Membrane-Based Inverse Transition Cycling: An Improved Means for Purifying Plant-Derived Recombinant Protein-Elastin-Like Polypeptide Fusions

Elastin-like peptide (ELP) was fused to two different avian flu H5N1 antigens and expressed in transgenic tobacco plants. The presence of the ELP tag enhanced the accumulation of the heterologous proteins in the tobacco leaves. An effective membrane-based Inverse Transition Cycling was developed to recover the ELPylated antigens and antibodies from plant material. The functionality of both the ELPylated neuraminidase and an ELPylated nanobody was demonstrated

Plant-based material, protein and biodegradable plastic

Fibrous proteins from spiders, proteins with synthetic multiple repeats and mammalian structural proteins such as collagen have been produced in transgenic plants. Recent advances in the production of biodegradable plastic in plants also show the potential of molecular farming for research into and production of materials. Selection of a growing variety of such products, optimization of expression, and the development of effective purification strategies will further promote this growing field of biotechnology. Introduction Plant materials such as cellulose, starch or even latex from rubber trees have traditionally been used for several purposes by mankind. In the past century, the chemical industry opened the way to modify plant-based materials, and products such as cellulose acetate or cellulose nitrate were invented. The development of genetic engineering technology and efficient transformation methods for several crops has opened the way for the genetic modification of traditional plant products; for example, the development of modified starch. Furthermore, interesting proteins originating from animals and novel polymers can now be synthesized in transgenic plants New protein-based materials from plants Naturally occurring proteins often exhibit interesting properties as fibers or adhesives. For example, larvae from insects produce fibrous glues in the form of sericin (produced by Bombyx mori for example Silk proteins from spiders and insects and their production in transgenic plants During evolution, spiders have become highly diverse in production and use of silks (for review see We believe that transgenic plants are powerful protein factories that can overcome the problems mentioned above. Therefore, we constructed stable transgenic tobacco and potato lines to express various synthetic spider-silk genes ranging from 420 to 3600 basepairs in size. The genes were assembled so as to achieve very high homology to the native MaSpI gene from Nephila clavipes (more than 90%). Accumulation of up to 2% spider-silk protein of total soluble protein (TSP) in the endoplasmic reticulum (ER) of tobacco leaves was achieved, and this production was relatively independent of the size of the spider-silk protein. The recombinant spidroins exhibited extreme heat stability. This property, together with resistance against acidification and fractionated ammonium sulfate precipitation, was used to purify the plantproduced spidroins by a simple and efficient procedure A protocol similar to that described by Scheller et al. A new purification strategy has been developed using the unique properties of ELP fusions. First, the spider silk proteins from plants were enriched by boiling. We used heat treatment at 95 8C for 60 min and clearance by centrifugation of tobacco leaf extracts to enrich the spider-silk-ELP fusion protein in the supernatant. For the selective precipitation of the SO1-100XELP, NaCl was added to a final concentration of 2 M and the temperature was raised to 60 8C. Under these conditions, the recombinant spider-silk-elastin fusion proteins aggregated and could be precipitated by centrifugation. Cellular proteins remained in the supernatant. The precipitated recombinant proteins were resolved at a lower temperature and without salt to a final concentration of 1 mg/ml of nearly homogenous product. Dialysis against water and drying led to the formation of silk-proteins in the form of storable membranes. Extraction of 1 kg tobacco leaf resulted in 80 mg pure recombinant spider-silk-elastin protein. Transgenic plants for spider silk and spider-silkelastin fusion proteins were phenotypically indistinguishable from wildtype plants. In addition, the expression of recombinant proteins in second generation plants was comparable to that in first-generation plants. In summary, expression and purification systems for plant-based silk proteins have been developed. Expression in storage organs such as tubers and seeds has been generally shown. The selection of suitable spidroin and fibroin sequences for defined applications and commercialization is still at an early stage. To date, there are no reports of the successful spinning of plant-derived silk proteins. The first example of possible medical use has, however, been reported Production of mammalian-derived structural proteins in plants Elastin is a strong elastic fiber that is present in ligaments and in arterial walls. Synthetic proteins that are constructed from multiple repeats also show elastic properties. Such polymers have also been shown to prevent postsurgical adhesions and scars in rats Collagens form a family of extra-cellular matrix proteins that are detectable in all connective tissues of mammals. They represent 30% of total body weight of proteins. Collagen molecules consist of three polypeptides called a chains, which assemble to form triple helical domains. The collagen a chains contain the repeating triple sequence Gly-X-Y, where X and Y are frequently proline and hydroxyproline residues, respectively. This sequence is necessary for the correct formation of the triple helix Biodegradable plastics and PHB from transgenic plants Until now, the commercial production of PHBs has been restricted by high production costs, making bacterial fermentation of PHB at least five times more expensive than chemical synthesis of polyethylene. Plants might be a suitable alternative for the low-cost production of PHBs. It was calculated that polymer concentrations in plants will need to reach at least 15% of dry weight for economically useful production. The general feasibility of the production of PHB in plants was shown in 1992 in pilot experiments in which PHB synthesis was directed into the cytoplasm of Arabidopsis thaliana. The accumulation of 0.1% of leaf dry weight as PHB was low in comparison to PHB accumulation in bacteria. Nevertheless, the plant-produced PHB formed granules whose size and appearance were comparable to those of the granules formed by bacteria. Unfortunately, these Arabidopsis plants displayed a stunted-growth phenotype, leading to the assumption that the redirection of cytoplasmic acetyl-CoA to PHB production results in insufficient production of isoprenoids and flavoids, which are needed for normal growth Major improvements in PHB accumulation were achieved by targeting the bacterial PHB-biosynthesis pathway to other compartments of plant cells in transgenic plants. In 1994, Nawrath et al. [58] produced constitutive expression of the enzymes of the PHB biosynthetic pathway in chloroplasts of A. thaliana by nuclear transformation and fusion of transit peptides to the three PHB enzymes. They reported PHB accumulation of up to 14% of leaf dry weight. In contrast to the cytoplasm, plastids are compartments in which there is a high flux of acetyl-CoA, which is mainly utilized as precursor for the fatty-acid biosynthesis of membrane lipids. Plants in which the bacterial PHBbiosynthesis pathway was targeted to plastids showed nearly wildtype growth and fertility but, in high PHB producers, chlorosis of the leaves was observed. One crucial issue that was not addressed by Nawrath et al. [58] is whether stable, high-level polymer production in plants could be maintained over many generations. Later, transgenic A. thaliana plants were directly screened for high accumulation of PHB in the plastids of leaves using gas chromatography and mass spectrometry (GC-MS). Accumulation of up to 40% of leaf dry weight as PHB Expression systems for the production of biopolymers in plants: ER Leaves Current Opinion in Plant Biology • Ubiquitous expression and expression of transgenic proteins in storage organs (e.g. tubers, seeds) • Accumulation in 'optimal' compartments (e.g. ER, chloroplast) • Production of plastic-like biomaterials in chloroplasts and peroxisomes The syntheses of PHB in transgenic cotton and flax were examples of introducing a new polymer into existing fibers There is an obvious need to further improve transgenic PHB production by producing higher accumulation without side-effects such as chlorosis, male sterility and growth retardation, but genetic engineering has already yielded the first examples of the production of PHAcopolymers in transgenic plants (SLC-and MCL-PHA). PHB is a highly crystalline polymer whose usefulness is restricted by its physical properties, being relatively stiff and brittle. SLC-and MCL-PHA are less stiff and brittle, even having rubber-like properties, and therefore exhibit a greater potential for commercial applications. In 1999, Slater and colleagues IlvA from E. coli encodes the protein threonine deaminase and converts threonine to 2-ketobutyrate. The endogenous pyruvate dehydrogenase complex catalyzes the synthesis of propionyl-CoA from 2-ketobutyrate. Acetyl-CoA and propionyl-CoA are then further processed and copolymerized into PHBV In contrast to peroxisomal fatty acids in mammals, those in plants can be completely oxidized by the acyl-CoA oxidase (SC-ACOX) to acetyl-CoA (a process known as b-oxidation

Passive extraction of Cabbage stem flea beetle larvae (Psylliodes chrysocephala L.)

Die Studie stellt eine einfache, aber nicht sehr weit verbreitete Methode vor, um quantitativ den Befall von Winterrapspflanzen mit Larven von Psylliodes chrysocephala zu bestimmen. Die Trichter-Methode kann als eine Abwandlung der Berlese-Trichter-Methode betrachtet werden, jedoch ohne Nutzung einer aktiven Wärmequelle. Durch die vorgestellte Methode konnten bei der Bonitur auf Rapserdflohlarven annähernd dieselben Ergebnisse generiert werden wie bei der manuellen Präparation. Der große Vorteil gegenüber der konventionellen Präpara­tion ist der geringere Arbeits- und Kostenaufwand, wohingegen der große Nachteil der passiven Austreibung die benötigte Zeit (bis zu 21 Tagen) ist, welche die zuletzt die Pflanzen verlassenden Larven benötigen, bis sie das Pflanzengewebe verlassen. Außerdem können die Larvenstadien für den Beprobungszeitpunkt nicht mehr bestimmt werden. Die Ergebnisse zeigen, dass die Trichter-Methode eine billige und einfache Methode für die praktische Anwendung darstellt, jedoch benötigt sie recht lang, bis die Ergebnisse verfügbar sind. DOI: 10.5073/JfK.2016.09.01, https://doi.org/10.5073/JfK.2016.09.01An easy but not widely used method to determine the number of Psylliodes chrysocephala larvae in winter oilseed rape plants was studied. The Funnel-Method can be considered as a modified Berlese-Funnel-Method with no heat supply. The passive extraction of cabbage stem flea larvae generated nearly the same results as the manual dissection. The big advantage of using such a Funnel-Method is that distinctly less labour time is needed; the disadvantage is that the extraction needs up to 21 days until all larvae have left the plant tissue and that the larval stages cannot be determined anymore for a specific plant collection date. The results of this study indicate that the Funnel-Method is a cheap and simple tool for practical use but with a time delay before results are available. DOI: 10.5073/JfK.2016.09.01, https://doi.org/10.5073/JfK.2016.09.0

Adults of Cabbage stem flea beetle (Psylliodes chrysocephala L.) in different types of yellow water traps in winter oilseed rape (Brassica napus L.)

Gelbschalen sind ein wichtiges Instrument zur Prognose der Abundanz des Rapserdflohs, Psylliodes chrysocephala, in Winterraps. Deutschlandweit gibt es zwei weit verbreitete Typen von Gelbschalen: eckiger (26 x 33 cm) und runder Typ (22,4 cm Durchmesser). Um Beifänge von Bienen und Hummeln zu vermeiden wird empfohlen, Gelbschalen nur mit Gitter aufzustellen. Für den Rapserdfloh hat sich in den meisten Gebieten Deutschlands ein einheitlicher Schwellenwert von 50 Käfern in drei Wochen etabliert. Ziel dieser Studie an 28 Standorten in Deutschland war es zu eruieren, inwieweit es Unterschiede in der Fängigkeit der beiden Gelbschalentypen (rund und eckig, mit und ohne Gitter) gibt und welchen Einfluss das Eingraben der Schale hat. Es konnte gezeigt werden, dass sich die Fängigkeit der Gelbschalensysteme zum Teil signifikant voneinander unterscheidet, wobei aber die Fangzahlen quantitativ nicht so weit voneinander liegen, dass angesichts der hohen Variabilität der Aufstellung in der landwirtschaftlichen Praxis unter­schiedliche Schwellenwerte für jeden Fallentyp notwendig werden. DOI: 10.5073/JfK.2016.08.01, https://doi.org/10.5073/JfK.2016.08.01Yellow water traps are a decisive factor in the forecasting system for Psylliodes chrysocephala, the Cabbage stem flea beetle. In most parts of Germany a threshold of 50 imagines per yellow water trap within three weeks is established. In Germany two types of yellow water traps are widely used: an angular (26 x 33 cm) and a round type (22.4 cm diameter). The use of a grid at the top of the yellow water traps is recommended to prevent bees from drowning. The aim of this study carried out on 28 fields all over Germany was to inves­tigate the effects of different types of yellow water traps on Cabbage stem flea beetle catches. Traps were used with and without grid and placed on the soil surface or dug into the soil. Beetle numbers differed significantly between some of the types of yellow water traps, but the difference in densities was in an acceptable range given the overall high variability induced by farmers. DOI: 10.5073/JfK.2016.08.01, https://doi.org/10.5073/JfK.2016.08.0

Expression and immunogenicity of the mycobacterial Ag85B/ESAT-6 antigens produced in transgenic plants by elastin-like peptide fusion strategy.

International audienceThis study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis

Подбор оборудования для эксплуатации скважин в условиях активного выноса песка на нефтяных месторождениях Западной Сибири

Цель работы – анализ известных представлений по проблеме пескопроявления в процессе эксплуатации скважин; анализ технологических и технических решений для борьбы с пескопроявлением. В результате даны рекомендации по использованию того или иного оборудования для получения наилучшего эффекта. Область применения: скважины, характеризующиеся высоким количеством песчаных частиц в извлекаемом флюиде, а также скважины, вскрывающие слабосцементированные песчаные породы.The purpose of the work is the analysis of well-known ideas on the problem of sand occurrence during the operation of wells; analysis of technologies and technical solutions for sand control. For the best effect. Scope: wells characterized by a high content of sand particles in the recovered fluid, as well as wells revealing weakly cemented sand formations

Arabidopsis NSE4 Proteins Act in Somatic Nuclei and Meiosis to Ensure Plant Viability and Fertility

The SMC 5/6 complex together with cohesin and condensin is a member of the structural maintenance of chromosome (SMC) protein family. In non-plant organisms SMC5/6 is engaged in DNA repair, meiotic synapsis, genome organization and stability. In plants, the function of SMC5/6 is still enigmatic. Therefore, we analyzed the crucial d-kleisin component NSE4 of the SMC5/6 complex in the model plant Arabidopsis thaliana. Two functional conserved Nse4 paralogs (Nse4A and Nse4B) are present in A. thaliana, which may have evolved via gene subfunctionalization. Due to its high expression level, Nse4A seems to be the more essential gene, whereas Nse4B appears to be involved mainly in seed development. The morphological characterization of A. thaliana T-DNA mutants suggests that the NSE4 proteins are essential for plant growth and fertility. Detailed investigations in wild-type and the mutants based on live cell imaging of transgenic GFP lines, fluorescence in situ hybridization (FISH), immunolabeling and super-resolution microscopy suggest that NSE4A acts in several processes during plant development, such as mitosis, meiosis and chromatin organization of differentiated nuclei, and that NSE4A operates in a cell cycle-dependent manner. Differential response of NSE4A and NSE4B mutants after induced DNA double strand breaks (DSBs) suggests their involvement in DNA repair processes

Slow solar wind sources

Context. The origin of the slow solar wind is still an open issue. One possibility that has been suggested is that upflows at the edge of an active region can contribute to the slow solar wind. Aims. We aim to explain how the plasma upflows are generated, which mechanisms are responsible for them, and what the upflow region topology looks like. Methods. We investigated an upflow region using imaging data with the unprecedented temporal (3 s) and spatial (2 pixels = 236 km) resolution that were obtained on 30 March 2022 with the 174 Å channel of the Extreme-Ultraviolet Imager (EUI)/High Resolution Imager (HRI) on board Solar Orbiter. During this time, the EUI and Earth-orbiting satellites (Solar Dynamics Observatory, Hinode, and the Interface Region Imaging Spectrograph, IRIS) were located in quadrature (∼92°), which provides a stereoscopic view with high resolution. We used the Hinode/EIS (Fe XII) spectroscopic data to find coronal upflow regions in the active region. The IRIS slit-jaw imager provides a high-resolution view of the transition region and chromosphere. Results. For the first time, we have data that provide a quadrature view of a coronal upflow region with high spatial resolution. We found extended loops rooted in a coronal upflow region. Plasma upflows at the footpoints of extended loops determined spectroscopically through the Doppler shift are similar to the apparent upward motions seen through imaging in quadrature. The dynamics of small-scale structures in the upflow region can be used to identify two mechanisms of the plasma upflow: Mechanism I is reconnection of the hot coronal loops with open magnetic field lines in the solar corona, and mechanism II is reconnection of the small chromospheric loops with open magnetic field lines in the chromosphere or transition region. We identified the locations in which mechanisms I and II work

Signatures of dynamic fibrils at the coronal base: Observations from Solar Orbiter/EUI

The solar chromosphere hosts a wide variety of transients, including dynamic fibrils (DFs) that are characterised as elongated, jet-like features seen in active regions, often through H$\alpha$ diagnostics. So far, these features have been difficult to identify in coronal images primarily due to their small size and the lower spatial resolution of the current EUV imagers. Here we present the first unambiguous signatures of DFs in coronal EUV data using high-resolution images from the Extreme Ultraviolet Imager (EUI) on board Solar Orbiter. Using the data acquired with the 174~{\AA} High Resolution Imager (HRI$_{EUV}$) of EUI, we find many bright dot-like features (of size 0.3-0.5 Mm) that move up and down (often repeatedly) in the core of an active region. In a space-time map, these features produce parabolic tracks akin to the chromospheric observations of DFs. Properties such as their speeds (14 km~s$^{-1}$), lifetime (332~s), deceleration (82 m~s$^{-2}$) and lengths (1293~km) are also reminiscent of the chromospheric DFs. The EUI data strongly suggest that these EUV bright dots are basically the hot tips (of the cooler chromospheric DFs) that could not be identified unambiguously before because of a lack of spatial resolution.Comment: Accepted for publication in A&A Letters. Event movie can be downloaded from https://drive.google.com/file/d/1o_4jHA5JbyQtrpUBtB3ItE_s3HjF6ncc/view?usp=sharin