The ideal vaccines for combating diseases that may emerge in the future require more than simply inactivating a few pathogenic strains. This study aims to provide a peptide-based multi-epitope vaccine effective against various severe acute respiratory syndrome coronavirus 2 strains. To design the vaccine, a library of peptides from the spike, nucleocapsid, membrane, and envelope structural proteins of various strains was prepared. Then, the final vaccine structure was optimized using the fully protected epitopes and the fynomer scaffold. Using bioinformatics tools, the antigenicity, allergenicity, toxicity, physicochemical properties, population coverage, and secondary and three-dimensional structures of the vaccine candidate were evaluated. The bioinformatic analyses confirmed the high quality of the vaccine. According to further investigations, this structure is similar to native protein and there is a stable and strong interaction between vaccine and receptors. Based on molecular dynamics simulation, structural compactness and stability in binding were also observed. In addition, the immune simulation showed that the vaccine can stimulate immune responses similar to real conditions. Finally, codon optimization and in silico cloning confirmed efficient expression in Escherichia coli. In conclusion, the fynomer-based vaccine can be considered as a new style in designing and updating vaccines to protect against coronavirus disease.

Summary: Cellulose is the most abundant unique biopolymer in nature with widespread applications in bioenergy and high‐value bioproducts. The large transmembrane‐localized cellulose synthase (CESA) complexes (CSCs) play a pivotal role in the biosynthesis and orientation of the para‐crystalline cellulose microfibrils during secondary cell wall (SCW) deposition. However, the hub CESA subunit with high potential homo/heterodimerization capacity and its functional effects on cell wall architecture, cellulose crystallinity, and saccharification efficiency remains unclear. Here, we reported the highly potent binding site containing four residues of Pro435, Trp436, Pro437, and Gly438 in the plant‐conserved region (P‐CR) of PalCESA4 subunit, which are involved in the CESA4‐CESA8 heterodimerization. The CRISPR/Cas9‐knockout mutagenesis in the predicted binding site results in physiological abnormalities, stunt growth, and deficient roots. The homozygous double substitution of W436Q and P437S and heterozygous double deletions of W436 and P437 residues potentially reduced CESA4‐binding affinity resulting in normal roots, 1.5–2‐fold higher plant growth and cell wall regeneration rates, 1.7‐fold thinner cell wall, high hemicellulose content, 37%–67% decrease in cellulose content, high cellulose DP, 25%–37% decrease in cellulose crystallinity, and 50% increase in saccharification efficiency. The heterozygous deletion of W436 increases about 2‐fold CESA4 homo/heterodimerization capacity led to the 50% decrease in plant growth and increase in cell walls thickness, cellulose content (33%), cellulose DP (20%), and CrI (8%). Our findings provide a strategy for introducing commercial CRISPR/Cas9‐mediated bioengineered poplars with promising cellulose applications. We anticipate our results could create an engineering revolution in bioenergy and cellulose‐based nanomaterial technologies.

Chelidonium majus is a traditional medicinal plant, which commonly known as a rich resource for the major benzylisoquinoline alkaloids (BIAs), including morphine, sanguinarine, and berberine. To understand the biosynthesis of C. majus BIAs, we performed de novo transcriptome sequencing of its leaf and root tissues using Illumina technology. Following comprehensive evaluation of de novo transcriptome assemblies produced with five programs including Trinity, Bridger, BinPacker, IDBA-tran, and Velvet/Oases using a series of k-mer sizes (from 25 to 91), BinPacker was found to produce the best assembly using a k-mer of 25. This study reports the results of differential gene expression (DGE), functional annotation, gene ontology (GO) analysis, classification of transcription factor (TF)s, and SSR and miRNA discovery. Our DGE analysis identified 6,028 transcripts that were up-regulated in the leaf, and 4,722 transcripts that were up-regulated in the root. Further investigations showed that most of the genes involved in the BIA biosynthetic pathway are significantly expressed in the root compared to the leaf. GO analysis showed that the predominant GO domain is “cellular component”, while TF analysis found bHLH to be the most highly represented TF family. Our study further identified 10 SSRs, out of a total of 39,841, that showed linkage to five unigenes encoding enzymes in the BIA pathway, and 10 conserved miRNAs that were previously not detected in this plant. The comprehensive transcriptome information presented herein provides a foundation for further explorations on study of the molecular mechanisms of BIA synthesis in C. majus.

Human papillomavirus (HPV), which is transmitted through sexual activity, is the primary cause of cervical cancer and the fourth most common type of cancer in women. In this study, an immunoinformatics approach was employed to predict immunodominant epitopes from a diverse array of antigens with the ultimate objective of designing a potent multiepitope vaccine against multiple HPV types. Immunodominant B cell, cytotoxic T cell (CTL), and helper T cell (HTL) epitopes were predicted using bioinformatics tools These epitopes were subsequently analyzed using various immunoinformatics tools, and those that exhibited high antigenicity, immunogenicity, non-allergenicity, non-toxicity, and excellent conservation were selected. The selected epitopes were linked with appropriate linkers and adjuvants to formulate a broad-spectrum multiepitope vaccine candidate against HPV. The stability of the multiepitope vaccine candidate was confirmed through structural analysis, and docking results indicated a high affinity for Toll-like receptors (TLR2 and TLR4). Molecular dynamics simulations demonstrated a persistent interaction of TLR2 and TLR4 with the multiepitope vaccine candidate. In silico immunological simulations showed that three injections of the multiepitope vaccine candidate resulted in high levels of B- and T-cell immune responses. Moreover, the in silico cloning results indicated that the multiepitope vaccine candidate could be expressed in substantial amounts in E. coli. The results of this study imply that designing a broad-spectrum vaccine against various HPV types using computational methods is plausible; however, experimental validation and safety testing to confirm the findings is essential.

Background: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. Results: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. Conclusion: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

Human metapneumovirus (hMPV) is a significant etiological agent of acute respiratory infections in children and immunocompromised individuals. Despite its growing clinical impact, no approved vaccines or targeted antiviral therapies are currently available. An immunoinformatic approach was employed to design a chimeric multi-epitope vaccine candidate against hMPV. Conserved and virulence-associated proteins were analyzed to predict highly antigenic B cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes. The selected epitopes were screened for antigenicity, non-toxicity, non-allergenicity, and lack of homology to human proteins. The final construct included six B cell epitopes, six CTL epitopes, and two HTL epitopes, linked with appropriate adjuvants and Toll-like receptor (TLR) agonists. Structural modeling, molecular docking, and molecular dynamics simulations were conducted to evaluate the stability and receptor binding. Immunogenicity and expression potential were assessed through in silico immune simulation and codon optimization for expression in Escherichia coli. All selected epitopes showed high antigenicity with no allergenic or toxicity. Structural validation indicated a stable vaccine construct with favorable physicochemical properties. Molecular docking analysis predicted a high binding affinity between the vaccine construct and TLR2/TLR4 receptors. Molecular dynamics (MD) simulations suggested that the docked complexes maintained stable interactions under simulated physiological conditions. In silico immune simulations predicted strong B- and T-cell responses following three doses. Codon adaptation analysis supported the high-level expression in E. coli. The proposed multi-epitope vaccine demonstrates strong potential against hMPV, as supported by comprehensive computational analyses. Further experimental studies are required to validate its efficacy and safety.