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Iron (Fe) storage in plant seeds is not only necessary for seedling establishment following germination but is also a major source of dietary Fe for humans and other animals. Accumulation of Fe in seeds is known to be low during early seed development. However, the underlying mechanism and biological significance remain elusive. Here, we show that reduced expression of Arabidopsis YABBY transcription factor INNER NO OUTER (INO) increases embryonic Fe accumulation, while transgenic overexpression of INO results in the opposite effect. INO is highly expressed during early seed development, and decreased INO expression increases the expression of NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1), which encodes a transporter that contributes to seed Fe loading. The relatively high embryonic Fe accumulation conferred by decreased INO expression is rescued by the nramp1 loss-of-function mutation. We further demonstrated that INO represses NRAMP1 expression by binding to NRAMP1-specific promoter region. Interestingly, we found that excessive Fe loading into developing seeds of ino mutants results in greater oxidative damage, leading to increased cell death and seed abortion, a phenotype that can be rescued by the nramp1 mutation. Taken together, these results indicate that INO plays an important role in safeguarding reproduction by reducing Fe loading into developing seeds by repressing NRAMP1 expression., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass ( Dicentrarchus labrax ), two different hepcidin types are found, with specialized roles: the single type 1 hepcidin is involved in iron homeostasis trough the regulation of ferroportin, the only known iron exporter; and the various type 2 hepcidins present antimicrobial activity against a number of different pathogens. In this study, we tested the administration of sea bass derived hepcidins in models of infection and iron overload. Administration with hamp2 substantially reduced fish mortalities and bacterial loads, presenting itself as a viable alternative to the use of antibiotics. On the other hand, hamp1 seems to attenuate the effects of iron overload. Further studies are necessary to test the potential protective effects of hamp2 against other pathogens, as well as to understand how hamp2 stimulate the inflammatory responses, leading to an increased fish survival upon infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Barroso, Carvalho, Nunes, Gonçalves, Rodrigues and Neves.)

Zinc and iron are essential micronutrients for plant growth, and their homeostasis must be tightly regulated. Previously, it has been shown that Zinc-Induced Facilitator 1 (ZIF1) is involved in basal Zn tolerance by controlling the vacuolar storage of nicotianamine (NA). However, knowledge of the functional roles of two ZIF1 paralogs, ZIF-LIKE1 (ZIFL1) and ZIFL2, in metal homeostasis remains limited. Here, we functionally characterized the roles of ZIF1, ZIFL1, and ZIFL2 in Zn and Fe homeostasis. Expression of ZIF1 and ZIFL1 was induced by both excess Zn and Fe-deficiency, and their loss-of-function led to hypersensitivity under excess Zn and Fe-deficiency, suggesting functional overlap between ZIF1 and ZIFL1. By contrast, the disruption of ZIFL2 resulted in no obvious phenotypic alteration under both conditions. Additionally, the expression of ZIFL1, but not that of ZIFL2, in the zif1 mutant partially restored the phenotype under excess Zn, suggesting that ZIF1 and ZIFL1 perform functionally redundant roles in Zn homeostasis., Competing Interests: Declaration of competing interest The author declares no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The demand for iron is high in pregnancy to meet the increased requirements for erythropoiesis. Even pregnant females with initially iron-replete stores develop iron-deficiency anemia, due to inadequate iron absorption. In anemic females, the maternal iron supply is dedicated to maintaining iron metabolism in the fetus and placenta. Here, using a mouse model of iron deficiency in pregnancy, we show that iron recycled from senescent erythrocytes becomes a predominant source of this microelement that can be transferred to the placenta in females with depleted iron stores. Ferroportin is a key protein in the molecular machinery of cellular iron egress. We demonstrate that under iron deficiency in pregnancy, levels of ferroportin are greatly reduced in the duodenum, placenta and fetal liver, but not in maternal liver macrophages and in the spleen. Although low expression of both maternal and fetal hepcidin predicted ferroportin up-regulation in examined locations, its final expression level was very likely correlated with tissue iron status. Our results argue that iron released into the circulation of anemic females is taken up by the placenta, as evidenced by high expression of iron importers on syncytiotrophoblasts. Then, a substantial decrease in levels of ferroportin on the basolateral side of syncytiotrophoblasts, may be responsible for the reduced transfer of iron to the fetus. As attested by the lowest decrease in iron content among analyzed tissues, some part is retained in the placenta. These findings confirm the key role played by ferroportin in tuning iron turnover in iron-deficient pregnant mouse females and their fetuses., (© 2021 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

In the present study, we demonstrate the coaction of thioredoxin and glutathione (GSH) systems in mouse liver against iron overload-induced oxidative stress (OS). Mice were injected intraperitoneally with an iron dextran solution twice a week for 3 weeks. Iron accumulation in mouse liver was demonstrated spectroscopically. To confirm the iron overload model in the liver, the increased gene expression levels of hepcidin (Hamp), ferroportin (Fpn1), and ferritin (Fth1), which regulate iron trafficking, were observed by a quantitative polymerase chain reaction. In the case of iron overload, the GSH level and the reduced glutathione/oxidized glutathione ratio, which represents a marker of OS, decreased significantly. An increase in the malondialdehyde level, one of the final products of the lipid peroxidation process, was observed. The gene expression of the thioredoxin system, including thioredoxin (Trx1) and thioredoxin reductase (TrxR1), was examined. Though TrxR1 expression decreased, no changes were observed in Trx1. The enzyme activity and semiquantitative protein expression of TRXR1 increased. The activity of GSH reductase and GSH peroxidase increased in the iron overload group. The gene and protein expressions of thioredoxininteracting protein, which is an indicator of the commitment of the cell to apoptosis, were elevated significantly. The increased protein expression of Bcl-2-related X protein and CASPASE-3, which is an indicator of apoptosis, increased significantly. In conclusion, excess iron accumulation in mouse liver tissue causes OS, which affects the redox state of the thioredoxin and GSH systems, inducing cell apoptosis and also ferroptosis due to increased lipid peroxidation and the depletion of GSH level., (© 2020 Wiley Periodicals LLC.)

Zinc constitutes the second most abundant transition metal in the human body, and it is implicated in numerous cellular processes, including cell division, DNA and protein synthesis as well as for the catalytic activity of many enzymes. Two major membrane protein families facilitate zinc homeostasis in the animal kingdom, i.e., Zrt/Irt-like proteins (ZIPs aka solute carrier 39, SLC39, family) and Zn transporters (ZnTs), essentially conducting zinc flux in the opposite directions. Human ZIPs (hZIPs) regulate import of extracellular zinc to the cytosol, being critical in preventing overaccumulation of this potentially toxic metal, and crucial for diverse physiological and pathological processes, including development of neurodegenerative disorders and several cancers. To date, our understanding of structure-function relationships governing hZIP-mediated zinc transport mechanism is scarce, mainly due to the notorious difficulty in overproduction of these proteins for biophysical characterization. Here we describe employment of a Saccharomyces cerevisiae -based platform for heterologous expression of hZIPs. We demonstrate that yeast is able to produce four full-length hZIP members belonging to three different subfamilies. One target (hZIP1) is purified in the high quantity and homogeneity required for the downstream biochemical analysis. Our work demonstrates the potential of the described production system for future structural and functional studies of hZIP transporters.

In mammals, 2 × 10 12 red blood cells (RBCs) are produced every day in the bone marrow to ensure a constant supply of iron to maintain effective erythropoiesis. Impaired iron absorption in the duodenum and inefficient iron reutilization from senescent RBCs by macrophages contribute to the development of anemia. Ferroportin (Fpn), the only known cellular iron exporter, as well as hephaestin (Heph) and ceruloplasmin, two copper-dependent ferroxidases involved in the above-mentioned processes, are key elements of the interaction between copper and iron metabolisms. Crosslinks between these metals have been known for many years, but metabolic effects of one on the other have not been elucidated to date. Neonatal iron deficiency anemia in piglets provides an interesting model for studying this interplay. In duodenal enterocytes of young anemic piglets, we identified iron deposits and demonstrated increased expression of ferritin with a concomitant decline in both Fpn and Heph expression. We postulated that the underlying mechanism involves changes in copper distribution within enterocytes as a result of decreased expression of the copper transporter-Atp7b. Obtained results strongly suggest that regulation of iron absorption within enterocytes is based on the interaction between proteins of copper and iron metabolisms and outcompetes systemic regulation.

Background: Hepatocellular carcinoma (HCC), the fourth leading cause of cancer-related deaths worldwide, has increased public concern. Data from previous work have validated that long noncoding RNAs are active participators in the malignant processes of a host of cancers. Small nucleolar RNA host gene 7 (SNHG7) has been revealed to act as a tumor promoter in several cancers and SNHG7 inhibition was revealed to suppress cell invasion in HCC. Nevertheless, the specific role of SNHG7 in HCC deserves deeper exploration. Aim of the Study: This work aimed to uncover the role and the regulatory mechanisms of SNHG7 in HCC. Materials and Methods: The expression of SNHG7 and cyclin mediator 1 (CNNM1) in HCC cells were analyzed by quantitative real-time polymerase chain reaction. The influences of SNHG7 on HCC occurrence were studied by cell counting kit-8 (CCK-8), colony formation, flow cytometry analysis, and Western blot assays. Luciferase reporter assay or RNA immunoprecipitation assay was conducted to confirm the relationship between miR-9-5p and SNHG7 (or CNNM1). Results: SNHG7 was overexpressed in HCC tissues and cell lines. SNHG7 facilitated cell proliferation, while suppressed cell apoptosis in HCC. Moreover, miR-9-5p expression was negatively modulated by SNHG7 and therefore was downregulated in HCC cells. We also found that CNNM1 existed in miR-9-5p induced RNA-induced silencing complex and a series of assays verified that CNNM1 acted as the target gene of miR-9-5p. Consequently, the messenger RNA and protein level of CNNM1 were detected to be inversely regulated by miR-9-5p. Moreover, rescue assays demonstrated that CNNM1 overexpression could countervail the SNHG7 depletion-mediated cellular functions of HCC cells. Conclusions: SNHG7 sponges miR-9-5p to upregulate CNNM1 in promoting HCC progression.

Type 2 diabetes is a chronic metabolic disease characterized by pancreatic β-cell dysfunction and peripheral insulin resistance. Among individuals with type 2 diabetes, ∼30% exhibit hypomagnesemia. Hypomagnesemia has been linked to insulin resistance through reduced tyrosine kinase activity of the insulin receptor; however, its impact on pancreatic β-cell function is unknown. In this study, through analysis of several single-cell RNA-sequencing data sets in tandem with quantitative PCR validation in both murine and human islets, we identified NIPAL1 (NIPA-like domain containing 1), encoding a magnesium influx transporter, as an islet-enriched gene. A series of immunofluorescence experiments confirmed NIPAL1 's magnesium-dependent expression and that it specifically localizes to the Golgi in Min6-K8 cells, a pancreatic β-cell-like cell line (mouse insulinoma 6 clone K8). Under varying magnesium concentrations, NIPAL1 knockdown decreased both basal insulin secretion and total insulin content; in contrast, its overexpression increased total insulin content. Although the expression, distribution, and magnesium responsiveness of NIPAL1 in α-TC6 glucagonoma cells (a pancreatic α-cell line) were similar to the observations in Min6-K8 cells, no effect was observed on glucagon secretion in α-TC6 cells under the conditions studied. Overall, these results suggest that NIPAL1 expression is regulated by extracellular magnesium and that down-regulation of this transporter decreases glucose-stimulated insulin secretion and intracellular insulin content, particularly under conditions of hypomagnesemia., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of the article., (© 2020 Manialawy et al.)

Background: miR-194-5p has been associated with drug resistance in many cancers. However, the role of miR-194-5p in cisplatin resistance in ovarian cancer is still unclear., Materials and Methods: To study the role and mechanism of miR-194-5p in cisplatin resistance, qRT-PCR was performed to determine the expression of miR-194-5p and SLC40A1 in ovarian cancer. Cell Counting Kit-8 (CCK8) assay was used to analyse cell viability after cisplatin treatment. Dual-luciferase reporter gene assay was performed to examine the relationship between miR-194-5p and SLC40A1. The genes downstream of SLC40A1 were investigated through bioinformatics analysis., Results: Compared to cisplatin-sensitive ovarian cancer cells, higher miR-194-5p expression and lower SLC40A1 expression were found in cisplatin-resistant ovarian cancer cells. Moreover, this study demonstrated that over-expression of miR-194-5p inhibited SLC40A1 expression, and knockdown of miR-194-5p promoted SLC40A1 expression. In addition, dual-luciferase reporter gene assay further confirmed the negative correlation between miR-194-5p and SLC40A1. Furthermore, we found that over-expression of miR-194-5p resulted in cisplatin resistance. When miR-194-5p and SLC40A1 were simultaneously up-regulated, cisplatin sensitivity increased, while down-regulation of miR-194-5p sensitised ovarian cancer cells to cisplatin. However, when miR-194-5p and SLC40A1 were simultaneously down-regulated, cisplatin sensitivity was decreased. These data suggested that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance. In addition, bioinformatics analysis indicated a positive correlation of SLC40A1 with hephaestin (HEPH), and homeostatic iron regulator (HFE). However, we found that HEPH and HFE were associated with cisplatin resistance, suggesting that their role in drug resistance is induced by miR-194-5p/SLC40A1., Conclusion: In conclusion, we found that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance in ovarian cancer. This study suggests that miR-194-5p could be a potential therapeutic target and a prognostic biomarker for ovarian cancer, with important implications for future research., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Objective: Colorectal cancer is a common malignancy and a common cause of tumor-related death. Long non-coding RNAs (lncRNAs) have become an important regulatory factor and tissue specific biomarker for a variety of cancers, including colorectal cancer. Recent evidence indicates that the novel lncRNA SLC30A10 plays an important role in tumor progression and metastasis. However, its role and molecular mechanisms in colorectal cancer are unclear., Patients and Methods: SLC30A10 expression was detected in 12 colorectal cancer and adjacent normal tissues by quantitative reverse transcription PCR. Insights into the underlying mechanisms of competitive endogenous RNAs (ceRNAs) were determined by transwell assay, CCK8 assay, and luciferase assay., Results: SLC30A10 was down-regulated in colorectal cancer tissues and cell lines, and its low expression was positively correlated with colorectal cancer progression and metastasis. Functionally, SLC30A10 depletion promotes cell proliferation and migration in colorectal cancer cells, while SLC30A10 overexpression has the opposite effect. Bioinformatics prediction and luciferase assay indicated that miR-21c is a direct target of SLC30A10, which plays the role of ceRNA in regulating colorectal cancer metastasis. In addition, miR-21c specifically targets APC gene., Conclusions: Our findings suggest that reduced expression of SLC30A10 is associated with aggressive tumor phenotypes and poor patient outcomes in colorectal cancer. SLC30A10 inhibits colorectal cancer progression and metastasis by acting as a ceRNA for miR-21c to regulate APC expression, suggesting that SLC30A10 may serve as a potential prognostic biomarker and anti-metastatic therapeutic target for colorectal cancer.

Morphine tolerance is a classic, challenging clinical issue. However, the mechanism underlying this phenomenon remains poorly understood. Recently, studies have shown that ferroptosis correlates with drug resistance. Therefore, this study investigated whether spinal cord ferroptosis contributes to morphine tolerance. C57BL/6 mice were continuously subcutaneously injected with morphine, with or without the ferroptosis inhibitor liproxstatin-1. We found that chronic morphine exposure led to morphine antinociception tolerance, accompanied by loss of spinal cord neurons, increase in the levels of iron, malondialdehyde, and reactive oxygen species, and decreases in the levels of superoxide dismutase. Additionally, inflammatory response and mitochondrial shrinkage, processes that are involved in ferroptosis, were observed. Simultaneously, we found that 10 mg/kg of liproxstatin-1 could alleviate iron overload by balancing transferrin receptor protein 1/ferroportin expression and attenuate morphine tolerance by increasing glutathione peroxidase 4 levels, while reducing the levels of malondialdehyde and reactive oxygen species. It also downregulated the expression of extracellularly regulated protein kinases that had been induced by chronic morphine exposure. Our results indicate that spinal cord ferroptosis contributes to morphine tolerance, while liproxstatin-1 attenuates the development of morphine tolerance. These findings suggest that ferroptosis may be a potential therapeutic target for morphine tolerance.

As a magnetic resonance imaging (MRI) reporter gene, MagA has become a powerful tool to monitor dynamic gene expression and allowed concomitant high resolution anatomical and functional imaging of subcellular genetic information. Here we establish a stably expressed MagA method for lung cancer MRI. The results show that MagA can not only enhance both in vitro and in vivo MRI contrast by specifically alternating the transverse relaxation rate of water, but also inhibit the malignant growth of lung tumor. In addition, MagA can regulate magnetic nanoparticle production in grafted tissues and also suppress transferrin receptor expression by acting as an iron transporter, and meanwhile can permit iron biomineralization in the presence of mammalian iron homeostasis. This work provides experimental evidence for the safe preclinical applications of MagA as both a potential inhibitor and an MRI-based tracing tool for iron ion-dependent lung cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Importance: Deviation from normal adolescent brain development precedes manifestations of many major psychiatric symptoms. Such altered developmental trajectories in adolescents may be linked to genetic risk for psychopathology., Objective: To identify genetic variants associated with adolescent brain structure and explore psychopathologic relevance of such associations., Design, Setting, and Participants: Voxelwise genome-wide association study in a cohort of healthy adolescents aged 14 years and validation of the findings using 4 independent samples across the life span with allele-specific expression analysis of top hits. Group comparison of the identified gene-brain association among patients with schizophrenia, unaffected siblings, and healthy control individuals. This was a population-based, multicenter study combined with a clinical sample that included participants from the IMAGEN cohort, Saguenay Youth Study, Three-City Study, and Lieber Institute for Brain Development sample cohorts and UK biobank who were assessed for both brain imaging and genetic sequencing. Clinical samples included patients with schizophrenia and unaffected siblings of patients from the Lieber Institute for Brain Development study. Data were analyzed between October 2015 and April 2018., Main Outcomes and Measures: Gray matter volume was assessed by neuroimaging and genetic variants were genotyped by Illumina BeadChip., Results: The discovery sample included 1721 adolescents (873 girls [50.7%]), with a mean (SD) age of 14.44 (0.41) years. The replication samples consisted of 8690 healthy adults (4497 women [51.8%]) from 4 independent studies across the life span. A nonsynonymous genetic variant (minor T allele of rs13107325 in SLC39A8, a gene implicated in schizophrenia) was associated with greater gray matter volume of the putamen (variance explained of 4.21% in the left hemisphere; 8.66; 95% CI, 6.59-10.81; P = 5.35 × 10-18; and 4.44% in the right hemisphere; t = 8.90; 95% CI, 6.75-11.19; P = 6.80 × 10-19) and also with a lower gene expression of SLC39A8 specifically in the putamen (t127 = -3.87; P = 1.70 × 10-4). The identified association was validated in samples across the life span but was significantly weakened in both patients with schizophrenia (z = -3.05; P = .002; n = 157) and unaffected siblings (z = -2.08; P = .04; n = 149)., Conclusions and Relevance: Our results show that a missense mutation in gene SLC39A8 is associated with larger gray matter volume in the putamen and that this association is significantly weakened in schizophrenia. These results may suggest a role for aberrant ion transport in the etiology of psychosis and provide a target for preemptive developmental interventions aimed at restoring the functional effect of this mutation.

Parkinson's disease (PD), a neurodegenerative disorder, is characterized by a loss of dopaminergic neurons in the substantia nigra (SN) of the brain and it is well known that the pathogenesis of PD is related to a number of risk factors including oxidative stress. Antioxidant 1 (ATOX1) protein plays a crucial role in various diseases as an antioxidant and chaperone. In this study, we determined whether Tat-ATOX1 could protect against 1-methyl-4-phenylpyridinium ion (MPP + )-induced SH-SY5Y cell death and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal model of PD. In the MPP + exposed SH-SY5Y cells, Tat-ATOX1 markedly inhibited cell death and toxicities. In addition, Tat-ATOX1 markedly suppressed the activation of Akt and mitogen activated protein kinases (MAPKs) as well as cleavage of caspase-3 and Bax expression levels. In a MPTP-induced animal model, Tat-ATOX1 transduced into brain and protected dopaminergic neuronal cell loss. Taken together, Tat-ATOX1 inhibits dopaminergic neuronal death through the suppression of MAPKs and apoptotic signal pathways. Thus, Tat-ATOX1 represents a potential therapeutic protein drug candidate for PD., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Cadmium (Cd) has been linked to a variety of cancers, including breast cancer; however, the molecular mechanism of its carcinogenic activity is not fully understood. To this end, the present study investigated the roles of ferroportin (FPN), a prognostic marker of breast cancer, in Cd-induced stimulation of cell proliferation and cell migration. Triple-negative MDA-MB-231 cells were treated with 1-3 μM Cd. The cells exhibited significant reduction in FPN expression and concomitant increase in iron concentration. Cells treated with Cd for 8 weeks displayed elevated proliferative and migratory activities which were inversely related with FPN expression. Reduced FPN expression also resulted in EMT as indicated by an increase in the expression of E-cadherin, and a decrease in the expression of N-cadherin, Twist and Slug. Further investigation revealed that Cd suppressed FPN expression at least partially by activating TGF-β, a known regulator of FPN expression. Taken together, these results indicate that Cd-induced stimulation of MDA-MB-231 cell proliferation, EMT, and migration is brought about by suppression of FPN expression and associated disruption of iron homeostasis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The antibacterial polymer ɛ-poly-L-lysine (ε-PL) has been widely used as a safe food preservative. As the synthesis of ε-PL requires a rich supply of nitrogen, the efficiency of nitrogen translocation and utilization is extremely important. The objective of this study was to improve the production of ε-PL by overexpressing the ammonium transporter gene amtB in Streptomyces albulus PD-1. Using the recombinant bacteria, the optimum carbon-to-nitrogen ratio in the synthesis stage of fermentation increased from 3 to 4.71, compared with that obtained using the wild-type strain, and the utilization efficiency of ammonium was improved too. Ultimately, the production of ε-PL increased from 22.7 to 35.7 g/L upon fed-batch cultivation in a 5 L bioreactor. Determination of the expression of the genes and enzymes associated with ammonium metabolism and ε-PL synthesis revealed that the overexpression of amtB in S. albulus PD-1 enhanced ε-PL biosynthesis by increasing the activity of the corresponding metabolic pathways. To the best of our knowledge, this is the first report on enhancing ε-PL production by overexpression of the amtB gene in an ε-PL-producing strain.

Mutations to the copper-dependent enzyme Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans, and transgenic overexpression of mutant SOD1 represents a robust murine model of the disease. We have previously shown that the copper-containing compound Cu II (atsm) phenotypically improves mutant SOD1 mice and delivers copper to copper-deficient SOD1 in the CNS to restore its physiological function. Cu II (atsm) is now in clinical trials for the treatment of ALS. In this study, we demonstrate that cuproenzyme dysfunction extends beyond SOD1 in SOD1 G37R mice to also affect the endogenous copper-dependent ferroxidase ceruloplasmin. We show that SOD1 and ceruloplasmin both accumulate progressively in the SOD1 G37R mouse spinal cord as the animals' ALS-like symptoms progress, yet the biochemical activity of the two cuproenzymes does not increase commensurately, indicating that, as per mutant SOD1, ceruloplasmin accumulates in a copper-deficient form. Consistent with this finding, we show that expression of the human copper transporter 1 (hCTR1) in SOD1 G37R mice increases copper levels in the spinal cord and concurrently restores SOD1 and ceruloplasmin activity. Soluble misfolded SOD1, a proposed driver of pathology in this model, is readily detectable in the SOD1 G37R mouse spinal cord. However, misfolded SOD1 G37R levels do not change in abundance with disease progression and are less abundant than misfolded SOD1 in the spinal cords of age-matched transgenic SOD1 WT mice which do not exhibit an evident ALS-like phenotype. Collectively, these outcomes support a copper malfunction phenomenon in mutant SOD1 mouse models of ALS and a copper-related mechanism of action for the therapeutic agent Cu II (atsm)., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Studies were undertaken to examine any role for the hepcidin/ferroportin axis in proliferative responses of human pulmonary artery smooth muscle cells (hPASMCs). Entirely novel findings have demonstrated the presence of ferroportin in hPASMCs. Hepcidin treatment caused increased proliferation of these cells most likely by binding ferroportin resulting in internalisation and cellular iron retention. Cellular iron content increased with hepcidin treatment. Stabilisation of ferroportin expression and activity via intervention with the therapeutic monoclonal antibody LY2928057 reversed proliferation and cellular iron accumulation. Additionally, IL-6 treatment was found to enhance proliferation and iron accumulation in hPASMCs; intervention with LY2928057 prevented this response. IL-6 was also found to increase hepcidin transcription and release from hPASMCs suggesting a potential autocrine response. Hepcidin or IL-6 mediated iron accumulation contributes to proliferation in hPASMCs; ferroportin mediated cellular iron excretion limits proliferation. Haemoglobin also caused proliferation of hPASMCs; in other novel findings, CD163, the haemoglobin/haptoglobin receptor, was found on these cells and offers a means for cellular uptake of iron via haemoglobin. Il-6 was also found to modulate CD163 on these cells. These data contribute to a better understanding of how disrupted iron homeostasis may induce vascular remodelling, such as in pulmonary arterial hypertension.

It is well established that tobacco smoking is associated with vascular endothelial dysfunction in a causative and dose dependent manner primarily related to the tobacco smoke (TS) content of reactive oxygen species (ROS), nicotine, and oxidative stress (OS) -driven inflammation. Preclinical studies have also shown that nicotine (the principal e-liquid's ingredient used in e-cigarettes (e-Cigs) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Likewise, chronic e-Cig vaping could be prodromal to vascular endothelial dysfunctions. Herein, we provide direct evidence that similarly to TS, e-Cig promotes mitochondrial depolarization in primary brain vascular endothelial cells as well as the vascular endothelial cell line bEnd3. In addition, both TS and e-Cig exposure upregulated the transmembrane iron exporter Slc40a1 (crucial to maintain cellular iron and redox homeostasis) and that of porphyrin importer Abcb6 (linked to accelerated atherosclerosis). We then investigated in vivo whether gender plays a role in how chronic TS affect vascular endothelial functions. Our results clearly show chronic TS exposure differentially impacts the expression levels of Phase-II enzymes as well as the iron transporters previously investigated in vitro. Although the physiological implications of the gender-specific differential responses to TS are not fully clear, they do demonstrate that gender is a risk factor that needs to be investigated when assessing the potential impact of chronic smoking and perhaps e-Cig vaping., (Published by Elsevier B.V.)

Magnesium (Mg) is an essential mineral element for normal plant growth and development, and the CorA/MRS2/MGT-type Mg transporters play a significant role in maintaining Mg homeostasis in plants. In total, 12 maize CorA-like Mg 2+ transporters have been identified, but none of them had been functionally characterized. Accordingly, we cloned and functionally characterized ZmMGT12 from the maize CorA-like gene family. ZmMGT12 exhibited the structure typical of Mg 2+ transporters, i.e., two conserved TM domains and a GMN tripeptide motif. ZmMGT12, Arabidopsis AtMGT6, and rice OsMRS2-6 shared high protein sequence identity and thus clustered in the same phylogenetic branch, suggesting that they could be homologs. A functional complementation assay in the Salmonella typhimurium MM281 mutant indicated that ZmMGT12 possessed Mg 2+ transport ability. ZmMGT12 was expressed in roots, stems, and leaves, with the highest expression in leaves. Moreover, ZmMGT12 expression was induced by light and exhibited a circadian expression pattern. In addition, the expression level of ZmMGT12 in leaf tissue was related to chlorophyll synthesis. Overexpression of ZmMGT12 in Arabidopsis caused no phenotypic change in transgenic plants, including in fresh shoot weight, chlorophyll content, shoot Mg 2+ content, and chloroplast Mg 2+ content. Together, these results suggest that ZmMGT12 is a Mg 2+ transporter and may play a role in Mg transport into chloroplasts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn-FLAG in LD infected J774 macrophage confirms that Fpn down-regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by 35 S-methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5'UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F-box and leucine-rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD-infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2-FBXL5 axis to inhibit host Fpn expression., (© 2018 John Wiley & Sons Ltd.)

Neuroblastoma is one of the leading causes of cancer‑associated mortality worldwide, particularly in children, partially due to the absence of effective therapeutic targets and diagnostic biomarkers. Therefore, novel molecular targets are critical to the development of therapeutic approaches for neuroblastoma. In the present study, the functions of zinc transporter ZIP8 (Zip8), a member of the zinc transporting protein family, were investigated as novel molecular targets in neuroblastoma cancer cells. The proliferation rates of neuroblastoma cancer cells were significantly decreased when Zip8 was knocked down by lentiviral‑mediated RNA interference. Study of the molecular mechanism suggested that Zip8 modulated the expression of key genes involved in the nuclear factor‑κB signaling pathway. Furthermore, Zip8 depletion suppressed the migratory potential of neuroblastoma cancer cells by reducing the expression levels of matrix metalloproteinases. In conclusion, the results of the present study suggested that Zip8 was an important regulator of neuroblastoma cell proliferation and migration, indicating that Zip8 may be a potential anticancer therapeutic target and a promising diagnostic biomarker for human neuroblastoma.

Using a hydroponics culture system, we monitored morphological, physiological, and molecular changes in Malus prunifolia seedlings when drought conditions induced by 5% polyethylene glycol (PEG) were combined with a low or normal supply of N (0.05 mM or 1 mM NH 4 NO 3 , respectively). Under either nutrient level, drought stress negatively inhibited seedling performance, as manifested by reduced photosynthesis and biomass production, decreased accumulations of total N, and inhibited root growth. Concentrations of NO 3 - and NH 4 + and the activities of enzymes involved in N metabolism (nitrate reductase, glutamine synthetase, and glutamate synthase) were also significantly decreased under drought stress. The net influx of NO 3 - at the surface of the fine roots declined while that of NH 4 + rose markedly, suggesting that the latter may play a more important role in improving drought tolerance in M. prunifolia. Consistently, two ammonium transporters (AMT1;2 and AMT4;2) were notably up-regulated in response to drought stress, whereas most genes related to nitrate uptake, reduction, and N metabolism were down-regulated. At the normal N level, PEG-treated plants showed higher values for biomass production, root growth, and N uptake/reduction when compared with plants exposed to the lower N supply. These results suggest that the negative effect of drought stress on M. prunifolia may be alleviated when more nitrogen is available., (Copyright © 2018. Published by Elsevier Masson SAS.)

Changes in bone matrix composition are frequently found with bone diseases and may be associated with increased fracture risk. Bone is rich in the trace element zinc. Zinc was established to play a significant role in the growth, development, and maintenance of healthy bones; however, the mechanisms underlying zinc effects on the integrity of the skeleton are poorly understood. Here, we show that the zinc receptor (ZnR)/Gpr39 is required for normal bone matrix deposition by osteoblasts. Initial analysis showed that Gpr39-deficient ( Gpr39 -/- ) mice had weaker bones as a result of altered bone composition. Fourier transform infrared spectroscopy analysis showed high mineral-to-matrix ratios in the bones of Gpr39 -/- mice. Histologic analysis showed abnormally high numbers of active osteoblasts but normal osteoclast numbers on the surfaces of bones from Gpr39 -/- mice. Furthermore, Gpr39 -/- osteoblasts had disorganized matrix deposition in vitro with cultures exhibiting abnormally low collagen and high mineral contents, findings that demonstrate a cell-intrinsic role for ZnR/Gpr39 in these cells. We show that both collagen synthesis and deposition by Gpr39 -/- osteoblasts are perturbed. Finally, the expression of the zinc transporter Zip13 and a disintegrin and metalloproteinase with thrombospondin motifs family of zinc-dependent metalloproteases that regulate collagen processing was downregulated in Gpr39 -/- osteoblasts. Altogether, our results suggest that zinc sensing by ZnR/Gpr39 affects the expression levels of zinc-dependent enzymes in osteoblasts and regulates collagen processing and deposition.-Jovanovic, M., Schmidt, F. N., Guterman-Ram, G., Khayyeri, H., Hiram-Bab, S., Orenbuch, A., Katchkovsky, S., Aflalo, A., Isaksson, H., Busse, B., Jähn, K., Levaot, N. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.

Purpose: In our previous global gene expression analysis, we identified NIPA-like domain containing 1 (NIPAL1), which encodes a magnesium transporter, as one of the most overexpressed genes in recurrent oral squamous cell carcinoma (OSCC). Although has been NIPAL1 linked with gout pathogenesis, little is known about its expression and function in human malignancies., Methods: In this study, we examined NIPAL1 expression in 192 cases of OSCC by immunohistochemistry and performed a functional analysis of human OSCC cells., Results: NIPAL1 immunostaining was observed in 39 of 192 OSCC patients (20.3%). NIPAL1 expression correlated significantly with cancer cell intravsation (P = 0.0062), as well as with poorer disease-free survival in a Kaplan-Meier analysis (P < 0.0001). Moreover, a multivariate Cox proportional hazards model analysis revealed that NIPAL1 expression was an independent predictor of disease-free survival in OSCC (P < 0.0001). In a functional analysis, NIPAL1 regulated the growth and adhesion of OSCC tumor cells and endothelial cells., Conclusions: Our findings suggest that NIPAL1 might be a novel factor promoting OSCC tumorigenesis, as well as a useful molecular marker of OSCC.

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes., (Copyright © 2018 by the American Society of Nephrology.)

Clinical and experimental studies have shown an association between intracellular free Zn 2+ ([Zn 2+ ] i )-dyshomeostasis and cardiac dysfunction besides [Ca 2+ ] i -dyshomeostasis. Since [Zn 2+ ] i -homeostasis is regulated through Zn 2+ -transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn 2+ -homeostasis via alteration in Zn 2+ -transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3β, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NFκB to the NFκB are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKCα and PKCα levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn 2+ -influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn 2+ ] i detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn 2+ ] i via decreased ER-[Zn 2+ ] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn 2+ -transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn 2+ ] i - besides [Ca 2+ ] i -dyshomeostasis.

Early evaluation of iron overload (IO) and prompt iron-chelation therapy reduce the haematopoietic damage wrought by IO-induced reactive oxygen species (ROS). We examined whether MagA could simultaneously increase the sensitivity of magnetic resonance imaging (MRI) for iron measurement and attenuate oxidative damage to the haematopoietic microenvironment. After generation of a transgenic (Tg) mouse model, MRI, transmission electron microscopy and cytotoxicity assays were used to assess various parameters in mesenchymal stem cells (MSCs). Transverse relaxation rate (R2*) of MagA-expressing MSCs in the presence of iron supplement was higher compared with that of control cells. Besides, R2* value of liver from IO magA Tg mice was higher than that of wild type mice. Moreover, MagA contributed to reduce the cytotoxicity of iron against MSCs, reduce expression of p-p38 mitogen-activated protein kinase and ferritin, and reduce inhibition of the osteogenic differentiation caused by IO. These data support the use of magA as a reporter gene for cell tracking with MRI and indicate exciting new possibilities for use of MagA in the attenuation of injury due to oxidative stress caused by exogenous iron.

The interaction among heart failure (HF), chronic kidney disease (CKD), and anemia is called cardio-renal anemia syndrome. The mechanism of anemia in cardio-renal anemia syndrome is complex and remains completely unknown. We have previously reported that impaired intestinal iron transporters may contribute to the mechanism of anemia in HF using in vivo HF model rats. In this study, we assessed intestinal iron transporters in CKD model rats to investigate the association of intestinal iron transporters in the mechanism of cardio-renal anemia syndrome. CKD was induced by 5/6 nephrectomy in Sprague-Dawley rats. Sham-operated rats served as a control. After 24-week surgery, CKD rats exhibited normocytic normochromic anemia and normal serum erythropoietin levels despite of anemia. Serum iron levels were decreased in CKD rats compared with the controls. Of interest, intestinal expression of critical iron importers, such as duodenal cytochrome b (Dcyt-b) and divalent metal transporter 1 (DMT-1), was decreased in CKD rats compared with the controls. On the other hand, intestinal expression of ferroportin, an intestinal iron exporter, was not different in the control and CKD groups. Moreover, hepatic expression of hepcidin, a regulator of iron homeostasis, did not differ between the control and CKD groups. These results suggest that impaired intestinal expression of Dcyt-b and DMT-1 might be associated with the reduction of an iron uptake in CKD. Taken together, impaired these intestinal iron transporters may become a novel therapeutic target for cardio-renal anemia syndrome.

Background: Iron deficiency anemia in celiac disease is related to impaired duodenal mucosal uptake, due to villous atrophy. Iron enters the enterocytes through an apical divalent metal transporter, DMT1. Different DMT1 transcripts have been identified, depending on the presence of an iron-responsive element that allows DMT1 up-regulation during iron starvation. An intronic DMT1 polymorphism, IVS4+44C>A, has been associated with metal toxicity, and the CC-carriers show high iron levels., Aims: This study investigates the association between DMT1 IVS4+44C>A and anemia in a cohort of 387 Italian celiac children, and the functional role of the polymorphism., Methods and Results: By association analysis, we found that DMT1 IVS4+44-AA genotype confers a four-fold risk of developing anemia, despite of atrophy degree. By analysis of mRNA from gastroesophageal biopsies, we found that total DMT1 is significantly upregulated in presence of mild, but not severe, atrophy, independently from IVS4+44C>A variant, and in normal but not in atrophic CC-biopsies. Moreover, we found that A-allele is associated to preferential expression of the DMT1 transcripts lacking the iron-responsive element, thus limiting the DMT1 overexpression that normally occurs to respond to iron starvation., Discussion: Possibly, the IVS4+44-AA-related dysregulation of the iron-induced changes in DMT1 expression is not able to impair iron absorption in physiological condition. However, if exacerbated by the concomitant massive loss of functional absorbing tissue paralleling worsened stages of villus atrophy, it might be ineffective in counteracting iron deficiency, despite of DMT1 overexpression., Conclusion: We suggest, for the first time, that celiac disease may unmask the contribution of the DMT1 IVS4+44C>A polymorphism to the risk of anemia.

In regulatory toxicology, the dose-response relationship is a key element towards fulfilling safety assessments and satisfying regulatory authorities. Conventionally, the larger the dose, the greater the response, following the dogma "the dose makes the poison". Many endocrine disrupting chemicals, including bisphenol-A (BPA), induce non-monotonic dose response (NMDR) relationships, which are unconventional and have tremendous implications in risk assessment. Although several molecular mechanisms have been proposed to explain NMDR relationships, they are largely undemonstrated. Using mouse pancreatic β-cells from wild-type and oestrogen receptor ERβ-/- mice, we found that exposure to increasing doses of BPA affected Ca 2+ entry in an NMDR manner. Low doses decreased plasma membrane Ca 2+ currents after downregulation of Cav2.3 ion channel expression, in a process involving ERβ. High doses decreased Ca 2+ currents through an ERβ-mediated mechanism and simultaneously increased Ca 2+ currents via oestrogen receptor ERα. The outcome of both molecular mechanisms explains the NMDR relationship between BPA and Ca 2+ entry in β-cells.

We studied physiological traits and Mn transporter genes expression in ryegrass genotypes (One-50, Banquet-II, Halo-AR1 and Nui) under increasing Mn (2.4-750 μM) at short-term (8-24 h) in nutrient solution. An increment in Mn concentration occurred early in roots of all genotypes at increased Mn doses relative to control. Banquet-II and Nui roots showed the greatest Mn concentration at the highest Mn supply. Net photosynthesis (Pn) of Banquet-II and Halo-AR1 were not perturbed by Mn doses, whereas One-50 and Nui, decayed strongly at the highest Mn dose, concomitant with reduced total chlorophyll concentration. A high accumulation of Mn in roots together the maintained Pn under increased Mn doses in Banquet-II and Halo-AR1 suggest a higher Mn resistance of these genotypes. Stomatal conductance (gs) of all genotypes did not vary in presence of Mn. In roots of Banquet-II an augment of lipid peroxidation (LP) by Mn excess was observed earlier decreasing afterwards, being attenuated by the augment of the radical scavenging activity (RSA) and total phenols (TP) of this genotype. Mn concentration and LP in tissues of One-50 and Nui genotypes rose together, may be due to its Mn sensitivity. Differential expression of Mn transporter genes were found in the studied genotypes grown under increasing supplies of Mn, being MTP8.1 expressed in shoots and NRAMP2-like in roots. We concluded that Banquet-II showed greater Mn concentration associated to high roots NRAMP2-like gene expression, without changes in photosynthetic performance. Despite, this genotype showed an increase of LP at the first hours of Mn-excess, it was decreased by the RSA and TP. Halo-AR1 appears to be Mn-resistant in the short-term due to its photosynthetic performance was unchanged by Mn-toxicity, whilst One-50 and Nui were Mn-sensitive., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Renal cell carcinoma, the most common neoplasm of adult kidney, accounts for about 3% of adult malignancies and is usually highly resistant to conventional therapy. MicroRNAs are a class of small non-coding RNAs, which have been previously shown to promote malignant initiation and progression. In this study, we focused our attention on miR-21, a well described oncomiR commonly upregulated in cancer. Using a cohort of 99 primary renal cell carcinoma samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues whereas no significant difference was observed with stages, grades, and metastatic outcome. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, using Boyden chambers and western blot techniques, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating renal cell carcinoma chemoresistance and further showed that miR-21 silencing significantly (1) increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin, and dovitinib; (2) decreased expression of multi-drug resistance genes; and (4) increased SLC22A1/OCT1, SLC22A2/OCT2, and SLC31A1/CTR1 platinum influx transporter expression. In conclusion, our results showed that miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs. In renal cell carcinoma, targeting miR-21 is a potential new therapeutic strategy to improve chemotherapy efficacy and consequently patient outcome.

Mosaic mutant mice displaying functional dysfunction of Atp7a copper transporter (the Menkes ATPase) are an established animal model of Menkes disease and constitute a convenient tool for investigating connections between copper and iron metabolisms. This model allows to explore changes in iron metabolism in suckling mutant mice suffering from systemic copper deficiency as well as in young and adult ones undergone copper therapy, which reduces lethal effect of the Atp7a gene mutation. Our recent study demonstrated that 14-day-old mosaic mutant males display blood cell abnormalities associated with intravascular hemolysis, and show disturbances in the functioning of the hepcidin-ferroportin regulatory axis, which controls systemic iron homeostasis. We thus aimed to check whether copper supplementation recovers mutants from hemolytic insult and rebalance systemic iron regulation. Copper supplementation of 14-day-old mosaic mutants resulted in the reestablishment of hematological status, attenuation of hepicidin and concomitant induction of the iron exporter ferroportin/Slc40a1 expression in the liver, down-regulated in untreated mutants. Interestingly, treatment of wild-type males with copper, induced hepcidin-independent up-regulation of ferroportin protein level in hepatic macrophages in both young and adult (6-month-old) animals. Stimulatory effect of copper on ferroportin mRNA and protein levels was confirmed in bone marrow-derived macrophages isolated from both wild-type and mosaic mutant males. Our study indicates that copper is an important player in the regulation of the Slc40a1 gene expression., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Studies have reported a positive effect of prebiotics on the bioavailability of iron. This study evaluated the effect of partially hydrolyzed guar gum (PHGG) on iron absorption mechanisms in anemic rats. Male Wistar rats were fed 75g American Institute of Nutrition Rodent Diets for growth, pregnancy and lactation (AIN93-G) without iron for three weeks in order to induce iron deficiency anemia. Then they were fed a control diet (n = 12; without fiber) or a diet with 7.5% of PHGG (n = 12), both without iron. Food intake, body growth and the feed efficiency coefficient (FEC) were measured. The animals were euthanized after two weeks of treatment. The weight of the organs, the pH of the cecal content, and the hepatic iron and ferroportin expression in the cecum, duodenum, and liver were assessed. The intake of PHGG reduced food intake without affecting body growth, and there was a difference between the groups regarding the FEC (p = 0.026), with the highest value found in the PHGG group. The weight of the cecal content increased (p ≤ 0.001) and the pH of the cecal content was significantly lower in the PHGG group. The intake of PHGG significantly increased ferroportin expression in the cecum; however, the difference was not significant in the duodenum and the liver. PHGG seems to have a positive influence on iron absorption through transporter expression, and structural and physiological changes in the colon of anemic growing animals.

When a ribosome translates mRNA sequences, the ribosome often stalls at certain codons because it is hard to translate. Consecutive proline codons are such examples that induce ribosome stalling and elongation factor P (EF-P) is required for the stalled ribosome to continue translation at those consecutive proline codons. We found that EF-P is required for translation of the mgtB gene encoding a Mg 2+ transporter in the mgtCBR virulence operon from the intracellular pathogen Salmonella enterica serovar Typhimurium. Salmonella lacking EF-P decreases MgtB protein levels in a manner dependent on consecutive proline codons located in the mgtB coding region despite increasing transcription of the mgtCBR operon via the mgtP open reading frame in the leader RNA, resulting in an altered ratio between MgtC and MgtB proteins within the operon. Substitution of the consecutive proline codons to alanine codons eliminates EF-P-mediated control of the mgtB gene during infection and thus contributes to Salmonella's survival inside macrophages where Salmonella experiences low levels of EF-P. This suggests that this pathogen utilizes a strategy to coordinate expression of virulence genes by an evolutionarily conserved translation factor., Competing Interests: The authors declare no competing financial interests.

Intracellular retinal iron accumulation has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness among individuals over the age of 50. Ceruloplasmin/hephaestin double knockout mice (Cp/Heph DKO) and hepcidin knockout mice (Hepc KO) accumulate retinal iron and model some features of AMD. Two canonical pathways govern cellular iron import - transferrin-bound iron import and non-transferrin bound iron import. In Cp/Heph DKO and Hepc KO iron-loaded retinas, transferrin-bound iron import is downregulated. Despite this effort to reduce cellular iron burden, iron continues to accumulate in these retinas in an age-dependent manner. Quantitative RT-PCR and Western analysis were used to quantify the expression of three ferrous iron importers, Dmt1, Zip8, and Zip14, in wild-type (Wt), Cp/Heph DKO, and Hepc KO retinas. Zip8 and Zip14 protein levels were analyzed using Western analysis in mice injected intravitreally with either apo- or holo-transferrin to elucidate one possible mechanism of Zip14 regulation in the retina. Both zip8 and zip14 were expressed in the mouse retina. Paradoxically, protein levels of non-transferrin bound iron importers were upregulated in both Cp/Heph DKO and Hepc KO retinas. Intravitreal holo-transferrin injection decreased Zip 14 protein levels. These data indicate that Zip8 and Zip14 may take up increasing amounts of non-transferrin bound iron in these two mouse models of retinal iron accumulation. Their upregulation in these already iron-loaded retinas suggests a vicious cycle leading to toxicity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Metal efflux pumps maintain ion homeostasis in the cell. The functions of the transporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. Although the copper ion transporter CopA has been known in Escherichia coli, no gene for its chaperone had been identified. We show that the CopA chaperone is expressed in E. coli from the same gene that encodes the transporter. Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a "slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The divalent cation zinc is associated with cortical plasticity. However, the mechanism of zinc in the pathophysiology of cortical injury-associated neurobehavioral damage following neonatal seizures is uncertain. We have previously shown upregulated expression of ZnT-3; MT-3 in hippocampus of neonatal rats submitted to flurothyl-induced recurrent seizures, which was restored by pretreatment with ketogenic diet (KD). In this study, utilizing a novel "twist" seizure model by coupling early-life flurothyl-induced seizures with later exposure to penicillin, we further investigated the long-term effects of KD on cortical expression of zinc homeostasis-related genes in a systemic scale. Ten Sprague-Dawley rats were assigned each averagely into the non-seizure plus normal diet (NS + ND), non-seizure plus KD (NS + KD), recurrent seizures plus normal diet (RS + ND) and recurrent seizures plus KD (RS + KD) group. Recurrent seizures were induced by volatile flurothyl during P9-P21. During P23-P53, rats in NS + KD and RS + KD groups were dieted with KD. Neurological behavioral parameters of brain damage (plane righting reflex, cliff avoidance reflex, and open field test) were observed at P43. At P63, we examined seizure threshold using penicillin, then the cerebral cortex were evaluated for real-time RT-PCR and western blot study. The RS + ND group showed worse performances in neurological reflex tests and reduced latencies to myoclonic seizures induced by penicillin compared with the control, which was concomitant with altered expressions of ZnT-7, MT-1, MT-2, and ZIP7. Specifically, there was long-term elevated expression of ZIP7 in RS + ND group compared with that in NS + ND that was restored by chronic ketogenic diet (KD) treatment in RS + KD group, which was quite in parallel with the above neurobehavioral changes. Taken together, these findings indicate that the long-term altered expression of the metal transporter ZIP7 in adult cerebral cortex might correlate with neurobehavioral damage and reduced seizure threshold following recurrent neonate seizures and further highlights ZIP7 as a candidate for therapeutic target of KD for the treatment of neonatal seizure-induced long-term brain damage.

Voltage-gated sodium channels (VGSCs), which are aberrantly expressed in several human cancers, affect cancer cell behavior; however, their role in gastric cancer (GC) and the link between these channels and tumorigenic signaling remain unclear. The aims of this study were to determine the clinicopathological significance and role of the VGSC Nav 1.7 in GC progression and to investigate the associated mechanisms. Here, we report that the SCN9A gene encoding Nav 1.7 was the most abundantly expressed VGSC subtype in GC tissue samples and two GC cell lines (BGC-823 and MKN-28 cells). SCN9A expression levels were also frequently found to be elevated in GC samples compared to nonmalignant tissues by real-time PCR. In the 319 GC specimens evaluated by immunohistochemistry, Nav 1.7 expression was correlated with prognosis, and transporter Na(+) /H(+) exchanger-1 (NHE1) and oncoprotein metastasis-associated in colon cancer-1 (MACC1) expression. Nav 1.7 suppression resulted in reduced voltage-gated sodium currents, decreased NHE1 expression, increased extracellular pH and decreased intracellular pH, and ultimately, reduced invasion and proliferation rates of GC cells and growth of GC xenografts in nude mice. Nav 1.7 inhibition led to reduced MACC1 expression, while MACC1 inhibition resulted in reduced NHE1 expression in vitro and in vivo. Mechanistically, the suppression of Nav 1.7 decreased NF-κB p65 nuclear translocation via p38 activation, thus reducing MACC1 expression. Downregulation of MACC1 decreased c-Jun phosphorylation and subsequently reduced NHE1 expression, whereas the addition of hepatocyte growth factor (HGF), a c-Met physiological ligand, reversed the effect. These results indicate that Nav 1.7 promotes GC progression through MACC1-mediated upregulation of NHE1. Therefore, Nav 1.7 is a potential prognostic marker and/or therapeutic target for GC., (© 2016 UICC.)

The nitrogen species available in the growth medium are key factors determining expression of xyl genes for biodegradation of aromatic compounds by Pseudomonas putida. Nitrogen compounds are frequently amended to promote degradation at polluted sites, but it remains unknown how regulation observed in the test tube is propagated into actual catabolism of, e.g. m-xylene in soil, the natural habitat of this bacterium. To address this issue, we have developed a test-tube-to-soil model system that exposes the end-effects of remediation practices influencing gene expression of P. putida mt-2. We found that NO 3 - compared with NH 4 + had a stimulating effect on xyl gene expression in pure culture as well as in soil, and that this stimulation was translated into increased m-xylene mineralization in soil. Furthermore, expression analysis of the nitrogen-regulated genes amtB and gdhA allowed us to monitor nitrogen sensing status in both experimental systems. Hence, for nitrogen sources, regulatory patterns that emerge in soil reflect those observed in liquid cultures. The current study shows how distinct regulatory traits can lead to discrete environmental consequences; and it underpins that attempts to improve bioremediation by nitrogen amendment should integrate knowledge on their effects on growth and on catabolic gene regulation under natural conditions., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Context: As the syndrome of hypogonadotropic hypogonadism (HH) is associated with anaemia and the administration of testosterone restores haematocrit to normal, we investigated the potential underlying mechanisms., Design: Randomized, double-blind, placebo-controlled trial., Methods: We measured basal serum concentrations of erythropoietin, iron, iron binding capacity, transferrin (saturated and unsaturated), ferritin and hepcidin and the expression of ferroportin and transferrin receptor (TR) in peripheral blood mononuclear cells (MNC) of 94 men with type 2 diabetes. Forty-four men had HH (defined as subnormal free testosterone along with low or normal LH concentrations) while 50 were eugonadal. Men with HH were randomized to testosterone or placebo treatment every 2 weeks for 15 weeks. Blood samples were collected at baseline, 3 and 15 weeks after starting treatment. Twenty men in testosterone group and 14 men in placebo group completed the study., Results: Haematocrit levels were lower in men with HH (41·1 ± 3·9% vs 43·8 ± 3·4%, P = 0·001). There were no differences in plasma concentrations of hepcidin, ferritin, erythropoietin, transferrin or iron, or in the expression of ferroportin or TR in MNC among HH and eugonadal men. Haematocrit increased to 45·3 ± 4·5%, hepcidin decreased by 28 ± 7% and erythropoietin increased by 21 ± 7% after testosterone therapy (P < 0·05). There was no significant change in ferritin concentrations, but transferrin concentration increased while transferrin saturation and iron concentrations decreased (P < 0·05). Ferroportin and TR mRNA expression in MNC increased by 70 ± 13% and 43 ± 10%, respectively (P < 0·01), after testosterone therapy., Conclusions: The increase in haematocrit following testosterone therapy is associated with an increase in erythropoietin, the suppression of hepcidin, and an increase in the expression of ferroportin and TR., (© 2016 John Wiley & Sons Ltd.)

Melatonin has recently been demonstrated to play important roles in the regulation of plant growth, development, and abiotic and biotic stress responses. However, the possible involvement of melatonin in Fe deficiency responses and the underlying mechanisms remained elusive in Arabidopsis thaliana . In this study, Fe deficiency quickly induced melatonin synthesis in Arabidopsis plants. Exogenous melatonin significantly increased the soluble Fe content of shoots and roots, and decreased the levels of root cell wall Fe bound to pectin and hemicellulose, thus alleviating Fe deficiency-induced chlorosis. Intriguingly, melatonin treatments induced a significant increase of nitric oxide (NO) accumulation in roots of Fe-deficient plants, but not in those of polyamine-deficient ( adc2-1 and d-arginine-treated) plants. Moreover, the melatonin-alleviated leaf chlorosis was blocked in the polyamine- and NO-deficient ( nia1nia2noa1 and c-PTIO-treated) plants, and the melatonin-induced Fe remobilization was largely inhibited. In addition, the expression of some Fe acquisition-related genes, including FIT1 , FRO2 , and IRT1 were significantly up-regulated by melatonin treatments, whereas the enhanced expression of these genes was obviously suppressed in the polyamine- and NO-deficient plants. Collectively, our results provide evidence to support the view that melatonin can increase the tolerance of plants to Fe deficiency in a process dependent on the polyamine-induced NO production under Fe-deficient conditions., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4+ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders., Competing Interests: The authors have declared that no competing interests exist.

Ferroportin 1 (FPN1) is an iron export protein found in mammals. FPN1 is important for the export of iron across the basolateral membrane of absorptive enterocytes and across the plasma membrane of macrophages. The expression of FPN1 is regulated by hepcidin, which binds to FPN1 and then induces its degradation. Previously, we demonstrated that divalent metal transporter 1 (DMT1) interacts with the intracellular iron chaperone protein poly(rC)-binding protein 2 (PCBP2). Subsequently, PCBP2 receives iron from DMT1 and then disengages from the transporter. In this study, we investigated the function of PCBP2 in iron export. Mammalian genomes encode four PCBPs (i.e. PCBP1-4). Here, for the first time, we demonstrated using both yeast and mammalian cells that PCBP2, but not PCBP1, PCBP3, or PCBP4, binds with FPN1. Importantly, iron-loaded, but not iron-depleted, PCBP2 interacts with FPN1. The PCBP2-binding domain of FPN1 was identified in its C-terminal cytoplasmic region. The silencing of PCBP2 expression suppressed FPN1-dependent iron export from cells. These results suggest that FPN1 exports iron received from the iron chaperone PCBP2. Therefore, it was found that PCBP2 modulates cellular iron export, which is an important physiological process., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mg(2+) is an essential cation to maintain cellular functions, and intracellular Mg(2+) concentration ([Mg(2+)]i) is regulated by Mg(2+) channels and transporters. In our previous study, we demonstrated that MPP(+) elicits Mg(2+) influx across the cell membrane and Mg(2+) mobilization from mitochondria, and the resulting [Mg(2+)]i is an important determinants of the cell viability in MPP(+) model of Parkinson's disease (PD). It indicates that cellular Mg(2+) transport is one of the important factors to determine the progress of PD. However, whether the expression levels of Mg(2+) transport proteins change in the progress of PD has still been obscure. In this study, we estimated the mRNA expression levels of Mg(2+) transport proteins upon the exposure to MPP(+). In thirteen Mg(2+) transport proteins examined, mRNA expression level of SLC41A2 was increased and that of ACDP2, NIPA1 and MMgT2 were decreased. Knockdown of SLC41A2, ACDP2 or NIPA1 accelerated the MPP(+)-induced cell degeneration, and overexpression attenuated it. The decrease in the mRNA expression levels of NIPA1 and MMgT2 were also elicited by rotenone, H2O2 and FCCP, indicating that mitochondrial dysfunction related to this down-regulation. The increase in that of SLC41A2 was induced by an uncoupler, FCCP, as well as MPP(+), suggesting that it is an intrinsic protection mechanism against depolarized mitochondrial membrane potential and/or cellular ATP depletion. Our results shown here indicate that alteration of Mg(2+) transport proteins is implicated in the MPP(+) model of PD, and it affects cell degeneration., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The human high-affinity copper transporter 1 (hCtr1) transports both Cu(I) and cisplatin (cDDP). Because Cu deficiency is lethal yet Cu overload is poisonous, hCtr1 expression is transcriptionally upregulated in response to Cu deficiency but is downregulated under Cu replete conditions in controlling Cu homeostasis. The up- and down-regulation of hCtr1 is regulated by Specific protein 1 (Sp1), which itself is also correspondingly regulated under these Cu conditions. hCtr1 expression is also upregulated by cDDP via upregulation of Sp1. The underlying mechanisms of these regulations are unknown. Using gel-electrophoretic mobility shift assays, we demonstrated here that Sp1-DNA binding affinity is reduced under Cu replete conditions but increased under reduced Cu conditions. Similarly, Sp1-DNA binding affinity is increased by cDDP treatment. This in vitro system demonstrated, for the first time, that regulation of Sp1/hCtr1 expression by these agents is modulated by the stability of Sp1-DNA binding, the first step in the Sp1-mediated transcriptional regulation process., (Copyright © 2016 Elsevier Inc. All rights reserved.)