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Books and chapters

Möller, B.; Zergiebel, L.; Bürstenbinder, K.; Quantitative and Comparative Analysis of Global Patterns of (Microtubule) Cytoskeleton Organization with CytoskeletonAnalyzer2D Cvrčková, F. & Žárský, V., eds. Methods Mol. Biol. 1992 151-171 (2019) ISBN:978-1-4939-9469-4 DOI: 10.1007/978-1-4939-9469-4_10
  • Abstract
  • BibText
  • RIS

The microtubule cytoskeleton plays important roles in cell morphogenesis. To investigate the mechanisms of cytoskeletal organization, for example, during growth or development, in genetic studies, or in response to environmental stimuli, image analysis tools for quantitative assessment are needed. Here, we present a method for texture measure-based quantification and comparative analysis of global microtubule cytoskeleton patterns and subsequent visualization of output data. In contrast to other approaches that focus on the extraction of individual cytoskeletal fibers and analysis of their orientation relative to the growth axis, CytoskeletonAnalyzer2D quantifies cytoskeletal organization based on the analysis of local binary patterns. CytoskeletonAnalyzer2D thus is particularly well suited to study cytoskeletal organization in cells where individual fibers are difficult to extract or which lack a clearly defined growth axis, such as leaf epidermal pavement cells. The tool is available as ImageJ plugin and can be combined with publicly available software and tools, such as R and Cytoscape, to visualize similarity networks of cytoskeletal patterns.

Books and chapters

Möller, B.; Poeschl, Y.; Klemm, S.; Bürstenbinder, K.; Morphological Analysis of Leaf Epidermis Pavement Cells with PaCeQuant Cvrčková, F. & Žárský, V., eds. Methods Mol. Biol. 1992 329-349 (2019) ISBN:978-1-4939-9469-4 DOI: 10.1007/978-1-4939-9469-4_22
  • Abstract
  • BibText
  • RIS

Morphological analysis of cell shapes requires segmentation of cell contours from input images and subsequent extraction of meaningful shape descriptors that provide the basis for qualitative and quantitative assessment of shape characteristics. Here, we describe the publicly available ImageJ plugin PaCeQuant and its associated R package PaCeQuantAna, which provides a pipeline for fully automatic segmentation, feature extraction, statistical analysis, and graphical visualization of cell shape properties. PaCeQuant is specifically well suited for analysis of jigsaw puzzle-like leaf epidermis pavement cells from 2D input images and supports the quantification of global, contour-based, skeleton-based, and pavement cell-specific shape descriptors.

Books and chapters

Marillonnet, S.; Werner, S.; Assembly of Complex Pathways Using Type IIs Restriction Enzymes Santos, C. N. S. & Ajikumar, P. K., eds. Methods Mol. Biol. 1927 93-109 (2019) ISBN:978-1-4939-9142-6 DOI: 10.1007/978-1-4939-9142-6_7
  • Abstract
  • BibText
  • RIS

Efficient DNA assembly methods are essential tools for synthetic biology and metabolic engineering. Among several recently developed methods that allow assembly of multiple DNA fragments in a single step, DNA assembly using type IIS enzymes provides many advantages for complex pathway engineering. In particular, it provides the ability for the user to quickly assemble multigene constructs using a series of simple one-pot assembly steps starting from libraries of cloned and sequenced parts. We describe here a protocol for assembly of multigene constructs using the modular cloning system (MoClo). Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. Basic parts that are not yet available need to be made. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Books and chapters

Janik, K.; Stellmach, H.; Mittelberger, C.; Hause, B.; Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC) Musetti, R. & Pagliari, L., eds. Methods Mol. Biol. 1875 321-331 (2019) ISBN:978-1-4939-8837-2 DOI: 10.1007/978-1-4939-8837-2_24
  • Abstract
  • BibText
  • RIS

Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.

Books and chapters

Hussain, H.; Ur Rehman, N.; Abbas, G.; Khattak, K. F.; Khan, A.; Green, I. R.; Recent Progress of Phenazines as Anticancer Agents Atta-ur-Rahman & Zaman, K., eds. Top. Anti-Cancer Res. 8 74-96 (2019) ISBN:978-981-14-0438-2 DOI: 10.2174/9789811404382119080006
  • Abstract
  • BibText
  • RIS

Phenazines are nitrogen-containing heterocycles which possess a wide range of biological activities and in particular, cytotoxic effects. Moreover, various phenazines have been prepared having alkyl, amide, carboxylic acid, aldehyde, and pyrano groups. These synthetic phenazines possess significant anticancer effects towards various cancers. On the other hand, only a few natural phenazines have been reported with anticancer effects. This chapter presents a comprehensive overview of the most recent patents related to the phenazines as anticancer agents.

Books and chapters

Hussain, H.; Schulz, B.; Green, I. R.; Fungal Polyketides: Chemical Diversity and Their Cytotoxic Effects Ramawat, K. G., ed. Sustain. Dev. Biodivers. 24 195-214 (2019) ISBN:978-3-030-30746-2 DOI: 10.1007/978-3-030-30746-2_9
  • Abstract
  • BibText
  • RIS

Compounds isolated from different natural sources have over the years played crucial roles in the treatment of a wide range of human diseases. Over the past six decades, microorganisms have provided valuable active compounds for the treatment of various diseases. Fungi, in general, produce diverse structural classes of natural products including polyketides, a major class of secondary metabolites obtained from various natural sources‚ which as a group have interesting chemical diversity. In addition, polyketides are known to possess a number of biological and pharmacological effects, viz. cytotoxic, antibacterial, antifungal, antiparasitic, and immunosuppressive effects. In this chapter the focus is on describing the cytotoxic effects of polyketides isolated from fungi and in particular their potential as cancerostatic pharmaceuticals.

Books and chapters

Hause, B.; Yadav, H.; Creation of composite plants – transformation of Medicago truncatula roots de Bruijn, F., ed. 1179-1184 (2019) ISBN:9781119409144 DOI: 10.1002/9781119409144.ch152
  • Abstract
  • BibText
  • RIS

Medicago truncatula, owing to its small diploid genome (∼500 Mbp), short life cycle, and high natural diversity makes it a good model plant and has opened the door of opportunities for scientists interested in studying legume biology. But over the years, challenges are also being faced for genetic manipulation of this plant. Many genetic manipulation protocols have been published involving Agrobacterium tumefaciens, a pathogen causing tumor disease in plants. These protocols apart from being difficult to achieve, are also time consuming. Nowadays, an easy, less time consuming and highly reproducible Agrobacterium rhizogenes based method is in use by many research groups. This method generates composite plants having transformed roots on a wild‐type shoot. Here, stable transformed lines that can be propagated over time are not achieved by this method, but for root‐development or root–microbe interaction studies this method has proven to be a useful tool for the community. In addition, transformed roots can be propagated by root organ cultures (ROCs), wherein transformed roots are propagated on sucrose containing media without any shoot part. Occasionally, even stable transgenic plants can be regenerated from transgenic roots. In this chapter, developments and improvements of various transformation protocols are discussed. The suitability of composite plants is highlighted by a study on mycorrhization of transformed and non‐transformed roots, which did not show differences in the mycorrhization rate and developmental stages of the arbuscular mycorrhizal (AM) fungus inside the roots as well as in transcript accumulation and metabolite levels of roots. Finally, applications of the A. rhizogenes based transformation method are discussed.

Books and chapters

Doell, S.; Arens, N.; Mock, H.; Liquid Chromatography and Liquid Chromatography–Mass Spectrometry of Plants: Techniques and Applications Meyers, R. A., ed. (2019) ISBN:9780470027318 DOI: 10.1002/9780470027318.a9912.pub2
  • Abstract
  • BibText
  • RIS

Mass spectrometry coupled with LC (liquid chromatography) separation has developed into a technique routinely applied for targeted as well as for nontargeted analysis of complex biological samples, not only in plant biochemistry. Earlier on, LC‐MS (liquid chromatography–mass spectrometry) was mostly part of the efforts for identification of one or few unknown metabolites of interest as part of a phytochemical study. As a major strategy, unknown compounds had to be purified in sufficient quantities. The purified fractions were then subjected to LC‐MS/MS as part of the structural elucidation, mostly complemented by NMR (nuclear magnetic resonance) analysis. With the advance of mass spectrometry instrumentation, LC‐MS is now widely applied for analysis of crude plant extracts and large numbers (100s to 1000s) of samples. It has become an essential part of metabolomic studies (see Metabolomics), aiming at the comprehensive coverage of the metabolite profiles of cells, tissues, or organs. Owing to the huge chemical diversity of small molecules, conditions for the extraction will restrict the subfraction of the metabolome, which can be actually analyzed. The conditions for LC have to be adjusted to allow good separation of the particular metabolites from the respective extract. Major consideration will be the selection of an appropriate column and suitable eluents, the establishment of gradient profiles, temperature conditions, and so on.

Books and chapters

Baseggio Conrado, A.; Capuozzo, E.; Mosca, L.; Francioso, A.; Fontana, M.; Thiotaurine: From Chemical and Biological Properties to Role in H2S Signaling Hu, J., et al., eds. Adv. Exp. Med. Biol. 1155 755-771 (2019) ISBN:978-981-13-8023-5 DOI: 10.1007/978-981-13-8023-5_66
  • Abstract
  • BibText
  • RIS

In the last decade thiotaurine, 2-aminoethane thiosulfonate, has been investigated as an inflammatory modulating agent as a result of its ability to release hydrogen sulfide (H2S) known to play regulatory roles in inflammation. Thiotaurine can be included in the “taurine family” due to structural similarity to taurine and hypotaurine, and is characterized by the presence of a sulfane sulfur moiety. Thiotaurine can be produced by different pathways, such as the spontaneous transsulfuration between thiocysteine – a persulfide analogue of cysteine – and hypotaurine as well as in vivo from cystine. Moreover, the enzymatic oxidation of cysteamine to hypotaurine and thiotaurine in the presence of inorganic sulfur can occur in animal tissues and last but not least thiotaurine can be generated by the transfer of sulfur from mercaptopyruvate to hypotaurine catalyzed by a sulfurtransferase. Thiotaurine is an effective antioxidant agent as demonstrated by its ability to counteract the damage caused by pro-oxidants in the rat. Recently, we observed the influence of thiotaurine on human neutrophils functional responses. In particular, thiotaurine has been found to prevent human neutrophil spontaneous apoptosis suggesting an alternative or additional role to its antioxidant activity. It is likely that the sulfane sulfur of thiotaurine may modulate neutrophil activation via persulfidation of target proteins. In conclusion, thiotaurine can represent a biologically relevant sulfur donor acting as a biological intermediate in the transport, storage and release of sulfide.

Preprints

Niemeyer, M.; Moreno Castillo, E.; Ihling, C.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A.; Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin receptor assemblies bioRxiv (2019) DOI: 10.1101/787770
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Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron′s vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.

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