We report a Cu^II peptide helicate that selectively binds DNA 3WJs in cells, damaging DNA replication foci by ROS production, thus demonstrating for the first time the selective cleavage of this noncanonical DNA structure with a chemical nuclease.

The accumulation of plastics in the environment has become a serious concern for the entire society. In recent years, enzyme-based biodegradation has emerged as a promising and sustainable strategy for the recycling of polyethylene terephthalate (PET), one of the most widely used polyester plastics. However, the translation of these technologies to the industrial field faces several underexplored challenges, including the immobilization and reusability of the biocatalysts. Here, we present the use of IC-Tagging as a novel one-step methodology for the "in cellulo" self-immobilization of the benchmark PET-degrading enzyme LCC^ICCG in protein nanospheres. The immobilized enzyme showed to be active against soluble substrates and exhibited improved thermal resistance and long-term storage stability, retaining 58 % of relative activity after 3 months at room temperature. Immobilized LCC^ICCG also demonstrates remarkable reusability, with minor activity loss up to 10 reuse cycles. Most importantly, nearly complete depolymerization (>90 %) of various untreated amorphous post-consumer PET materials was achieved at a wide range of temperatures (50-70 °C) by removing the products and reusing the enzyme repeatedly. Furthermore, reutilization led to almost full degradation of two consecutive batches of post-consumer PET in 6 days, outperforming all immobilized biocatalysts reported at laboratory scale. Overall, IC-Tagging emerges as a promising and versatile platform for the production, immobilization and reutilization of top-performing PET hydrolases, contributing to sustainable management of plastic waste.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that cause important economic losses in the livestock and equine industries, respectively. In addition to these, another arthropod-transmitted orbivirus known as epizootic hemorrhagic disease virus (EHDV) entails a major threat as there is a conducive landscape that nurtures its emergence in non-endemic countries. To date, only vaccinations with live attenuated or inactivated vaccines permit the control of these three viral diseases, although important drawbacks, e.g., low safety profile and effectiveness, and lack of DIVA (differentiation of infected from vaccinated animals) properties, constrain their usage as prophylactic measures. Moreover, a substantial number of serotypes of BTV, AHSV and EHDV have been described, with poor induction of cross-protective immune responses among serotypes. In the context of next-generation vaccine development, antigen delivery systems based on nano- or microparticles have gathered significant attention during the last few decades. A diversity of technologies, such as virus-like particles or self-assembled protein complexes, have been implemented for vaccine design against these viruses. In this work, we offer a comprehensive review of the nano- and microparticulated vaccine candidates against these three relevant orbiviruses. Additionally, we also review an innovative technology for antigen delivery based on the avian reovirus nonstructural protein muNS and we explore the prospective functionality of the nonstructural protein NS1 nanotubules as a BTV-based delivery platform.

A methodology that programs eukaryotic or bacterial cells to encapsulate proteins of any kind inside micro/nanospheres formed by muNS-Mi viral protein was developed in our laboratory. In the present study such “in cellulo” encapsulation technology is utilized for immobilizing a protein with an enzymatic activity of industrial interest, CotA laccase. The encapsulation facilitates its purification, resulting in a cost-effective, one-step way of producing immobilized enzymes for industrial use. In addition to the ability to be recycled without activity loss, the encapsulated protein showed an increased pH working range and high resistance to chemical inactivation. Also, its activity was almost unaffected after 30 min incubation at 90 °C and 15 min at the almost-boiling temperature of 95 °C. Furthermore, the encapsulated laccase was able to efficiently decolorate the recalcitrant dye RB19 at room temperature.

Cu(II) coordination complexes are emerging as promising anticancer agents due to their ability to induce oxidative stress through reactive oxygen species (ROS) generation. In this study, we synthesized and characterized two novel Cu(II) metallopeptide systems, 1/Cu(II) and 2/Cu(II), derived from the oligocationic bipyridyl cyclopeptides 1 and 2, and designed to enhance the transport of Cu(II) into cells and increase ROS levels. Spectroscopic and electrochemical analyses confirmed the formation of stable metallopeptide species in aqueous media. Inductively coupled plasma mass spectrometry (ICP-MS) studies demonstrated that both metallopeptides significantly increase intracellular Cu(II) accumulation in NCI/ADR-RES cancer cells, highlighting their role as efficient Cu(II) transporters. Additionally, ROS generation assays revealed that 1/Cu(II) induces a substantial increase in intracellular ROS levels, supporting the hypothesis of oxidative stress-induced cytotoxicity. Cell-viability assays further confirmed that both 1/Cu(II) and 2/Cu(II) exhibit strong anticancer activity in a number of cancer cell lines, with IC₅₀ values significantly lower than those of their free cyclopeptide counterparts or Cu(II) alone, showing an order of activity higher than that of cisplatin. Finally, molecular modeling studies provided further insights into the structural stability and coordination environment of Cu(II) within the metallopeptide complexes. These findings suggest that these Cu(II) cyclometallopeptide systems hold potential as novel metal-based therapeutic agents, leveraging Cu(II) transport and ROS increase as key strategies for cancer treatment.

Gene therapy provides exciting new therapeutic opportunities beyond the reach of traditional treatments. Despite the tremendous progress of viral vectors, their high cost, complex manufacturing, and side effects have encouraged the development of nonviral alternatives, including cationic polymers. However, these are less efficient in overcoming cellular barriers, resulting in lower transfection efficiencies. Although the exquisite structural tunability of polymers might be envisaged as a versatile tool for improving transfection, the need to fine-tune several structural parameters represents a bottleneck in current screening technologies. By taking advantage of the fast-forming and strong boronate ester bond, an archetypal example of dynamic covalent chemistry, a highly adaptable gene delivery platform is presented, in which the polycation synthesis and pDNA complexation occur in situ. The robustness of the strategy entitles the simultaneous evaluation of several structural parameters at will, enabling the accelerated screening and adaptive optimization of lead polymeric vectors using dynamic covalent libraries.