Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

Dopamine­iron homeostasis interaction rescues mitochondrial fitness in Parkinson's disease.

Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.

Genetic Determined Iron Starvation Signature in Friedreich's Ataxia.

BACKGROUND: Early studies in cellular models suggested an iron accumulation in Friedreich's ataxia (FA), yet findings from patients are lacking. OBJECTIVES: The objective is to characterize systemic iron metabolism, body iron storages, and intracellular iron regulation in FA patients. METHODS: In FA patients and matched healthy controls, we assessed serum iron parameters, regulatory hormones as well as the expression of regulatory proteins and iron distribution in peripheral blood mononuclear cells (PBMCs). We applied magnetic resonance imaging with R2*-relaxometry to quantify iron storages in the liver, spleen, and pancreas. Across all evaluations, we assessed the influence of the genetic severity as expressed by the length of the shorter GAA-expansion (GAA1). RESULTS: We recruited 40 FA patients (19 women). Compared to controls, FA patients displayed lower serum iron and transferrin saturation. Serum ferritin, hepcidin, mean corpuscular hemoglobin and mean corpuscular volume in FA inversely correlated with the GAA1-repeat length, indicating iron deficiency and restricted availability for erythropoiesis with increasing genetic severity. R2*-relaxometry revealed a reduction of splenic and hepatic iron stores in FA. Liver and spleen R2* values inversely correlated with the GAA1-repeat length. FA PBMCs displayed downregulation of ferritin and upregulation of transferrin receptor and divalent metal transporter-1 mRNA, particularly in patients with >500 GAA1-repeats. In FA PBMCs, intracellular iron was not increased, but shifted toward mitochondria. CONCLUSIONS: We provide evidence for a previously unrecognized iron starvation signature at systemic and cellular levels in FA patients, which is related to the underlying genetic severity. These findings challenge the use of systemic iron lowering therapies in FA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Comparative analysis of oral and intravenous iron therapy in rat models of inflammatory anemia and iron deficiency.

Anemia is a major health issue and associated with increased morbidity. Iron deficiency anemia (IDA) is the most prevalent, followed by anemia of chronic disease (ACD). IDA and ACD often co-exist, challenging diagnosis and treatment. While iron supplementation is the first-line therapy for IDA, its optimal route of administration and the efficacy of different repletion strategies in ACD are elusive. Female Lewis rats were injected with group A streptococcal peptidoglycan-polysaccharide (PG-APS) to induce inflammatory arthritis with associated ACD and/or repeatedly phlebotomized and fed with a low iron diet to induce IDA, or a combination thereof (ACD/IDA). Iron was either supplemented by daily oral gavage of ferric maltol or by weekly intravenous (i.v.) injection of ferric carboxymaltose for up to 4 weeks. While both strategies reversed IDA, they remained ineffective to improve hemoglobin (Hb) levels in ACD, although oral iron showed slight amelioration of various erythropoiesis-associated parameters. In contrast, both iron treatments significantly increased Hb in ACD/IDA. In ACD and ACD/IDA animals, i.v. iron administration resulted in iron trapping in liver and splenic macrophages, induction of ferritin expression and increased circulating levels of the iron hormone hepcidin and the inflammatory cytokine interleukin-6, while oral iron supplementation reduced interleukin-6 levels. Thus, oral and i.v. iron resulted in divergent effects on systemic and tissue iron homeostasis and inflammation. Our results indicate that both iron supplements improve Hb in ACD/IDA, but are ineffective in ACD with pronounced inflammation, and that under the latter condition, i.v. iron is trapped in macrophages and may enhance inflammation.

A fully human anti-BMP6 antibody reduces the need for erythropoietin in rodent models of the anemia of chronic disease.

Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.

One-pot synthesis of brewer's spent grain-supported superabsorbent polymer for highly efficient uranium adsorption from wastewater.

High-efficient and fast adsorption of uranium is important to reduce the hazards caused by the uranium contamination of water environment due to the increased human activities. Herein, brewer's spent grain (BSG)-supported superabsorbent polymers (SAP) with different cross-linking densities are prepared as cheap and eco-friendly adsorbents for the first time via one-pot swelling and graft polymerization. A 7 wt% NaOH solution is used to swell BSG before grafting and subsequently neutralize the acrylic acid to control the reaction rate without producing alkaline wastewater. Compared with the traditional methods, swelling improves the grafting density and the utilization of raw materials due to the increased disorder degree of the BSG fibers. This results in the grafting of abundant carboxyl and amide groups onto the BSG backbone, forming a strongly hydrophilic polymer network of the BSG-SAP. Compared with the reference polymers without BSG, BSG-SAP presents higher adsorption capacity and enhanced reusability. The highly cross-linked BSG-SAP (BSG-SAP-H) shows an outstanding adsorption capacity of U(VI) (1465 mg/g at pH0 = 4.6), a fast adsorption rate (81% of equilibrium adsorption capacity in 15 min), and a high selectivity in the presence of competing ions. Adsorption mechanism studies reveal the involvement of amide groups, a bidentate binding structure between UO22+ and the carboxyl groups, and a cation exchange between Na+ and UO22+. More importantly, the adsorption capacity of BSG-SAP-H reaches 254.4 mg/g in the fixed-bed column experiment at a low initial concentration (c0(U) = 30 mg/L) and keeps 80% of the adsorption capacity after four cycles, indicating a great potential for uranium removal from wastewater. This work shows a suitable approach to explore the untreated biomass to prepare SAP with enhanced adsorption performance via a general and low-cost strategy.

DMT1 Protects Macrophages from Salmonella Infection by Controlling Cellular Iron Turnover and Lipocalin 2 Expression.

Macrophages are at the center of innate pathogen control and iron recycling. Divalent metal transporter 1 (DMT1) is essential for the uptake of non-transferrin-bound iron (NTBI) into macrophages and for the transfer of transferrin-bound iron from the endosome to the cytoplasm. As the control of cellular iron trafficking is central for the control of infection with siderophilic pathogens such as Salmonella Typhimurium, a Gram-negative bacterium residing within the phagosome of macrophages, we examined the potential role of DMT1 for infection control. Bone marrow derived macrophages lacking DMT1 (DMT1fl/flLysMCre(+)) present with reduced NTBI uptake and reduced levels of the iron storage protein ferritin, the iron exporter ferroportin and, surprisingly, of the iron uptake protein transferrin receptor. Further, DMT1-deficient macrophages have an impaired control of Salmonella Typhimurium infection, paralleled by reduced levels of the peptide lipocalin-2 (LCN2). LCN2 exerts anti-bacterial activity upon binding of microbial siderophores but also facilitates systemic and cellular hypoferremia. Remarkably, nifedipine, a pharmacological DMT1 activator, stimulates LCN2 expression in RAW264.7 macrophages, confirming its DMT1-dependent regulation. In addition, the absence of DMT1 increases the availability of iron for Salmonella upon infection and leads to increased bacterial proliferation and persistence within macrophages. Accordingly, mice harboring a macrophage-selective DMT1 disruption demonstrate reduced survival following Salmonella infection. This study highlights the importance of DMT1 in nutritional immunity and the significance of iron delivery for the control of infection with siderophilic bacteria.

The haemochromatosis gene Hfe and Kupffer cells control LDL cholesterol homeostasis and impact on atherosclerosis development.

AIMS: Imbalances of iron metabolism have been linked to the development of atherosclerosis. However, subjects with hereditary haemochromatosis have a lower prevalence of cardiovascular disease. The aim of our study was to understand the underlying mechanisms by combining data from genome-wide association study analyses in humans, CRISPR/Cas9 genome editing, and loss-of-function studies in mice. METHODS AND RESULTS: Our analysis of the Global Lipids Genetics Consortium (GLGC) dataset revealed that single nucleotide polymorphisms (SNPs) in the haemochromatosis gene HFE associate with reduced low-density lipoprotein cholesterol (LDL-C) in human plasma. The LDL-C lowering effect could be phenocopied in dyslipidaemic ApoE-/- mice lacking Hfe, which translated into reduced atherosclerosis burden. Mechanistically, we identified HFE as a negative regulator of LDL receptor expression in hepatocytes. Moreover, we uncovered liver-resident Kupffer cells (KCs) as central players in cholesterol homeostasis as they were found to acquire and transfer LDL-derived cholesterol to hepatocytes in an Abca1-dependent fashion, which is controlled by iron availability. CONCLUSION: Our results disentangle novel regulatory interactions between iron metabolism, KC biology and cholesterol homeostasis which are promising targets for treating dyslipidaemia but also provide a mechanistic explanation for reduced cardiovascular morbidity in subjects with haemochromatosis.

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents.

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.

Association of mitochondrial iron deficiency and dysfunction with idiopathic restless legs syndrome.

BACKGROUND: Restless legs syndrome is a sensorimotor neurological disorder of the limbs that impairs quality of life and disturbs sleep. However, there has been progress in understanding the disease involving the dopaminergic system as well as iron metabolism. The exact pathophysiological mechanisms of restless legs syndrome remain elusive. We tried to elucidate the underlying mechanisms in iron metabolism in restless legs syndrome subjects on a systemic, cellular, and mitochondrial level. METHODS: We conducted a study prospectively recruiting 168 restless legs syndrome patients and 119 age-matched healthy controls focusing on iron metabolism using human monocytes as surrogates. RESULTS: Evaluation of systemic iron metabolism parameters in the circulation showed no significant difference between patients and controls. We observed a significant reduction in mRNA levels of heme oxygenase 1 and mitochondrial iron genes like mitoferrin 1 and 2 in monocytes isolated from restless legs syndrome patients, indicating mitochondrial iron deficiency. Interestingly, we also observed reduced expression of iron regulatory protein 2 along with impaired activity of mitochondrial aconitase and reduced mitochondrial superoxide formation in restless legs syndrome subjects. Along this line, patients had reduced mitochondrial respiratory capacity that improved in restless legs syndrome subjects under treatment with dopaminergic drugs compared with untreated patients. CONCLUSIONS: Our data suggest that restless legs syndrome is linked to mitochondrial iron deficiency and associated impairment of mitochondrial function. This is partly corrected by treatment with dopaminergic drugs compared with untreated patients, which may be linked to an effect of dopamine on cellular iron homeostasis. © 2018 International Parkinson and Movement Disorder Society.

Salmonella Utilizes Zinc To Subvert Antimicrobial Host Defense of Macrophages via Modulation of NF-κB Signaling.

Zinc sequestration by macrophages is considered a crucial host defense strategy against infection by the intracellular bacterium Salmonella enterica serovar Typhimurium. However, the underlying mechanisms remain elusive. In this study, we found that zinc favors pathogen survival within macrophages. Salmonella-hosting macrophages contained higher free zinc levels than did uninfected macrophages and cells that successfully eliminated bacteria, which was paralleled by the impaired production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in bacterium-harboring cells. A profound, zinc-mediated inhibition of NF-κB p65 transcriptional activity affecting the expression of the ROS- and RNS-forming enzymes phos47 and inducible nitric oxide synthase (iNOS) provided a mechanistic explanation for this phenomenon. Macrophages responded to infection by enhancing the expression of zinc-scavenging metallothioneins 1 and 2, whose genetic deletion caused increased free zinc levels, reduced ROS and RNS production, and increased the survival of Salmonella Our data suggest that Salmonella invasion of macrophages results in a bacterium-driven increase in the intracellular zinc level, which weakens antimicrobial defense and the ability of macrophages to eradicate the pathogen. Thus, limitation of cytoplasmic zinc levels may help to control infection by intracellular bacteria.

Hypoxia induced downregulation of hepcidin is mediated by platelet derived growth factor BB.

OBJECTIVE: Hypoxia affects body iron homeostasis; however, the underlying mechanisms are incompletely understood. DESIGN: Using a standardised hypoxia chamber, 23 healthy volunteers were subjected to hypoxic conditions, equivalent to an altitude of 5600 m, for 6 h. Subsequent experiments were performed in C57BL/6 mice, CREB-H knockout mice, primary hepatocytes and HepG2 cells. RESULTS: Exposure of subjects to hypoxia resulted in a significant decrease of serum levels of the master regulator of iron homeostasis hepcidin and elevated concentrations of platelet derived growth factor (PDGF)-BB. Using correlation analysis, we identified PDGF-BB to be associated with hypoxia mediated hepcidin repression in humans. We then exposed mice to hypoxia using a standardised chamber and observed downregulation of hepatic hepcidin mRNA expression that was paralleled by elevated serum PDGF-BB protein concentrations and higher serum iron levels as compared with mice housed under normoxic conditions. PDGF-BB treatment in vitro and in vivo resulted in suppression of both steady state and BMP6 inducible hepcidin expression. Mechanistically, PDGF-BB inhibits hepcidin transcription by downregulating the protein expression of the transcription factors CREB and CREB-H, and pharmacological blockade or genetic ablation of these pathways abrogated the effects of PDGF-BB toward hepcidin expression. CONCLUSIONS: Hypoxia decreases hepatic hepcidin expression by a novel regulatory pathway exerted via PDGF-BB, leading to increased availability of circulating iron that can be used for erythropoiesis.

Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients.

In transfusional iron overload, extra-hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non-transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond-Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin-iron utilization (DBA).

Hepcidin as a predictive factor and therapeutic target in erythropoiesis-stimulating agent treatment for anemia of chronic disease in rats.

Anemia of chronic disease is a multifactorial disorder, resulting mainly from inflammation-driven reticuloendothelial iron retention, impaired erythropoiesis, and reduced biological activity of erythropoietin. Erythropoiesis-stimulating agents have been used for the treatment of anemia of chronic disease, although with varying response rates and potential adverse effects. Serum concentrations of hepcidin, a key regulator of iron homeostasis, are increased in patients with anemia of chronic disease and linked to the pathogenesis of this disease, because hepcidin blocks cellular iron egress, thus limiting availability of iron for erythropoiesis. We tested whether serum hepcidin levels can predict and affect the therapeutic efficacy of erythropoiesis-stimulating agent treatment using a well-established rat model of anemia of chronic disease. We found that high pre-treatment hepcidin levels correlated with an impaired hematologic response to an erythropoiesis-stimulating agent in rats with anemia of chronic disease. Combined treatment with an erythropoiesis-stimulating agent and an inhibitor of hepcidin expression, LDN-193189, significantly reduced serum hepcidin levels, mobilized iron from tissue stores, increased serum iron levels and improved hemoglobin levels more effectively than did the erythropoiesis-stimulating agent or LDN-193189 monotherapy. In parallel, both the erythropoiesis-stimulating agent and erythropoiesis-stimulating agent/LDN-193189 combined reduced the expression of cytokines known to inhibit erythropoiesis. We conclude that serum hepcidin levels can predict the hematologic responsiveness to erythropoiesis-stimulating agent therapy in anemia of chronic disease. Pharmacological inhibition of hepcidin formation improves the erythropoiesis-stimulating agent's therapeutic efficacy, which may favor a reduction of erythropoiesis-stimulating agent dosages, costs and side effects.

Recycling of Spent FCC Catalysts: Conversion of Leached Residues to Zeolite ZSM-5.

The commercial recycling of spent FCC catalyst typically focuses on recovering only 1-3 % of rare-earth elements, with the remaining residues often disposed of in landfills. Here, we present a novel method to close a recycling loop for spent FCC catalyst. The method involves a series of leaching steps: Firstly, the spent catalyst material is leached with HNO3 to remove rare-earth elements such as La; second, solvothermal leaching with HCl removes most of Al and impurities like Fe, Ni and V; finally, a third leaching with H2SO4 removes Ti. The solid residues are then used to synthesize ZSM-5 without the addition of any extra silicon or aluminum sources after mild activation. The impurities in the synthesis gel strongly modify the properties of the zeolite, with ZSM-5 crystals containing higher levels of impurities exhibiting lower crystallinities, surface areas, acidities, cracking activities, as well as larger particle sizes.

Quantification of Macrophage Cellular Ferrous Iron (Fe2+) Content Using a Highly Specific Fluorescent Probe in a Plate Reader.

Macrophages are at the center of innate immunity and iron metabolism. In the case of an infection, macrophages adapt their cellular iron metabolism to deprive iron from invading bacteria to combat intracellular bacterial proliferation. A concise evaluation of the cellular iron content upon an infection with bacterial pathogens and diverse cellular stimuli is necessary to identify underlying mechanisms concerning iron homeostasis in macrophages. For the characterization of cellular iron levels during infection, we established an in vitro infection model where the murine macrophage cell line J774A.1 is infected with Salmonella enterica serovar Typhimurium (S.tm), the mouse counterpart to S. enterica serovar Typhi, under normal and iron-overload conditions using ferric chloride (FeCl3) treatment. To evaluate the effect of infection and iron stimulation on cellular iron levels, the macrophages are stained with FerroOrange. This fluorescent probe specifically detects Fe2+ ions and its fluorescence can be quantified photometrically in a plate reader. Importantly, FerroOrange fluorescence does not increase with chelated iron or other bivalent metal ions. In this protocol, we present a simple and reliable method to quantify cellular Fe2+ levels in cultured macrophages by applying a highly specific fluorescence probe (FerroOrange) in a TECAN Spark microplate reader. Compared to already established techniques, our protocol allows assessing cellular iron levels in innate immune cells without the use of radioactive iron isotopes or extensive sample preparation, exposing the cells to stress. Key features • Easy quantification of Fe2+ in cultured macrophages with a fluorescent probe. • Analysis of iron in living cells without the need for fixation. • Performed on a plate reader capable of 540 nm excitation and 585 nm emission by trained employees for handling biosafety level 2 bacteria.