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The expanding cultivation of grains to meet agro-industry demands, the implementation of more efficient, sustainable, and integrative management practices capable of mitigating the selection of resistant pests and weeds becomes necessary. Due to the intensive application of chemical herbicides, in addition to environmental impacts, led to the development of resistance mechanisms in weed populations, such as sourgrass ( Digitaria insularis ). These mechanisms complicate management efforts, escalate production costs, and diminish productivity. In this context, RNA interference (RNAi) technology has emerged as a promising molecular tool for the targeted control of weeds, owing to its specificity in the post-transcriptional silencing of vital genes. This study investigated the application of RNAi technology for the suppression of 5-enolpyruvylshikimate-3-phosphate synthase ( EPSPS ) gene expression in D. insularis , a weed species of significant agronomic importance in Latin America. A double-stranded RNA (dsRNA) sequence, specifically designed for the EPSPS gene, was synthesized via bacterial fermentation with a strain of E. coli HT115, extracted using TRIzol™ and purified with phenol: chloroform: isoamyl alcohol, and applied topically in D. insularis leaves. The subsequent phenotypic and molecular effects were evaluated. The spray application of the dsRNA resulted in a 44% reduction in the plant's shoot dry mass and a 75% reduction in the number of tillers, thereby indicating consistent physiological impacts due to gene silencing. Quantitative reverse transcription PCR (qRT-PCR) analysis confirmed a significant suppression of EPSPS transcript levels following treatment, suggesting partial gene silencing. These findings collectively demonstrate the efficacy of RNAi in modulating gene expression in D. insularis , thereby underscoring its potential as a sustainable biotechnological strategy for the development of novel weed control methodologies.

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein‐based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large‐scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. Silks exhibit strength and elasticity and are biocompatible and biodegradable protein–based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large scale production of recombinant silks for various biomedical and materials science applications. This topic is reviewed in the present paper.

Background Recombinant DNA technology has been extensively employed to generate a variety of products from genetically modified organisms (GMOs) over the last decade, and the development of technologies capable of analyzing these products is crucial to understanding gene expression patterns. Liquid chromatography coupled with mass spectrometry is a powerful tool for analyzing protein contents and possible expression modifications in GMOs. Specifically, the NanoUPLC-MS E technique provides rapid protein analyses of complex mixtures with supported steps for high sample throughput, identification and quantization using low sample quantities with outstanding repeatability. Here, we present an assessment of the peptide and protein identification and quantification of soybean seed EMBRAPA BR16 cultivar contents using NanoUPLC-MS E and provide a comparison to the theoretical tryptic digestion of soybean sequences from Uniprot database. Results The NanoUPLC-MS E peptide analysis resulted in 3,400 identified peptides, 58% of which were identified to have no miscleavages. The experiment revealed that 13% of the peptides underwent in-source fragmentation, and 82% of the peptides were identified with a mass measurement accuracy of less than 5 ppm. More than 75% of the identified proteins have at least 10 matched peptides, 88% of the identified proteins have greater than 30% of coverage, and 87% of the identified proteins occur in all four replicates. 78% of the identified proteins correspond to all glycinin and beta-conglycinin chains. The theoretical Uniprot peptide database has 723,749 entries, and 548,336 peptides have molecular weights of greater than 500 Da. Seed proteins represent 0.86% of the protein database entries. At the peptide level, trypsin-digested seed proteins represent only 0.3% of the theoretical Uniprot peptide database. A total of 22% of all database peptides have a pI value of less than 5, and 25% of them have a pI value between 5 and 8. Based on the detection range of typical NanoUPLC-MS E experiments, i.e., 500 to 5000 Da, 64 proteins will not be identified. Conclusions NanoUPLC-MS E experiments provide good protein coverage within a peptide error of 5 ppm and a wide MW detection range from 500 to 5000 Da. A second digestion enzyme should be used depending on the tissue or proteins to be analyzed. In the case of seed tissue, trypsin protein digestion results offer good databank coverage. The Uniprot database has many duplicate entries that may result in false protein homolog associations when using NanoUPLC-MS E analysis. The proteomic profile of the EMBRAPA BR-16 seed lacks certain described proteins relative to the profiles of transgenic soybeans reported in other works.

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.

Summary Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high‐mortality coronavirus infections and diseases that disproportionately affect the developing world.

Soybean is one of the main sources of vegetable protein used in animal feed, but its nutritional value is limited by the presence of antinutritional factors, such as protease inhibitors (Kunitz and Bowman–Birk), lectins, phytic acid, raffinose family oligosaccharides (RFOs), and saponins, which reduce the digestibility and absorption of nutrients. In recent decades, advances in metabolic engineering and functional genomics have allowed the targeting of biochemical pathways to increase the content and quality of proteins while simultaneously reducing these undesirable compounds. This work reviews the main progress achieved through transgenesis, induced mutagenesis, and precision gene editing, highlighting the role of tools such as RNAi, CRISPR/Cas9, and AlphaFold2-guided gene editing in modifying genes involved in carbon and nitrogen metabolism and storage proteins. Recent studies demonstrate that the silencing of negative regulatory genes, such as CIF1 and AIP2 , can elevate the protein content of seeds, while the editing of sugar transporters SWEET10a and SWEET10b allows the modulation of the oil-protein balance. Simultaneously, the inactivation of genes related to antinutritional factors has significantly reduced the expression of compounds such as phytate and protease inhibitors. The integration of new approaches, such as promoter engineering and Prime Editing, promises to further enhance the precision of genetic modifications, minimizing pleiotropic effects. Taken together, these strategies consolidate metabolic engineering as a promising tool for the development of soybean cultivars with higher protein content and quality, and with lower content of antinutritional factors, optimizing their use in animal feed

Spider silks are well known for their extraordinary mechanical properties. This characteristic is a result of the interplay of composition, structure and self-assembly of spider silk proteins (spidroins). Advances in synthetic biology have enabled the design and production of spidroins with the aim of biomimicking the structure-property-function relationships of spider silks. Although in nature only fibers are formed from spidroins, in vitro , scientists can explore non-natural morphologies including nanofibrils, particles, capsules, hydrogels, films or foams. The versatility of spidroins, along with their biocompatible and biodegradable nature, also placed them as leading-edge biological macromolecules for improved drug delivery and various biomedical applications. Accordingly, in this review, we highlight the relationship between the molecular structure of spider silk and its mechanical properties and aims to provide a critical summary of recent progress in research employing recombinantly produced bioengineered spidroins for the production of innovative bio-derived structural materials.

Agricultural Biotechnology and Bioeconomy Unit (ABBU), Universitat de Lleida, Spain, EU Pharma-Factory grant agreement 77,4078 to P.C. Research in NI-U’s lab is supported by the Spanish Ministry of Science and Innovation (grant PID2020-117145RB-I00),EU HORIZON-HLTH-2021-CORONA-01 (grant 101046118) and by institutional funding of Grifols,Pharma Mar,HIPRA,Amassence,and Palobiofarma. E.L.R. is supported by Embrapa Genetic Resources and Biotechnology/National Institute of Science and Technology in Synthetic Biology, National Council for Scientific and Technological Development (465603/2014-9), Research Support Foundation of the Federal District (0193.001.262/2017), and Coordination for the Improvement of Higher Education Personnel. This research has been supported in part by the Intramural Research Program of the NIH, NCI, Center for Cancer Research and with federal funds from the NCI, NIH, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S.Government.All animal experiments were approved by

Living organisms are masters at designing outstanding self-assembled nanostructures through a hierarchical organization of modular proteins. Protein-based biopolymers improved and selected by the driving forces of molecular evolution are among the most impressive archetypes of nanomaterials. One of these biomacromolecules is the myriad of compound fibroins of spider silks, which combine surprisingly high tensile strength with great elasticity. However, no consensus on the nano-organization of spider silk fibres has been reached. Here we explore the biodiversity of spider silk fibres, focusing on nanoscale characterization with high-resolution atomic force microscopy. Our results reveal an evolution of the nanoroughness, nanostiffness, nanoviscoelastic, nanotribological and nanoelectric organization of microfibres, even when they share similar sizes and shapes. These features are related to unique aspects of their molecular structures. The results show that combined nanoscale analyses of spider silks may enable the screening of appropriate motifs for bioengineering synthetic fibres from recombinant proteins. Spider silk fibre is known to be composed of arrangements of structural domains. Here, the authors implement multiple atomic force microscopy modes to study the nanoscale morphology and mechanics of these fibres from nine spiders, and relate them to their molecular structures.

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7–10 months.