The biomarkers' landscape of post-COVID-19 patients can suggest selective clinical interventions.

In COVID-19 clinical symptoms can persist even after negativization also in individuals who have had mild or moderate disease. We here investigated the biomarkers that define the post-COVID-19 clinical state analyzing the exhaled breath condensate (EBC) of 38 post COVID-19 patients and 38 sex and age-matched healthy controls via nuclear magnetic resonance (NMR)-based metabolomics. Predicted gene-modulated microRNAs (miRNAs) related to COVID-19 were quantified from EBC of 10 patients and 10 controls. Finally, clinical parameters from all post-COVID-19 patients were correlated with metabolomic data. Post-COVID-19 patients and controls showed different metabolic phenotype ("metabotype"). From the metabolites, by using enrichment analysis we identified miRNAs that resulted up-regulated (hsa-miR146a-5p) and down-regulated (hsa-miR-126-3p and hsa-miR-223-3p) in post-COVID-19. Taken together, our multiomics data indicate that post-COVID-19 patients before rehabilitation are characterized by persistent inflammation, dysregulation of liver, endovascular thrombotic and pulmonary processes, and physical impairment, which should be the primary clinical targets to contrast the post-acute sequelae of COVID-19.

Fatty Acids in Waste Tissues: The Nutraceutical Value of Gonads and Livers from the Moroccan Hypophthalmichthys molitrix and Cyprinus carpio Fishes.

Fishes are an important component of human nutrition, mainly acting as source of essential fatty acids in the prevention of cardiovascular disorders. The increase in their consumption has led to a growth of fishes waste; therefore, the disposal and recycling of waste has become a key issue to address, in accordance with circular economy principles. The Moroccan Hypophthalmichthys molitrix and Cyprinus carpio fishes, living in freshwater and marine environments, were collected at mature and immature stages. The fatty acid (FA) profiles of liver and ovary tissues were investigated by GC-MS and compared with edible fillet tissues. The gonadosomatic index, the hypocholesterolemic/hypercholesterolemic ratio, and the atherogenicity and thrombogenicity indexes were measured. Polyunsaturated fatty acids were found to be abundant in the mature ovary and fillet of both species, with a polyunsaturated fatty acids/saturated fatty acids ratio ranging from 0.40 to 1.06 and a monounsaturated fatty acids/polyunsaturated fatty acids ratio between 0.64 and 1.84. Saturated fatty acids were found to be highly abundant in the liver and gonads of both species (range 30-54%), as well as monounsaturated fatty acids (range 35-58%). The results suggested that the exploitation of fish wastes, such as the liver and ovary, may represent a sustainable strategy for the achievement of high value-added molecules with nutraceutical potential.

Metabolomics of COPD Pulmonary Rehabilitation Outcomes via Exhaled Breath Condensate.

Chronic obstructive pulmonary disease (COPD) is characterized by different phenotypes and clinical presentations. Therefore, a single strategy of pulmonary rehabilitation (PR) does not always yield the expected clinical outcomes as some individuals respond excellently, others discreetly, or do not respond at all. Fifty consecutive COPD patients were enrolled. Of them, 35 starting a 5-week PR program were sampled at admission (T0), after 2 (T2W) and 5 (T5W) weeks, while 15 controls not yet on PR were tested at T0 and T5W. Nuclear magnetic resonance (NMR) profiling of exhaled breath condensate (EBC) and multivariate statistical analysis were applied to investigate the relationship between biomarkers and clinical parameters. The model including the three classes correctly located T2W between T0 and T5W, but 38.71% of samples partially overlapped with T0 and 32.26% with T5W, suggesting that for some patients PR is already beneficial at T2W (32.26% overlapping with T5W), while for others (38.71% overlapping with T0) more time is required. Rehabilitated patients presented several altered biomarkers. In particular, methanol from T0 to T5W decreased in parallel with dyspnea and fatigue, while the walk distance increased. Methanol could be ascribed to lung inflammation. We demonstrated that the metabolic COPD phenotype clearly evolves during PR, with a strict relationship between clinical and molecular parameters. Methanol, correlating with clinical parameters, represents a useful biomarker for monitoring personalized outcomes and establishing more targeted protocols.

Identification of biomarkers in COPD by metabolomics of exhaled breath condensate and serum/plasma.

Chronic obstructive pulmonary disease (COPD) is the third cause of death worldwide, presenting poor long-term outcomes and chronic disability. COPD is a condition with a wide spectrum of clinical presentations because its pathophysiological determinants relate to tobacco smoke, genetic factors, alteration of several metabolic pathways, and oxidative stress. Consequently, patients present different phenotypes even with comparable degrees of airflow limitation. Because of the increasing social and economic costs of COPD, a growing attention is currently paid to "omics" techniques for more personalized treatments and patient-tailored rehabilitation programs. In this regard, the systematic investigation of the metabolome (i.e., the whole set of endogenous molecules) in biomatrices, namely metabolomics, has become indispensable for phenotyping respiratory diseases. The metabolomic profiling of biological samples contains the small molecules produced during biological processes and their identification and quantification help in the diagnosis, comprehension of disease outcome and treatment response. Exhaled breath condensate (EBC), plasma and serum are biofluids readily available, with negligible invasiveness, and, therefore, suitable for metabolomics investigations. In this paper, we describe the latest advances on metabolomic profiling of EBC, plasma and serum in COPD patients.

Metabolomic Profiling of Exhaled Breath Condensate and Plasma/ Serum in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is an increasing cause of global morbidity and mortality, with poor long-term outcomes and chronic disability. COPD is a condition with a wide spectrum of clinical presentations, with different phenotypes being identified even among patients with comparable degrees of airflow limitation. Considering the burden of COPD in terms of social and economic costs, in recent years growing attention has been given to the need for more personalized approaches and patienttailored rehabilitation programs. In this regard, the systematic analysis of metabolites in biological matrices, namely metabolomics, may become an essential tool in phenotyping diseases. Through the identification and quantification of the small molecules produced during biological processes, metabolomic profiling of biological samples has thus been proposed as an opportunity to identify novel biomarkers of disease outcome and treatment response. Exhaled breath condensate (EBC) and plasma/serum are fluid pools, which can be easily extracted and analyzed. In this review, we discuss the potential clinical applications of the metabolomic profiling of EBC and plasma/serum in COPD.

Comparative Fatty Acid Profiling of Edible Fishes in Kuala Terengganu, Malaysia.

The aim of this study was to compare the relative nutritional benefit of edible Malaysian fishes from the coast of Terengganu in Malaysia, as well as to perform a taxonomical characterization and metal assessment. Discrimination between species was carried out by a morphological and molecular approach by evaluating the total concentrations of metals by ICP-MS analyses and the fatty acids (FA) composition using the GC-MS approach on the fish fillet tissues. The taxonomical studies detected fishes of 11 families and 13 species. The heavy metal assessment showed that all detected elements did not exceed the regulatory limit stated by Malaysian Food Regulations. The proportion of saturated fatty acids (SFA) ranged from 33 to 58.34%, followed by the polyunsaturated fatty acids (PUFA) values from 24 to 51.8%, and the lowest proportion was of monounsaturated fatty acids (MUFA), ranging from 12.7 to 35.9%. The ω-3/ω-6 PUFA and PUFA/SFA ratios were determined in the range 1.1 to 7.4 and 0.35 to 1.6, respectively. The C20:5 ω-3 and C22:6 ω-3 acids were detected at levels comparable to those found in the corresponding species from similar tropical marine ecosystems. The high FA values can be useful biochemical tools for comparing the relative nutritional benefits of these biodiverse and non-toxic edible Malaysian fishes.

Chemistry of Tropical Eucheumatoids: Potential for Food and Feed Applications.

The use of seaweeds as additives in animal nutrition may be a valid option to traditional feed as they represent a rich source of minerals, carbohydrates and antioxidants. The aim of this study was to analyze the chemical composition and in vitro antioxidant capacity of two tropical eucheumatoids, Kappaphycus alvarezii and Kappaphycus striatus, in Malaysian wild offshore waters. The chemical analysis was performed via inductively coupled plasma-optical emission spectroscopy for evaluating the concentration of toxic (Cd, Pb, Hg, As) and essential elements (Mn, Fe, Cu, Ni, Zn, Se); NMR spectroscopy was used for carrageenans investigation. Furthermore, the soluble and fat-soluble antioxidant capacities were determined by FRAP, DPPH and ABTS assays. The chemical analysis revealed a higher content of trace elements in K. alvarezii as compared to K. striatus, and both exhibited a high mineral content. No significant differences in metal concentrations were found between the two species. Both samples showed a mixture of prevailing κ- and t-carrageenans. Finally, the levels of soluble and fat-soluble antioxidants in K. alvarezii were significantly higher than in K. striatus. Our findings suggest that K. alvarezii could be used as a potential feed additive because of its favorable chemical and nutritional features.

Involvement of a Quorum Sensing Signal Molecule in the Extracellular Amylase Activity of the Thermophilic Anoxybacillus amylolyticus.

Anoxybacillus amylolyticus is a moderate thermophilic microorganism producing an exopolysaccharide and an extracellular α-amylase able to hydrolyze starch. The synthesis of several biomolecules is often regulated by a quorum sensing (QS) mechanism, a chemical cell-to-cell communication based on the production and diffusion of small molecules named "autoinducers", most of which belonging to the N-acyl homoserine lactones' (AHLs) family. There are few reports about this mechanism in extremophiles, in particular thermophiles. Here, we report the identification of a signal molecule, the N-butanoyl-homoserine lactone (C4-HSL), from the milieu of A. amylolyticus. Moreover, investigations performed by supplementing a known QS inhibitor, trans-cinnamaldehyde, or exogenous C4-HSL in the growth medium of A. amylolyticus suggested the involvement of QS signaling in the modulation of extracellular α-amylase activity. The data showed that the presence of the QS inhibitor trans-cinnamaldehyde in the medium decreased amylolytic activity, which, conversely, was increased by the effect of exogenous C4-HSL. Overall, these results represent the first evidence of the production of AHLs in thermophilic microorganisms, which could be responsible for a communication system regulating thermostable α-amylase activity.

Extremophilic Natrinema versiforme Against Pseudomonas aeruginosa Quorum Sensing and Biofilm.

Pseudomonas aeruginosa is an opportunistic pathogen that causes high morbidity and mortality rates due to its biofilm form. Biofilm formation is regulated via quorum sensing (QS) mechanism and provides up to 1000 times more resistance against conventional antibiotics. QS related genes are expressed according to bacterial population density via signal molecules. QS inhibitors (QSIs) from natural sources are widely studied evaluating various extracts from extreme environments. It is suggested that extremely halophilic Archaea may also produce QSI compounds. For this purpose, we tested QS inhibitory potentials of ethyl acetate extracts from cell free supernatants and cells of Natrinema versiforme against QS and biofilm formation of P. aeruginosa. To observe QS inhibition, all extracts were tested on P. aeruginosa lasB-gfp, rhlA-gfp, and pqsA-gfp biosensor strains and biofilm inhibition was studied using P. aeruginosa PAO1. According to our results, QS inhibition ratios of cell free supernatant extract (CFSE) were higher than cell extract (CE) on las system, whereas CE was more effective on rhl system. In addition, anti-biofilm effect of CFSE was higher than CE. Structural analysis revealed that the most abundant compound in the extracts was trans 4-(2-carboxy-vinyl) benzoic acid.

Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate.

Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed of a subunit of 8 kDa and another of 50 kDa. The two protein subunits are noncovalently associated. The cloning and expression of the two protein subunits in Escherichia coli and their subsequent purification to homogeneity under native conditions result in the production of an active HPSE enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the nonreducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on HPSE, as the HPSE cleavage of fluorogenic tag would result in a measurable response.

Degradative actions of microbial xylanolytic activities on hemicelluloses from rhizome of Arundo donax.

Polysaccharidases from extremophiles are remarkable for specific action, resistance to different reaction conditions and other biotechnologically interesting features. In this article the action of crude extracts of thermophilic microorganisms (Thermotoga neapolitana, Geobacillus thermantarcticus and Thermoanaerobacterium thermostercoris) is studied using as substrate hemicellulose from one of the most interesting biomass crops, the giant reed (Arundo donax L.). This biomass can be cultivated without competition and a huge amount of rhizomes remains in the soil at the end of cropping cycle (10-15 years) representing a further source of useful molecules. Optimization of the procedure for preparation of the hemicellulose fraction from rhizomes of Arundo donax, is studied. Polysaccharidases from crude extracts of thermophilic microorganisms revealed to be suitable for total degradative action and/or production of small useful oligosaccharides from hemicelluloses from A. donax. Xylobiose and interesting tetra- and pentasaccharide are obtained by enzymatic action in different conditions. Convenient amount of raw material was processed per mg of crude enzymes. Raw hemicelluloses and pretreated material show antioxidant activity unlike isolated tetra- and pentasaccharide. The body of results suggest that rhizomes represent a useful raw material for the production of valuable industrial products, thus allowing to increase the economic efficiency of A. donax cultivation.

Enzymatic routes for the production of mono- and di-glucosylated derivatives of hydroxytyrosol.

In this work, a new eco-friendly procedure for the synthesis of hydroxytyrosol and tyrosol α-glycosidic derivatives was proposed by using the marine α-glucosidase from Aplysia fasciata, and a commercial tyrosinase from mushroom for the bioconversion of tyrosol glycosidic derivatives into the corresponding hydroxytyrosol products. New hydroxytyrosol mono- and di-saccharide derivatives were synthesized at final concentrations of 9.35 and 10.8 g/l of reaction, respectively, and their antioxidant activity was evaluated by DPPH test. The best antioxidant agent resulted the (3,4-dihydroxyphenyl) ethyl-α-D-glucopyranoside; it showed a radical scavenging activity similar to that of the hydroxytyrosol, together with an increased hydrosolubility. This molecule could be a good response to many food industry demands, always in search of cheap antioxidants with nutritional properties to improve the nutritional value and the quality of foods.

Anti-biofilm activity of an exopolysaccharide from a sponge-associated strain of Bacillus licheniformis.

BACKGROUND: Secondary metabolites ranging from furanone to exo-polysaccharides have been suggested to have anti-biofilm activity in various recent studies. Among these, Escherichia coli group II capsular polysaccharides were shown to inhibit biofilm formation of a wide range of organisms and more recently marine Vibrio sp. were found to secrete complex exopolysaccharides having the potential for broad-spectrum biofilm inhibition and disruption. RESULTS: In this study we report that a newly identified ca. 1800 kDa polysaccharide having simple monomeric units of α-D-galactopyranosyl-(1→2)-glycerol-phosphate exerts an anti-biofilm activity against a number of both pathogenic and non-pathogenic strains without bactericidal effects. This polysaccharide was extracted from a Bacillus licheniformis strain associated with the marine organism Spongia officinalis. The mechanism of action of this compound is most likely independent from quorum sensing, as its structure is unrelated to any of the so far known quorum sensing molecules. In our experiments we also found that treatment of abiotic surfaces with our polysaccharide reduced the initial adhesion and biofilm development of strains such as Escherichia coli PHL628 and Pseudomonas fluorescens. CONCLUSION: The polysaccharide isolated from sponge-associated B. licheniformis has several features that provide a tool for better exploration of novel anti-biofilm compounds. Inhibiting biofilm formation of a wide range of bacteria without affecting their growth appears to represent a special feature of the polysaccharide described in this report. Further research on such surface-active compounds might help developing new classes of anti-biofilm molecules with broad spectrum activity and more in general will allow exploring of new functions for bacterial polysaccharides in the environment.

Hydrosoluble antioxidants by enzymatic glucosylation of a vitamin E derivative using marine α-D-glucosidase from Aplysia fasciata.

Relevant examples of polyglycosylating exo-glycosidases were reported among enzymes of marine origin (Aplysia fasciata, Geobacillus, and Pecten maximus). Herein we describe the enzymatic polyglucosylation of a chromane-methanol (2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-ol) performed by using the α-D-glucosidase from the sea hare Aplysia fasciata. New di-, tri-, and tetrasaccharide derivatives were synthesized and their antioxidant activities were evaluated by DPPH test. High enzymatic substrate conversion was assessed by NMR spectroscopy, and the products were easily purified. These findings suggest that the proposed procedure is an effective process both for the molecular diversity of products and for the peculiar stereochemistry of the enzyme. At the beginning of the enzymatic reaction, only (S)-diastereomer of the monoglucoside was obtained. The isomaltoside was the most abundant disaccharide obtained and showed a radical scavenging activity similar to that of the chromane-methanol. The disaccharide can be considered a new hydrosoluble antioxidant agent useful for various technological applications (cosmetics, food industry, etc.). A relationship between the interglycosidic linkage present in disaccharides and trisaccharides and their scavenging activity was also pointed out.

2/4-Substituted-9-fluorenones and their O-glucosides as potential immunomodulators and anti-herpes simplex virus-2 agents. Part 5.

In pursuing a research on the antiviral and immunomodulatory activity of tilorone congeners, two new series of compounds were prepared and pharmacologically explored: 9-fluorenone carboxyhydroxyesters, indicated as AG, and 9-fluorenone carboxyhydroxamides, indicated as MG. Two of them, AG17 and MG3, were used as sugar acceptors in the transglycosylation reactions performed by alpha- and beta-glucosidases extracted from the marine mollusc Aplysia fasciata providing different alpha- and beta-, mono- and oligosaccharides. Then aglycons and saccharides were assayed for cytotoxicity, for anti-herpes virus-2 properties on peripheral blood mononuclear cells (PBMC) and for their capability to trigger human cells to produce antiviral cytokines such as IFNalpha and TNFalpha. Some promising compounds were individuated whereas the utility of the biocatalytic procedures in the preparation of pure anomeric material was further focused.

Facile biocatalytic access to 9-fluorenylmethyl polyglycosides: evaluation of antiviral activity on immunocompetent cells.

The biological activities of a series of mono- and oligosaccharides (beta-xylosides and alpha-glucosides) of 9-fluorenylmethanol were investigated together with mono-beta-galactoside and beta-glucoside of this aglycone, produced by biocatalytic routes. By using marine glycoside hydrolases and inexpensive donors such as maltose or xylan, access to mono-alpha-glucoside or mono-beta-xyloside of 9-fluorenylmethanol was obtained. Additionally, interesting polyglycoside derivatives were isolated. Biological testing indicated that in vitro treatment with these carbohydrate derivatives may influence the balance of cytokines in the environment of human peripheral blood mononuclear cells (PBMC), restricting the harmful effect of herpes simplex type 2 replication. In fact, these carbohydrate derivatives tested in WISH cells did not show any significant antiviral activity.

Direct enzymatic glucosylation of naringin in grapefruit juice by alpha-D-glucosidase from the marine mollusc Aplysia fasciata.

The enzymatic glucosylations of naringin, performed using alpha-D-glucosidase, identified in the Mediterranean mollusc Aplysia fasciata is reported. The enzyme actively operates on maltose and has an interesting transglycosylation potential using this donor. We also investigated the use of this marine alpha-glucosidase for a food-compatible glucosylation of naringin to produce new enzymatically modified carbohydrate possessing naringin derivatives. The regioselective formations of the beta-gluco-C6 alpha-glucosyl derivative and of the corresponding isomaltosyl diglucoside of naringin were obtained in high yield and efficiency of reaction. Suspensions of naringin can be used up to approximately 90 mg/mL initial acceptor concentration. In different experiments it was demonstrated that the enzyme was still active after 48 h in presence of this high amount of acceptor and that one of the diasteromers of the naringin is preferred by the enzyme from A. fasciata during glucosylation/deglucosylation enzymatic steps. Finally, the feasibility of efficient naringin glucosylation in grapefruit juice was also demonstrated at optimal pH of the enzyme and low maltose concentrations.

Glycoside hydrolases in Aplysia fasciata: analysis and applications.

Glycosylation is considered to be an important reaction for the chemical modification of compounds with useful biological activities. Glycoside hydrolases are biotechnologically attractive enzymes which can be used in synthetic reactions for assembling glycosidic linkages with absolute stereoselectivity at an anomeric centre. Most of these enzymes are commercially available but there is great interest in the search for new biocatalysts with original catalytic characteristics. The marine environment has shown to be a very interesting source for new glycosyl hydrolases for both hydrolytic and synthetic aspects. In particular, Aplysia fasciata a marine herbivorous mollusc has been shown to be a potent producer of a library of glycoside hydrolases applied to the synthesis of glycosidic bonds. The impressive assortment of glycosidases in marine organisms clearly indicates that the potential biodiversity of these enzymes is still largely unexplored and that potential applications of biocatalysts from the sea will increase in the near future.

Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides.

The marine ecosystem can be considered a rather unexplored source of biological material (e.g. natural substances with therapeutic activity) and can also be a surprising source of enzymes carrying new and interesting catalytic activities to be applied in biocatalysis. The use of glycosyl hydrolases from marine environments dates back to the end of the 1960s and was mainly focused on the development of sensitive and reliable hydrolytic methods for the analysis of sugar chains. As a result not all the benefits of a particular enzymatic activity have been investigated, especially regarding the transglycosylation potential of these enzymes for the synthesis of glycosidic bonds. In this review, the potential of marine sources will be demonstrated reporting on the few examples found in literature for the synthesis and hydrolysis of biologically relevant oligosaccharides catalyzed by glycosyl hydrolases of marine origin. Particular emphasis is given to the synthesis of glycosidic bonds, which is easy by the use of glycosyl hydrolases. Further aspects considered in this review are applications of these biocatalysts for vegetal waste treatment in recovering useful materials, for structural identification and for preparation of target materials from new purified polysaccharides, for the synthesis or modification of food-related compounds and for glycobiology related studies.