Molecular Biotechnology最新文献

Tomato brown rugose fruit virus (Tobamovirus fructirugosum, ToBRFV) is an emerging tobamovirus threatening tomato and pepper production worldwide. Accurate and scalable detection methods are essential to limit its agricultural impact. In this study, we report the recombinant production and characterization of ToBRFV capsid (CP^ToBRFV) and movement (MP^ToBRFV) proteins as candidate antigens for serological detection. Both coding sequences were amplified from infected tomato tissue and heterologously expressed in Escherichia coli BL21(DE3). CP^ToBRFV exhibited partial solubility under low-temperature induction, allowing purification under native conditions, whereas MP^ToBRFV accumulated in inclusion bodies and was purified under denaturing conditions. Purified proteins were validated by SDS-PAGE and immunoblotting. In silico analyses indicated the presence of multiple B- and T-cell epitopes in both proteins. Immunization of mice with purified proteins elicited IgG responses. Interestingly, MP^ToBRFV induced a higher humoral response, but antisera raised against CP^ToBRFV showed superior ELISA reactivity with crude extracts from ToBRFV-infected plants. Together, these results support CP^ToBRFV as a robust antigen for serological detection and provide a reproducible framework for the development of recombinant protein-based diagnostics for ToBRFV.

Begomovirus, the largest genus within the family Geminiviridae, poses a significant threat to global agriculture, resulting in substantial economic losses across diverse cropping systems. The increasing incidence and geographic spread of begomoviruses are driven by both the emergence of novel viral strains and the adaptation of existing ones, complicating efforts to manage the disease effectively. Traditional techniques, such as PCR and ELISA, have long been the primary methods used for detecting begomoviruses. However, recent advancements have introduced modern detection techniques designed to improve speed and accuracy. Additionally, some techniques can also be applied in the field with commercial products, enhancing the practical efficiency of begomoviruses detection. This review summarizes the most used methods for begomoviruses detection and provides a comparative assessment of the strengths and limitations of each technique. Based on this, it will assist researchers in gaining a comprehensive overview of detection methods and their applications in studying this plant virus genus. Consequently, researchers can select the optimal detection method for their studies without expending excessive time and resources.

Virus-like particles (VLPs) are protein-based nanoscale assemblies derived from structural proteins of viruses. They are non-infectious scaffolds and are highly immunogenic; thus, they widely used as scaffolds to display and enhance the immunogenicity of less immunogenic foreign antigens. Different approaches, including genetic insertions, can be used to display foreign antigens on VLPs; however, some of these approaches have limitations, i.e., inability of viral coat proteins to tolerate a foreign insertion. In this study, we assessed the tolerability of bacteriophage Qβ and PP7 coat proteins to insertions of Spytag003 or Spytag peptides; we assessed whether the insertions of these peptides to the coat proteins will affect the ability of the recombinant coat proteins to assemble into VLPs; chimeric VLPs displaying these peptides can be used to conjugate antigens for vaccine studies. While the insertion of Spytag003 peptide at either the N or C termini of the coat protein of Qβ and at the C terminus of the coat protein of PP7 did not affect the expression of the recombinant proteins, the expressed proteins were not soluble. In contrast, insertion of the shorter Spytag at either the N terminus or AB-loop of PP7 gave rise to soluble proteins that assembled into VLPs. PP7-Spytag VLPs were successfully conjugated with SpyCatcher003 and a fungal antigen. Immunization studies revealed that Spycatcher003 protein conjugated on PP7-SpyTag VLPs elicited significantly higher anti-Spycatcher003 antibody titers compared to unconjugated Spycatcher003 (p = 0.0286). Together, these findings establish proof of concept that PP7-Spytag VLPs should be explored as a platform to conjugate foreign proteins.

Mesenchymal stem/stromal stem cells (MSCs) are promising therapeutic candidates in regenerative medicine and tissue engineering. MSCs have been applied in many medical conditions and their therapeutic effects, safety, and efficacy have been well established over the past few decades. However, several challenges exist when utilizing whole cells in research and the clinical settings. As a result, researchers have turned their attention to cell-free alternatives to overcome these challenges. One promising approach that can achieve the desired therapeutic effects without the need of whole-cell transplantation is the use of MSC-conditioned medium and secretome. This article gives an overview of the advantages of using cell-free strategies over cell-based strategies and the various types of cell-free alternatives available. It also critically examines the various cell-free approaches in MSC research and therapy and provides an in-depth discussion on the opportunities and challenges of using these strategies, with an emphasis on recent advances in the field.

Carbapenems are the last-resort antibiotic option against A. baumannii infections, and Carbapenem resistance leads to the emergence of CRAB strains, which are difficult to treat. The CRAB novel reported mutation in the blaOXA-58, whose resistance mechanism was not explored. Here, we conducted molecular docking and molecular dynamics simulation to unveil and compare the molecular mechanisms of blaOXA-58 wild and mutant types (I87M, A88L, and double-mutated). The crystal structure of blaOXA-58 was retrieved from PDB, mutations were induced using the AlphaFold online server, and the structures of Carbapenem drugs (DOR, ETP, MEM, and IPM) were retrieved from PubChem database. Using PyRx 0.8, molecular docking was performed between Carbapenem drugs and protein blaOXA-58, both in wild-type and mutant variants. The results were validated through molecular dynamics simulations lasting 100 ns. Docking results showed the binding score of Carbapenem drugs with wild-type blaOXA-58 protein, I87M, A88L, and double-mutated forms, which revealed that the single mutation enhanced the binding affinity of Carbapenems toward the protein. Meanwhile, the decreased binding affinity of Carbapenem toward the double-mutated protein may be due to combined effect of the mutations on the protein structure. Overall, Ertapenem showed good binding scores (-8.1, -8.8, -8.8, and -6.1 kcal/mol), and Imipenem showed weak affinity (-6.3, -5.6, -6.3, and -5 kcal/mol) toward wild-type protein, I87M, A88L, and the double-mutated blaOXA-58 protein, respectively. Wild and double-mutated complexes were subjected to molecular dynamics simulation which revealed mutant-ETP was the most stable complex with low RMSD, RMSF, RoG, and B-factor values. PCA showed reduced flexibility, mutation affected surface loop arrangements in mutant complexes, raising SASA in DOR/IPM, reducing it in ETP, minimal effect in MEM. In contrast, mutant-IPM had higher RMSD, RMSF, RoG, lower hydrogen bonding, and better flexibility. Mutant-DOR exhibited a loss of α-helices and β-strands. The outcomes of the current analysis demonstrated that slight mutation in blaOXA-58 affects working of carbapenem antibiotic. The complicated impact of the double mutation on stability and binding should improve antibiotic-resistant pathogen research by enhancing targeted medication or inhibitor design.

Microbial alginate lyases are essential biocatalysts for analyzing alginate structure and sustainably producing bioactive alginate oligosaccharides (AOS). In this study, we characterized Aly94, a novel alginate lyase from the polysaccharide lyase family 6 (PL6) family, identified from a marine sediment metagenomic library. Biochemical analyses showed Aly94 exhibits optimal activity at 40 ℃ in 50 mM NaH₂PO₄-Na₂HPO₄ buffer (pH 7.0). Adding 20 mM NaCl significantly increases its catalytic efficiency. The enzyme exhibits a strong preference for polyguluronate (polyG) over polymannuronate (polyM), with specific activities of 4.19 U/mg (polyG), 0.25 U/mg (polyM), and 2.45 U/mg (alginate). When degrading substrates-particularly polyG-Aly94 primarily generates trisaccharides. Although Aly94 acts as an endolytic alginate lyase, it also could digest the monosaccharides from small oligosaccharide chains (∆G3, ∆G4). These catalytic properties, combined with its polyG-specific depolymerization, made Aly94 a promising candidate for biotechnological applications requiring controlled alginate saccharification and high-value AOS production.

The global incidence of acute pancreatitis (AP) is progressively increasing with rising risk factors such as hyperlipidemia and alcoholism. Berberine (BBR), a quaternary ammonium alkaloid and primary antibacterial agent isolated from the traditional Chinese herb, Coptis chinensis, was investigated for its therapeutic mechanisms in AP. Cell viability was first assessed using the MTT assay at various BBR concentrations, with lactate dehydrogenase (LDH) levels measured in parallel. Then, ELISA quantified inflammatory mediators (caspase-1, IL-1β, IL-18, TNF-α, and IL-6), while western blot analyzed pyroptosis markers (NLRP3 and N-GSDMD). ROS and Fe²⁺ levels were detected using commercial kits. Moreover, molecular docking was performed to determine the interaction between BBR and Vanin 1 (VNN1). In vivo experiments were further conducted to assess BBR's role in AP. MTT assays confirmed BBR's non-cytotoxicity at therapeutic concentrations. BBR attenuated caerulein-induced cellular damage and pyroptosis in HPDE6-C7 cells. Molecular docking revealed strong BBR-VNN1 binding affinity (less than -6 kcal/mol). Moreover, VNN1 knockdown attenuated caerulein-induced HPDE6-C7 cell injury and pyroptosis; BBR achieved this effect by inhibiting VNN1 protein expression. BBR suppressed VNN1 expression and NF-κB pathway activation (p-p65/p65 ratio). In vivo validation confirmed BBR-mediated AP suppression via VNN1 inhibition. BBR mitigates AP by inhibiting VNN1 expression, thereby suppressing NF-κB pathway activation and attenuating caerulein-induced cellular damage and pyroptosis.

Plant-based oral vaccines are now becoming a more transformative medical form in immunotherapy; they are cheap and needle-free. These systems use natural bioencapsulation in plant cell walls, which preserves antigen stability within the gastrointestinal tract and promotes targeted release to mucosal immune tissues. Tests indicate that plant-derived antigens preserved their structure after exposure and they produced robust mucosal IgA and systemic IgG responses. From the early trial, such as seroconversion among human subjects consuming hepatitis B surface antigen (HBsAg)-expressing potatoes and high mucosal immunity generated by CTB-producing plants, promising results show their immunogenic potential. These technologies including chloroplast transformation that can produce more than 100 times higher antigen yields, CRISPR/Cas9 precision editing, and AI-equipped antigen design, further strengthen this platform. Although their relative strengths in standardization and poor digestibility are not widely available, plant-based oral vaccines have proven clear success with significant potential for immunization through functional foods, especially for resource-limited areas.

Over the past two decades, genome editing has advanced dramatically from Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) to more refined systems such as CRISPR-Cas9, prime editing, and nanoCas technologies. These innovations have opened new frontiers in cancer treatment. This review aims to critically examine and compare recent advances in these genome editing platforms, with a focus on their molecular mechanisms, delivery challenges, oncological applications, and clinical prospects. We systematically explore how CRISPR-Cas9 enables gene knockouts, high-throughput functional genomic screens, and immune editing, while acknowledging its limitations due to off-target effects and genotoxicity. In contrast, base and prime editors offer precise, double-strand breaks (DSBs) free alternatives, suitable for correcting oncogenic mutations such as TP53, KRAS, and EGFR. Prime editing, although versatile, faces delivery and efficiency challenges. The emergence of nanoCas systems, derived from compact Cas orthologs, provides promising delivery advantages for in vivo applications. We also examine how tumor microenvironment, cell-type specificity, and immune barriers impact editing efficacy and safety. Strategies such as high-fidelity variants, optimized guide RNAs, and stimuli-responsive nanoparticles are discussed to enhance precision and minimize risk. Conclusively, integrating these genome editing tools into oncology requires addressing translational barriers while harnessing their precision and therapeutic potential for next-generation cancer treatments.

This study investigated the antiviral effect of bee venom (BV) and chitosan nanoparticles (CNPs) obtained from Chrysomya albiceps maggots as natural mixture in vitro against herpes simplex viruses, HSV-1 and HSV-2 using TCID₅₀ assay. Also, investigation of molecular interactions of chitosan nanoparticles and BV proteins (Melittin and phospholipase A2) with viral enzymes using docking simulations was done. CNPs were prepared successfully in nanoscale (< 100 nm) and DDA of 85%. The mixture reduced the titers of HSV-1 and HSV-2 with 26.47% and 26.74%, respectively. In molecular docking simulations, CNPs exhibited strong binding affinity (- 9.9906 kcal/mol) to the Protease enzyme (2wpo), outperforming the co-crystallized ligand (- 8.1957 kcal/mol), aligning with in vitro results. Notably, CNPs did not bind to Thymidine kinase (2uz3), prompting further docking of BV proteins, which showed exceptional binding energies (- 14.9780 kcal/mol for melittin; - 16.9570 kcal/mol for phospholipase A2 with 2uz3, while failing to bind effectively to 2wpo. These findings strongly support a synergistic antiviral mechanism, where CNPs target Protease, while BV proteins inhibit Thymidine kinase, collectively enhancing antiviral efficacy against HSV-1 and HSV-2.