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

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 β-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

Diatom Polysaccharides: Extracellular Production, Isolation and Molecular Characterization

The extracellular polysaccharide production by marine diatoms is a significant route by which photosynthetically produced organic carbon enters the trophic web and may influence the physical environment in the sea as observed for example when massive aggregation events on basin scale occur. Many papers showed that the aldose signatures of marine DOM obtained from different seawater samples around the world is similar to that determined on cultured phytoplankton DOM and that the carbohydrate production could be very different among the species selected, growth and environmental conditions. These results are very important in order to understand the role of algal exudation in the aggregation processes observed in all of the seas and in general in carbon cycling in the euphotic zone. Many authors showed that cultured diatoms growth in P-limiting condition determines an increase of polysaccharides exudated by different diatoms species both pelagic and benthic

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 bro-mosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cya-nidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indo-methacin 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

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

Valutazione della qualit\ue0 della vita e stress ossidativo in ambiente urbano limitrofo a insediamenti industriali

Lo scopo della ricerca \ue8 quello di valutare la qualit\ue0 della vita, la percezione della salubrit\ue0 dell\u2019ambiente, l\u2019attaccamento al quartiere e lo stress ossidativo di una popolazione residente in un quartiere urbano vicino a insediamenti industriali. Sono stati individuati due campioni di popolazione, 200 esposti (gruppo sperimentale), 200 non esposti (gruppo di controllo) residenti in due quartieri della citt\ue0 di Trieste, aventi caratteristiche socio-anagrafiche simili, ma uno limitrofo a impianti industriali con significativo impatto ambientale, l'altro lontano da aree industriali. Sono stati somministrati il WHQOL-BREEF e due scale tratte dall\u2019IQURP (Indicatori di Qualit\ue0 Urbana Residenziale Percepita) tramite interviste dirette; l\u2019analisi dello stress ossidativo \ue8 stata condotta su campioni di urina dei soggetti intervistati attraverso la determinazione della malondialdeide urinaria (MDA) un biomarcatore di ossidazione lipidica e l\u20198-idrossi-2\u2019-deossiguanosina (8-OHdG) un biomarcatore di ossidazione del DNA. La qualit\ue0 della vita \ue8 simile nei due campioni considerati tranne per alcuni aspetti relativi all\u2019area dei rapporti con l\u2019ambiente; differenze statisticamente significative si riscontrano nella percezione di salubrit\ue0 ambientale e nell\u2019attaccamento al quartiere, entrambe negative per il quartiere limitrofo agli insediamenti industriali. Anche se \ue8 presente una marcata variabilit\ue0 dei risultati, l\u2019analisi dell\u2019MDA mostra un valore medio per il quartiere esposto doppio rispetto al quartiere non esposto, mentre dall'analisi del 8-OHdG la differenza \ue8 di un ordine di grandezza maggiore. Le due misure utilizzate in questo studio, l'una di natura percettivo-affettivo-emotiva e l'altra bio-fisiologica permettono di ottenere una valutazione integrata di impatto sul benessere complessivo delle persone in quartieri urbani caratterizzati da presenza di inquinamento di origine industriale. The aim of this study is to evaluate quality of life, perception of environmental health, neighborhood attachment and oxidative stress in a population living in an urban neighborhood close to industrial settlements. Two population samples were identified: 200 exposed individuals (case group) and 200 non-exposed individuals (control group) residing in two different neighborhoods in the city of Trieste. These groups display similar age and social features, however the former resides close to industrial plants with significant environmental impact, whereas the latter is located far from industrial areas. A WHOQOL-BREEF survey and two Perceived Urban Residential Quality Indicators-derived scales were submitted through direct interviews. An oxidative stress analysis was conducted on urine samples through the assessment of urinary malondialdehyde (MDA), a lipid peroxidation biomarker, and of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a DNA oxidative stress biomarker. In most of the parameters considered in this study, the quality of life of the two groups is alike, except for some environment-related aspects. Statistically significant differences were found in the perception of environmental health and neighborhood attachment. Both parameters are negative in the exposed group living in the vicinity of industrial plants. Despite a noticeable variability in the results, the MDA analysis shows an average value two times higher in the exposed group than in the non-exposed group, whereas the 8-OHdG analysis shows an even greater variation. The perceptional-affectional-emotional (WHOQOL-BREEF survey and IQURP-derived scales) and bio-physiological (MDA and 8-OHdG assessments) measures used in this study allow to obtain an integrated impact evaluation of oxidative stress on the overall health of people living in urban neighborhoods characterized by the presence of industrial pollution

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 cinetospora 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

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

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

Polysaccharide Characterisation of Marine Organic Matter in a Coastal Station of the Gulf of Trieste (Northern Adriatic Sea, Italy)

In the last two decades the presence and distribution of large aggregates in the Mediterranean Sea have encouraged to study the macromolecular features of dissolved organic matter and the implication of molecular characteristics of biopolymers on the aggregation processes. Monthly monitoring of marine carbohydrates in a coastal station of the Northern Adriatic sea during years 2001–2003 at 0.5, 10 and 15 m depth showed different pattern for dissolved and total carbohydrates in concurrence of the massive formation of aggregates. Since March to June 2004 marine water was collected for the partition experiment. The polysaccharide component was isolated by ultrafiltration. The monosaccharide compositions were very similar to those obtained from Adriatic pelagic mucilage. In June a significant reduction of the concentration of the low molecular weight polysaccharidic fraction was observed, in conjunction with the rapid increase of the high-molecular weight polysaccharides (HMW). In addition, the concentrations of deoxysugar (rhamnose and fucose) in the particulate matter decreased, and the fucose disappeared in June. These two monosaccharides and galactose may be considered bioindicators of the different degradation steps of marine polysaccharidic matter. This enrichment suggested that galactose-rich polysaccharides are able to undergo aggregate formation. Scanning electron microscopy and AFM imaging on extracted fractions showed the presence of fibrillar structures forming a highly branched tri-dimensional network similar to those found in Adriatic pelagic aggregates