Combined fluorometric analysis of biliverdin and bilirubin by the recombinant protein HUG.

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.

Standardized lab-scale production of the recombinant fusion protein HUG for the nanoscale analysis of bilirubin.

The recombinant bifunctional protein HELP-UnaG (HUG) is a fusion product of the Human Elastin-like Polypeptide (HELP) with the bilirubin-binding fluorescent protein UnaG. HUG is used for the fluorometric detection of bilirubin in serum and a variety of biological fluids and extracts. Here we describe a detailed method for the standardized production and purification of HUG from E. coli extracts on a laboratory scale. This method takes advantage of the HELP-specific thermoreactive behavior that enables the separation of HUG from complex E. coli extracts by repeated precipitation/re-dissolution steps at near physiological temperature.•The method is based on the inverse thermal transition process.•The "green" method is affordable for basic laboratories and can be easily transferred to new users.

Materials derived from the human elastin-like polypeptide fusion with an antimicrobial peptide strongly promote cell adhesion.

Protein and peptide materials have attracted great interest in recent years, especially for biological applications, in light of their possibility to easily encode bioactivity whilst maintaining cytocompatibility and biodegradability. Heterologous recombinant expression to produce antimicrobial peptides is increasingly considered a convenient alternative for the transition from conventional methods to more sustainable production systems. The human elastin-like polypeptide (HELP) has proven to be a valuable fusion carrier, and due to its cutting-edge properties, biomimetic materials with antimicrobial capacity have been successfully developed. In this work, we have taken advantage of this platform to produce a difficult-to-synthesise sequence as that of the human ß-defensin 1 (hBD1), an amphipathic cationic peptide with structural folding constraints relevant to its bioactivity. In the design of the gene, highly specific endoproteinases recognition sites were introduced to release the active forms of hBD1. After the expression and purification of the new fusion construct, its biological activity was evaluated. It was found that both the fusion biopolymer and the released active forms can inhibit the growth of Escherichia coli in redox environments. Remarkably, 2D and 3D materials derived from the biopolymer showed a strong cell adhesion-promoting activity. These results suggest that HELP represents a multitasking platform that not only facilitates the production of bioactive domains and derived materials but could also pave the way for the development of new approaches to study biological interactions at the molecular level.

1,4-dihydroxy quininib activates ferroptosis pathways in metastatic uveal melanoma and reveals a novel prognostic biomarker signature.

Uveal melanoma (UM) is an ocular cancer, with propensity for lethal liver metastases. When metastatic UM (MUM) occurs, as few as 8% of patients survive beyond two years. Efficacious treatments for MUM are urgently needed. 1,4-dihydroxy quininib, a cysteinyl leukotriene receptor 1 (CysLT1) antagonist, alters UM cancer hallmarks in vitro, ex vivo and in vivo. Here, we investigated the 1,4-dihydroxy quininib mechanism of action and its translational potential in MUM. Proteomic profiling of OMM2.5 cells identified proteins differentially expressed after 1,4-dihydroxy quininib treatment. Glutathione peroxidase 4 (GPX4), glutamate-cysteine ligase modifier subunit (GCLM), heme oxygenase 1 (HO-1) and 4 hydroxynonenal (4-HNE) expression were assessed by immunoblots. Biliverdin, glutathione and lipid hydroperoxide were measured biochemically. Association between the expression of a specific ferroptosis signature and UM patient survival was performed using public databases. Our data revealed that 1,4-dihydroxy quininib modulates the expression of ferroptosis markers in OMM2.5 cells. Biochemical assays validated that GPX4, biliverdin, GCLM, glutathione and lipid hydroperoxide were significantly altered. HO-1 and 4-HNE levels were significantly increased in MUM tumor explants from orthotopic patient-derived xenografts (OPDX). Expression of genes inhibiting ferroptosis is significantly increased in UM patients with chromosome 3 monosomy. We identified IFerr, a novel ferroptosis signature correlating with UM patient survival. Altogether, we demontrated that in MUM cells and tissues, 1,4-dihydroxy quininib modulates key markers that induce ferroptosis, a relatively new type of cell death driven by iron-dependent peroxidation of phospholipids. Furthermore, we showed that high expression of specific genes inhibiting ferroptosis is associated with a worse UM prognosis, thus, the IFerr signature is a potential prognosticator for which patients develop MUM. All in all, ferroptosis has potential as a clinical biomarker and therapeutic target for MUM.

A Versatile Elastin-Like Carrier for Bioactive Antimicrobial Peptide Production and Delivery.

Elastin-like polypeptides are biotechnological protein and peptide carriers that offer a vast scope of applicability. This work aims to build a model for the expression of antimicrobial peptides (AMPs) by genetically engineering the Human Elastin-like Polypeptide platform developed in the lab. The well-characterized AMP indolicidin is selected as an example of an antimicrobial domain for the recombinant fusion at the C-terminus of the carrier. The fusion construct has been designed to allow the release of the antimicrobial domain. The expression product has been purified and its physicochemical and antimicrobial properties has been characterized. Taking advantage of the self-assembling and matrix-forming properties of the recombinant biopolymer, the materials that are obtained have been evaluated for antimicrobial activity toward bacterial-strain models. This approach represents a cost-effective strategy for the production of smart components and materials endowed with antimicrobial capacity triggered by external stimuli.

Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG.

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.

Physicochemical Characterization of a Biomimetic, Elastin-Inspired Polypeptide with Enhanced Thermoresponsive Properties and Improved Cell Adhesion.

Genetic engineering allows fine-tuning and controlling protein properties, thus exploiting the new derivatives to obtain novel materials and systems with improved capacity to actively interact with biological systems. The elastin-like polypeptides are tunable recombinant biopolymers that have proven to be ideal candidates for realizing bioactive interfaces that can interact with biological systems. They are characterized by a thermoresponsive behavior that is strictly related to their peculiar amino acid sequence. We describe here the rational design of a new biopolymer inspired by elastin and the comparison of its physicochemical properties with those of another already characterized member of the same protein class. To assess the cytocompatibility, the behavior of cells of different origins toward these components was evaluated. Our study shows that the biomimetic strategy adopted to design new elastin-based recombinant polypeptides represents a versatile and valuable tool for the development of protein-based materials with improved properties and advanced functionality.

Cyanidin 3-glucoside targets a hepatic bilirubin transporter in rats.

One of the organ-specific functions of the liver is the excretion of bilirubin into the bile. Membrane transport of bilirubin from the blood to the liver is not only an orphan function, because there is no link to the protein/gene units that perform this function, but also a poorly characterised function. The aim of this study was to investigate the pharmacology of bilirubin uptake in the liver of the female Wistar rat to improve basic knowledge in this neglected area of liver physiology. We treated isolated perfused livers of female rats with repeated single-pass, albumin-free bilirubin boli. We monitored both bilirubin and bilirubin glucuronide in perfusion effluent with a bio-fluorometric assay. We tested the ability of nine molecules known as substrates or inhibitors of sinusoidal membrane transporters to inhibit hepatic uptake of bilirubin. We found that cyanidin 3-glucoside and malvidin 3-glucoside were the only molecules that inhibited bilirubin uptake. These dietary anthocyanins resemble bromosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cyanidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indomethacin and ketoprofen were inactive. We demonstrated the existence of a bilirubin-glucuronide transporter inhibited by bilirubin, a fact reported only once in the literature. The data suggest that bilirubin and bilirubin glucuronide are transported to the liver via pharmacologically distinct membrane transport pathways. Some dietary anthocyanins may physiologically modulate the uptake of bilirubin into the liver.

Macromolecular and Solution Properties of the Recombinant Fusion Protein HUG.

The recombinant fusion protein HELP-UnaG (HUG) is a bifunctional product that exhibits human elastin-like polypeptide (HELP)-specific thermal behavior, defined as a reverse phase transition, and UnaG-specific bilirubin-dependent fluorescence emission. HUG provides an interesting model to understand how its two domains influence each other's properties. Turbidimetric, calorimetric, and light scattering measurements were used to determine different parameters for the reverse temperature transition and coacervation behavior. This shows that the UnaG domain has a measurable but limited effect on the thermal properties of HELP. Although the HELP domain decreased the affinity of UnaG for bilirubin, HUG retained the property of displacing bilirubin from bovine serum albumin and thus remains one of the strongest bilirubin-binding proteins known to date. These data demonstrate that HELP can be used to create new bifunctional fusion products that pave the way for expanded technological applications.

Organic aggregates formed by benthopleustophyte brown alga Acinetospora crinita (Acinetosporaceae, Ectocarpales).

This work presents the elemental, polysaccharide, and fatty acid compositions of benthic aggregates formed by the filamentous brown alga Acinetospora crinita, which are widely spread on the rocky bottoms of the Mediterranean Sea. The aggregates can be characterized as mineralized centers in which regeneration of nutrients and recycling of dissolved organic matter actively occur and favor the development of an abundant phytoplankton community. Analyses of the stable isotopes of C and N display their marine origin and could provide evidence of the processes that occur inside/outside of the aggregates. The monosaccharide compositions of Adriatic and Tyrrhenian mucilages produced by brown alga A. crinita were quite similar. In particular, the Adriatic sample compositions resembled the average composition of the Tyrrhenian high molecular weight exopolymers, and the observed differences could be ascribed to different degradation stages. The fatty acid patterns found for the aggregates were similar to those observed in the isolated A. crinita algae with variable contributions from embedded diatom species. The bacterial contribution to the fatty acid pool was quite low, most likely due to the known poor conditions for their heterotrophic growth.

[Particulate matter and atmospheric pollutants exposure for outdoor workers in the Muggia area (Trieste, Italy)]. / Indagine sull'esposizione a polveri ed inquinanti atmosferici di lavoratori outdoor nell'area di Muggia (Trieste).

UNLABELLED: Urban air pollution originates from traffic, heating and industrial activities and it exposes several categories of workers to noxious chemicals; in recent years various surveys have been carried on taxi drivers, bus drivers, policemen, newspaper kiosks, fuel station workers and general population, for assessing exposure to main contaminants, benzene and PAHs in particular. AIM: defining a specific sampling strategy for collecting an experimental data base for the assessment of potential population exposure to atmospheric particulate matter in the Muggia area (Trieste, North-East Italy). MATERIALS AND METHODS: A sample of 14 urban police officers working mainly on the roads and potentially subject to occupational exposure has been analyzed, as well as a sample of the municipal administrative personnel (13 people) working in indoor environments during the same sampling periods. Exposure has been monitored by personal samplers and high volume environmental samples; urinary levels of 1 OH-pyrene and creatinine were collected for each participant at the end of each sampling period. RESULTS: PAHs analyses provided diagnostic ratios for identification of the sources of pollution. In general experimental BaP values stay below the environmental target value, with satisfactory agreement between environmental and personal monitoring. Levels of urinary 1 OH-pyrene for administrative personnel was detected always below the background value (0.089 micromoles/mole creatinine), while they are above the background and below the attention value for most of urban police officers. CONCLUSIONS: Municipal police officers participating to the present study, while belonging to one of the categories of people most exposed to outdoor PM, do not show any worrying exposure to the considered chemicals associated to their occupational activity, in comparison to other factors, possibly associated to personal lifestyles.

Spontaneous patterning obtained by evaporation of Human Elastin-like Polypeptide solutions.

The potential of producing patterned, bioactive coatings with Human Elastin-Like Polypeptides (HELPs) has been investigated. The physicochemical features of these compounds have evidenced some differences between the two recombinantly expressed products. By a device-free, simple route and avoiding the use of chemically unfriendly compounds, micropatterned surfaces with the ability to control cell behavior could be obtained. Thus, HELPs represent a very promising class of macromolecule for future applications in surface engineering.

Comparison of thermal behavior of two recombinantly expressed human elastin-like polypeptides for cell culture applications.

Two synthetic genes that code for artificial proteins have been constructed that were modeled on the most regularly repeated hydrophobic domain of human tropoelastin. We compare the physicochemical properties of the recombinant products that differ in their primary structure; the alanine/lysine-rich cross-linking domains, which are highly conserved in mammalian tropoelastin, were either present or absent in the recombinant products. Both biopolymers showed thermoresponsive properties, and variations were observed that were dependent on solution conditions. Cell compatibility was assayed using the biopolymers as coating agents in culture experiments with a neuroblastoma cell line; cell adhesion and proliferation effects were evaluated. The cells were found to retain their neural differentiation potential. The data presented in our work support the usefulness of these versatile biopolymers for a variety of applications related to biotechnology and biomedicine.

Relevance of the dinoflagellate Gonyaulax fragilis in mucilage formations of the Adriatic Sea.

Oceanographic cruises were carried out monthly from June 1999 to July 2002 to follow the mucilage formation process in the Northern Adriatic Sea. Results show that in correspondence with these events the dinoflagellate Gonyaulax fragilis (Schütt) Kofoid was observed both in the water column and within mucilage aggregates. In the water column, increasing abundances were observed from May until July, with values never exceeding 8500 cells l(-1). Much higher densities were observed within superficial gelatinous aggregates (22800-3400000 cells l(-1)). In mucilage samples, a large number of decomposing cells were present, together with abundant alive cells, enveloped in exudates. G. fragilis isolated from mucilage samples was cultured in three different culture media; it was characterized by a low growth rate but it produced a high amount of polysaccharides. The highest yield both in terms of cell number and carbohydrate production was observed in the medium having the highest nitrogen and phosphorus content and the lowest N/P ratio. The monomeric composition of G. fragilis carbohydrates, compared with that of mucilage samples, showed that in both natural and cultured samples galactose was the most abundant sugar; in addition, an overall good correlation, especially between the monomeric carbohydrate composition of G. fragilis grown in f/2 medium and that of a mucilage sample in which this species was present in high density, was observed.

Extracellular enzyme activity and dynamics of bacterial community in mucilaginous aggregates of the northern Adriatic Sea.

Bacterial degradation of mucilaginous aggregates (creamy layers, stringers and macroflocs) collected during two summer events (2001-2002) was tested. The objective was to describe the temporal trend of the bacterial activity, abundance and composition in the aggregated and dissolved organic matter under different trophic conditions. In the native aggregates proteins and organic phosphorous were actively hydrolyzed as aminopeptidase and alkaline phosphatase activities represented up to 87% and 25% of total activity, respectively; polysaccharides were less hydrolyzed and the highest activities were observed for beta-glucosidase (5% of the total). This hydrolysation pattern tends to a progressive accumulation of long persistent polysaccharides. During short term incubations nutrient addition (P, N and Glucose) differently stimulated bacterial growth in the seawater: P played the main role in stimulating bacterial production from 3 to 6 folds higher than in the control, whereas a secondary C-limitation was observed only for bacteria growing on seawater from macroflocs. This scarce dissolved organic carbon (DOC) bioavailability was confirmed by the lower DOC removal (13% macroflocs, 36% stringers). The total amount of carbon incorporated by bacteria living on aggregates was similar (0.58 mg C L(-1)) both in the control and under P enrichments showing a more balanced condition with respect to the seawater. Hence the well-known P limitation in the Northern Adriatic Sea affects only dissolved organic carbon uptake without influencing the uptake of aggregated organic matter. Organic matter limitation was observed only on stringers--total C incorporated raised to 0.96 mg C L(-1) after PNG addition. Macroflocs release of refractory compounds leads to DOC accumulation (73 microM DOC) contributing to inflate the pool of refractory DOC in the surrounding waters. Several evidences, including different monosaccharide composition of stringers and macroflocs (glucose 15% and 56% on stringers and macroflocs, respectively), bring to the conclusion that stringers are in an older stage in comparison with macroflocs. Community composition described by fluorescence in situ hybridization did not show significant differences between free-living and attached bacteria but it was modified by the different enrichment conditions: Cytophaga-Flavobacteria increased after inorganic nutrients enrichments while organics advantaged gamma-Proteobacteria.

Chemical characterization of different typologies of mucilaginous aggregates in the Northern Adriatic Sea.

The chemical composition of mucilage aggregates found during summer 2000, 2001 and 2002 in the North Adriatic Sea depends on the nature of the organic matter during aggregation, on the environmental conditions of the site of formation and on the transformations during ageing. The mucilages were composed of organic matter, together with a significant inorganic fraction. Elemental analysis revealed 12.5-32.2% of organic carbon, 0-7.3% of inorganic carbon and 1.0-3.7% of nitrogen. The C(org)/N ratios of most aggregates were between 7.5 and 12.6, values close to those found in the suspended matter; higher ratios were found in large-size (>5 m) aggregates which are probably older. The content of carbohydrates and proteins determined in the aggregates, respectively, 15.4+/-8.9% and 7.9+/-4.8%, w/w, showed a prevalence of carbohydrates over proteins. Neutral carbohydrate analysis of purified polysaccharides from mucilage samples showed very similar signatures with high relative abundance of galactose and glucose. Humic, fulvic and humin substances extracted from the mucilages constitute an important fraction of the organic matter in the aggregates. The humin (a fraction insoluble in acidic and basic media) was present in all mucilage samples, indicating the refractory nature of a part of the organic matter in the mucilage. The iron and calcium could play a role during the aggregation process to form a complex with polysaccharides and humic fractions. The C(org)/N ratio 10+/-2 found in the humic acids extracted from the Adriatic aggregates disclosed a marine origin. The low phosphorus content and the high C(org)/P ratio found in the aggregates might depend from high bacteria activity or from the aggregation of organic fractions depleted of phosphorus. The principal inorganic species contained aluminium and silicon, part of which was of biogenic origin and was more significant in the offshore mucilage aggregates than in the coastal ones. The Si(biog)/C(org) ratio showed that diatoms were always present in the aggregates, although it cannot be established whether these are the producers or these develop within the aggregates.

Extracellular carbohydrates released by the marine diatoms Cylindrotheca closterium, Thalassiosira pseudonana and Skeletonema costatum: effect of P-depletion and growth status.

A laboratory study was performed on the extracellular production of carbohydrates by the marine diatoms Cylindrotheca closterium, Thalassiosira pseudonana and Skeletonema costatum. The investigation was aimed at elucidating the role of P-starvation and growth status on abundance and chemical characteristics of the released non-attached polysaccharides. Inorganic phosphorus depletion determined an increase of total polysaccharides in all species examined compared to nutrient-replete (complete f/2) conditions. The highest abundance of polysaccharides per unit cell was found in T. pseudonana (28.4 micromol C 10(-6) cells), followed by C. closterium (2.56 micromol C 10(-6) cells) and S. costatum (1.18 micromol C 10(-6) cells). Maximum production rates were found at the transition between exponential and stationary growth phase. Gas-chromatographic analysis of the dissolved fraction showed glucose to be the most abundant monomer in exponentially growing, P-replete cultures (81.6%, 90% and 32% as molar percentage of total aldoses in C. closterium, T. pseudonana and S. costatum, respectively). A strong reduction in glucose was found in C. closterium, but not in T. pseudonana and S. costatum, under P-depleted conditions. Species-specific variations in the amount and aldose signatures of the released polysaccharides according to nutrient status and growth conditions can provide useful insights on the production and persistence of these organic compounds in the water column.

Conformational features of cepacian: the exopolysaccharide produced by clinical strains of Burkholderia cepacia.

Conformational energy calculations and molecular dynamics investigations, both in water and in dimethyl sulfoxide, were carried out on the exopolysaccharide cepacian produced by the majority of the clinical strains of Burkholderia cepacia, an opportunistic pathogen causing serious lung infection in patients affected by cystic fibrosis, The investigation was aimed at defining the structural and conformational features, which might be relevant for clarification of the structure-function relationships of the polymer. The molecular dynamics calculations were carried out by Ramachandran-type energy plots of the disaccharides that constitute the polymer repeating unit. The dynamics of an oligomer composed of three repeating units were investigated in water and in Me2SO, a non-aggregating solvent. Analysis of the time persistence of hydrogen bonds showed the presence of a large number of favourable interactions in water, which were less evident in Me2SO. The calculations on the cepacian chain indicated that polymer conformational features in water were affected by the lateral chains, but were also largely dictated by the presence of solvent. Moreover, the large number of intra-chain hydrogen bonds in water disappeared in Me2SO solution, increasing the average dimension of the polymer chains.

Macromolecular and solution properties of Cepacian: the exopolysaccharide produced by a strain of Burkholderia cepacia isolated from a cystic fibrosis patient.

Light scattering and viscosity measurements were carried out on the previously chemically characterised exopolysaccharide produced by a strain of Burkholderia cepacia isolated from a cystic fibrosis patient. The same exopolysaccharide was also produced by other clinical strains in different laboratories. Therefore, the name Cepacian is now proposed for this exopolysaccharide. Experiments performed as a function of the ionic strength on the native polymer revealed a change in the overall shape of the polymer at low ionic strength. This behaviour was absent in the de-acetylated sample. Potentiometric titrations and light scattering experiments carried out on the acidic form of the native polymer revealed the formation of macromolecular aggregates with a stoichiometry n and 2n stabilised by interactions involving the uronic acid residues.