The biology of mammalian multi-copper ferroxidases.

The mammalian multicopper ferroxidases (MCFs) ceruloplasmin (CP), hephaestin (HEPH) and zyklopen (ZP) comprise a family of conserved enzymes that are essential for body iron homeostasis. Each of these enzymes contains six biosynthetically incorporated copper atoms which act as intermediate electron acceptors, and the oxidation of iron is associated with the four electron reduction of dioxygen to generate two water molecules. CP occurs in both a secreted and GPI-linked (membrane-bound) form, while HEPH and ZP each contain a single C-terminal transmembrane domain. These enzymes function to ensure the efficient oxidation of iron so that it can be effectively released from tissues via the iron export protein ferroportin and subsequently bound to the iron carrier protein transferrin in the blood. CP is particularly important in facilitating iron release from the liver and central nervous system, HEPH is the major MCF in the small intestine and is critical for dietary iron absorption, and ZP is important for normal hair development. CP and HEPH (and possibly ZP) function in multiple tissues. These proteins also play other (non-iron-related) physiological roles, but many of these are ill-defined. In addition to disrupting iron homeostasis, MCF dysfunction perturbs neurological and immune function, alters cancer susceptibility, and causes hair loss, but, despite their importance, how MCFs co-ordinately maintain body iron homeostasis and perform other functions remains incompletely understood.

A Decrease in Maternal Iron Levels Is the Predominant Factor Suppressing Hepcidin during Pregnancy in Mice.

In order to supply adequate iron during pregnancy, the levels of the iron regulatory hormone hepcidin in the maternal circulation are suppressed, thereby increasing dietary iron absorption and storage iron release. Whether this decrease in maternal hepcidin is caused by changes in factors known to regulate hepcidin expression, or by other unidentified pregnancy factors, is not known. To investigate this, we examined iron parameters during pregnancy in mice. We observed that hepatic iron stores and transferrin saturation, both established regulators of hepcidin production, were decreased in mid and late pregnancy in normal and iron loaded dams, indicating an increase in iron utilization. This can be explained by a significant increase in maternal erythropoiesis, a known suppressor of hepcidin production, by mid-pregnancy, as indicated by an elevation in circulating erythropoietin and an increase in spleen size and splenic iron uptake. Iron utilization increased further in late pregnancy due to elevated fetal iron demand. By increasing maternal iron levels in late gestation, we were able to stimulate the expression of the gene encoding hepcidin, suggesting that the iron status of the mother is the predominant factor influencing hepcidin levels during pregnancy. Our data indicate that pregnancy-induced hepcidin suppression likely occurs because of reductions in maternal iron reserves due to increased iron requirements, which predominantly reflect stimulated erythropoiesis in mid-gestation and increased fetal iron requirements in late gestation, and that there is no need to invoke other factors, including novel pregnancy factor(s), to explain these changes.

Inferring the Genetic Basis of Sex Determination from the Genome of a Dioecious Nightshade.

Dissecting the genetic mechanisms underlying dioecy (i.e., separate female and male individuals) is critical for understanding the evolution of this pervasive reproductive strategy. Nonetheless, the genetic basis of sex determination remains unclear in many cases, especially in systems where dioecy has arisen recently. Within the economically important plant genus Solanum (∼2,000 species), dioecy is thought to have evolved independently at least 4 times across roughly 20 species. Here, we generate the first genome sequence of a dioecious Solanum and use it to ascertain the genetic basis of sex determination in this species. We de novo assembled and annotated the genome of Solanum appendiculatum (assembly size: ∼750 Mb scaffold N50: 0.92 Mb; ∼35,000 genes), identified sex-specific sequences and their locations in the genome, and inferred that males in this species are the heterogametic sex. We also analyzed gene expression patterns in floral tissues of males and females, finding approximately 100 genes that are differentially expressed between the sexes. These analyses, together with observed patterns of gene-family evolution specific to S. appendiculatum, consistently implicate a suite of genes from the regulatory network controlling pectin degradation and modification in the expression of sex. Furthermore, the genome of a species with a relatively young sex-determination system provides the foundational resources for future studies on the independent evolution of dioecy in this clade.

A Novel Ferritin-Core Analog Is a Safe and Effective Alternative to Oral Ferrous Iron for Treating Iron Deficiency during Pregnancy in Mice.

BACKGROUND: Many women enter pregnancy with iron stores that are insufficient to maintain maternal iron balance and support fetal development and consequently, often require iron supplements. However, the side effects associated with many currently available iron supplements can limit compliance. OBJECTIVE: This study aimed to test the safety and efficacy of a novel nanoparticulate iron supplement, a dietary ferritin analog termed iron hydroxide adipate tartrate (IHAT), in pregnant mice. METHODS: Female C57BL/6 mice were maintained on either an iron-deficient or a control diet for 2 wk prior to timed mating to develop iron-deficient or iron-sufficient pregnancy models, respectively. Mice from each model were then gavaged daily with 10 mg iron/kg body weight as either IHAT or ferrous sulfate, or with water only, beginning on embryonic day (E) 4.5. Mice were killed on E18.5 and maternal iron and hematological parameters were measured. The expression of genes encoding iron transporters and oxidative stress markers in the duodenum and placenta were determined, along with hepatic expression of the gene encoding the iron regulatory hormone hepcidin and fetal iron. RESULTS: Oral IHAT and ferrous sulfate were equally effective at increasing maternal hemoglobin (20.2% and 16.9%, respectively) and hepatic iron (30.2% and 29.3%, respectively), as well as total fetal iron (99.7% and 83.8%, respectively), in iron-deficient pregnant mice compared with those gavaged with water only, with no change in oxidative stress markers seen with either treatment. However, there was a significant increase in the placental expression of the oxidative stress marker heme oxygenase 1 in iron-replete pregnant mice treated with ferrous sulfate when compared with iron-replete pregnant mice gavaged with IHAT (96.9%, P <0.05). CONCLUSIONS: IHAT has proved a safe and effective alternative to oral ferrous sulfate in mice, and it has potential for treating iron deficiency in human pregnancy.

Supplementation with Sucrosomial® iron leads to favourable changes in the intestinal microbiome when compared to ferrous sulfate in mice.

Iron deficiency is one of the most common nutritional deficiencies worldwide and is often treated with oral iron supplements. However, commonly used supplements, including those based on ferrous iron salts, are associated with gastrointestinal side effects and unfavorable changes in the intestinal microbiome. Sucrosomial® iron is a novel iron formulation that is effective at treating iron deficiency, and with fewer gastrointestinal side effects, yet its effect on the gut microbiome has not been examined previously. Thus, we treated mice for two weeks with diets containing either Sucrosomial® iron or ferrous sulfate as the sole iron source and examined bacterial communities in the intestine using 16S Microbial Profiling of DNA extracted from feces collected both prior to and following dietary treatment. Mice treated with Sucrosomial® iron showed an increase in Shannon diversity over the course of the study. This was associated with a decrease in the abundance of the phylum Proteobacteria, which contains many pathogenic species, and an increase in short chain fatty acid producing bacteria such as Lachnospiraceae, Oscillibacter and Faecalibaculum. None of these changes were observed in mice treated with ferrous sulfate. These results suggest that Sucrosomial® iron may have a beneficial effect on the intestinal microbiome when compared to ferrous sulfate and that this form of iron is a promising alternative to ferrous iron salts for the treatment of iron deficiency.

Biallelic HEPHL1 variants impair ferroxidase activity and cause an abnormal hair phenotype.

Maintenance of the correct redox status of iron is functionally important for critical biological processes. Multicopper ferroxidases play an important role in oxidizing ferrous iron, released from the cells, into ferric iron, which is subsequently distributed by transferrin. Two well-characterized ferroxidases, ceruloplasmin (CP) and hephaestin (HEPH) facilitate this reaction in different tissues. Recently, a novel ferroxidase, Hephaestin like 1 (HEPHL1), also known as zyklopen, was identified. Here we report a child with compound heterozygous mutations in HEPHL1 (NM_001098672) who presented with abnormal hair (pili torti and trichorrhexis nodosa) and cognitive dysfunction. The maternal missense mutation affected mRNA splicing, leading to skipping of exon 5 and causing an in-frame deletion of 85 amino acids (c.809_1063del; p.Leu271_ala355del). The paternal mutation (c.3176T>C; p.Met1059Thr) changed a highly conserved methionine that is part of a typical type I copper binding site in HEPHL1. We demonstrated that HEPHL1 has ferroxidase activity and that the patient's two mutations exhibited loss of this ferroxidase activity. Consistent with these findings, the patient's fibroblasts accumulated intracellular iron and exhibited reduced activity of the copper-dependent enzyme, lysyl oxidase. These results suggest that the patient's biallelic variants are loss-of-function mutations. Hence, we generated a Hephl1 knockout mouse model that was viable and had curly whiskers, consistent with the hair phenotype in our patient. These results enhance our understanding of the function of HEPHL1 and implicate altered ferroxidase activity in hair growth and hair disorders.

The Placental Ferroxidase Zyklopen Is Not Essential for Iron Transport to the Fetus in Mice.

BACKGROUND: The ferroxidase zyklopen (Zp) has been implicated in the placental transfer of iron to the fetus. However, the evidence for this is largely circumstantial. OBJECTIVES: This study aimed to determine whether Zp is essential for placental iron transfer. METHODS: A model was established using 8- to 12-wk-old pregnant C57BL/6 mice on standard rodent chow in which Zp was knocked out in the fetus and fetal components of the placenta. Zp was also disrupted in the entire placenta using global Zp knockout mice. Inductively coupled plasma MS was used to measure total fetal iron, an indicator of the amount of iron transferred by the placenta to the fetus, at embryonic day 18.5 of gestation. Iron transporter expression in the placenta was measured by Western blotting, and the expression of Hamp1, the gene encoding the iron regulatory hormone hepcidin, was determined in fetal liver by real-time PCR. RESULTS: There was no change in the amount of iron transferred to the fetus when Zp was disrupted in either the fetal component of the placenta or the entire placenta. No compensatory changes in the expression of the iron transport proteins transferrin receptor 1 or ferroportin were observed, nor was there any change in fetal liver Hamp1 mRNA. Hephl1, the gene encoding Zp, was expressed mainly in the maternal decidua of the placenta and not in the nutrient-transporting syncytiotrophoblast. Disruption of Zp in the whole placenta resulted in a 26% increase in placental size (P < 0.01). CONCLUSIONS: Our data indicate that Zp is not essential for the efficient transfer of iron to the fetus in mice and is localized predominantly in the maternal decidua. The increase in placental size observed when Zp is knocked out in the entire placenta suggests that this protein may play a role in placental development.

Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis.

BACKGROUND: Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. RESULTS: Here, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator deferoxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane. CONCLUSION: These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis.

Dihydrolipoic Acid-Gold Nanoclusters Regulate Microglial Polarization and Have the Potential To Alter Neurogenesis.

Microglia-mediated neuroinflammation is one of the most significant features in a variety of central nervous system (CNS) disorders such as traumatic brain injury, stroke, and many neurodegenerative diseases. Microglia become polarized upon stimulation. The two extremes of the polarization are the neuron-destructive proinflammatory M1-like and the neuron-regenerative M2-like phenotypes. Thus, manipulating microglial polarization toward the M2 phenotype is a promising therapeutic approach for CNS repair and regeneration. It has been reported that nanoparticles are potential tools for regulating microglial polarization. Gold nanoclusters (AuNCs) could penetrate the blood-brain barrier and have neuroprotective effects, suggesting the possibility of utilizing AuNCs to regulate microglial polarization and improve neuronal regeneration in CNS. In the current study, AuNCs functionalized with dihydrolipoic acid (DHLA-AuNCs), an antioxidant with demonstrated neuroprotective roles, were prepared, and their effects on polarization of a microglial cell line (BV2) were examined. DHLA-AuNCs effectively suppressed proinflammatory processes in BV2 cells by inducing polarization toward the M2-like phenotype. This was associated with a decrease in reactive oxygen species and reduced NF-kB signaling and an improvement in cell survival coupled with enhanced autophagy and inhibited apoptosis. Conditioned medium from DHLA-AuNC-treated BV2 cells was able to enhance neurogenesis in both the neuronal cell line N2a and in an ex vivo brain slice stroke model. The direct treatment of brain slices with DHLA-AuNCs also ameliorated stroke-related tissue injury and reduced astrocyte activation (astrogliosis). This study suggests that by regulating neuroinflammation to improve neuronal regeneration, DHLA-AuNCs could be a potential therapeutic agent in CNS disorders.

Modularly Designed Peptide Nanoprodrug Augments Antitumor Immunity of PD-L1 Checkpoint Blockade by Targeting Indoleamine 2,3-Dioxygenase.

The limited efficacy of single-agent immune checkpoint inhibitors in treating tumors has prompted investigations on their combination partners. Here, a tumor-homing indoleamine 2,3-dioxygenase (IDO) nanoinhibitor is reported to selectively inhibit immunosuppressive IDO pathway in the tumor microenvironment. It is self-assembled from a modularly designed peptide-drug conjugate containing a hydrophilic targeting motif (arginyl-glycyl-aspartic acid; RGD), two protonatable histidines, and an ester bond-linked hydrophobic IDO inhibitor, which exhibits pH-responsive disassembly and esterase-catalyzed drug release. Markedly, it achieved potent and persistent inhibition of intratumoral IDO activity with a reduced systemic toxicity, which greatly enhanced the therapeutic efficacy of programmed cell death-ligand 1 blockade in vivo. Overall, this study provides a promising paradigm of combinatorial normalization immunotherapy by exploiting a targeted IDO nanoinhibitor to augment the antitumor immunity of checkpoint inhibitors.

Regression of Fibrosis Stage With Treatment Reduces Long-Term Risk of Liver Cancer in Patients With Hemochromatosis Caused by Mutation in HFE.

BACKGROUND & AIMS: Fibrosis stage can decrease following treatment in patients with hemochromatosis caused by mutations in the homeostatic iron regulator gene (HFE), but the effects on cirrhosis are not clear. We assessed regression of severe fibrosis and the ensuing risk of liver cancer after treatment. METHODS: We performed a retrospective analysis of data from 106 patients in France or Australia who were homozygous for the C282Y mutation in HFE with F3 fibrosis (n = 40) or F4 fibrosis (n = 66) at diagnosis and from whom at least 1 liver biopsy was collected during follow up. We collected data from the time of first biopsy and during follow-up period on patient demographics, treatment, smoking habits, alcohol consumption, infection with hepatitis B or C viruses, and other diseases. The median time between first and last liver biopsy was 9.5 years (range, 3.5-15.6 years). We collected results of tests for liver function, markers of iron stores, and platelet levels. Patients were followed for a median 17.6 years (range, 9.8-24.1 years) for development of liver cancer occurrence. RESULTS: At last liver biopsy, 41 patients (38.6%) had fibrosis scores of F2 or less. Liver cancer occurred in 34 patients (52.3%) with F3 or F4 fibrosis at last liver biopsy vs 2 patients (4.8%) with fibrosis scores of F2 or less at last liver biopsy (P < .001). Liver cancer incidences were 32.8 per 1000 person-years (95% CI, 22.7-45.9 per 1000 person-years) in patients with F3 or F4 fibrosis and 2.3 per 1000 person-years (95% CI, 0.2-8.6 per 1000 person-years) in patients with fibrosis scores of F2 or less (P < .001). In multivariate analysis, male sex (hazard ratio [HR], 6.09; 95% CI, 1.21-30.4), age at diagnosis (HR, 1.16; 95% CI, 1.09-1.25), presence of diabetes (HR, 3.07; 95% CI, 1.35-6.97), excess alcohol consumption (HR, 3.1; 95% CI, 1.47-6.35), serum level of ferritin at diagnosis (P < .01), and regression to fibrosis scores of F2 or less (HR, 0.08; 95% CI, 0.01-0.62) were significantly associated with risk of liver cancer. CONCLUSIONS: In a retrospective analysis of patients with hemochromatosis caused by the C282Y mutation in HFE, we found that severe liver fibrosis can regress with treatment. In patients with fibrosis regression to a stage F2 or less, the long-term risk for liver cancer is significantly reduced.

High Dietary Iron Disrupts Iron Homeostasis and Induces Amyloid-ß and Phospho-τ Expression in the Hippocampus of Adult Wild-Type and APP/PS1 Transgenic Mice.

BACKGROUND: Brain iron deposition is a feature of Alzheimer disease and may contribute to its development. However, the relative contribution of dietary iron remains unclear. OBJECTIVES: We investigated the impact of high dietary iron on brain pathological changes and cognitive function in adult wild-type (WT) mice and amyloid precursor protein/presenilin 1 (APP/PS1) double transgenic mice. METHODS: Male WT mice and APP/PS1 mice aged 10 wk were fed either a control diet (66 mg Fe/kg) (WT-Ctrl and APP/PS1-Ctrl) or a high iron diet (14 g Fe/kg) (WT-High Fe and APP/PS1-High Fe) for 20 wk. Iron concentrations in brain regions were measured by atomic absorption spectrophotometry. Brain iron staining and amyloid-ß (Aß) immunostaining were performed. Protein expressions in the hippocampus were determined by immunoblotting. Superoxide dismutase (SOD) activity and malondialdehyde concentration were examined. Cognitive functions were tested with the Morris water maze system. RESULTS: In the hippocampus, APP/PS1-High Fe mice had significantly higher iron concentration (2.5-fold) and ferritin (2.0-fold) than APP/PS1-Ctrl mice (P < 0.001), and WT-High Fe mice had significantly higher ferritin (2.0-fold) than WT-Ctrl mice (P < 0.001). Interestingly, APP/PS1 mice had significantly higher iron concentration (2-3-fold) and ferritin (2-2.5-fold) than WT mice fed either diet (P < 0.001). Histological analysis indicated that iron accumulated in the hippocampal dentate gyrus region in APP/PS1 mice, consistent with the pattern of Aß deposition. For both mouse strains, iron treatment induced Aß and phospho-τ expression (1.5-3-fold) in the hippocampus, but had little impact on oxidative stress and cognitive function. Furthermore, APP/PS1 mice had significantly lower SOD activity and higher malondialdehyde concentration than WT mice in the hippocampus (P < 0.0001), paralleled by apparent cognitive dysfunction. CONCLUSIONS: Dietary iron overload induces iron disorder and Aß and phospho-τ expression in the hippocampus of adult WT and APP/PS1 transgenic mice.

Food deprivation increases hepatic hepcidin expression and can overcome the effect of Hfe deletion in male mice.

Iron-loading disorders, such as hereditary hemochromatosis, are associated with inappropriately low expression of the iron regulatory hormone, hepcidin. A recent study has demonstrated that food deprivation can increase hepcidin production in mice. We have examined this effect in more detail to determine whether the pathway(s) that are responsible might provide novel targets for pharmaceutical intervention in disorders of iron homeostasis. C57BL/6 mice were deprived of food for 5, 10, 16, or 24 h before euthanasia, then blood and tissue samples were collected for analysis. The effect of food deprivation was also examined in Hfe-/- mice, a model of hereditary hemochromatosis, as well as mice that were maintained on an iron-deficient diet or injected with erythropoietin. Food deprivation increased the hepatic expression of the gene that encodes hepcidin, hepcidin antimicrobial peptide 1 ( Hamp1), with maximal expression observed after 16 h, and was able to overcome the reduction in Hamp1 expression associated with Hfe deficiency. Food deprivation also increased Hamp1 expression in response to stimuli that more strongly suppress the gene, such as iron deficiency and erythropoietin treatment, but the effects were not significant. These results indicate that Hamp1 induction by food deprivation is independent of HFE and suggest that targeting the pathway regulated by food deprivation could have clinical benefit in iron-loading conditions.-Mirciov, C. S. G., Wilkins, S. J., Anderson, G. J., Frazer, D. M. Food deprivation increases hepatic hepcidin expression and can overcome the effect of Hfe deletion in male mice.

Dietary iron absorption during early postnatal life.

Inadequate iron levels during early life can have adverse consequences for the developing infant. Iron deficiency during this critical period of growth can affect brain development and cognitive function, problems that can be lifelong despite subsequent correction of the iron deficit. Therefore, it is critical that the suckling infant has sufficient iron for their developmental needs. Much of the iron used in the immediate post-natal period is stored iron that was acquired from the mother in the final trimester of pregnancy, however, despite having low iron levels, breast milk can also make a significant contribution to infant iron needs. This reflects the ability of the suckling infant to absorb dietary iron far more efficiently than is possible after weaning. The mechanisms underlying this enhanced iron absorption are poorly understood. The iron export protein ferroportin is essential for this process, as it is in adults, however, the role of other molecules normally involved in iron absorption following weaning is less clear. The composition and distribution of iron in breast milk may be important, as could the contribution of more distal parts of the gastrointestinal tract. This review discusses the potential role of each of the above components in intestinal iron absorption during suckling and highlights the need for further research into this important process.

Polymeric Nanoparticles Enhance the Ability of Deferoxamine To Deplete Hepatic and Systemic Iron.

Chelators are commonly used to remove excess iron in iron-loading disorders. Deferoxamine (DFO) is an effective and safe iron chelator but an onerous parenteral administration regimen limits its routine use. To develop more effective methods for delivering iron chelators, we examined whether amphiphilic copolymer nanoparticles (NPs) could deliver DFO more efficiently. Physical characterization showed a uniform and stable preparation of DFO nanoparticles (DFO-NPs) with an average diameter of 105.3 nm. In macrophage (RAW264.7) and hepatoma (HepG2) cell lines, DFO-NPs proved more effective at depleting iron than free DFO. In wild-type mice previously loaded with iron dextran, as well as Hbb th3 /+ and Hfe -/- mice, which are predisposed to iron loading, DFO-NPs (40 mg/kg DFO; alternate days; 4 weeks) reduced hepatic iron levels by 71, 46, and 37%, respectively, whereas the equivalent values for free DFO were 53, 7, and 15%. Staining for tissue iron and urinary iron excretion confirmed these findings. Pharmacokinetic analysis showed that NP-encapsulated DFO had a much longer elimination half-life than free DFO (48.63 ± 28.80 vs 1.46 ± 0.59 h), and that DFO-NPs could be readily taken up by tissues and in particular by hepatic Kupffer cells. In vitro, DFO-NPs were less toxic to several cell lines than free DFO, and in vivo they did not elicit any specific inflammatory responses or histological changes. Our results suggest that using a nanoformulation of DFO is a valuable strategy for improving its efficiency as an iron chelator and that this could broaden its clinical use for the treatment of human iron overload disorders.

Sequentially Responsive Therapeutic Peptide Assembling Nanoparticles for Dual-Targeted Cancer Immunotherapy.

Combination therapeutic regimen is becoming a primary direction for current cancer immunotherapy to broad the antitumor response. Functional nanomaterials offer great potential for steady codelivery of various drugs, especially small molecules, therapeutic peptides, and nucleic acids, thereby realizing controllable drug release, increase of drug bioavailability, and reduction of adverse effects. Herein, a therapeutic peptide assembling nanoparticle that can sequentially respond to dual stimuli in the tumor extracellular matrix was designed for tumor-targeted delivery and on-demand release of a short d-peptide antagonist of programmed cell death-ligand 1 (DPPA-1) and an inhibitor of idoleamine 2,3-dioxygenase (NLG919). By concurrent blockade of immune checkpoints and tryptophan metabolism, the nanoformulation increased the level of tumor-infiltrated cytotoxic T cells and in turn effectively inhibited melanoma growth. To achieve this, an amphiphilic peptide, consisting of a functional 3-diethylaminopropyl isothiocyanate (DEAP) molecule, a peptide substrate of matrix metalloproteinase-2 (MMP-2), and DPPA-1, was synthesized and coassembled with NLG919. The nanostructure swelled when it encountered the weakly acidic tumor niche where DEAP molecules were protonated, and further collapsed due to the cleavage of the peptide substrate by MMP-2 that is highly expressed in tumor stroma. The localized release of DPPA-1 and NLG919 created an environment which favored the survival and activation of cytotoxic T lymphocytes, leading to the slowdown of melanoma growth and increase of overall survival. Together, this study offers new opportunities for dual-targeted cancer immunotherapy through functional peptide assembling nanoparticles with design features that are sequentially responsive to the multiple hallmarks of the tumor microenvironment.

Copper Ions and Coordination Complexes as Novel Carbapenem Adjuvants.

Carbapenem-resistant Enterobacteriaceae are urgent threats to global human health. These organisms produce ß-lactamases with carbapenemase activity, such as the metallo-ß-lactamase NDM-1, which is notable due to its association with mobile genetic elements and the lack of a clinically useful inhibitor. Here we examined the ability of copper to inhibit the activity of NDM-1 and explored the potential of a copper coordination complex as a mechanism to efficiently deliver copper as an adjuvant in clinical therapeutics. An NDM-positive Escherichia coli isolate, MS6192, was cultured from the urine of a patient with a urinary tract infection. MS6192 was resistant to antibiotics from multiple classes, including diverse ß-lactams (penicillins, cephalosporins, and carbapenems), aminoglycosides, and fluoroquinolones. In the presence of copper (range, 0 to 2 mM), however, the susceptibility of MS6192 to the carbapenems ertapenem and meropenem increased markedly. In standard checkerboard assays, copper decreased the MICs of ertapenem and meropenem against MS6192 in a dose-dependent manner, suggesting a synergistic mode of action. To examine the inhibitory effect of copper in the absence of other ß-lactamases, the blaNDM-1 gene from MS6192 was cloned and expressed in a recombinant E. coli K-12 strain. Analysis of cell extracts prepared from this strain revealed that copper directly inhibited NDM-1 activity, which was confirmed using purified recombinant NDM-1. Finally, delivery of copper at a low concentration of 10 µM by using the FDA-approved coordination complex copper-pyrithione sensitized MS6192 to ertapenem and meropenem in a synergistic manner. Overall, this work demonstrates the potential use of copper coordination complexes as novel carbapenemase adjuvants.

Circulating iron levels influence the regulation of hepcidin following stimulated erythropoiesis.

The stimulation of erythrocyte formation increases the demand for iron by the bone marrow and this in turn may affect the levels of circulating diferric transferrin. As this molecule influences the production of the iron regulatory hormone hepcidin, we hypothesized that erythropoiesis-driven changes in diferric transferrin levels could contribute to the decrease in hepcidin observed following the administration of erythropoietin. To examine this, we treated mice with erythropoietin and examined diferric transferrin at various time points up to 18 hours. We also investigated the effect of altering diferric transferrin levels on erythropoietin-induced inhibition of Hamp1, the gene encoding hepcidin. We detected a decrease in diferric transferrin levels 5 hours after erythropoietin injection and prior to any inhibition of the hepatic Hamp1 message. Diferric transferrin returned to control levels 12 hours after erythropoietin injection and had increased beyond control levels by 18 hours. Increasing diferric transferrin levels via intravenous iron injection prevented the inhibition of Hamp1 expression by erythropoietin without altering hepatic iron concentration or the expression of Erfe, the gene encoding erythroferrone. These results suggest that diferric transferrin likely contributes to the inhibition of hepcidin production in the period shortly after injection of erythropoietin and that, under the conditions examined, increasing diferric transferrin levels can overcome the inhibitory effect of erythroferrone on hepcidin production. They also imply that the decrease in Hamp1 expression in response to an erythropoietic stimulus is likely to be mediated by multiple signals.

Multi-Copper Ferroxidase-Deficient Mice Have Increased Brain Iron Concentrations and Learning and Memory Deficits.

Background: The accumulation of iron occurs in the central nervous system (CNS) in several neurodegenerative diseases. Although multi-copper ferroxidases (MCFs) play an important role in cellular iron metabolism and homeostasis, the mechanism of MCFs in the CNS remains unclear. Objective: The aim was to study the role of MCFs in CNS iron metabolism and homeostasis by using hephaestin/ceruloplasmin (Heph/Cp) double knockout (KO) mice. Methods: Heph/Cp double KO male mice were generated by crossing both single KO mice. In Heph/Cp KO and wild-type (WT) control mice at 4 wk and 6 mo of age, iron concentrations of selected brain regions were measured by atomic absorption spectrophotometry, and gene expressions of Heph, Cp, ferroportin 1 (Fpn1) [+ iron responsive element (IRE)], L-ferritin, H-ferritin, transferrin receptor 1 (Tfrc), and divalent metal transporter 1 (Dmt1) (+IRE) were quantitated by quantitative reverse transcriptase-polymerase chain reaction. Brain region L-ferritin protein concentration, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and malondialdehyde (MDA) concentration were also determined. Learning and memory abilities in Heph/Cp KO and WT control mice at 6 mo of age were tested by the IntelliCage system (New Behavior). Results: Iron concentration was significantly higher in Heph/Cp KO mice than in WT control mice at 4 wk of age in the cortex (50%), hippocampus (120%), brainstem (35%), and cerebellum (220%) and at 6 mo of age in the cortex (140%), hippocampus (420%), brainstem (560%), and cerebellum (340%). L-Ferritin and MDA concentrations were significantly higher and SOD and GPx activities were significantly lower in the cortex, hippocampus, brainstem, and cerebellum of KO mice than in those of WT controls at both 4 wk and 6 mo of age. Iron-related gene expressions also differed significantly between groups. Learning and memory deficits occurred in Heph/Cp KO mice at 6 mo of age. Conclusion: Mutation of both MCFs in mice induces iron accumulation in brain regions, oxidative damage, and learning and memory defects.

Iron supplementation has minor effects on gut microbiota composition in overweight and obese women in early pregnancy.

Fe is an essential nutrient for many bacteria, and Fe supplementation has been reported to affect the composition of the gut microbiota in both Fe-deficient and Fe-replete individuals outside pregnancy. This study examined whether the dose of Fe in pregnancy multivitamin supplements affects the overall composition of the gut microbiota in overweight and obese pregnant women in early pregnancy. Women participating in the SPRING study with a faecal sample obtained at 16 weeks' gestation were included in this substudy. For each subject, the brand of multivitamin used was recorded. Faecal microbiome composition was assessed by 16S rRNA sequencing and analysed with the QIIME software suite. Dietary intake of Fe was assessed using a FFQ at 16 weeks' gestation. Women were grouped as receiving low (<60 mg/d, n 94) or high (≥60 mg/d; n 65) Fe supplementation. The median supplementary Fe intake in the low group was 10 (interquartile range (IQR) 5-10) v. 60 (IQR 60-60) mg/d in the high group (P<0·001). Dietary Fe intake did not differ between the groups (10·0 (IQR 7·4-13·3) v. 9·8 (IQR 8·2-13·2) mg/d). Fe supplementation did not significantly affect the composition of the faecal microbiome at any taxonomic level. Network analysis showed that the gut microbiota in the low Fe supplementation group had a higher predominance of SCFA producers. Pregnancy multivitamin Fe content has a minor effect on the overall composition of the gut microbiota of overweight and obese pregnant women at 16 weeks' gestation.