Seed-produced anti-globulin VHH-Fc antibodies retrieve globulin precursors in the insoluble fraction and modulate the Arabidopsis thaliana seed subcellular morphology

Key message Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins. Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination

Russell-like bodies in plant seeds share common features with prolamin bodies and occur upon recombinant protein production

Although many recombinant proteins have been produced in seeds at high yields without adverse effects on the plant, endoplasmic reticulum (ER) stress and aberrant localization of endogenous or recombinant proteins have also been reported. The production of murine interleukin-10 (mIL-10) in Arabidopsis thaliana seeds resulted in the de novo formation of ER-derived structures containing a large fraction of the recombinant protein in an insoluble form. These bodies containing mIL-10 were morphologically similar to Russell bodies found in mammalian cells. We confirmed that the compartment containing mIL-10 was enclosed by ER membranes, and 3D electron microscopy revealed that these structures have a spheroidal shape. Another feature shared with Russell bodies is the continued viability of the cells that generate these organelles. To investigate similarities in the formation of Russell-like bodies and the plant-specific protein bodies formed by prolamins in cereal seeds, we crossed plants containing ectopic ER-derived prolamin protein bodies with a line accumulating mIL-10 in Russell-like bodies. This resulted in seeds containing only one population of protein bodies in which mIL-10 inclusions formed a central core surrounded by the prolamin-containing matrix, suggesting that both types of protein aggregates are together removed from the secretory pathway by a common mechanism. We propose that, like mammalian cells, plant cells are able to form Russell-like bodies as a self-protection mechanism, when they are overloaded with a partially transport-incompetent protein, and we discuss the resulting challenges for recombinant protein production

Разработка инструментов продвижения инновационного продукта на рынке B2B

Тема данной работы - "Разработка инструментов продвижения инновационного продукта на рынке B2B" - в рамках которой были разработаны инструменты продвижения которые будут использоваться компанией ОАО "Манотомь". Задача данной работы заключается в анализе продукции выпускаемой компанией и инструментов продвижения используемых компанией. С целью модернизации и разработкой новых инструментов продвижения. Исходя из поставленных задач, в дипломной работе последовательно рассмотрены ряд вопросов, такие как конкурентоспособность продукции ОАО "Манотомь" а так же используемые инструменты продвижения. Разработан новый инструмент продвижения такой как контекстная реклама. Разработаны предложения по модернизации сайта. И рассчитаны оптимальные экономические затраты на внедрения данных инструментов.The topic of this work is "Development of tools for promoting an innovative product in the B2B market" - within the framework of which the promotion tools were developed that will be used by the company "Manotom". The task of this work is to analyze the products produced by the company and the promotion tools used by the company. In order to modernize and develop new promotion tools. Based on the tasks set, the thesis consistently considers a number of issues, such as the competitiveness of the products of JSC "Manotom" as well as the promotion tools used. A new promotion tool, such as contextual advertising, has been developed. Proposals for the modernization of the site have been developed. And the optimal economic costs for the implementation of these tools are calculated

Communication Skills in Foreign Languages in Engineering

This article is devoted to innovative methods of teaching ESL. The factors of development of engineering activities are shown and the prerequisites for the component composition of a foreign language professional communicative competence are identified. The first positions in achieving the effectiveness of business communication are the verbal activities of the engineer

Generation of multi-layered protein bodies in N. benthamiana for the encapsulation of vaccine antigens

The ability of plants to assemble particulate structures such as virus-like particles and protein storage organelles allows the direct bioencapsulation of recombinant proteins during the manufacturing process, which holds promise for the development of new drug delivery vehicles. Storage organelles found in plants such as protein bodies (PBs) have been successfully used as tools for accumulation and encapsulation of recombinant proteins. The fusion of sequences derived from 27-kDa-γ-zein, a major storage protein of maize, with a protein of interest leads to the incorporation of the chimeric protein into the stable and protected environment inside newly induced PBs. While this procedure has proven successful for several, but not all recombinant proteins, the aim of this study was to refine the technology by using a combination of PB-forming proteins, thereby generating multi-layered protein assemblies in N. benthamiana. We used fluorescent proteins to demonstrate that up to three proteinaceous components can be incorporated into different layers. In addition to 27-kDa-γ-zein, which is essential for PB initiation, 16-kDa-γ-zein was identified as a key element to promote the incorporation of a third zein-component into the core of the PBs. We show that a vaccine antigen could be incorporated into the matrix of multi-layered PBs, and the protein microparticles were characterized by confocal and electron microscopy as well as flow cytometry. In future, this approach will enable the generation of designer PBs that serve as drug carriers and integrate multiple components that can be functionalized in different ways

Extracellular vesicles in human skin: cross-talk from senescent fibroblasts to keratinocytes by miRNAs

Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging

Increased efficacy of influenza virus vaccine candidate through display of recombinant neuraminidase on virus like particles

Vaccination against influenza virus can reduce the risk of influenza by 40% to 60%, they rely on the production of neutralizing antibodies specific to influenza hemagglutinin (HA) ignoring the neuraminidase (NA) as an important surface target. Vaccination with standardized NA concentration may offer broader and longer-lasting protection against influenza infection. In this regard, we aimed to compare the potency of a NA displayed on the surface of a VLP with a soluble NA. The baculovirus expression system (BEVS) and the novel virus-free Tnms42 insect cell line were used to express N2 NA on gag-based VLPs. To produce VLP immunogens with high levels of purity and concentration, a two-step chromatography purification process combined with ultracentrifugation was used. In a prime/boost vaccination scheme, mice vaccinated with 1 µg of the N2-VLPs were protected from mortality, while mice receiving the same dose of unadjuvanted NA in soluble form succumbed to the lethal infection. Moreover, NA inhibition assays and NA-ELISAs of pre-boost and pre-challenge sera confirm that the VLP preparation induced higher levels of NA-specific antibodies outperforming the soluble unadjuvanted NA

Progressive Aggregation of 16 kDa Gamma-Zein during Seed Maturation in Transgenic Arabidopsis thaliana

Prolamins constitute a unique class of seed storage proteins, present only in grasses. In the lumen of the endoplasmic reticulum (ER), prolamins form large, insoluble heteropolymers termed protein bodies (PB). In transgenic Arabidopsis (Arabidopsis thaliana) leaves, the major maize (Zea mays) prolamin, 27 kDa γ-zein (27γz), assembles into insoluble disulfide-linked polymers, as in maize endosperm, forming homotypic PB. The 16 kDa γ-zein (16γz), evolved from 27γz, instead forms disulfide-bonded dispersed electron-dense threads that enlarge the ER lumen without assembling into PB. We have investigated whether the peculiar features of 16γz are also maintained during transgenic seed development. We show that 16γz progressively changes its electron microscopy appearance during transgenic Arabidopsis embryo maturation, from dispersed threads to PB-like, compact structures. In mature seeds, 16γz and 27γz PBs appear very similar. However, when mature embryos are treated with a reducing agent, 27γz is fully solubilized, as expected, whereas 16γz remains largely insoluble also in reducing conditions and drives insolubilization of the ER chaperone BiP. These results indicate that 16γz expressed in the absence of the other zein partners forms aggregates in a storage tissue, strongly supporting the view that 16γz behaves as the unassembled subunit of a large heteropolymer, the PB, and could have evolved successfully only following the emergence of the much more structurally self-sufficient 27γz