Immunoinformatics design of a structural proteins driven multi-epitope candidate vaccine against different SARS-CoV-2 variants based on fynomer.

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.

A new trivalent recombinant protein for type 2 diabetes mellitus with oral delivery potential: design, expression, and experimental validation.

Glucagon-like peptide-1 (GLP-1) receptor agonists are increasingly used in clinical practice for the management of type 2 diabetes mellitus. However, the extremely short half-life of GLP-1 and the need for subcutaneous administration limit its clinical application. Thus, half-life extension and alternative delivery methods are highly desired. DARPin domains with high affinity for human serum albumin (HSA) have been selected for the half-life extension of therapeutic peptides and proteins. In the present study, novel trivalent fusion proteins as long-acting GLP-1 receptor agonists with potential for oral delivery were computationally engineered by incorporating a protease-resistant modified GLP-1, an anti-human serum albumin DARPin, and an approved cell-penetrating peptide (Penetratin, Tat, and Polyarginine) linked either by rigid or flexible linkers. Theoretical studies and molecular dynamics simulation results suggested that mGLP1-DARPin-Pen has acceptable quality and stability. Moreover, the potential affinity of the selected fusion proteins for GLP-1 receptor and human serum albumin was explored by molecular docking. The recombinant construct was cloned into the pET28a vector and expressed in Escherichia coli. SDS-PAGE analysis of the purified fusion protein matched its molecular size and was confirmed by western blot analysis. The results demonstrated that the engineered fusion protein could bind HSA with high affinity. Importantly, insulin secretion assays using a mouse pancreatic ß-cell line (ß-TC6) revealed that the engineered trivalent fusion protein retained the ability to stimulate cellular insulin secretion. Immunofluorescence microscopy analysis indicated the CPP-dependent cellular uptake of mGLP1-DARPin-Pen. These findings demonstrated that mGLP1-DARPin-Pen is a highly potent oral drug candidate that could be particularly useful in the treatment of type 2 diabetes mellitus.Communicated by Ramaswamy H. Sarma.

Designing and computational analyzing of chimeric long-lasting GLP-1 receptor agonists for type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is an intestinally derived incretin that plays a vital role in engineering the biological circuit involved in treating type 2 diabetes. Exceedingly short half-life (1-2 min) of GLP-1 limits its therapeutic applicability, and the implication of its new variants is under question. Since albumin-binding DARPin as a mimetic molecule has been reported to increase the serum half-life of therapeutic compounds, the interaction of new variants of GLP-1 in fusion with DARPin needs to be examined against the GLP-1 receptor. This study was aimed to design stable and functional fusion proteins consisting of new protease-resistant GLP-1 mutants (mGLP1) genetically fused to DARPin as a critical step toward developing long-acting GLP-1 receptor agonists. The stability and solubility of the engineered fusion proteins were analyzed, and their secondary and tertiary structures were predicted and satisfactorily validated. Molecular dynamics simulation studies revealed that the predicted structures of engineered fusion proteins remained stable throughout the simulation. The relative binding affinity of the engineered fusion proteins' complex with human serum albumin and the GLP-1 receptor individually was assessed using molecular docking analyses. It revealed a higher affinity compared to the interaction of the individual GLP-1 and HSA-binding DARPin with the GLP-1 receptor and human serum albumin, respectively. The present study suggests that engineered fusion proteins can be used as a potential molecule in the treatment of type 2 diabetes, and this study provides insight into further experimental use of mimetic complexes as alternative molecules to be evaluated as new bio-breaks in the engineering of biological circuits in the treatment of type 2 diabetes.

CRISPR/Cas9-mediated P-CR domain-specific engineering of CESA4 heterodimerization capacity alters cell wall architecture and improves saccharification efficiency in poplar.

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.

Novel Recombinant Traceable c-Met Antagonist-Avimer Antibody Mimetic Obtained by Bacterial Expression Analysis.

BACKGROUND: Avimers are originally types of artificial proteins with multiple binding sites for specific binding to certain antigens. Various radioisotopes and nanoparticles link these molecules, which are widely used in early detection in tissue imaging, treatment and study on carcinogenesis. Among these, c-Met antagonist avimer (C426 avimer), with ability to bind the c-Met receptor of tyrosine kinase (RTK) is an attractive candidate for targeted cancer therapy. In this study, a novel traceable C426 avimer gene was designed and introduced by adding the 12nt tracer binding site encoded four specific amino acid residues at the C-terminal region of C426 avimer coding sequence. METHODS: The 282 bp DNA sequence encoded 94aa avimer protein was synthesized and sub-cloned into prokaryotic pET26b expression vector. The expression of the mature peptide encoding the traceable avimer molecule was carried out in Escherichia coli strain BL21 using IPTG (Isopropyl ß-D-1-thiogalactopyranoside) induction process. The expression level of the 11 kDa traceable avimer was studied by SDS-PAGE, western blot and ELISA analysis. RESULTS: Docking analysis of C426 avimer protein and its ligand c-Met showed that the traceability related changes happened at the best conformation and optimal energy. The SDS-PAGE, western blotting and ELISA analysis results demonstrated that the expression of the 11 kDa C426 avimer molecule was detectable without any degradation compared with the control group. CONCLUSION: Concerning the consequences of this work, this new approach can be widely used in the medical field and provide an opportunity to evaluate the affinity and traceability features.

In Silico Identification of Novel microRNAs and Targets Using EST Analysis in Allium cepa L.

microRNAs (miRNAs) are a newly discovered class of non-coding small RNAs roughly 22 nucleotides long. Increasing evidence has shown that miRNAs play multiple roles in biological processes, including development, cell proliferation, apoptosis and stress responses. The identification of miRNAs and their targets is an important need to understand their roles in the development and physiology of sweet onion (Allium cepa). In this research, several computational approaches were combined to make concise prediction of the potential miRNAs and their targets. We used previously known miRNAs from other plant species against Expressed Sequence Tags (EST) database to search for the potential miRNAs. As a result, nine potential miRNAs were identified in eight ESTs of A. cepa, belonging to eight families. We could further BLAST the mRNA database and found total 154 number of the potential targets in A. cepa based on these potential miRNAs. According to the mRNA target information provided by NCBI, most of the target mRNAs appeared to be involved in plant growth, signal transduction, development, and stress responses. Gene ontology (GO) analysis implicated these targets in 32 biological processes such as protein ubiquitination, plant hormone signalling pathways and heme biosynthesis.

Design, simplified cloning, and in-silico analysis of multisite small interfering RNA-targeting cassettes.

Multiple gene silencing is being required to target and tangle metabolic pathways in eukaryotes and researchers have to develop a subtle method for construction of RNA interference (RNAi) cassettes. Although, several vectors have been developed due to different screening and cloning strategies but still some potential limitations remain to be dissolved. Here, we worked out a simple cloning strategy to develop multisite small interfering RNA (siRNA) cassette from different genes by two cloning steps. In this method, effective siRNA sites in the target messenger RNAs (mRNAs) were determined using in silico analysis and consecutively arranged to reduce length of inverted repeats. Here, we used one-step (polymerase chain reaction) PCR by designed long primer sets covering the selected siRNA sites. Rapid screening, cost-effective and shorten procedure are advantages of this method compare to PCR classic cloning. Validity of constructs was confirmed by optimal centroid secondary structures with high stability in plants.