ASPECTS OF CELLULAR IRON HOMEOSTASIS: NRAMP TRANSPORTER FUNCTION AND ERYPTOSIS

The present study focus attention on \u201cAspects of cellular iron homeostasis: NRAMP transporter function and eryptosis\u201d combining aspects of both basic (first chapter: \u201cIron transporters NRAMP1 and NRAMP2 from Dictyostelium discoideum as a model of cellular iron homeostasis\u201d) and applied physiology (second chapter: \u201cEffects of xenobiotics on the suicidal death of erythrocytes\u201d). Iron plays a central role in a large number of essential cellular functions but it is also potentially toxic being able to generate reactive oxygen species (ROS). SLC11 and SLC40 families are involved in iron transport and play an important role in the maintenance of iron homeostasis. The SLC11 family is comprised of two members, SLC11A1 and SLC11A2. SLC11A1 is expressed in the phagolysosome of macrophages and in the tertiary granules of neutrophils and it contributes to the innate resistance against bacterial infection. SLC11A2 (also known as DMT1) is expressed in the proximal duodenum, immature erythroid cells, brain, placenta and kidney and is a key player in iron metabolism. Intestinal iron absorption is indeed mediated by SLC11A2 at the apical membrane of enterocytes and is followed by basolateral exit via SLC40A1. D. discoideum represents a model for the study of cellular iron homeostasis, showing subcellular localization of iron transporters resembling that of macrophages. The Dictyostelium genome shares with mammals many genes regulating iron homeostasis; in particular, D. discoideum expresses the ortholog of SLC11A1 transporter in phagolysosomes and that of SLC11A2 in the contractile vacuole. To better understand the function of Dictyostelium NRAMP proteins, they were expressed in Xenopus laevis oocytes by cRNA injection and functionally tested by radiochemical techniques and by a novel assay based on metal-induced changes in calcein fluorescence. Radiochemical assays showed that NRAMP1 induced iron transport is proton-dependent and it is inhibited by Mn2+, Cd2+, Co2+, Ni2+, Cu2+ and to a lesser extent by Zn2+. In calcein-injected oocytes expressing NRAMP1 and analyzed using confocal microscopy, Fe2+, Mn2+ and but not Fe3+ or Cu2+ led to fluorescence quenching due to their transport and accumulation into the cytoplasm of the oocytes. Therefore Dictyostelium NRAMP1 is an electrogenic proton-dependent divalent metal ion transporter with a cation selectivity comparable to that of rat DMT1. NRAMP1 colocalizes with V-ATPase in the membrane of phagolysosomes. Thus, it exploits the proton gradient maintained by the V-ATPase to mediate the efflux of iron from the phagolysosomes to the cytosol after bacterial engulfment. Preliminary studies showed that D. discoideum NRAMP2 can transport ferrous iron at neutral pH and it appears independent from proton gradient but dependent on Na+, nevertheless its transport activity is strongly reduced compared with that observed for NRAMP1. The second topic of this PhD thesis is eryptosis, the suicidal death of erythrocytes. Mutations that reduce DMT1 activity in human are associated with a severe defect in erythroid iron utilization and are correlated with several diseases. DMT1 deficiency causes an impaired erythroid differentiation hallmarked by accumulation of immature forms of erythroblast, accelerated death of erythroid precursors and a decreased survival in the erythroid progenitors. Iron deficiency is associated with shortened life span of erythrocytes. The accelerated clearance of erythrocytes can be attributed to excessive hemolysis or induction of programmed cell death of erythrocytes, called eryptosis. Eryptosis is fostered by an increase in cytosolic calcium. Iron deficient erythrocytes when exposed to stress conditions has been demonstrated to activate Ca2+-permeable cation channel allowing Ca2+ entry. Ca2+ entry through this channels leads to activation of a scramblase with subsequent phosphatidylserine exposure and to activation of the Gardos channels leading to KCl loss and cell shrinkage. This study was conducted in order to investigate prospective antitumoral products which are employed against tumor growth in humans, in particular CA4P or Pazopanib, or compounds used in vitro, such as Nocodazole, Terfenadine, Piceatannol, Ceranib-2 and Sclareol, in order to unveil the effects on erythrocytes survival and to clarify the mechanisms and signalling involved in their action. Human erythrocytes drawn from healthy individual were incubated in vitro at a hematocrit of 0.4% in Ringer solution. Where indicated, RBCs were exposed for 48 hours to the drugs at the indicated concentrations. The main hallmarks of eryptosis were investigated by flow cytometry. Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from DCF-dependent fluorescence, GSH levels by CMF-dependent fluorescence and ceramide abundance utilizing specific antibodies. Hemoglobin concentration in the supernatant was taken as measure of hemolysis. ATP levels following CA4P treatment were measured using luciferin-luciferase assay kit. For studying the effect of Nocodazole on tubulin in human erythrocytes, TubulinTracker\u2122 Green reagent was used. A 48 hours exposure of human erythrocytes to CA4P, Pazopanib, Nocodazole, Terfenadine, Piceatannol, Ceranib-2 or Sclareol treatment increased the percentage of annexin-V-binding cells. Furthermore, the effect of Sclareol on annexin-V-binding was significantly blunted in the presence of p38 kinase inhibitor skepinone and in the presence of casein kinase 1\u3b1 inhibitor D4476. CA4P and Piceatannol significantly decreased forward scatter. CA4P, Nocodazole, Terfenadine, Ceranib-2 and Sclareol treatment significantly increased Fluo3-fluorescence. CA4P significantly decreased GSH abundance and ATP levels. In addition, Pazopanib, Terfenadine, Ceranib-2 and Sclareol further resulted in significant hemolysis. Pazopanib, Nocodazole and Ceranib-2 significantly increased DCF-fluorescence and ceramide abundance. Nocodazole treatment reduced total tubulin abundance. In conclusion, these xenobiotics trigger eryptosis through different mechanisms

A review on the efficient catalysts for algae transesterification to biodiesel

The depletion of fossil fuel resources and increasing environmental pollution led to a trend for using alternative, clean, green, and sustainable fuel and energy resources. To attain this aim, using biomass as an alternative resource for diesel production has been a hotspot among researchers. Biodiesel has several advantages, such as being lower toxic and more renewable, and eco-friendlier than diesel from fossil fuel resources. Several edible and non-edible bio-sources were used for the production of biodiesel from the transesterification process. Algal oil as a non-edible source is considered an abundant, low cost and green substrate for biodiesel production. Various factors such as reaction conditions and the type of catalyst affect the biodiesel production process. Different catalytic systems such as basic and acidic homogeneous and heterogeneous catalysts and biocatalysts were introduced for the process in the literature, and each proposed catalyst has its own advantages and disadvantages. For instance, in spite of the lower cost and better mass transfer of base and acid homogeneous catalysts, reaction system corrosion, non-reusability, and soap formation are serious challenges of these catalysts at an industrial scale. On the other hand, acid and base heterogenous catalysts overcame the issues of corrosion and recovery, but some matters such as mass transfer limitation, high cost, and weak performance in catalyzing both esterification of FFAs and transesterification of lipids must be taken into account. In addition, bio-catalysis as a high-cost process led to a purer product formation with less side reaction. Therefore, several significant factors should be considered for transesterification catalysts such as availability, cost, reusability, stability, mass transfer, and the possibility to manage both the transesterification of triglycerides and the esterification of FFAs, selecting a catalyst with predominant pros is viable. Here, a review of the biodiesel production from algal biomass focusing on the efficient catalyst of the process is presented

Catalytic Production of Levulinic Acid (LA) from Actual Biomass

Catalytic conversion of actual biomass to valuable chemicals is a crucial issue in green chemistry. This review discusses on the recent approach in the levulinic acid (LA) formation from three prominent generations of biomasses. Our paper highlights the impact of the nature of different types of biomass and their complex structure and impurities, different groups of catalyst, solvents, and reaction system, and condition and all related pros and cons for this process

Stimulating effect of sclareol on suicidal death of human erythrocytes

Background/Aims: The diterpene alcohol Sclareol has been proposed for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, p38 kinase and casein kinase 1\u3b1. The present study explored, whether Sclareol induces eryptosis and, if so, shed light on the mechanisms involved. Methods: Phosphatidylserine abundance at the erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was estimated from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Sclareol ( 65 50 \u3bcM) significantly increased the percentage of annexin-V-binding cells without significantly modifying the average forward scatter, DCF-fluorescence or ceramide abundance. Sclareol ( 65 50 \u3bcM) further triggered hemolysis. Sclareol (100 \u3bcM) significantly increased Fluo3-fluorescence, but the effect of Sclareol on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+. Instead, the effect of Sclareol on annexin-V-binding was significantly blunted in the presence of p38 kinase inhibitor skepinone (2 \u3bcM) and in the presence of casein kinase 1\u3b1 inhibitor D4476 (10 \u3bcM). Conclusions: Sclareol triggers phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to activation of p38 kinase and casein kinase 1\u3b1

Activated biochars as sustainable and effective supports for hydrogenations

Activated biochars were obtained from pyrolysis and CO2-physical activation of four different biomasses including tannery shaving waste (T), vine wood waste (W), barley waste (B) and Sargassum, brown macroalgae of Venice lagoon (A). The potential of obtained carbonaceous materials as the supports of Ni,Al catalysts was investigated in levulinic acid (LA) conversion to γ-valerolactone (GVL) as a model hydrogenation reaction. Al-containing species as the Lewis acid sites for the dehydration step were incorporated to the supports using wet impregnation or precipitation. Ni as a hydrogenation active phase was added to the supports via wet impregnation. Biochar-based supports and catalysts were characterized by AAS, elemental analysis, FTIR, N2 physisorption, XRD, SEM, EDS, TEM, He-TPD, NH3-TPD and TPR techniques. The catalysts were tested for LA hydrogenation to GVL in a batch system and aqueous medium. The results showed that Ni supported on activated biochar was not active due to a lack of Lewis acid sites for dehydration. Precipitated Al-containing species on the biochar-based supports demonstrated a better catalytic performance in the reaction compared to impregnated one because of different interactions with the support and Ni species. Among different supports, the activated biochars obtained from T and W acted as the best ones. A higher catalytic efficiency was strongly influenced by the chemical (aromaticity and stability, presence of N,O-doped and functional groups), textural (the porous texture and surface area), and morphological (higher dispersion of active phases) properties of activated biochars obtained from different biomasses with different natures

Triggering of Suicidal Erythrocyte Death by Psammaplin A

Background/Aims: Psammaplin A, a natural product isolated from marine sponges, triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. In analogy to apoptosis of nucleated tumor cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Psammaplin A induces eryptosis and to possibly shed some light on the underlying mechanisms. Methods: Phosphatidylserine exposing erythrocytes were identified utilizing annexin-V-binding, cell volume was estimated from forward scatter, [Ca2+]i determined utilizing Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) quantified with DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface detected with specific antibodies. Results: A 48 hours exposure of human erythrocytes to Psammaplin A (2-8 \u3bcg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Psammaplin A significantly increased Fluo3-fluorescence, the effect of Psammaplin A on annexin-V-binding and forward scatter was, however, not significantly blunted by removal of extracellular Ca2+. Psammaplin A significantly increased DCFDA fluorescence and ceramide abundance. Conclusions: Psammaplin A triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by increase of [Ca2+]i, induction of oxidative stress and enhanced appearance of ceramide

Funzionalizzazione di Nanoparticelle con mAbSp17 per la cura del carcinoma ovarico

Nonostante l\u2019avvento di nuovi agenti chemioterapici nella terapia contro il carcinoma ovarico, la mortalit\ue0 causata da questo tumore maligno rimane invariata. Il problema maggiore \ue8 che il tumore viene diagnosticato in fase tardiva e la sopravvivenza, a 5 anni dalla diagnosi, \ue8 del 27% contro il 45,6% se la diagnosi \ue8 precoce. Sperm Protein 17 (SP17) \ue8 una proteina altamente conservata nei mammiferi ed \ue8 implicata nel legame dello spermatozoo alla zona pellucida. SP17 fa parte della famiglia dei cancer testis antigen ed \ue8 considerato un possibile target per l\u2019immunoterapia. La presenza di questa proteina \ue8 stata osservata nelle linee cellulari di OC umano, considerandolo come un possibile biomarker per questo tumore. Studi pre-clinici, in modello murino con OC, hanno dimostrato che la proteina SP17 funziona come vaccino prevenendo la formazione del tumore. Lo scopo di questo lavoro \ue8 quello di legare l\u2019anticorpo monoclonale contro SP17 (mAbSP17) a nanoparticelle di ossido di ferro (Fe3O4NP), iniettare il sistema in un modello murino con carcinoma ovarico umano, per valutare se l\u2019anticorpo legato direzioni le NP verso il tumore. In parallelo si vuole anche valutare la biodistribuzione delle Fe3O4NP. A tale scopo, le Fe3O4NP sono state rivestite con amminopropiltrietossisilano (APTES) e coniugate all'anticorpo, usando EDC (1-Etil-3-(3-dimetilamminopropil)-carbodiimmide cloridrato) e NHS (N-idrossisulfosuccinimide). Il legame covalente dell\u2019APTES con le NP \ue8 stato confermato mediante spettroscopia FT-IR, mentre il legame dell\u2019anticorpo alle NP \ue8 stata determinata mediante saggio ELISA. La capacit\ue0 di internalizzazione del sistema [email protected] \ue8 stata valutata, mediante microscopio confocale, trattando la linea cellulare di carcinoma ovarico umano (SKOV3). I sistemi, NP@APTES-mAbSP17 e [email protected], sono stati testati in vivo su modello murino con carcinoma ovarico indotto da SKOV3. NP@APTES-mAbSP17 e [email protected] sono stati iniettati, per via intraperitoneale, dopo 7 giorni dall\u2019induzione del tumore. Nelle immagini acquisite, utilizzando lo strumento IVIS-LUMINA (Caliper, LifeSciences), si osserva una forte fluorescenza nella zona tumorale, che indica che le [email protected] sono localizzate nel tumore. La nostra ipotesi ancora da confermare \ue8 che le NP siano state internalizzate dalle cellule tumorali, come gi\ue0 dimostrato in vitro. Per quanto riguarda il sistemaNP@APTES-mAbSP17, il lavoro \ue8 ancora in corso. Ad oggi si ha solo una valutazione macroscopica del fenomeno, ma le NP non sembrano aver causato risposta immunitaria o tossicit\ue0. Ovaio, polmone e fegato verranno valutati sia, per via immunoistochimica, per verificare la presenza di SP17 e quindi del tumore, che analizzati al TEM per osservare l\u2019eventuale presenza delle NP

From seaweeds to cosmeceutics: A multidisciplinar approach

Macroalgae are widespread on the coasts of all the globe and lead to a negative ecological impact, requiring expensive remediations. Therefore, the valorization of invasive seaweed as a renewable source of bioactive products could represent a valid solution. In this context, three algal biomasses, belonging to brown, green, and red families (Sargassum muticum, Ulva lactuca, Solieria filiformis), collected in the venetian Laguna, were investigated as a source of active compounds for the formulation of cosmeceutics. Microwave (MW) and ultrasound (US) were applied to enhance the algae extraction by means of a hydroalcoholic solution. According to total phenolic content (TPC) evaluation, MW demonstrated the best performing outcomes, resulting in 19.77, 22.02, and 16.94 mgGAE/gExtr (30 min at 90◦C) for brown, green, and red algae, respectively. Antioxidant activity was tested as well, showing comparable trends (49.19, 26.24, and 3.02 mmolTrolox eq./gExtr for brown, green, and red algae, respectively). Due to natural algae predisposition to absorb contaminants, the metal content analysis helped to screen the applicability of these extracts, identifying Ulva lactuca as the most suitable source of antioxidants for cosmetic formulations. This MW extract was then adopted to formulate two different preparations, namely a gel and an emulsion. Thermal and mechanical tests confirmed the stability of each formulation, together with neutral organoleptic characteristics. Finally, the actives release was investigated by means of a tape stripping essay, showing an efficient controlled release for gel formulation, even after 7 h of test. The produced cosmeceutics merged non-conventional extraction technologies with formulation expertise, offering a valuable alternative to solve the macroalgae disposal issue

Lithium-Metal Free Sulfur Battery Based on Waste Biomass Anode and Nano-Sized Li2S Cathode

The realization of a stable lithium-metal free (LiMF) sulfur battery based on amorphous carbon anode and lithium sulfide (Li2S) cathode is here reported. In particular, a biomass waste originating full-cell combining a carbonized brewer's spent grain (CBSG) biochar anode with a Li2S-graphene composite cathode (Li2S70Gr30) is proposed. This design is particularly attractive for applying a cost-effective, high performance, environment friendly, and safe anode material, as an alternative to standard graphite and metallic lithium in emerging battery technologies. The anodic and cathodic materials are characterized in terms of structure, morphology and composition through X-ray diffraction, scanning and transmission electron microscopy, X-ray photoelectron and Raman spectroscopies. Furthermore, an electrochemical characterization comprising galvanostatic cycling, rate capability and cyclic voltammetry tests were carried out both in half-cell and full-cell configurations. The systematic investigation reveals that unlike graphite, the biochar electrode displays good compatibility with the electrolyte typically employed in sulfur batteries. The CBSG/Li2S70Gr30 full-cell demonstrates an initial charge and discharge capacities of 726 and 537 mAh g−1, respectively, at 0.05C with a coulombic efficiency of 74%. Moreover, it discloses a reversible capacity of 330 mAh g−1 (0.1C) after over 300 cycles. Based on these achievements, the CBSG/Li2S70Gr30 battery system can be considered as a promising energy storage solution for electric vehicles (EVs), especially when taking into account its easy scalability to an industrial level. © 2022 The Authors. Energy & Environmental Materials published by John Wiley & Sons Australia, Ltd on behalf of Zhengzhou University