has been attributed to an innate higher competitive ability and release from co-evolved specialist enemies. Specifically, invasive success in the new range might be explained by release from biotic negative soil-feedbacks, which control potentially dominant species in their native range. To test this hypothesis, we grew individuals from sixteen phylogenetically paired European grassland species that became either invasive or naturalized in new ranges, in either sterilized soil or in sterilized soil with unsterilized soil inoculum from their native home range. We found that although the native members of invasive species generally performed better than those of naturalized species, these native members of invasive species also responded more negatively to native soil inoculum than did the native members of naturalized species. This supports our hypothesis that potentially invasive species in their native range are held in check by negative soil-feedbacks. However, contrary to expectation, negative soil-feedbacks in potentially invasive species were not much increased by interspecific competition. There was no significant variation among families between invasive and naturalized species regarding their feedback response (negative vs. neutral). Therefore, we conclude that the observed negative soil feedbacks in potentially invasive species may be quite widespread in European families of typical grassland species.
Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two (4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional NFe-4S) cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the L-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production. IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes.
The c-Fosc-Jun complex forms the activator protein 1 transcription factor, a therapeutic target in the treatment of cancer. Various synthetic peptides have been designed to try to selectively disrupt the interaction between c-Fos and c-Jun at its leucine zipper domain. To evaluate the binding affinity between these synthetic peptides and c-Fos, polarizable and nonpolarizable molecular dynamics (MD) simulations were conducted, and the resulting conformations were analyzed using the molecular mechanics generalized Born surface area (MM/GBSA) method to compute free energies of binding. In contrast to empirical and semiempirical approaches, the estimation of free energies of binding using a combination of MD simulations and the MM/GBSA approach takes into account dynamical properties such as conformational changes, as well as solvation effects and hydrophobic and hydrophilic interactions. The predicted binding affinities of the series of c-Jun-based peptides targeting the c-Fos peptide show good correlation with experimental melting temperatures. This provides the basis for the rational design of peptides based on internal, van der Waals, and electrostatic interactions.
Background: Protein kinases constitute a particularly large protein family in Arabidopsis with important functions in cellular signal transduction networks. At the same time Arabidopsis is a model plant with high frequencies of gene duplications. Here, we have conducted a systematic analysis of the Arabidopsis kinase complement, the kinome, with particular focus on gene duplication events. We matched Arabidopsis proteins to a Hidden-Markov Model of eukaryotic kinases and computed a phylogeny of 942 Arabidopsis protein kinase domains and mapped their origin by gene duplication.
Results: The phylogeny showed two major clades of receptor kinases and soluble kinases, each of which was divided into functional subclades. Based on this phylogeny, association of yet uncharacterized kinases to families was possible which extended functional annotation of unknowns. Classification of gene duplications within these protein kinases revealed that representatives of cytosolic subfamilies showed a tendency to maintain segmentally duplicated genes, while some subfamilies of the receptor kinases were enriched for tandem duplicates. Although functional diversification is observed throughout most subfamilies, some instances of functional conservation among genes transposed from the same ancestor were observed. In general, a significant enrichment of essential genes was found among genes encoding for protein kinases.
Conclusions: The inferred phylogeny allowed classification and annotation of yet uncharacterized kinases. The prediction and analysis of syntenic blocks and duplication events within gene families of interest can be used to link functional biology to insights from an evolutionary viewpoint. The approach undertaken here can be applied to any gene family in any organism with an annotated genome.
In high-value sweet cherry (Prunus avium), the red coloration - determined by the anthocyanins content - is correlated with the fruit ripeness stage and market value. Non-destructive spectroscopy has been introduced in practice and may be utilized as a tool to assess the fruit pigments in the supply chain processes. From the fruit spectrum in the visible (Vis) wavelength range, the pigment contents are analyzed separately at their specific absorbance wavelengths.
A drawback of the method is the need for re-calibration due to varying optical properties of the fruit tissue. In order to correct for the scattering differences, most often the spectral intensity in the visible spectrum is normalized by wavelengths in the near infrared (NIR) range, or pre-processing methods are applied in multivariate calibrations.
In the present study, the influence of the fruit scattering properties on the Vis/NIR fruit spectrum were corrected by the effective pathlength in the fruit tissue obtained from time-resolved readings of the distribution of time-of-flight (DTOF). Pigment analysis was carried out according to Lambert-Beer law, considering fruit spectral intensities, effective pathlength, and refractive index. Results were compared to commonly applied linear color and multivariate partial least squares (PLS) regression analysis. The approaches were validated on fruits at different ripeness stages, providing variation in the scattering coefficient and refractive index exceeding the calibration sample set.
In the validation, the measuring uncertainty of non-destructively analyzing fruits with Vis/NIR spectra by means of PLS or Lambert-Beer in comparison with combined application of Vis/NIR spectroscopy and DTOF measurements showed a dramatic bias reduction as well as enhanced coefficients of determination when using both, the spectral intensities and apparent information on the scattering influence by means of DTOF readings. Corrections for the refractive index did not render improved results.
Polymeric matrices mimicking multiple functions of the ECM are expected to enable a material induced regeneration of tissues. Here, we investigated the adipogenic differentiation of human adipose derived mesenchymal stem cells (hADSCs) in a 3D architectured gelatin based hydrogel (ArcGel) prepared from gelatin and L-lysine diisocyanate ethyl ester (LDI) in an one-step process, in which the formation of an open porous morphology and the chemical network formation were integrated. The ArcGel was designed to support adipose tissue regeneration with its 3D porous structure, high cell biocompatibility, and mechanical properties compatible with human subcutaneous adipose tissue. The ArcGel could support initial cell adhesion and survival of hADSCs. Under static culture condition, the cells could migrate into the inner part of the scaffold with a depth of 840 +/- 120 mu m after 4 days, and distributed in the whole scaffold (2mm in thickness) within 14 days. The cells proliferated in the scaffold and the fold increase of cell number after 7 days of culture was 2.55 +/- 0.08. The apoptotic rate of hADSCs in the scaffold was similar to that of cells maintained on tissue culture plates. When cultured in adipogenic induction medium, the hADSCs in the scaffold differentiated into adipocytes with a high efficiency (93 +/- 1%). Conclusively, this gelatin based 3D scaffold presented high cell compatibility for hADSC cultivation and differentiation, which could serve as a potential implant material in clinical applications for adipose tissue reparation and regeneration.
Downscaling of microfluidic cell culture and detection devices for electrochemical monitoring has mostly focused on miniaturization of the microfluidic chips which are often designed for specific applications and therefore lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line electrode cleaning, sterilization, surface functionalization, cell seeding, cultivation and electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in both applications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well as the effect of mechanical stimulation on dopamine release were demonstrated using the programmable fluid handling capability. The here presented platform is aimed at applications utilizing cell based assays, ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation, and screening of genetically modified microorganisms to environmental monitoring.
Three cDNAs encoding purple acid phosphatase (PAP) were cloned from potato (Solanum tuberosum L. cv. Desiree) and expression of the corresponding genes was characterised. StPAP1 encodes a low-molecular weight PAP clustering with mammalian, cyanobacterial, and other plant PAPs. It was highly expressed in stem and root and its expression did not change in response to phosphorus (P) deprivation. StIPAP2 and StPAP3 code for high-molecular weight PAPs typical for plants. Corresponding gene expression was shown to be responsive to the level of P supply, with transcripts of StPAP2 and StPAP3 being most abundant in P-deprived roots or both stem and roots, respectively. Root colonisation by arbuscular mycorrhizal fungi had no effect on the expression of any of the three PAP genes. StIPAP1 mRNA is easily detectable along the root axis, including root hairs, but is barely detectable in root tips. In contrast, both StPAP2 and StPAP3 transcripts are abundant along the root axis, but absent in root hairs, and are most abundant in the root tip. All three PAPs described contain a predicted N-terminal secretion signal and could play a role in extracellular P scavenging, P mobilisation from the rhizosphere, or cell wall regeneration
The Fram Strait is an area with a relatively low and irregular distribution of diatom microfossils in surface sediments, and thus microfossil records are scarce, rarely exceed the Holocene, and contain sparse information about past richness and taxonomic composition. These attributes make the Fram Strait an ideal study site to test the utility of sedimentary ancient DNA (sedaDNA) metabarcoding. Amplifying a short, partial rbcL marker from samples of sediment core MSM05/5-712-2 resulted in 95.7% of our sequences being assigned to diatoms across 18 different families, with 38.6% of them being resolved to species and 25.8% to genus level. Independent replicates show a high similarity of PCR products, especially in the oldest samples. Diatom sedaDNA richness is highest in the Late Weichselian and lowest in Mid- and Late Holocene samples. Taxonomic composition is dominated by cold-water and sea-ice-associated diatoms and suggests several reorganisations - after the Last Glacial Maximum, after the Younger Dryas, and after the Early and after the Mid-Holocene. Different sequences assigned to, amongst others, Chaetoceros socialis indicate the detectability of intra-specific diversity using sedaDNA. We detect no clear pattern between our diatom sedaDNA record and the previously published IP25 record of this core, although proportions of pennate diatoms increase with higher IP25 concentrations and proportions of Nitzschia cf. frigida exceeding 2% of the assemblage point towards past sea-ice presence.
Ecosystem boundaries, such as the Arctic-Boreal treeline, are strongly coupled with climate and were spatially highly dynamic during past glacial-interglacial cycles. Only a few studies cover vegetation changes since the last interglacial, as most of the former landscapes are inundated and difficult to access. Using pollen analysis and sedimentary ancient DNA (sedaDNA) metabarcoding, we reveal vegetation changes on Bol’shoy Lyakhovsky Island since the last interglacial from permafrost sediments. Last interglacial samples depict high levels of floral diversity with the presence of trees (Larix, Picea, Populus) and shrubs (Alnus, Betula, Ribes, Cornus, Saliceae) on the currently treeless island. After the Last Glacial Maximum, Larix re-colonised the island but disappeared along with most shrub taxa. This was probably caused by Holocene sea-level rise, which led to increased oceanic conditions on the island. Additionally, we applied two newly developed larch-specific chloroplast markers to evaluate their potential for tracking past population dynamics from environmental samples. The novel markers were successfully re-sequenced and exhibited two variants of each marker in last interglacial samples. SedaDNA can track vegetation changes as well as genetic changes across geographic space through time and can improve our understanding of past processes that shape modern patterns.
Larix populations at the tundra-taiga ecotone in northern Siberia are highly under-represented in population genetic studies, possibly due to the remoteness of these regions that can only be accessed at extraordinary expense. The genetic signatures of populations in these boundary regions are therefore largely unknown. We aim to generate organelle reference genomes for the detection of single nucleotide polymorphisms (SNPs) that can be used for paleogenetic studies. We present 19 complete chloroplast genomes and mitochondrial genomic sequences of larches from the southern lowlands of the Taymyr Peninsula (northernmost range of Larix gmelinii (Rupr.) Kuzen.), the lower Omoloy River, and the lower Kolyma River (both in the range of Larix cajanderi Mayr). The genomic data reveal 84 chloroplast SNPs and 213 putatively mitochondrial SNPs. Parsimony-based chloroplast haplotype networks show no spatial structure of individuals from different geographic origins, while the mitochondrial haplotype network shows at least a slight spatial structure with haplotypes from the Omoloy and Kolyma populations being more closely related to each other than to most of the haplotypes from the Taymyr populations. Whole genome alignments with publicly available complete chloroplast genomes of different Larix species show that among official plant barcodes only the rcbL gene contains sufficient polymorphisms, but has to be sequenced completely to distinguish the different provenances. We provide 8 novel mitochondrial SNPs that are putatively diagnostic for the separation of L. gmelinii and L. cajanderi, while 4 chloroplast SNPs have the potential to distinguish the L. gmelinii/ L. cajanderi group from other Larix species. Our organelle references can be used for a targeted primer and probe design allowing the generation of short amplicons. This is particularly important with regard to future investigations of, for example, the biogeographic history of Larix by screening ancient sedimentary DNA of Larix.
Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that microcystin fulfils an important function within cells of its natural producer Microcystis. The microcystin deficient mutant Delta mcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of microcystin in the wild type. Finally, the mutant defective in microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites.
Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium Microcystis via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene mdnA encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in Microcystis aeruginosa strain NIES843 and heterologously produced variants.
Ranging behavior of European rabbits (Oryctolagus cuniculus) in urban and suburban landscapes
(2020)
Various mammals, particularly carnivores, reportedly establish smaller home ranges in urban compared with rural areas. This may be because urban environments provide optimal resources within a small area, negating the requirement to range further, or because habitat fragmentation constrains ranging behavior. Comparable information on urban populations of herbivorous mammalian species (such as European rabbits) is scarce. To fill this knowledge gap, we radio-tracked 13 individuals (seven females and six males) equipped with radio collars in a suburban and an urban study site in the city of Frankfurt am Main in Germany during the reproductive season (March to September) of 2012. The study sites differed in levels of habitat fragmentation. We report the smallest home ranges ever described for this species, with mean 95% minimum convex polygons (MCPs) covering 0.50 ha, while no consistent differences between sites were uncovered. We occasionally tracked individuals crossing streets underground (in burrows), suggesting that streets may restrict the ranging behavior of rabbits-and possibly other burrowing species-to a much lesser extent than previously thought. We conclude that heterogeneous landscape structures, made up of a diverse mosaic of buildings, parks, and gardens, provide sufficient food and shelter in close proximity to burrows at both study sites. Therefore, our data support the hypothesis that optimal resources constrain ranges in this case rather than habitat fragmentation.
Multidirectional communicative interactions in social networks can have a profound effect on mate choice behavior. Male Atlantic molly Poecilia mexicana exhibit weaker mating preferences when an audience male is presented. This could be a male strategy to reduce sperm competition risk: interacting more equally with different females may be advantageous because rivals might copy mate choice decisions. In line with this hypothesis, a previous study found males to show a strong audience effect when being observed while exercising mate choice, but not when the rival was presented only before the choice tests. Audience effects on mate choice decisions have been quantified in poeciliid fishes using association preference designs, but it remains unknown if patterns found from measuring association times translate into actual mating behavior. Thus, we created five audience treatments simulating different forms of perceived sperm competition risk and determined focal males' mating preferences by scoring pre-mating (nipping) and mating behavior (gonopodial thrusting). Nipping did not reflect the pattern that was found when association preferences were measured, while a very similar pattern was uncovered in thrusting behavior. The strongest response was observed when the audience could eavesdrop on the focal male's behavior. A reduction in the strength of focal males' preferences was also seen after the rival male had an opportunity to mate with the focal male's preferred mate. In comparison, the reduction of mating preferences in response to an audience was greater when measuring association times than actual mating behavior. While measuring direct sexual interactions between the focal male and both stimulus females not only the male's motivational state is reflected but also females' behavior such as avoidance of male sexual harassment.
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.
The all-female Amazon molly (Poecilia formosa) is the result of a hybridization of the Atlantic molly (P. mexicana) and the sailfin molly (P. latipinna) approximately 120,000 years ago. As a gynogenetic species, P. formosa needs to copulate with heterospecific males including males from one of its bisexual ancestral species. However, the sperm only triggers embryogenesis of the diploid eggs. The genetic information of the sperm donor typically will not contribute to the next generation of P. formosa. Hence, P. formosa possesses generally one allele from each of its ancestral species at any genetic locus. This raises the question whether both ancestral alleles are equally expressed in P. formosa. Allele-specific expression (ASE) has been previously assessed in various organisms, e.g., human and fish, and ASE was found to be important in the context of phenotypic variability and disease. In this study, we utilized Real-Time PCR techniques to estimate ASE of the androgen receptor alpha (arα) gene in several distinct tissues of Amazon mollies. We found an allelic bias favoring the maternal ancestor (P. mexicana) allele in ovarian tissue. This allelic bias was not observed in the gill or the brain tissue. Sequencing of the promoter regions of both alleles revealed an association between an Indel in a known CpG island and differential expression. Future studies may reveal whether our observed cis-regulatory divergence is caused by an ovary-specific trans-regulatory element, preferentially activating the allele of the maternal ancestor.
The all-female Amazon molly (Poecilia formosa) originated from a single hybridization of two bisexual ancestors, Atlantic molly (Poecilia mexicana) and sailfin molly (Poecilia latipinna). As a gynogenetic species, the Amazon molly needs to copulate with a heterospecific male, but the genetic information of the sperm-donor does not contribute to the next generation, as the sperm only acts as the trigger for the diploid eggs’ embryogenesis. Here, we study the sequence evolution and gene expression of the duplicated genes coding for androgen receptors (ars) and other pathway-related genes, i.e., the estrogen receptors (ers) and cytochrome P450, family19, subfamily A, aromatase genes (cyp19as), in the Amazon molly, in comparison to its bisexual ancestors. Mollies possess–as most other teleost fish—two copies of the ar, er, and cyp19a genes, i.e., arα/arβ, erα/erβ1, and cyp19a1 (also referred as cyp19a1a)/cyp19a2 (also referred to as cyp19a1b), respectively. Non-synonymous single nucleotide polymorphisms (SNPs) among the ancestral bisexual species were generally predicted not to alter protein function. Some derived substitutions in the P. mexicana and one in P. formosa are predicted to impact protein function. We also describe the gene expression pattern of the ars and pathway-related genes in various tissues (i.e., brain, gill, and ovary) and provide SNP markers for allele specific expression research. As a general tendency, the levels of gene expression were lowest in gill and highest in ovarian tissues, while expression levels in the brain were intermediate in most cases. Expression levels in P. formosa were conserved where expression did not differ between the two bisexual ancestors. In those cases where gene expression levels significantly differed between the bisexual species, P. formosa expression was always comparable to the higher expression level among the two ancestors. Interestingly, erβ1 was expressed neither in brain nor in gill in the analyzed three molly species, which implies a more important role of erα in the estradiol synthesis pathway in these tissues. Furthermore, our data suggest that interactions of steroid-signaling pathway genes differ across tissues, in particular the interactions of ars and cyp19as.
The all-female Amazon molly (Poecilia formosa) originated from a single hybridization of two bisexual ancestors, Atlantic molly (Poecilia mexicana) and sailfin molly (Poecilia latipinna). As a gynogenetic species, the Amazon molly needs to copulate with a heterospecific male, but the genetic information of the sperm-donor does not contribute to the next generation, as the sperm only acts as the trigger for the diploid eggs’ embryogenesis. Here, we study the sequence evolution and gene expression of the duplicated genes coding for androgen receptors (ars) and other pathway-related genes, i.e., the estrogen receptors (ers) and cytochrome P450, family19, subfamily A, aromatase genes (cyp19as), in the Amazon molly, in comparison to its bisexual ancestors. Mollies possess–as most other teleost fish—two copies of the ar, er, and cyp19a genes, i.e., arα/arβ, erα/erβ1, and cyp19a1 (also referred as cyp19a1a)/cyp19a2 (also referred to as cyp19a1b), respectively. Non-synonymous single nucleotide polymorphisms (SNPs) among the ancestral bisexual species were generally predicted not to alter protein function. Some derived substitutions in the P. mexicana and one in P. formosa are predicted to impact protein function. We also describe the gene expression pattern of the ars and pathway-related genes in various tissues (i.e., brain, gill, and ovary) and provide SNP markers for allele specific expression research. As a general tendency, the levels of gene expression were lowest in gill and highest in ovarian tissues, while expression levels in the brain were intermediate in most cases. Expression levels in P. formosa were conserved where expression did not differ between the two bisexual ancestors. In those cases where gene expression levels significantly differed between the bisexual species, P. formosa expression was always comparable to the higher expression level among the two ancestors. Interestingly, erβ1 was expressed neither in brain nor in gill in the analyzed three molly species, which implies a more important role of erα in the estradiol synthesis pathway in these tissues. Furthermore, our data suggest that interactions of steroid-signaling pathway genes differ across tissues, in particular the interactions of ars and cyp19as.
Melanoma represents a prime example demonstrating the success of targeted therapy in cancer. Nevertheless, it remained a deadly disease until now, and the identification of new, independent strategies as well as the understanding of their molecular mechanisms may help to finally overcome the high mortality. Both indirubins and TNF-related apoptosis-inducing ligand (TRAIL) represent promising candidates. Here, the indirubin derivative DKP-073 is shown to trigger apoptosis in melanoma cells, which is enhanced by the combination with TRAIL and is accompanied by complete loss of cell viability. Addressing the signaling cascade, characteristic molecular steps were identified as caspase-3 activation, downregulation of XIAP, upregulation of p53 and TRAIL receptor 2, loss of mitochondrial membrane potential, and STAT-3 dephosphorylation. The decisive step, however, turned out to be the early production of ROS already at 1 h. This was proven by antioxidant pretreatment, which completely abolished apoptosis induction and loss of cell viability as well as abrogated all signaling effects listed above. Thus, ROS appeared as upstream of all proapoptotic signaling. The data indicate a dominant role of ROS in apoptosis regulation, and the new pathway may expose a possible Achilles heel of melanoma.
The process of leaf senescence consists of the final stage of leaf development. It has evolved as a mechanism to degrade macromolecules and micronutrients and remobilize them to other developing parts of the plant; hence it plays a central role for the survival of plants and crop production. During senescence, a range of physiological, morphological, cellular, and molecular events occur, which are generally referred to as the senescence syndrome that includes several hallmarks such as visible yellowing, loss of chlorophyll and water content, increase of ion leakage and cell death, deformation of chloroplast and cell structure, as well as the upregulation of thousands of so-called senescence-associated genes (SAGs) and downregulation of photosynthesis-associated genes (PAGs). This chapter is devoted to methods characterizing the onset and progression of leaf senescence at the morphological, physiological, cellular, and molecular levels. Leaf senescence normally progresses in an age-dependent manner but is also induced prematurely by a variety of environmental stresses in plants. Focused on the hallmarks of the senescence syndrome, a series of protocols is described to asses quantitatively the senescence process caused by developmental cues or environmental perturbations. We first briefly describe the senescence process, the events associated with the senescence syndrome, and the theories and methods to phenotype senescence. Detailed protocols for monitoring senescence in planta and in vitro, using the whole plant and the detached leaf, respectively, are presented. For convenience, most of the protocols use the model plant species Arabidopsis and rice, but they can be easily extended to other plants.
RNA-based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre-mRNAs into mature mRNAs ready for translation and RNA-based silencing processes, such as RNA-directed DNA methylation (RdDM). Polyadenylation of pre-mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence-labelling strategy, we have characterized this defect in more detail using RNA and small-RNA sequencing. In addition to global defects in the expression of pollen-differentiation genes, paps1 null-mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21- and particularly 24-nucleotide-long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double-mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double-mutant of paps1 and rna-dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast.
Changes in carbon flow and sink/source activities can affect floral, architectural, and reproductive traits of plants. In potato, overexpression (OE) of the purple acid phosphatase 2 of Arabidopsis (AtPAP2) resulted in earlier flowering, faster growth rate, increased tubers and tuber starch content, and higher photosynthesis rate. There was a significant change in sucrose, glucose and fructose levels in leaves, phloem and sink biomass of the OE lines, consistent with an increased expression of sucrose transporter 1 (StSUT1). Furthermore, the expression levels and enzyme activity of sucrose-phosphate synthase (SPS) were also significantly increased in the OE lines. These findings strongly suggest that higher carbon supply from the source and improved sink strength can improve potato tuber yield. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.
Molecularly imprinted polymer (MP) nanofilrns for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of similar to 5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered.
The epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.
The persulfide sulfur formed on an active site cysteine residue of pyridoxal 5'-phosphate-dependent cysteine desulfurases is subsequently incorporated into the biosynthetic pathways of a variety of sulfur-containing cofactors and thionucleosides. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of molybdopterin (MPT) synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. It has been shown that three cysteine desulfurases (CsdA, SufS, and IscS) of Escherichia coli can transfer sulfur from L-cysteine to the thiocarboxylate of MoaD in vitro. Here, we demonstrate by surface plasmon resonance analyses that IscS, but not CsdA or SufS, interacts with MoeB and MoaD. MoeB and MoaD can stimulate the IscS activity up to 1.6-fold. Analysis of the sulfuration level of MoaD isolated from strains defective in cysteine desulfurases shows a largely decreased sulfuration level of the protein in an iscS deletion strain but not in a csdA/sufS deletion strain. We also show that another iscS deletion strain of E. coli accumulates compound Z, a direct oxidation product of the immediate precursor of MPT, to the same extent as an MPT synthase-deficient strain. In contrast, analysis of the content of compound Z in Delta csdA and Delta sufS strains revealed no such accumulation. These findings indicate that IscS is the primary physiological sulfur-donating enzyme for the generation of the thiocarboxylate of MPT synthase in MPT biosynthesis.
Aldol reactions play an important role in organic synthesis, as they belong to the class of highly beneficial C-C-linking reactions. Aldol-type reactions can be efficiently and stereoselectively catalyzed by the enzyme 2-deoxy-D-ribose-5-phosphate aldolase (DERA) to gain key intermediates for pharmaceuticals such as atorvastatin. The immobilization of DERA would open the opportunity for a continuous operation mode which gives access to an efficient, large-scale production of respective organic intermediates. In this contribution, we synthesize and utilize DERA/polymer conjugates for the generation and fixation of a DERA bearing thin film on a polymeric membrane support. The conjugation strongly increases the tolerance of the enzyme toward the industrial relevant substrate acetaldehyde while UV-cross-linkable groups along the conjugated polymer chains provide the opportunity for covalent binding to the support. First, we provide a thorough characterization of the conjugates followed by immobilization tests on representative, nonporous cycloolefinic copolymer supports. Finally, immobilization on the target supports constituted of polyacrylonitrile (PAN) membranes is performed, and the resulting enzymatically active membranes are implemented in a simple membrane module setup for the first assessment of biocatalytic performance in the continuous operation mode using the combination hexanal/acetaldehyde as the substrate.
Collagen-based biomaterials with oriented fibrils have shown great application potential in medicine. However, it is still challenging to control the type I collagen fibrillogenesis in ultrathin films. Here, we report an approach to produce cohesive and well-organized type I collagen ultrathin films of about 10 nm thickness using the Langmuir-Blodgett technique. Ellipsometry, rheology, and Brewster angle microscopy are applied to investigate in situ how the molecules behave at the air-water interface, both at room temperature and 37 degrees C. The interfacial storage modulus observed at room temperature vanishes upon heating, indicating the existence and disappearance of the network structure in the protein nanosheet. The films were spanning over holes as large as 1 mm diameter when transferred at room temperature, proving the strong cohesive interactions. A highly aligned and fibrillar structure was observed by atomic force microscopy (AFM) and optical microscopy.
Biodegradation of polyester polyurethane by the marine fungus Cladosporium halotolerans 6UPA1
(2022)
Lack of degradability and the accumulation of polymeric wastes increase the risk for the health of the environment. Recently, recycling of polymeric waste materials becomes increasingly important as raw materials for polymer synthesis are in short supply due to the rise in price and supply chain disruptions. As an important polymer, polyurethane (PU) is widely used in modern life, therefore, PU biodegradation is desirable to avoid its accumulation in the environment. In this study, we isolated a fungal strain Cladosporium halotolerans from the deep sea which can grow in mineral medium with a polyester PU (Impranil DLN) as a sole carbon source. Further, we demonstrate that it can degrade up to 80% of Impranil PU after 3 days of incubation at 28 celcius by breaking the carbonyl groups (1732 cm(-1)) and C-N-H bonds (1532 cm(-1) and 1247 cm(-1)) as confirmed by Fourier-transform infrared (FTIR) spectroscopy analysis. Gas chromatography-mass spectrometry (GC-MS) analysis revealed polyols and alkanes as PU degradation intermediates, indicating the hydrolysis of ester and urethane bonds. Esterase and urease activities were detected in 7 days-old cultures with PU as a carbon source. Transcriptome analysis showed a number of extracellular protein genes coding for enzymes such as cutinase, lipase, peroxidase and hydrophobic surface binding proteins A (HsbA) were expressed when cultivated on Impranil PU. The yeast two-hybrid assay revealed that the hydrophobic surface binding protein ChHsbA1 directly interacts with inducible esterases, ChLip1 (lipase) and ChCut1 (cutinase). Further, the KEGG pathway for "fatty acid degradation " was significantly enriched in Impranil PU inducible genes, indicating that the fungus may use the degradation intermediates to generate energy via this pathway. Taken together, our data indicates secretion of both esterase and hydrophobic surface binding proteins by C. halotolerans plays an important role in Impranil PU absorption and subsequent degradation. Our study provides a mechanistic insight into Impranil PU biodegradation by deep sea fungi and provides the basis for future development of biotechnological PU recycling.
Morphological and genetic evidence for early Holocene cattle management in northeastern China
(2013)
The domestication of cattle is generally accepted to have taken place in two independent centres: around 10,500 years ago in the Near East, giving rise to modern taurine cattle, and two millennia later in southern Asia, giving rise to zebu cattle. Here we provide firmly dated morphological and genetic evidence for early Holocene management of taurine cattle in northeastern China. We describe conjoining mandibles from this region that show evidence of oral stereotypy, dated to the early Holocene by two independent C-14 dates. Using Illumina high-throughput sequencing coupled with DNA hybridization capture, we characterize 15,406 bp of the mitogenome with on average 16.7-fold coverage. Phylogenetic analyses reveal a hitherto unknown mitochondrial haplogroup that falls outside the known taurine diversity. Our data suggest that the first attempts to manage cattle in northern China predate the introduction of domestic cattle that gave rise to the current stock by several thousand years.
The retinitis pigmentosa 2 polypeptide (RP2) functions as a GTPase-activating protein (GAP) for ARL3 (Arf-like protein 3), a small GTPase. ARL3 is an effector of phosphodiesterase 6 Delta (PDE6D), a prenyl-binding protein and chaperone of prenylated protein in photoreceptors. Mutations in the human RP2 gene cause X-linked retinitis pigmentosa (XLRP) and cone-rod dystrophy (XL-CORD). To study mechanisms causing XLRP, we generated an RP2 knockout mouse. The RP2h(-/-) mice exhibited a slowly progressing rod-cone dystrophy simulating the human disease. RP2h(-/-) scotopic a-wave and photopic b-wave amplitudes declined at 1 mo of age and continued to decline over the next 6 mo. Prenylated PDE6 subunits and G-protein coupled receptor kinase 1 (GRK1) were unable to traffic effectively to the RP2h(-/-) outer segments. Mechanistically, absence of RP2 GAP activity increases ARL3-GTP levels, forcing PDE6D to assume a predominantly "closed" conformation that impedes binding of lipids. Lack of interaction disrupts trafficking of PDE6 and GRK1 to their destination, the photoreceptor outer segments. We propose that hyperactivity of ARL3-GTP in RP2 knockout mice and human patients with RP2 null alleles leads to XLRP resembling recessive rod-cone dystrophy.
In eukaryotes, the transcription of tRNA genes is initiated by the concerted action of transcription factors IIIC (TFIIIC) and IIIB (TFIIIB) which direct the recruitment of polymerase III. While TFIIIC recognizes highly conserved, intragenic promoter elements, TFIIIB binds to the non-coding 5'-upstream regions of the tRNA genes. Using a systematic bioinformatic analysis of 11 multicellular eukaryotic genomes we identified a highly conserved TATA motif followed by a CAA-motif in the tRNA upstream regions of all plant genomes. Strikingly, the 5'-flanking tRNA regions of the animal genomes are highly heterogeneous and lack a common conserved sequence signature. Interestingly, in the animal genomes the tRNA species that read the same codon share conserved motifs in their upstream regions. Deep-sequencing analysis of 16 human tissues revealed multiple splicing variants of two of the TFIIIB subunits, Bdp1 and Brf1, with tissue-specific expression patterns. These multiple forms most likely modulate the TFIIIB-DNA interactions and explain the lack of a uniform signature motif in the tRNA upstream regions of animal genomes. The anticodon-dependent 5'-flanking motifs provide a possible mechanism for independent regulation of the tRNA transcription in various human tissues.
Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein Sufl can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding.
The translation of genetic information according to the sequence of the mRNA template occurs with high accuracy and fidelity. Critical events in each single step of translation are selection of transfer RNA (tRNA), codon reading and tRNA-regeneration for a new cycle. We developed a model that accurately describes the dynamics of single elongation steps, thus providing a systematic insight into the sensitivity of the mRNA translation rate to dynamic environmental conditions. Alterations in the concentration of the aminoacylated tRNA can transiently stall the ribosomes during translation which results, as suggested by the model, in two outcomes: either stress-induced change in the tRNA availability triggers the premature termination of the translation and ribosomal dissociation, or extensive demand for one tRNA species results in a competition between frameshift to an aberrant open-reading frame and ribosomal drop-off. Using the bacterial Escherichia coli system, we experimentally draw parallels between these two possible mechanisms.
The most crucial step in data processing from high-throughput sequencing applications is the accurate and sensitive alignment of the sequencing reads to reference genomes or transcriptomes. The accurate detection of insertions and deletions (indels) and errors introduced by the sequencing platform or by misreading of modified nucleotides is essential for the quantitative processing of the RNA-based sequencing (RNA-Seq) datasets and for the identification of genetic variations and modification patterns. We developed a new, fast and accurate algorithm for nucleic acid sequence analysis, FANSe, with adjustable mismatch allowance settings and ability to handle indels to accurately and quantitatively map millions of reads to small or large reference genomes. It is a seed-based algorithm which uses the whole read information for mapping and high sensitivity and low ambiguity are achieved by using short and non-overlapping reads. Furthermore, FANSe uses hotspot score to prioritize the processing of highly possible matches and implements modified Smith-Watermann refinement with reduced scoring matrix to accelerate the calculation without compromising its sensitivity. The FANSe algorithm stably processes datasets from various sequencing platforms, masked or unmasked and small or large genomes. It shows a remarkable coverage of low-abundance mRNAs which is important for quantitative processing of RNA-Seq datasets.
Light switching of the activity of a coiled-coil protein, the AP-1 transcription factor, in living cells was made possible by the introduction of a designed azobenzene-cross-linked dominant negative peptide, XAFosW (red and yellow in the picture). In the dark, XAFosW showed decreased helical content and decreased affinity for target Jun proteins (green); irradiation at 365 nm enhanced helicity and target affinity.
A direct electron transfer (DET) based sulphite/oxygen biofuel cell is reported that utilises human sulphite oxidase (hSOx) and Myrothecium verrucaria bilirubin oxidase (MvBOx) and nanostructured gold electrodes. For bioanode construction, the nanostructured gold microelectrodes were further modified with 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) to which polyethylene imine was covalently attached. hSOx was adsorbed onto this chemically modified nanostructured electrode with high surface loading of electroactive enzyme and in presence of sulphite high anodic bioelectrocatalytic currents were generated with an onset potential of 0.05 V vs. NHE. The biocathode contained MyBOx directly adsorbed to the deposited gold nanoparticles for cathodic oxygen reduction starting at 0.71 V vs. NHE. Both enzyme electrodes were integrated to a DET-type biofuel cell. Power densities of 8 and 1 mu W cm(-2) were achieved at 0.15 V and 0.45 V of cell voltages, respectively, with the membrane based biodevices under aerobic conditions. (C) 2014 Elsevier B.V. All rights reserved.
Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes
(2017)
Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a “closed” conformation, suitable for internal electron transfer, and an “open” conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.
The bioelectrocatalytic sulfite oxidation by human sulfite oxidase (hSO) on indium tin oxide (ITO) is reported, which is facilitated by functionalizing of the electrode surface with polyethylenimine (PEI)-entrapped CdS nanoparticles and enzyme. hSO was assembled onto the electrode with a high surface loading of electroactive enzyme. In the presence of sulfite but without additional mediators, a high bioelectrocatalytic current was generated. Reference experiments with only PEI showed direct electron transfer and catalytic activity of hSO, but these were less pronounced. The application of the polyelectrolyte-entrapped quantum dots (QDs) on ITO electrodes provides a compatible surface for enzyme binding with promotion of electron transfer. Variations of the buffer solution conditions, e.g., ionic strength, pH, viscosity, and the effect of oxygen, were studied in order to understand intramolecular and heterogeneous electron transfer from hSO to the electrode. The results are consistent with a model derived for the enzyme by using flash photolysis in solution and spectroelectrochemistry and molecular dynamic simulations of hSO on monolayer-modified gold electrodes. Moreover, for the first time a photoelectrochemical electrode involving immobilized hSO is demonstrated where photoexcitation of the CdS/hSO-modified electrode lead to an enhanced generation of bioelectrocatalytic currents upon sulfite addition. Oxidation starts already at the redox potential of the electron transfer domain of hSO and is greatly increased by application of a small overpotential to the CdS/hSO-modified ITO.
Recent investigations propose the acid sphingomyelinase (ASM)/ceramide system as a novel target for antidepressant action. ASM catalyzes the breakdown of the abundant membrane lipid sphingomyelin to the lipid messenger ceramide. This ASM‐induced lipid modification induces a local shift in membrane properties, which influences receptor clustering and downstream signaling. Canonical transient receptor potential channels 6 (TRPC6) are non‐selective cation channels located in the cell membrane that play an important role in dendritic growth, synaptic plasticity and cognition in the brain. They can be activated by hyperforin, an ingredient of the herbal remedy St. John’s wort for treatment of depression disorders. Because of their role in the context of major depression, we investigated the crosstalk between the ASM/ceramide system and TRPC6 ion channels in a pheochromocytoma cell line 12 neuronal cell model (PC12 rat pheochromocytoma cell line). Ca2+ imaging experiments indicated that hyperforin‐induced Ca2+ influx through TRPC6 channels is modulated by ASM activity. While antidepressants, known as functional inhibitors of ASM activity, reduced TRPC6‐mediated Ca2+ influx, extracellular application of bacterial sphingomyelinase rebalanced TRPC6 activity in a concentration‐related way. This effect was confirmed in whole‐cell patch clamp electrophysiology recordings. Lipidomic analyses revealed a decrease in very long chain ceramide/sphingomyelin molar ratio after ASM inhibition, which was connected with changes in the abundance of TRPC6 channels in flotillin‐1–positive lipid rafts as visualized by western blotting. Our data provide evidence that the ASM/ceramide system regulates TRPC6 channels likely by controlling their recruitment to specific lipid subdomains and thereby fine‐tuning their physical properties.
The presence of partially folded intermediates along the folding funnel of proteins has been suggested to be a signature of potentially aggregating systems. Many studies have concluded that metastable, highly flexible intermediates are the basic elements of the aggregation process. In a previous paper, we demonstrated how the choice between aggregation and folding behavior was influenced by hydrophobicity distribution patterning along the sequence, as quantified by recurrence quantification analysis (RQA) of the Myiazawa-Jernigan coded primary structures. In the present paper, we tried to unify the "partially folded intermediate" and "hydrophobicity/charge" models of protein aggregation verifying the ability of an empirical relation, developed for rationalizing the effect of different mutations on aggregation propensity of acyl-phosphatase and based on the combination of hydrophobicity RQA and charge descriptors, to discriminate in a statistically significant way two different protein populations: (a) proteins that fold by a process passing by partially folded intermediates and (b) proteins that do not present partially folded intermediates
Influence of tyrosine-derived moieties and drying conditions on the formation of helices in gelatin
(2011)
The single and triple helical organization of protein chains strongly influences the mechanical properties of gelatin-based materials. A chemical method for obtaining different degrees of helical organization in gelatin is covalent functionalization, while a physical method for achieving the same goal is the variation of the drying conditions of gelatin solutions. Here we explored how the introduction of desaminotyrosine (DAT) and desaminotyrosyl tyrosine (DATT) linked to lysine residues of gelatin influenced the kinetics and thermodynamic equilibrium of the helicalization process of single and triple helices following different drying conditions. Drying at a temperature above. the helix-to-coil transition temperature of gelatin (T > T-c, called nu(short)) generally resulted in gelatins with relatively lower triple helical content (X-c,X-t = 1-2%) than lower temperature drying (T < T-c, called nu(long)) (X-c,X-t = 8-10%), where the DAT(T) functional groups generally disrupted helix formation. While different helical contents affected the thermal transition temperatures only slightly, the mechanical properties were strongly affected for swollen hydrogels (E = 4-13 kPa for samples treated by nu(long) and E = 120-700 kPa for samples treated by nu(short)). This study shows that side group functionalization and different drying conditions are viable options to control the helicalization and macroscopic properties of gelatin-based materials.
Chirocephalus ruffoi is a fairy shrimp endemic to the Italian peninsula, where it is known only from thirteen high mountain locations. Twelve of these are in the Northern Apennines while the thirteenth is about 700 km away in the Calabrian Apennines (Southern Italy). We quantified degree of genetic divergence within the species by sequencing a fragment of the mitochondrial DNA encoding for Cytochrome Oxidase I. We then combined genetic data with the re-analysis of two different datasets on the morphometrics of the resting eggs (cysts) produced by the species as an adaptation to survive seasonal droughts. Genetic data revealed profound divergence; we identified four clusters of haplotypes within the species phylogeography, three in the Northern Apennines and one in the Calabrian Apennines with most of the genetic variation (a parts per thousand 70%) being apportioned among haplogroups. We found high variability in cyst morphometrics, especially in cyst size and height of the spines ornamenting the surface. Genetic and morphometric data are decoupled suggesting that cyst morphology is either under selection or a plastic trait. We, therefore, caution against using cyst morphology for taxonomic purposes in anostracans.
Ecosystems with highly pulsed water supply must be better understood as climate change may increase frequency and severity of intense storms, droughts and floods. Here we collected data over 3 years (2016-2018) in the episodic wetland outflow channel (Aluize), Banhine National Park, in which the system state changed from dry to wet to dry. Field sampling included vegetation records, small-scale vegetation zoning, the seed bank and water and soil quality. The same main plant species were found in both dry and wet conditions across the riverbed of the outflow channel. We found only very few diaspores of plants in the soil after prolonged drought. In the subsequent flooded state, we examined very dense vegetation on the water surface, which was dominated by the gramineous species Paspalidium obtusifolium. This species formed a compact floating mat that was rooted to the riverbed. The Cyperaceae Bolboschoenus glaucus showed high clonal growth in the form of root tubers, which likely serve as important food reservoir during drought. Soil and water analyses do not indicate a limitation by nutrients. We outline how resident people may change the plant community structure with an increasing practice of setting fire to the meadows in the dried-up riverbed to facilitate plant regrowth as food for their livestock.
Venomous snakes often display extensive variation in venom composition both between and within species. However, the mechanisms underlying the distribution of different toxins and venom types among populations and taxa remain insufficiently known. Rattlesnakes (Crotalus, Sistrurus) display extreme inter-and intraspecific variation in venom composition, centered particularly on the presence or absence of presynaptically neurotoxic phospholipases A2 such as Mojave toxin (MTX). Interspecific hybridization has been invoked as a mechanism to explain the distribution of these toxins across rattlesnakes, with the implicit assumption that they are adaptively advantageous. Here, we test the potential of adaptive hybridization as a mechanism for venom evolution by assessing the distribution of genes encoding the acidic and basic subunits of Mojave toxin across a hybrid zone between MTX-positive Crotalus scutulatus and MTX-negative C. viridis in southwestern New Mexico, USA. Analyses of morphology, mitochondrial and single copy-nuclear genes document extensive admixture within a narrow hybrid zone. The genes encoding the two MTX subunits are strictly linked, and found in most hybrids and backcrossed individuals, but not in C. viridis away from the hybrid zone. Presence of the genes is invariably associated with presence of the corresponding toxin in the venom. We conclude that introgression of highly lethal neurotoxins through hybridization is not necessarily favored by natural selection in rattlesnakes, and that even extensive hybridization may not lead to introgression of these genes into another species.
Time hierarchies, arising as a result of interactions between system's components, represent a ubiquitous property of dynamical biological systems. In addition, biological systems have been attributed switch-like properties modulating the response to various stimuli across different organisms and environmental conditions. Therefore, establishing the interplay between these features of system dynamics renders itself a challenging question of practical interest in biology. Existing methods are suitable for systems with one stable steady state employed as a well-defined reference. In such systems, the characterization of the time hierarchies has already been used for determining the components that contribute to the dynamics of biological systems. However, the application of these methods to bistable nonlinear systems is impeded due to their inherent dependence on the reference state, which in this case is no longer unique. Here, we extend the applicability of the reference-state analysis by proposing, analyzing, and applying a novel method, which allows investigation of the time hierarchies in systems exhibiting bistability. The proposed method is in turn used in identifying the components, other than reactions, which determine the systemic dynamical properties. We demonstrate that in biological systems of varying levels of complexity and spanning different biological levels, the method can be effectively employed for model simplification while ensuring preservation of qualitative dynamical properties (i.e., bistability). Finally, by establishing a connection between techniques from nonlinear dynamics and multivariate statistics, the proposed approach provides the basis for extending reference-based analysis to bistable systems.
We have analyzed the O-antigen polysaccharide of the previously uncharacterized Escherichia coli strain TD2158 which is a host of bacteriophage HK620. This bacteriophage recognizes and cleaves the polysaccharide with its tailspike protein (TSP). The polysaccharide preparation as well as oligosaccharides obtained from HK620TSP endoglycosidase digests were analyzed with NMR spectroscopy. Additionally, sugar analysis was performed on the O-antigen polysaccharide and MALDI-TOF MS was used in oligosaccharide analysis. The present study revealed a heterogeneous polysaccharide with a hexasaccharide repeating unit of the following structure:
alpha-D-Glcp-(1 -> 6) vertical bar vertical bar 2)-alpha-L-Rhap-(1 -> 6)-alpha-D-Glcp-(1 -> 4)-alpha-D-Galp-(1 -> 3)-alpha-D-GlcpNAc- (1 ->vertical bar beta-D-Glcp/beta-D-GlcpNAc-(1 -> 3)
A repeating unit with a D-GlcNAc substitution of D-Gal has been described earlier as characteristic for serogroup O18A1. Accordingly, we termed repeating units with D-Glc substitution at D-Gal as O18A2. NMR analyses of the polysaccharide confirmed that O18A1- and O18A2-type repeats were present in a 1:1 ratio. However, HK620TSP preferentially bound the D-GlcNAc- substituted O18A1-type repeating units in its high affinity binding pocket with a dissociation constant of 140 mu M and disfavored the O18A2-type having a beta-D-Glcp-(1 -> 3)-linked group. As a result, in hexasaccharide preparations, O18A1 and O18A2 repeats were present in a 9: 1 ratio stressing the clear preference of O18A1- type repeats to be cleaved by HK620TSP.
Due to the low cutaneous bioavailability of tacrolimus (TAC), penetration enhancers are used to improve its penetration into the skin. However, poor loading capacity, non-biodegradability, toxicity, and in some cases inefficient skin penetration are challenging issues that hamper their applications for the dermal TAC delivery. Here we present poly(lactide-co-glycerol) (PLG) as a water soluble, biodegradable, and biocompatible TAC-carrier with high loading capacity (14.5% w/w for TAC) and high drug delivery efficiencies into the skin. PLG was synthesized by cationic ring-opening copolymerization of a mixture of glycidol and lactide and showed 35 nm and 300 nm average sizes in aqueous solutions before and after loading of TAC, respectively. Delivery experiments on human skin, quantified by fluorescence microscopy and LC-MS/MS, showed a high ability for PLG to deposit Nile red and TAC into the stratum corneum and viable epidermis of skin in comparison with Protopic (R) (0.03% w/w, TAC ointment). The cutaneous distribution profile of delivered TAC proved that 80%, 16%, and 4% of the cutaneous drug level was deposited in the stratum corneum, viable epidermis, and upper dermis, respectively. TAC delivered by PLG was able to efficiently decrease the IL-2 and TSLP expressions in human skin models. Taking advantage of the excellent physicochemical and biological properties of PLG, it can be used for efficient dermal TAC delivery and potential treatment of inflammatory skin diseases.
Penicillin amidase from Alacaligenes faecalis is an attractive biocatalyst for hydrolysis of penicillin G for production of 6-aminopenicillanic acid, which is used in the synthesis of semi-synthetic beta-lactam antibiotics. Recently a mutant of this enzyme with extended C-terminus of the A-chain comprising parts of the connecting linker peptide was constructed. Its turnover number for the hydrolysis of penicillin G was 140 s(-1), about twice of the value for the wild-type enzyme (80 s(-1)). At the same time the specificity constant was improved about three-fold. The wild- type and the mutant enzymes showed similar pH stability suggesting that the linker peptide fragment covalently attached to the A-chain does not alter the electrostatic interactions in the protein core. Although the global stability of A. faecalis wild-type enzyme and the T206GS213G variant does not differ, the presence of the linker fragment stabilizes the domains interface, as evidenced by the monophasic transition of the mutant enzyme from folded to unfolded state during urea-induced denaturation. The high stability and activity of the mutant enzyme provides a rationale to use it as a biocatalyst in the industrial processes, where the enzyme must be more robust to fluctuations in the operational conditions.
Mitochondrial genomes of Late Pleistocene caballine horses from China belong to a separate clade
(2020)
There were several species of Equus in northern China during the Late Pleistocene, including Equus przewalskii and Equus dalianensis. A number of morphological studies have been carried out on E. przewalskii and E. dalianensis, but their evolutionary history is still unresolved. In this study, we retrieved near-complete mitochondrial genomes from E. dalianensis and E. przewalskii specimens excavated from Late Pleistocene strata in northeastern China. Phylogenetic analyses revealed that caballoid horses were divided into two subclades: the New World and the Old World caballine horse subclades. The Old World caballine horses comprise of two deep phylogenetic lineages, with modern and ancient Equus caballus and modern E. przewalskii forming lineage I, and the individuals in this study together with one Yakut specimen forming lineage II. Our results indicate that Chinese Late Pleistocene caballoid horses showed a closer relationship to other Eurasian caballine horses than that to Pleistocene horses from North America. In addition, phylogenetic analyses suggested a close relationship between E. dalianensis and the Chinese fossil E. przewalskii, in agreement with previous researches based on morphological analyses. Interestingly, E. dalianensis and the fossil E. przewalskii were intermixed rather than split into distinct lineages, suggesting either that gene flow existed between these two species or that morphology-based species assignment of palaeontological specimens is not always correct. Moreover, Bayesian analysis showed that the divergence time between the New World and the Old World caballoid horses was at 1.02 Ma (95% CI: 0.86-1.24 Ma), and the two Old World lineages (I & II) split at 0.88 Ma (95% CI: 0.69-1.13 Ma), which indicates that caballoid horses seem to have evolved into different populations in the Old World soon after they migrated from North America via the Bering Land Bridge. Finally, the TMRCA of E. dalianensis was estimated at 0.20 Ma (95% CI: 0.15-0.28 Ma), and it showed a relative low genetic diversity compared with other Equus species.
Molecular identification of late and terminal Pleistocene Equus ovodovi from northeastern China
(2019)
The extant diversity of horses (family Equidae) represents a small fraction of that occurring over their evolutionary history. One such lost lineage is the subgenus Sussemionus, which is thought to have become extinct during the Middle Pleistocene. However, recent molecular studies and morphological analysis have revealed that one of their representatives, E. ovodovi, did exist in Siberia during the Late Pleistocene. Fossil materials of E. ovodovi have thus far only been found in Russia. In this study, we extracted DNA from three equid fossil specimens excavated from northeastern China dated at 12,770-12,596, 29,525-28,887 and 40,201-38,848 cal. yBP, respectively, and retrieved three near-complete mitochondrial genomes from the specimens. Phylogenetic analyses cluster the Chinese haplotypes together with previously published Russian E. ovodovi, strongly supporting the assignment of these samples to this taxon. The molecular identification of E. ovodovi in northeastern China extends the known geographical range of this fossil species by several thousand kilometers to the east. The estimated coalescence time of all E. ovodovi haplotypes is approximately 199 Kya, with the Chinese haplotypes coalescing approximately 130 Kya. With a radiocarbon age of 12,770-12,596 cal. yBP, the youngest sample in this study represents the first E. ovodovi sample dating to the terminal Pleistocene, moving the extinction date of this species forwards considerably compared to previously documented fossils. Overall, comparison of our three mitochondrial genomes with the two published ones suggests a genetic diversity similar to several extant species of the genus Equus.
Cotton (Gossypium hirsutum) fibres consist of single cells that grow in a highly polarized manner, assumed to be controlled by the cytoskeleton(1-3). However, how the cytoskeletal organization and dynamics underpin fibre development remains unexplored. Moreover, it is unclear whether cotton fibres expand via tip growth or diffuse growth(2-4). We generated stable transgenic cotton plants expressing fluorescent markers of the actin and microtubule cytoskeleton. Live-cell imaging revealed that elongating cotton fibres assemble a cortical filamentous actin network that extends along the cell axis to finally form actin strands with closed loops in the tapered fibre tip. Analyses of F-actin network properties indicate that cotton fibres have a unique actin organization that blends features of both diffuse and tip growth modes. Interestingly, typical actin organization and endosomal vesicle aggregation found in tip-growing cell apices were not observed in fibre tips. Instead, endomembrane compartments were evenly distributed along the elongating fibre cells and moved bi-directionally along the fibre shank to the fibre tip. Moreover, plus-end tracked microtubules transversely encircled elongating fibre shanks, reminiscent of diffusely growing cells. Collectively, our findings indicate that cotton fibres elongate via a unique tip-biased diffuse growth mode.
Ecological and evolutionary dynamics can occur on similar timescales. However, theoretical predictions of how rapid evolution can affect ecological dynamics are inconclusive and often depend on untested model assumptions. Here we report that rapid prey evolution in response to oscillating predator density affects predator-prey (rotifer-algal) cycles in laboratory microcosms. Our experiments tested explicit predictions from a model for our system that allows prey evolution. We verified the predicted existence of an evolutionary tradeoff between algal competitive ability and defence against consumption, and examined its effects on cycle dynamics by manipulating the evolutionary potential of the prey population. Single-clone algal cultures (lacking genetic variability) produced short cycle periods and typical quarter-period phase lags between prey and predator densities, whereas multi-clonal (genetically variable) algal cultures produced long cycles with prey and predator densities nearly out of phase, exactly as predicted. These results confirm that prey evolution can substantially alter predator-prey dynamics, and therefore that attempts to understand population oscillations in nature cannot neglect potential effects from ongoing rapid evolution.
TusA is a versatile protein that links translation efficiency to cell division in Escherichia coli
(2021)
To enable accurate and efficient translation, sulfur modifications are introduced posttranscriptionally into nucleosides in tRNAs. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems for the incorporation of sulfur atoms in different nucleosides of tRNA. One of the proteins that is involved in inserting the sulfur for 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modifications in tRNAs is the TusA protein. TusA, however, is a versatile protein that is also involved in numerous other cellular pathways. Despite its role as a sulfur transfer protein for the 2-thiouridine formation in tRNA, a fundamental role of TusA in the general physiology of Escherichia coli has also been discovered. Poor viability, a defect in cell division, and a filamentous cell morphology have been described previously for tusA-deficient cells. In this report, we aimed to dissect the role of TusA for cell viability. We were able to show that the lack of the thiolation status of wobble uridine (U-34) nucleotides present on Lys, Gln, or Glu in tRNAs has a major consequence on the translation efficiency of proteins; among the affected targets are the proteins RpoS and Fis. Both proteins are major regulatory factors, and the deregulation of their abundance consequently has a major effect on the cellular regulatory network, with one consequence being a defect in cell division by regulating the FtsZ ring formation. <br /> IMPORTANCE More than 100 different modifications are found in RNAs. One of these modifications is the mnm(5)s(2)U modification at the wobble position 34 of tRNAs for Lys, Gln, and Glu. The functional significance of U34 modifications is substantial since it restricts the conformational flexibility of the anticodon, thus providing translational fidelity. We show that in an Escherichia coli TusA mutant strain, involved in sulfur transfer for the mnm(5)s(2)U34 thio modifications, the translation efficiency of RpoS and Fis, two major cellular regulatory proteins, is altered. Therefore, in addition to the transcriptional regulation and the factors that influence protein stability, tRNA modifications that ensure the translational efficiency provide an additional crucial regulatory factor for protein synthesis.
An electrochemical assay for the indication of the activity of the cell bound differentiation marker alkaline phosphatase (ALP) is proposed using voltammetry on an in-vitro cell culture. The basis of the assay is cultivation of cells on gold microelectrodes in wells of a microplate, catalytic hydrolysis of p-aminophenyl phosphate by ALP and indication of p-aminophenol oxidation by square wave voltammetry (SWV) with the sensors onto which the cells attached. The morphology of the bone marrow stromal cell line (MBA-15) on the electrode surface was investigated and it exhibited in vitro osteogenic characteristics. Since ALP is expressed on the cell surface in early differentiation stage of osteoblastic cells, its activity was followed after different culture times over a period of 144 h by recording repetitive voltammograms at different time points upon addition of the substrate p-aminophenyl phosphate. The ALP activity was estimated from the signal increase related to formation rate of p-aminophenol and the number of cells. The highest value was measured at 120 h, when the cells reached confluence. The results of the electrochemical activity assay are consistent with the colorimetric acquired value from p-nitrophenol formation rate.
Increasing evidence shows that anthropogenic climate change is affecting biodiversity. Reducing or stabilizing greenhouse gas emissions may slow global warming, but past emissions will continue to contribute to further unavoidable warming for more than a century. With obvious signs of difficulties in achieving effective mitigation worldwide in the short term at least, sound scientific predictions of future impacts on biodiversity will be required to guide conservation planning and adaptation. This is especially true in Mediterranean type ecosystems that are projected to be among the most significantly affected by anthropogenic climate change, and show the highest levels of confidence in rainfall projections. Multiple methods are available for projecting the consequences of climate change on the main unit of interest - the species - with each method having strengths and weaknesses. Species distribution models (SDMs) are increasingly applied for forecasting climate change impacts on species geographic ranges. Aggregation of models for different species allows inferences of impacts on biodiversity, though excluding the effects of species interactions. The modelling approach is based on several further assumptions and projections and should be treated cautiously. In the absence of comparable approaches that address large numbers of species, SDMs remain valuable in estimating the vulnerability of species. In this review we discuss the application of SDMs in predicting the impacts of climate change on biodiversity with special reference to the species-rich South West Australian Floristic Region and South African Cape Floristic Region. We discuss the advantages and challenges in applying SDMs in biodiverse regions with high levels of endemicity, and how a similar biogeographical history in both regions may assist us in understanding their vulnerability to climate change. We suggest how the process of predicting the impacts of climate change on biodiversity with SDMs can be improved and emphasize the role of field monitoring and experiments in validating the predictions of SDMs.
Drosophila melanogaster photoreceptors are highly polarized cells and their plasma membrane is organized into distinct domains. Zonula adherens junctions separate a smooth peripheral surface, the equivalent of the basolateral surface in other epithelial cells, from the central surface (cong apical surface). The latter consists of the microvillar rhabdomere and the juxtarhabdomeric domain, a nonmicrovillar area between the rhabdomere and the zonulae adherens. The distribution of Na/K-ATPase over these domains was examined by immunocytochemical, developmental, and genetic approaches. Immunofluorescence and immunogold labeling of adult compound eyes reveal that the distribution of Na/ K-ATPase is concentrated at the peripheral surface in the photoreceptors R1-R6, but extends over the juxtarhabdomeric domain to the rhabdomere in the photoreceptors R7/R8. Developmental analysis demonstrates further that Na/K-ATPase is localized over the entire plasma membrane in all photoreceptors in early pupal eyes. Redistribution of Na/K-ATPase in R1- R6 occurs at about 78% of pupal life, coinciding with the onset of Rh1-rhodopsin expression on the central surface of these cells. Despite the essential role of Rh1 in structural development and intracellular trafficking, Rh1 mutations do not affect the distribution of Na/K-ATPase. These results suggest that Na/K-ATPase and rhodopsin are involved in distinct intracellular localization mechanisms, which are maintained independent of each other.
For the first time the direct electron transfer of an enzyme - cellobiose dehydrogenase, CDH - has been coupled with the hexokinase catalyzed competition for glucose in a sensor for ATP. To enhance the signal output for ATP, pyruvate kinase was coimmobilized to recycle ADP by the phosphoenolpyruvate driven reaction. The new sensor overcomes the limit of 1:1 stoichiometry of the sequential or competitive conversion of ATP by effective enzymatic recycling of the analyte. The anodic oxidation of the glucose converting CDH proceeds at electrode potentials below 0 mV vs. Ag vertical bar AgCl thus potentially interfering substances like ascorbic acid or catecholamines do not influence the measuring signal. The combination of direct electron transfer of CDH with the enzymatic recycling results in an interference-free and oxygen-independent measurement of ATP in the lower mu molar concentration range with a lower limit of detection of 63.3 nM (S/N=3).
We present an electrochemical MIP sensor for tamoxifen (TAM)-a nonsteroidal anti-estrogen-which is based on the electropolymerisation of an O-phenylenediamine. resorcinol mixture directly on the electrode surface in the presence of the template molecule. Up to now only. bulk. MIPs for TAM have been described in literature, which are applied for separation in chromatography columns. Electro-polymerisation of the monomers in the presence of TAM generated a film which completely suppressed the reduction of ferricyanide. Removal of the template gave a markedly increased ferricyanide signal, which was again suppressed after rebinding as expected for filling of the cavities by target binding. The decrease of the ferricyanide peak of the MIP electrode depended linearly on the TAM concentration between 1 and 100 nM. The TAM-imprinted electrode showed a 2.3 times higher recognition of the template molecule itself as compared to its metabolite 4-hydroxytamoxifen and no cross-reactivity with the anticancer drug doxorubucin was found. Measurements at + 1.1 V caused a fouling of the electrode surface, whilst pretreatment of TAM with peroxide in presence of HRP generated an oxidation product which was reducible at 0 mV, thus circumventing the polymer formation and electrochemical interferences.
A new electrochemical MIP sensor for the most frequently used drug paracetamol (PAR) was prepared by electropolymerization of mixtures containing the template molecule and the functional monomers ophenylenediamine, resorcinol and aniline. The imprinting factor of 12 reflects the effective target binding to the MIP as compared with the non-imprinted electropolymer. Combination of the MIP with a nonspecific esterase allows the measurement of phenacetin - another analgesic drug. In the second approach the PAR containing sample solution was pretreated with tyrosinase in order to prevent electrochemical interferences by ascorbic acid and uric acid. Interference-free indication at a very low electrode potential without fouling of the electrode surface was achieved with the o-phenylenediamine: resorcinol-based MIP.
Simple and robust
(2021)
A spectrum of 7562 publications on Molecularly Imprinted Polymers (MIPs) has been presented in literature within the last ten years (Scopus, September 7, 2020). Around 10 % of the papers published on MIPs describe the recognition of proteins. The straightforward synthesis of MIPs is a significant advantage as compared with the preparation of enzymes or antibodies. MIPs have been synthesized from only one up to six functional monomers while proteins are made up of 20 natural amino acids. Furthermore, they can be synthesized against structures of low immunogenicity and allow multi-analyte measurements via multi-target synthesis. Electrochemical methods allow simple polymer synthesis, removal of the template and readout. Among the different sensor configurations electrochemical MIP-sensors provide the broadest spectrum of protein analytes. The sensitivity of MIP-sensors is sufficiently high for biomarkers in the sub-nanomolar region, nevertheless the cross-reactivity of highly abundant proteins in human serum is still a challenge. MIPs for proteins offer innovative tools not only for clinical and environmental analysis, but also for bioimaging, therapy and protein engineering.
The aromatic peroxygenase (APO; EC 1.11.2.1) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of -278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe(2+)/Fe(3+) redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed.
Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11
(2011)
Microperoxidase-11 (MR-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline. 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-II and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.
The first molecularly imprinted polymer (MIP) for the recognition of the copper-enzyme laccase was successfully prepared by electropolymerizing scopoletin in the presence of alkaline-inactivated enzyme. Laccase-MIP and the control polymer without laccase (nonimprinted polymer, NIP) were characterized by voltammetry using the redox marker ferricyanide. After electropolymerization, the signals for ferricyanide for both the MIP and the NIP were almost completely suppressed and increased after removal of the target from the polymer layer. Rebinding of both inactivated and active laccase decreased the ferricyanide peak currents to almost equal extent. The relative decrease of signal suppression approached saturation above 10 nM. Furthermore, the surface activity of rebound laccase toward the oxidation of catechol was investigated. The surface activity approached saturation above 10 nM, a value close to the value of the measurements with ferricyanide. Interaction of NIP with laccase brought about a six times smaller signal of catechol oxidation.
For the first time a molecularly imprinted polymer (MIP)-based sensor for tyrosinase is described. This sensor is based on the electropolymerization of scopoletin or o-phenylenediamine in the presence of tyrosinase from mushrooms, which has a high homology to the human enzyme. The template was removed either by treatment with proteinase Kor by alkaline treatment. The measuring signal was generated either by measuring the formation of a product by the target enzyme or by evaluation of the permeability of the redox marker ferricyanide. The o-phenylenediamine-based MIP sensor has a linear measuring range up to 50 nM of tyrosinase with a limit of detection of 3.97 nM (R 2 = 0.994) and shows good discrimination towards other proteins, e.g., bovine serum albumin and cytochrome c.
QuestionDisturbed areas offer great opportunities for restoring native biodiversity, but they are also prone to invasion by alien plants. Following the limiting similarity hypothesis, we address the question of whether or not similarity of plant functional traits helps developing seed mixtures of native communities with high resistance to invasive species at an early stage of restoration. LocationCentre of Greenhouses and Laboratories Durnast, Technische Universitat Munchen, Freising, Germany. MethodsUsing a system of linear equations, we designed native communities maximizing the similarity between the native and two invasive species according to ten functional traits. We used native grassland plants, two invasive alien species that are often problematic in disturbed areas (i.e., Ambrosia artemisiifolia and Solidago gigantea) and trait information obtained from databases. The two communities were then tested for resistance against establishment of the two invaders separately in a greenhouse experiment. We measured height of the invasive species and above-ground biomass, along with leaf area index, 4 and 8months after sowing respectively. ResultsBoth invasive species were successfully reduced by the native community designed to suppress S. gigantea dominated by small-seeded species. These results could be considered as partial support for the limiting similarity hypothesis. However, given the success of this mixture against both invasive species, suppression was better explained by a seed density effect resulting from the smaller seed mass of the native species included in this mixture. Further, the dominance of a fast-developing competitive species could also contribute to its success. ConclusionsThere was no unequivocal support for the limiting similarity hypothesis in terms of the traits selected. Instead we found that increasing seeding density of native species and selecting species with a fast vegetative development is an effective way to suppress invasive plants during early stages of restoration. If limiting similarity is used to design communities for restoration, early life-history traits should be taken into account.
In plants, transcripts move to distant body parts to potentially act as systemic signals regulating development and growth. Thousands of messenger RNAs (mRNAs) are transported across graft junctions via the phloem to distinct plant parts. Little is known regarding features, structural motifs, and potential base modifications of transported transcripts and how these may affect their mobility. We identified Arabidopsis thalianam RNAs harboring the modified base 5-methylcytosine (m(5)C) and found that these are significantly enriched in mRNAs previously described as mobile, moving over graft junctions to distinct plant parts. We confirm this finding with graft-mobile methylated mRNAs TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1 (TCTP1) and HEAT SHOCK COGNATE PROTEIN 70.1 (HSC70.1), whose mRNA transport is diminished in mutants deficient in m(5)C mRNA methylation. Together, our results point toward an essential role of cytosine methylation in systemic mRNA mobility in plants and that TCTP1 mRNA mobility is required for its signaling function.
Enhancers are critical for developmental stage-specific gene expression, but their dynamic regulation in plants remains poorly understood. Here we compare genome-wide localization of H3K27ac, chromatin accessibility and transcriptomic changes during flower development in Arabidopsis. H3K27ac prevalently marks promoter-proximal regions, suggesting that H3K27ac is not a hallmark for enhancers in Arabidopsis. We provide computational and experimental evidence to confirm that distal DNase. hypersensitive sites are predictive of enhancers. The predicted enhancers are highly stage-specific across flower development, significantly associated with SNPs for flowering-related phenotypes, and conserved across crucifer species. Through the integration of genome-wide transcription factor (TF) binding datasets, we find that floral master regulators and stage-specific TFs are largely enriched at developmentally dynamic enhancers. Finally, we show that enhancer clusters and intronic enhancers significantly associate with stage-specific gene regulation by floral master TFs. Our study provides insights into the functional flexibility of enhancers during plant development, as well as hints to annotate plant enhancers.
Background
The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment.
Results
Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression.
Conclusions
Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
Results: Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression. Conclusions: Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
The variation of the molecular architecture of multiblock copolymers has enabled the introduction of functional behaviour and the control of key mechanical properties. In the current study, we explore the synergistic relationship of two structural components in a shape-memory material formed of a multiblock copolymer with crystallizable poly(epsilon-caprolactone) and crystallizable polyfoligo(3S-iso-butylmorpholine-2,5-dione) segments (PCL-PIBMD). The thermal and structural properties of PCL-PIBMD films were compared with PCI.-PU and PMMD-PU investigated by means of DSC, SAXS and WARS measurements. The shape-memory properties were quantified by cyclic, thermomechanical tensile tests, where deformation strains up to 900% were applied for programming PCL-PIBMD films at 50 degrees C. Toluene vapor treatment experiments demonstrated that the temporary shape was fixed mainly by glassy PIBMD domains at strains lower than 600% with the PCL contribution to fixation increasing to 42 +/- 2% at programming strains of 900% This study into the shape-memory mechanism of PCL-PIBMD provides insight into the structure function relation in multiblock copolymers with both crystallizable and glassy switching segments.
The Eukaryotic-Specific ISD11 Is a Complex-Orphan Protein with Ability to Bind the Prokaryotic IscS
(2016)
The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction.
Human sulfite oxidase (hSO) is a homodimeric two-domain enzyme central in the biological sulfur cycle. A pyranopterin molybdenum cofactor (Moco) is the catalytic site and a heme b(5) group located in the N-terminal domain. The two domains are connected by a flexible linker region. Electrons produced at the Moco in sulfite oxidation, are relayed via heme b(5) to electron acceptors or an electrode surface. Inter-domain conformational changes between an open and a closed enzyme conformation, allowing "gated" electron transfer has been suggested. We first recorded cyclic voltammetry (CV) of hSO on single-crystal Au(111)-electrode surfaces modified by self-assembled monolayers (SAMs) both of a short rigid thiol, cysteamine and of a longer structurally flexible thiol, omega-amino-octanethiol (AOT). hSO on cysteamine SAMs displays a well-defined pair of voltammetric peaks around -0.207 V vs. SCE in the absence of sulfite substrate, but no electrocatalysis. hSO on AOT SAMs displays well-defined electrocatalysis, but only "fair" quality voltammetry in the absence of sulfite. We recorded next in situ scanning tunnelling spectroscopy (STS) of hSO on AOT modified Au(111)-electrodes, disclosing, a 2-5 % surface coverage of strong molecular scale contrasts, assigned to single hSO molecules, notably with no contrast difference in the absence and presence of sulfite. In situ STS corroborated this observation with a sigmoidal tunnelling current/overpotential correlation.
Nitric oxide synthase (NOS) plays a critical role in a number of key physiological and pathological processes. Investigation of electron-transfer reactions in NOS would contribute to a better understanding of the nitric oxide (NO) synthesis mechanism. Herein, we describe an electrochemically driven catalytic strategy, using a nanocomposite that consisted of the oxygenase domain of neuronal NOS (D290nNOSoxy), indium tin oxide (ITO) nanopartides and polyvinyl alcohol (PVA). Fast direct electron transfer between electrodes and D290nNOSoxy was observed with the heterogeneous electron transfer rate constant (k(er)) of 154.8 +/- 0.1 s(-1) at the scan rate of 5 V s(-1). Moreover, the substrate IV-hydroxy-L-arginine (NHA) was used to prove the concept of electrochemically driven biocatalysis of D290nNOSoxy. In the presence of the oxygen cosubstrate and tetrahydrobiopterin (BH4) cofactor, the addition of NHA caused the decreases of both oxidation current at + 0.1 V and reduction current at potentials ranging from -0.149 V to -0.549 V vs Ag/AgCl. Thereafter, a series of control experiments such as in the absence of BH4 or D290nNOSoxy were performed. All the results demonstrated that D290nNOSoxy biocatalysis was successfully driven by electrodes in the presence of BH4 and oxygen. This novel bioelectronic system showed potential for further investigation of NOS and biosensor applications. (C) 2013 Elsevier B.V. All rights reserved.