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- Arabidopsis thaliana (58)
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Institute
- Institut für Biochemie und Biologie (5356) (remove)
Research on novel and advanced biomaterials is an indispensable step towards their applications in desirable fields such as tissue engineering, regenerative medicine, cell culture, or biotechnology. The work presented here focuses on such a promising material: polyelectrolyte multilayer (PEM) composed of hyaluronic acid (HA) and poly(L-lysine) (PLL). This gel-like polymer surface coating is able to accumulate (bio-)molecules such as proteins or drugs and release them in a controlled manner. It serves as a mimic of the extracellular matrix (ECM) in composition and intrinsic properties. These qualities make the HA/PLL multilayers a promising candidate for multiple bio-applications such as those mentioned above. The work presented aims at the development of a straightforward approach for assessment of multi-fractional diffusion in multilayers (first part) and at control of local molecular transport into or from the multilayers by laser light trigger (second part).
The mechanism of the loading and release is governed by the interaction of bioactives with the multilayer constituents and by the diffusion phenomenon overall. The diffusion of a molecule in HA/PLL multilayers shows multiple fractions of different diffusion rate. Approaches, that are able to assess the mobility of molecules in such a complex system, are limited. This shortcoming motivated the design of a novel evaluation tool presented here.
The tool employs a simulation-based approach for evaluation of the data acquired by fluorescence recovery after photobleaching (FRAP) method. In this approach, possible fluorescence recovery scenarios are primarily simulated and afterwards compared with the data acquired while optimizing parameters of a model until a sufficient match is achieved. Fluorescent latex particles of different sizes and fluorescein in an aqueous medium are utilized as test samples validating the analysis results. The diffusion of protein cytochrome c in HA/PLL multilayers is evaluated as well.
This tool significantly broadens the possibilities of analysis of spatiotemporal FRAP data, which originate from multi-fractional diffusion, while striving to be widely applicable. This tool has the potential to elucidate the mechanisms of molecular transport and empower rational engineering of the drug release systems.
The second part of the work focuses on the fabrication of such a spatiotemporarily-controlled drug release system employing the HA/PLL multilayer. This release system comprises different layers of various functionalities that together form a sandwich structure. The bottom layer, which serves as a reservoir, is formed by HA/PLL PEM deposited on a planar glass substrate. On top of the PEM, a layer of so-called hybrids is deposited. The hybrids consist of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) -based hydrogel microparticles with surface-attached gold nanorods. The layer of hybrids is intended to serve as a gate that controls the local molecular transport through the PEM–solution-interface. The possibility of stimulating the molecular transport by near-infrared (NIR) laser irradiation is being explored.
From several tested approaches for the deposition of hybrids onto the PEM surface, the drying-based approach was identified as optimal. Experiments, that examine the functionality of the fabricated sandwich at elevated temperature, document the reversible volume phase transition of the PEM-attached hybrids while sustaining the sandwich stability. Further, the gold nanorods were shown to effectively absorb light radiation in the tissue- and cell-friendly NIR spectral region while transducing the energy of light into heat. The rapid and reversible shrinkage of the PEM-attached hybrids was thereby achieved. Finally, dextran was employed as a model transport molecule. It loads into the PEM reservoir in a few seconds with the partition constant of 2.4, while it spontaneously releases in a slower, sustained manner. The local laser irradiation of the sandwich, which contains the fluorescein isothiocyanate tagged dextran, leads to a gradual reduction of fluorescence intensity in the irradiated region.
The release system fabricated employs renowned photoresponsivity of the hybrids in an innovative setting. The results of the research are a step towards a spatially-controlled on-demand drug release system that paves the way to spatiotemporally controlled drug release.
The approaches developed in this work have the potential to elucidate the molecular dynamics in ECM and to foster engineering of multilayers with properties tuned to mimic the ECM. The work aims at spatiotemporal control over the diffusion of bioactives and their presentation to the cells.
Ćwiek-Kupczyńska, Hanna ; Altmann, Thomas ; Arend, Daniel ; Arnaud, Elizabeth ; Chen, Dijun ; Cornut, Guillaume ; Fiorani, Fabio ; Frohmberg, Wojciech ; Junker, Astrid ; Klukas, Christian ; Lange, Matthias ; Mazurek, Cezary ; Nafissi, Anahita ; Neveu, Pascal ; van Oeveren, Jan ; Pommier, Cyril ; Poorter, Hendrik ; Rocca-Serra, Philippe ; Sansone, Susanna-Assunta ; Scholz, Uwe ; van Schriek, Marco ; Seren, Ümit ; Usadel, Björn ; Weise, Stephan ; Kersey, Paul ; Krajewski, Paweł
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 combin‑
ing 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 docu‑
ment 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.
The Proteasome Acts as a Hub for Plant Immunity and Is Targeted by Pseudomonas Type III Effectors
(2016)
Üstün, Suayib ; Sheikh, Arsheed ; Gimenez-Ibanez, Selena ; Jones, Alexandra ; Ntoukakis, Vardis ; Börnke, Frederik
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.
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)
´Cwiek-Kupczynska, Hanna ; Altmann, Thomas ; Arend, Daniel ; Arnaud, Elizabeth ; Chen, Dijun ; Cornut, Guillaume ; Fiorani, Fabio ; Frohmberg, Wojciech ; Junker, Astrid ; Klukas, Christian ; Lange, Matthias ; Mazurek, Cezary ; Nafissi, Anahita ; Neveu, Pascal ; van Oeveren, Jan ; Pommier, Cyril ; Poorter, Hendrik ; Rocca-Serra, Philippe ; Sansone, Susanna-Assunta ; Scholz, Uwe ; van Schriek, Marco ; Seren, Ümit ; Usadel, Bjorn ; Weise, Stephan ; Kersey, Paul ; Krajewski, Pawel
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.
Zór, K. ; Heiskanen, A. ; Caviglia, Claudia ; Vergani, M. ; Landini, E. ; Shah, F. ; Carminati, Marco ; Martínez-Serrano, A. ; Ramos Moreno, T. ; Kokaia, M. ; Benayahu, Dafna ; Keresztes, Zs. ; Papkovsky, D. ; Wollenberger, Ursula ; Svendsen, W. E. ; Dimaki, M. ; Ferrari, G. ; Raiteri, R. ; Sampietro, M. ; Dufva, M. ; Emnéus, J.
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.
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.