Release of a novel peptide from ferritin nanocages: A new tool for therapeutic applications.

The development of new drug delivery systems for targeted chemotherapy release in cancer cells represents a very promising tool. In this contest, protein-based nanocages have considerable potential as drug delivery devices. Notably, ferritin has emerged as an excellent candidate due to its unique architecture, surface properties and high biocompatibility. A promising strategy might then involve ferritin cargos for specifical release of AntiMicrobial Peptides endowed with anticancer activity to cancer cells. In this paper, we encapsulated the TRIL analogue of Temporin-L peptide within a ferritin nanocage and evaluated the cargo biological properties. The results demonstrated a reduced haemolytic activity of the peptide and a selective cytotoxicity activity on cancer cells likely mediated by oxidative stress while having no effects on non-tumoral cells. The combination of the properties of ferritin with TRIL, might open up the way to the development of novel peptide delivery systems for future pharmaceutical applications.

A new and efficient procedure to load bioactive molecules within the human heavy-chain ferritin nanocage.

For their easy and high-yield recombinant production, their high stability in a wide range of physico-chemical conditions and their characteristic hollow structure, ferritins (Fts) are considered useful scaffolds to encapsulate bioactive molecules. Notably, for the absence of immunogenicity and the selective interaction with tumor cells, the nanocages constituted by the heavy chain of the human variant of ferritin (hHFt) are optimal candidates for the delivery of anti-cancer drugs. hHFt nanocages can be disassembled and reassembled in vitro to allow the loading of cargo molecules, however the currently available protocols present some relevant drawbacks. Indeed, protein disassembly is achieved by exposure to extreme pH (either acidic or alkaline), followed by incubation at neutral pH to allow reassembly, but the final protein recovery and homogeneity are not satisfactory. Moreover, the exposure to extreme pH may affect the structure of the molecule to be loaded. In this paper, we report an alternative, efficient and reproducible procedure to reversibly disassemble hHFt under mild pH conditions. We demonstrate that a small amount of sodium dodecyl sulfate (SDS) is sufficient to disassemble the nanocage, which quantitatively reassembles upon SDS removal. Electron microscopy and X-ray crystallography show that the reassembled protein is identical to the untreated one. The newly developed procedure was used to encapsulate two small molecules. When compared to the existing disassembly/reassembly procedures, our approach can be applied in a wide range of pH values and temperatures, is compatible with a larger number of cargos and allows a higher protein recovery.

A bioinformatic study of antimicrobial peptides identified in the Black Soldier Fly (BSF) Hermetia illucens (Diptera: Stratiomyidae).

Antimicrobial peptides (AMPs) play a key role in the innate immunity, the first line of defense against bacteria, fungi, and viruses. AMPs are small molecules, ranging from 10 to 100 amino acid residues produced by all living organisms. Because of their wide biodiversity, insects are among the richest and most innovative sources for AMPs. In particular, the insect Hermetia illucens (Diptera: Stratiomyidae) shows an extraordinary ability to live in hostile environments, as it feeds on decaying substrates, which are rich in microbial colonies, and is one of the most promising sources for AMPs. The larvae and the combined adult male and female H. illucens transcriptomes were examined, and all the sequences, putatively encoding AMPs, were analysed with different machine learning-algorithms, such as the Support Vector Machine, the Discriminant Analysis, the Artificial Neural Network, and the Random Forest available on the CAMP database, in order to predict their antimicrobial activity. Moreover, the iACP tool, the AVPpred, and the Antifp servers were used to predict the anticancer, the antiviral, and the antifungal activities, respectively. The related physicochemical properties were evaluated with the Antimicrobial Peptide Database Calculator and Predictor. These analyses allowed to identify 57 putatively active peptides suitable for subsequent experimental validation studies.

Antimicrobial and Antibiofilm Peptides.

The increasing onset of multidrug-resistant bacteria has propelled microbiology research towards antimicrobial peptides as new possible antibiotics from natural sources. Antimicrobial peptides are short peptides endowed with a broad range of activity against both Gram-positive and Gram-negative bacteria and are less prone to trigger resistance. Besides their activity against planktonic bacteria, many antimicrobial peptides also show antibiofilm activity. Biofilms are ubiquitous in nature, having the ability to adhere to virtually any surface, either biotic or abiotic, including medical devices, causing chronic infections that are difficult to eradicate. The biofilm matrix protects bacteria from hostile environments, thus contributing to the bacterial resistance to antimicrobial agents. Biofilms are very difficult to treat, with options restricted to the use of large doses of antibiotics or the removal of the infected device. Antimicrobial peptides could represent good candidates to develop new antibiofilm drugs as they can act at different stages of biofilm formation, on disparate molecular targets and with various mechanisms of action. These include inhibition of biofilm formation and adhesion, downregulation of quorum sensing factors, and disruption of the pre-formed biofilm. This review focuses on the proprieties of antimicrobial and antibiofilm peptides, with a particular emphasis on their mechanism of action, reporting several examples of peptides that over time have been shown to have activity against biofilm.

Ecdysteroidogenesis in Heliothis virescens (Lepidoptera: Noctuidae): Recombinant Prothoracicotropic Hormone and Brain Extract Show Comparable Effects.

Prothoracicotropic hormone (PTTH) is a neuropeptide that triggers a cascade of events within the prothoracic gland (PG) cells, leading to the activation of all the crucial enzymes involved in ecdysone biosynthesis, the main insect steroid hormone. Studies concerning ecdysteroidogenesis predicted PTTH action using brain extract (BE), consisting in a complex mixture in which some components positively or negatively interfere with PTTH-stimulated ecdysteroidogenesis. Consequently, the integration of these opposing factors in steroidogenic tissues leads to a complex secretory pattern. A recombinant form of prothoracicotropic hormone (rPTTH) from the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) was expressed and purified to perform in vitro tests in a standard and repeatable manner. A characterization of rPTTH primary and secondary structures was performed. The ability of rPTTH and H. virescens BE to stimulate ecdysteroidogenesis was investigated on the third day of fifth larval stage. rPTTH activity was compared with the BE mixture by enzyme immunoassay and western blot, revealing that they equally stimulate the production of significant amount of ecdysone, through a transduction cascade that includes the TOR pathway, by the phosphorylation of 4E binding protein (4E-BP) and S6 kinase (S6K), the main targets of TOR protein. The results of these experiments suggest the importance of obtaining a functional pure hormone to perform further studies, not depending on the crude brain extract, composed by different elements and susceptible to different uncontrollable variables.

PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment.

PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation.