In recent years, antimicrobial peptides (AMPs) have enjoyed a renaissance, as the world is currently facing an emergency in terms of severe infections that evade antibiotics’ treatment. This is due to the increasing emergence and spread of resistance mechanisms. Covalent conjugation with polymers is an interesting strategy to modulate the pharmacokinetic profile of AMPs and enhance their biocompatibility profile. It can also be an effective approach to develop active coatings for medical implants and devices, and to avoid biofilm formation on their surface. In this concise review, we focus on the last 5 years’ progress in this area, pertaining in particular to AMPs that contain d-amino acids, as well as their role, and the advantages that may arise from their introduction into AMPs.

Bilirubin is a toxicological biomarker for hemolysis and liver diseases. The current automated diazo method used in clinical chemistry has limited applicability in rodent models and cannot be used in small animals relevant to toxicology, microphysiological systems, cell cultures, and kinetic studies. Here, we present a versatile fluorometric method for nanoscale analysis of bilirubin based on its highly specific binding to the recombinant bifunctional protein HELP–UnaG (HUG). The assay is sensitive (LoQ = 1.1 nM), accurate (4.5% relative standard error), and remarkably robust, allowing analysis at pH 7.4–9.5, T = 25–37 °C, in various buffers, and in the presence of 0.4–4 mg × L⁻¹ serum albumin or 30% DMSO. It allows repeated measurements of bilirubinemia in murine models and small animals, fostering the 3Rs principle. The assay determines bilirubin in human plasma with a relative standard error of 6.7% at values that correlate and agree with the standard diazo method. Furthermore, it detects differences in human bilirubinemia related to sex and UGT1A1 polymorphisms, thus demonstrating its suitability for the uniform assessment of bilirubin at the nanoscale in translational and precision medicine.

Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins such as hemoglobin, myoglobin, cytochromes, and catalase into biliverdin, iron (II) and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase. Biliverdin and bilirubin form a redox couple and are important for the redox homeostasis of cells. Heme oxygenase-1 is an inducible enzyme that is induced by hypoxic conditions, increased availability of heme or proinflammatory mechanisms such as LPS, UV radiation, etc. In addition, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis by modulating specific metabolic pathways at the transcriptional level. There is a need for affordable assays to analyze these bile pigments in biological and clinical samples. Here we present a method for the combined determination of biliverdin and bilirubin that utilizes the specific binding of bilirubin to the fluorescent recombinant fusion protein HUG and the enzymatic conversion of biliverdin to bilirubin. • This method enables the combined measurement of bilirubin and biliverdin in the nM range. • The method does not require solvent extraction or protein precipitation of the samples.

Bilirubin (BR) is the product of cellular heme catabolism and the major bile pigment in animal blood. It is an established biomarker of hemolysis and liver function. In the last decade, mild hyperbilirubinemia has been shown to be a biomarker of lower cardiovascular disease risk, attributed to its antioxidant and anti-inflammatory effects. To use bilirubin as a predictive biomarker, new powerful methods are currently being developed for its direct analysis in human blood. To harmonize the different methods, it is essential to use high-quality BR standard solutions for assay calibration. We present here a protocol for the preparation of stable standard solutions in the range of 10^-9- 10^-5 M BR at pH 7.4 that can facilitate a universal approach to assay calibration.

HUG is the HELP-UnaG recombinant fusion protein featuring the typical functions of both HELP and UnaG. In HUG, the HELP domain is a thermoresponsive human elastin-like polypeptide. It forms a shield enwrapping the UnaG domain that emits bilirubin-dependent fluorescence. Here, we recapitulate the technological development of this bifunctional synthetic protein from the theoretical background of its distinct protein moieties to the detailed characterization of its macromolecular and functional properties. These pieces of knowledge are the foundations for HUG production and application in the fluorometric analysis of bilirubin and its congeners, biliverdin and bilirubin glucuronide. These bile pigments are metabolites that arise from the catabolism of heme, the prosthetic group of cytochromes, hemoglobin and several other intracellular enzymes engaged in electron transfer, oxygen transport and protection against oxygen free radicals. The HUG assay is a powerful, user-friendly and affordable analytical tool that alone supports research at each level of complexity or taxonomy of living entities, from enzymology, cell biology and pathophysiology to veterinary and clinical sciences.

We present a protocol for the study of bilirubin uptake in the isolated and perfused rat liver. The liver is perfused with an albumin-free saline buffered solution supplemented with glucose, pyruvate and lactate, in the absence of oxygen, at a physiologically low flow rate. Fractions of the venous effluent are collected and analyzed for bilirubin, bilirubin glucuronide and biomarkers of liver integrity. The liver preparation is viable and intact for 1 h after isolation from the general circulation, with constant levels of both bilirubin and bilirubin glucuronide (< 2 nM) in the effluent. Up to 12 boli of 10 nmol bilirubin can be sequentially injected into the portal vein without and with molecules that target sinusoidal membrane transporters of organic anions. Selective inhibition of bilirubin or bilirubin glucuronide uptake is detected as transient peaks in the effluent (C_max up 6 to 60 nM). This protocol allows collecting repeated observations in the same liver, thus reducing the animal number by a factor of 10.