1) MOLECULAR NUTRITION Bilirubin Diet Interactions
Bilirubin is an endogenous antioxidant derived from heme metabolism. It circulates in the blood, bound to albumin, and is excreted by the liver through bile. Mild hyperbilirubinemia is associated with a lower risk of cardiovascular disease and other chronic diseases. A diet rich in plant foods also reduces the risk of age-related chronic diseases. Some phytochemicals, such as flavonoids and including anthocyanins, could cause a slight increase in bilirubinemia by acting on hepatic bilirubin transport or bilirubin synthesis enzymes.
To investigate the factors that modulate bilirubinemia, we have developed a kit for fluorometric analysis of bile pigments that we can use in a variety of experimental models as well as in clinical and veterinary analyses. The kit uses a specific bilirubin bioreceptor developed and manufactured in our group called HUG. It is the fusion product of a HELP polypeptide with the UnaG protein.
2) Elastin-like polypeptides for diagnosis and controlled release of biodrugs?
Recombinantly produced biomimetic proteins represent a promising alternative to synthetic polymers for biomedical and biotechnological applications. Elastin-like polypeptides (ELPs) are artificial repetitive protein biopolymers derived from the hydrophobic domains of elastin. Like the latter, they exhibit interesting thermosensitive behavior in that they are completely soluble below a certain temperature, whereas they aggregate and undergo phase separation upon a temperature rise. The research activity has led to the creation of a prototype macromolecule called HELP (Human Elastin-like Polypeptide), whose sequence is based on the repeated hexapeptide motif and the cross-linking domains of human elastin. Subsequently, an enzymatic method for the preparation of hydrogel-like matrices from HELP was developed, which led to the filing of a patent application. Of extreme interest is the possibility of creating "tailor-made" biomaterials with precisely tunable specific functional properties. So far, about twenty constructs based on HELP with different functionalities have been fabricated in our laboratory. This branch of research is characterized by the extremely innovative content, both in terms of approach, aimed at creating new macromolecules that do not exist in nature but are assembled in the laboratory, as well as in terms of potential, since it opens the door to new scenarios that need to be explored and have great potential for application. Being in the field of biotechnology, a cutting-edge and distinctly multidisciplinary sector, these studies benefit greatly from the contributions of several disciplines such as molecular biology, physics, engineering, biochemistry, etc., while offering opportunities for collaboration between partners that have developed these expertises.
Ongoing projects
A) Precision diagnostics of bile pigments in biological matrices. We have developed a kit for the analysis of bile pigments in animal blood. The kit is based on the polypeptide HUG and allows nanoscale analysis of biliverdin, bilirubin and bilirubin glucuronide.
B) New blood biomarkers for farmed fish welfare assessment. Within the AdriAquaNet project, we have elaborated the operational protocols for the analysis of whole blood samples from experimental animals (rat), farmed fish and human subjects. We have analysed groups of fish reared under different conditions and on experimental diets. The results show that bile pigments are useful biomarkers for quantifying stress (or well-being) in farmed fish.
C) Screening of bile pigments in human plasma biobanks. As part of the iNEST project, we will screen plasma biobanks from patients recruited to clinical trials to identify novel biomarkers for the efficacy of dietary strategies to reduce the risk of chronic disease.
D) Heme catabolism in cell cultures. Heme is the coenzyme of cytochromes and many other key enzymes for cell growth and differentiation. In the iNEST project, we are developing an approach for assessing cell growth in vitro based on the production of intracellular and extracellular heme catabolites. This approach can be applied to microphysiological systems (organ-on-chip).
E) Development of HELP polypeptides and lo-conjugates. The polypeptide HELP has been conjugated to the epidermal growth factor (EGF) and to the RGD sequences that promote cell adhesion.however, it can also be conjugated to other proteins that play a key rolein binding to membrane receptors and activating intracellular signaling pathways required for growth and differentiation. In the iNEST project, we will use these polypeptides to obtain composite materials suitable for the development of in vitro micro-physiological systems.
F) Components with antimicrobial capacitythat prevent antibiotic resistance.HELP Polypeptides are still underexploited fusion partners for the production of antimicrobial peptides. These latter are currently considered promising biodrugs that can replace conventional antibiotics. In the European Union-funded projects AIMED and STOP, the HELP platform is used to produce fusion proteins with antibiotic domains derived from human b-defensins. They will be used to produce biocompatible materials and surfaces that cannot be colonized by bacteria and do not induce resistance.
