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Supplemental material, sj-docx-1-nmi-10.1177_11786388211065372 for Modulation of Human Hydrogen Sulfide Metabolism by Micronutrients, Preliminary Data by Maurizio Dattilo, Carolina Fontanarosa, Michele Spinelli, Vittorio Bini and Angela Amoresano in Nutrition and Metabolic Insights

In this work, we assess the potential of waste products of Phlegrean mandarin (Citrus reticulata Blanco), namely seeds and peel, to be reutilized as a source of bioactive compounds beneficial for the human diet. Starting from the evidence that the by-products of this specific cultivar are the most powerful sources of antioxidants compared to pulp, we have investigated if and how the bioactive compounds in peel and seeds may be affected by fruit ripening. Three stages of fruit ripening have been considered in our study: unripe fruits = UF, semi-ripe fruits = SRF, ripe fruits = RF. The overall results indicated that RF showed the highest concentration of antioxidants. Among fruit components, peel was the richest in total antioxidant capacity, total polyphenol content, total flavonoids, total chlorophylls and carotenoids, while seeds exhibited the highest concentration of total condensed tannins and ascorbic acid. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay indicates the occurrence, in peel extracts, of 28 phenolic compounds, mainly flavonoids (FLs); in seeds, 34 derivatives were present in the first stage (UF), which diminish to 24 during the ripening process. Our data indicated that the content of phytochemicals in citrus strongly varies among the fruit components and depends on the ripening stage. The higher antioxidant activity of peel and seeds, especially in RF, encourage a potential use of by-products of this specific citrus cultivar for industrial or pharmacological applications. However, to maximize the occurrence of desired bioactive compounds, it is important also to consider the ripening stage at which fruits must be collected.

To investigate whether short term fructose-rich diet induces changes in the gut microbiota as well as in skeletal muscle and adipose tissue physiology and verify whether they persist even after fructose withdrawal, young rats of 30 d of age were fed for 3 weeks a fructose-rich or control diet. At the end of the 3-weeks period, half of the rats from each group were maintained for further 3 weeks on a control diet. Metagenomic analysis of gut microbiota and short chain fatty acids levels (faeces and plasma) were investigated. Insulin response was evaluated at the whole-body level and both in skeletal muscle and epididymal adipose tissue, together with skeletal muscle mitochondrial function, oxidative stress, and lipid composition. In parallel, morphology and physiological status of epididymal adipose tissue was also evaluated. Reshaping of gut microbiota and increased content of short chain fatty acids was elicited by the fructose diet and abolished by switching back to control diet. On the other hand, most metabolic changes elicited by fructose-rich diet in skeletal muscle and epididymal adipose tissue persisted after switching to control diet. Increased dietary fructose intake even on a short-time basis elicits persistent changes in the physiology of metabolically relevant tissues, such as adipose tissue and skeletal muscle, through mechanisms that go well beyond the reshaping of gut microbiota. This picture delineates a harmful situation, in particular for the young populations, posed at risk of metabolic modifications that may persist in their adulthood.

The green synthesis of highly conductive polyaniline by using two biological macromolecules, i.e laccase as biocatalyst, and DNA as template/dopant, was achieved in this work. Trametes versicolor laccase B (TvB) was found effective in oxidizing both aniline and its less toxic/mutagenic dimer N-phenyl-p-phenylenediamine (DANI) to conductive polyaniline. Reaction conditions for synthesis of conductive polyanilines were set-up, and structural and electrochemical properties of the two polymers were extensively investigated. When the less toxic aniline dimer was used as substrate, the polymerization reaction was faster and gave less-branched polymer. DNA was proven to work as hard template for both enzymatically synthesized polymers, conferring them a semi-ordered morphology. Moreover, DNA also acts as dopant leading to polymers with extraordinary conductive properties (∼6 S/cm). It can be envisaged that polymer properties are magnified by the concomitant action of DNA as template and dopant. Herein, the developed combination of laccase and DNA represents a breakthrough in the green synthesis of conductive materials.

The setup of an economic and sustainable method to increase the production and commercialization of products from microalgae, beyond niche markets, is a challenge. Here, a cascade approach has been designed to optimize the recovery of high valuable bioproducts starting from the wet biomass of Galdieria phlegrea. This unicellular thermo-acidophilic red alga can accumulate high-value compounds and can live under conditions considered hostile to most other species. Extractions were performed in two sequential steps: a conventional high-pressure procedure to recover phycocyanins and a solvent extraction to obtain fatty acids. Phycocyanins were purified to the highest purification grade reported so far and were active as antioxidants on a cell-based model. Fatty acids isolated from the residual biomass contained high amount of PUFAs, more than those recovered from the raw biomass. Thus, a simple, economic, and high effective procedure was set up to isolate phycocyanin at high purity levels and PUFAs.

Cerebral tissues in human remains are rare archaeological discoveries. These tissues are typically saponified, meaning that their triglycerides have been converted to glycerol and fatty acid salts, or soap. In c.e. 79, a volcanic hot ash avalanche rapidly killed the inhabitants of Pompeii and Herculaneum. In the 1960s, at the Collegium Augustalium in Herculaneum, a human victim of the avalanche was found lying on a wooden bed, buried by volcanic ash. In this victim’s skull, we discovered apparent brain remains that were vitrified instead of saponified.

Mild endurance exercise has been shown to compensate for declined muscle quality and may positively affect the brain under conditions of energy restriction. Whether this involves brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) activation in relation to central and peripheral tissue levels of associated factors such as beta hydroxy butyrate (BHB), branched-chain amino acids (BCAA) and thyroid hormone (T3) has not been studied. Thus, a subset of male Wistar rats housed at thermoneutrality that were fed or fasted was submitted to 30-min-mild treadmill exercise bouts (five in total, twice daily, 15 m/min, 0° inclination) over a period of 66 h. Prefrontal cortex and gastrocnemius muscle BHB, BCAA, and thyroid hormone were measured by LC-MS/MS analysis and were related to BDNF and mammalian target of rapamycin (mTOR) signaling. In gastrocnemius muscle, mild endurance exercise during fasting maintained the fasting-induced elevated BHB levels and BDNF-CREB activity and unlocked the downstream Akt-mTORC1 pathway associated with increased tissue BCAA. Consequently, deiodinase 3 mRNA levels decreased whereas increased phosphorylation of the mTORC2 target FOXO1 was associated with increased deiodinase 2 mRNA levels, accounting for the increased T3 tissue levels. These events were related to increased expression of CREB and T3 target genes beneficial for muscle quality previously observed in this condition. In rat L6 myoblasts, BHB directly induced BDNF transcription and maturation. Mild endurance exercise during fasting did not increase prefrontal cortex BHB levels nor was BDNF activated, whereas increased leucine levels were associated with Akt-independent increased phosphorylation of the mTORC1 target P70S6K. The associated increased T3 levels modulated the expression of known T3-target genes involved in brain tissue maintenance. Our observation that mild endurance exercise modulates BDNF, mTOR and T3 during fasting provides molecular clues to explain the observed beneficial effects of mild endurance exercise in settings of energy restriction.

In the context of the vascular effects of hydrogen sulfide (H2S), it is known that this gaseous endogenous biological modulator of inflammation, oxidative stress, etc. is a potent vasodilator. Chronic renal failure, a common disease affecting the aging population, is characterized by low levels of H2S in plasma and tissues, which could mediate their typical hypertensive pattern, along with other abnormalities. Lanthionine and homolanthionine, natural non-proteinogenic amino acids, are formed as side products of H2S production. Also in consideration of the intrinsic difficulties in H2S measuring, these compounds have been proposed as reliable and stable markers of H2S synthesis. However, in the setting of chronic renal failure patients on hemodialysis, they represent typical retention products (without ruling out the possibility of an increased intestinal synthesis) and prospective novel uremic toxins. Here, a method utilizing liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring ion mode has been developed and evaluated for the determination of these key H2S metabolites in plasma, by using a triple quadrupole mass spectrometer.

In the last years, laccases have become leading biocatalysts in green bioprocesses for dye synthesis. Among fungal laccases, the high-redox potential laccase POXA1b from Pleurotus ostreatus is a valuable candidate since it brings together several key-elements: stability and activity in a wide range of pHs and temperatures and the availability of a cost-effective bioprocess for its production. In this work POXA1b catalyzed the coupling of a p-phenylenediamine and α-naphtol, resulting in the production of a new dyeing product, named SIC-RED. The dye was structurally characterized and its performances in protein gel staining tested. The economic feasibility of the process was demonstrated in the case of an Italian small medium enterprise. The results let envisage a real exploitation of the herein enzymatically synthesized dye for protein gel staining.

Diatoms are among the most successful primary producers in ocean and freshwater environments. Deriving from a secondary endosymbiotic event, diatoms have a mixed genome containing bacterial, animal and plant genes encoding for metabolic pathways that may account for their evolutionary success. Studying the transcriptomes of two strains of the diatom Skeletonema marinoi, we report, for the first time in microalgae, an active animal-like prostaglandin pathway that is differentially expressed in the two strains. Prostaglandins are hormone-like mediators in many physiological and pathological processes in mammals, playing a pivotal role in inflammatory responses. They are also present in macroalgae and invertebrates, where they act as defense and communication mediators. The occurrence of animal-like prostaglandins in unicellular photosynthetic eukaryotes opens up new intriguing perspectives on the evolution and role of these molecules in the marine environment as possible mediators in cell-to-cell signaling, eventually influencing population dynamics in the plankton.The ISME Journal advance online publication, 28 March 2017; doi:10.1038/ismej.2017.27.

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses. Several studies have suggested that free D-Asp has a crucial role in N-methyl D-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free D-Asp, L-Asp and N-methyl D-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing L-Asp, D-Asp and N-methyl D-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for D-Asp, 0.46 pg/μl for L-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for D-Asp, 1.41 pg/μl for L-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of D-Asp, L-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free D-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.

Diatoms are dominant photosynthetic organisms in the world's oceans and are considered essential in the transfer of energy to higher trophic levels. However, these unicellular organisms produce secondary metabolites deriving from the oxidation of fatty acids, collectively termed oxylipins, with negative effects on predators, such as copepods, that feed on them (e.g. reduction in survival, egg production and hatching success) and, indirectly, on higher trophic levels. Here, a multidisciplinary study (oxylipin measurements, copepod fitness, gene expression analyses, chlorophyll distribution, phytoplankton composition, physico-chemical characteristics) was carried out at the end of the spring diatom bloom in April 2011 in the Northern Adriatic Sea (Mediterranean Sea) in order to deeply investigate copepod diatom interactions, chemical communication and response pathways. The results show that the transect with the lowest phytoplankton abundance had the lowest copepod egg production and hatching success, but the highest oxylipin concentrations. In addition, copepods in both the analyzed transects showed increased expression levels of key stress-related genes (e.g. heat-shock proteins, catalase, glutathione S-transferase, aldehyde dehydrogenase) compared to control laboratory conditions where copepods were fed with the dinoflagellate Prorocentrum minimum which does not produce any oxylipins. New oxylipins that have never been reported before for microalgae are described for the first time, giving new insights into the complex nature of plant-animal signaling and communication pathways at sea. This is also the first study providing insights on the copepod response during a diatom bloom at the molecular level. (C) 2016 Elsevier B.V. All rights reserved.