Biophysical Probes of Iron Metabolism in Yeast Cells, Mitochondria, and Mouse Brains

Iron is essential in nearly all organisms. It is a cofactor in many proteins and enzymes. This transition metal can also be toxic because it participates in reactions which produce reactive oxygen species. To avoid these toxic effects while still being used for essential processes, the cell must regulate tightly iron import, metabolism, trafficking, and homeostasis. These processes were studied using biophysical methods centered on Mossbauer spectroscopy supplemented by electron paramagnetic resonance, electronic absorption spectroscopy, and inductively coupled plasma mass spectrometry. This integrated biophysical approach was applied to yeast cells, isolated yeast mitochondria, and mouse brains. We determined the concentration of Fe, and the proportion of that Fe present as iron-sulfur clusters, heme centers, mononuclear nonheme centers, and as Fe3+ oxyhydroxide (phosphate) nanoparticles for each system. In yeast, the dependence of metabolic mode of growth and iron in the growth medium on this distribution was studied. Approximately three-quarters of the iron in fermenting cells was located in vacuoles, where it was present as high-spin mononuclear Fe3+ species with rhombic symmetry. The remaining quarter was present in the mitochondria. In fermenting mitochondria 4 distinct species of iron were observed, including [Fe4S4]2+ clusters and low-spin Fe2+ hemes arising from respiratory complexes, non-heme high spin (NHHS) Fe2+ species, high spin nonheme Fe3+ species, and nanoparticles. These distributions (in both the cells and mitochondria) change when the cells are grown on iron deficient medium but remained relatively unaltered as iron in the growth medium was increased. Respiring cells had less Fe associated with vacuoles, and more Fe present as HS Fe2+. Respiring mitochondria contain more [Fe4S4]2+ clusters and low-spin Fe2+ hemes, more S = 1/2 [Fe2S2]1+ clusters, and less NHHS Fe2+, HS Fe3+ species and Fe3+ nanoparticles. These changes were rationalized by assuming that the NHHS Fe2+ and Fe3+ species, and the nanoparticles were in equilibrium within the matrix of the mitochondria, and that the Fe2+ species served as feedstock for the synthesis of iron-sulfur clusters and heme centers. The iron in the mouse brain consisted mostly of [Fe4S4]2+ clusters and Fe2+ hemes from mitochondria respiratory complexes, and of ferritin, an Fe storage protein complex. NHHS Fe2+ and Fe3+ species were also observed. The ratio of stored Fe to mitochondrial Fe was sensitive to age. The brains of prenatal animals were dominated by ferritin. Following birth up to the first 4 weeks of life, there was an increase in mitochondrial Fe and a decline of ferritin Fe. Beyond 4 weeks up to 58 weeks, levels of ferritin increased and mitochondrial Fe remained constant. The brains of mice fed an Fe-deficient diet were also studied; most of the Fe in these brains was present as mitochondrial Fe, with little stored as ferritin. A model was developed to explain these changes

Dimeric ferrochelatase bridges ABCB7 and ABCB10 homodimers in an architecturally defined molecular complex required for heme biosynthesis

Loss-of-function mutations in the ATP-binding cassette (ABC) transporter of the inner mitochondrial membrane, ABCB7, cause X-linked sideroblastic anemia with ataxia, a phenotype that remains largely unexplained by the proposed role of ABCB7 in exporting a special sulfur species for use in cytosolic iron-sulfur (Fe-S) cluster biogenesis. Here, we generated inducible ABCB7-knockdown cell lines to examine the time-dependent consequences of loss of ABCB7. We found that knockdown of ABCB7 led to significant loss of mitochondrial Fe-S proteins, which preceded the development of milder defects in cytosolic Fe-S enzymes. In erythroid cells, loss of ABCB7 altered cellular iron distribution and caused mitochondrial iron overload due to activation of iron regulatory proteins 1 and 2 in the cytosol and to upregulation of the mitochondrial iron importer, mitoferrin-1. Despite the exceptionally large amount of iron imported into mitochondria, erythroid cells lacking ABCB7 showed a profound hemoglobinization defect and underwent apoptosis triggered by oxidative stress. In ABCB7-depleted cells, defective heme biosynthesis resulted from translational repression of ALAS2 by iron regulatory proteins and from decreased stability of the terminal enzyme ferrochelatase. By combining chemical crosslinking, tandem mass spectrometry and mutational analyses, we characterized a complex formed of ferrochelatase, ABCB7 and ABCB10, and mapped the interfaces of interactions of its components. A dimeric ferrochelatase physically bridged ABCB7 and ABCB10 homodimers by binding near the nucleotide-binding domains of each ABC transporter. Our studies not only underscore the importance of ABCB7 for mitochondrial Fe-S biogenesis and iron homeostasis, but also provide the biochemical characterization of a multiprotein complex required for heme biosynthesis

Post-diagnosis dietary factors and survival after invasive breast cancer.

Little is known about the effects of diet after breast cancer diagnosis on survival. We prospectively examined the relation between post-diagnosis dietary factors and breast cancer and all-cause survival in women with a history of invasive breast cancer diagnosed between 1987 and 1999 (at ages 20-79 years). Diet after breast cancer diagnosis was measured using a 126-item food frequency questionnaire. Among 4,441 women without a history of breast cancer recurrence prior to completing the questionnaire, 137 subsequently died from breast cancer within 7 years of enrollment. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated for intake of macronutrients as well as selected micronutrients and food groups from Cox proportional hazards regression models. After adjustment for factors at diagnosis (age, state of residence, menopausal status, smoking, breast cancer stage, alcohol, history of hormone replacement therapy), interval between diagnosis and diet assessment, and at follow-up (energy intake, breast cancer treatment, body mass index, and physical activity), women in the highest compared to lowest quintile of intake of saturated fat and trans fat had a significantly higher risk of dying from any cause (HR = 1.41, 95% CI = 1.06-1.87, P trend = 0.03) for saturated fat; (HR = 1.78, 95% CI = 1.35-2.32, P trend = 0.01) for trans fat intake. Associations were similar, though did not achieve statistical significance, for breast cancer survival. This study suggests that lower intake of saturated and trans fat in the post-diagnosis diet is associated with improved survival after breast cancer diagnosis

Combining scales to assess suicide risk

Authors posting Accepted Author Manuscript online should later add a citation for the Published Journal Article indicating that the Article was subsequently published, and may mention the journal title provided they add the following text at the beginning of the document: “NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Cardiovascular Echography. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Psychiatric Research, [VOL#, ISSUE#, (DATE)] DOI#”A major interest in the assessment of suicide risk is to develop an accurate instrument, which could be easily adopted by clinicians. This article aims at identifying the most discriminative items from a collection of scales usually employed in the assessment of suicidal behavior. Methods: The answers to the Barrat Impulsiveness Scale, International Personality Disorder Evaluation Screening Questionnaire, BrowneGoodwin Lifetime History of Aggression, and Holmes & Rahe Social Readjustment Rating Scale provided by a group of 687 subjects (249 suicide attempters, 81 non-suicidal psychiatric inpatients, and 357 healthy controls) were used by the Lars-en algorithm to select the most discriminative items. Results: We achieved an average accuracy of 86.4%, a specificity of 89.6%, and a sensitivity of 80.8% in classifying suicide attempters using 27 out of the 154 items from the original scales. Conclusions: The 27 items reported here should be considered a preliminary step in the development of a new scale evaluating suicidal risk in settings where time is scarce.This article was supported by the National Alliance for Research on Schizophrenia and Affective Disorders (NARSAD), Fondo de Investigacion Sanitaria (FIS) PI060092, Fondo de Investigacion Sanitaria FIS RD06/0011/0016, ETES (PI07/90207), the Conchita Rabago Foundation, and the Spanish Ministry of Health, Instituto de Salud Carlos III, CIBERSAM (Intramural 521 Project, P91B; SCO/3410/2004)

Urokinaseâ deficient and urokinase receptorâ deficient mice have impaired neutrophil antimicrobial activation in vitro

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141382/1/jlb0648.pd

Structure and function of the vacuolar Ccc1/VIT1 family of iron transporters and its regulation in fungi

Iron is an essential micronutrient for most living beings since it participates as a redox active cofactor in many biological processes including cellular respiration, lipid biosynthesis, DNA replication and repair, and ribosome biogenesis and recycling. However, when present in excess, iron can participate in Fenton reactions and generate reactive oxygen species that damage cells at the level of proteins, lipids and nucleic acids. Organisms have developed different molecular strategies to protect themselves against the harmful effects of high concentrations of iron. In the case of fungi and plants, detoxification mainly occurs by importing cytosolic iron into the vacuole through the Ccc1/VIT1 iron transporter. New sequenced genomes and bioinformatic tools are facilitating the functional characterization, evolution and ecological relevance of metabolic pathways and homeostatic networks across the Tree of Life. Sequence analysis shows that Ccc1/VIT1 homologs are widely distributed among organisms with the exception of animals. The recent elucidation of the crystal structure of a Ccc1/VIT1 plant ortholog has enabled the identification of both conserved and species-specific motifs required for its metal transport mechanism. Moreover, recent studies in the yeast Saccharomyces cerevisiae have also revealed that multiple transcription factors including Yap5 and Msn2/Msn4 contribute to the expression of CCC1 in high-iron conditions. Interestingly, Malaysian S. cerevisiae strains express a partially functional Ccc1 protein that renders them sensitive to iron. Different regulatory mechanisms have been described for non-Saccharomycetaceae Ccc1 homologs. The characterization of Ccc1/VIT1 proteins is of high interest in the development of biofortified crops and the protection against microbial-derived diseases

Induction of Biogenic Magnetization and Redox Control by a Component of the Target of Rapamycin Complex 1 Signaling Pathway

Most organisms are simply diamagnetic, while magnetotactic bacteria and migratory animals are among organisms that exploit magnetism. Biogenic magnetization not only is of fundamental interest, but also has industrial potential. However, the key factor(s) that enable biogenic magnetization in coordination with other cellular functions and metabolism remain unknown. To address the requirements for induction and the application of synthetic bio-magnetism, we explored the creation of magnetism in a simple model organism. Cell magnetization was first observed by attraction towards a magnet when normally diamagnetic yeast Saccharomyces cerevisiae were grown with ferric citrate. The magnetization was further enhanced by genetic modification of iron homeostasis and introduction of ferritin. The acquired magnetizable properties enabled the cells to be attracted to a magnet, and be trapped by a magnetic column. Superconducting quantum interference device (SQUID) magnetometry confirmed and quantitatively characterized the acquired paramagnetism. Electron microscopy and energy-dispersive X-ray spectroscopy showed electron-dense iron-containing aggregates within the magnetized cells. Magnetization-based screening of gene knockouts identified Tco89p, a component of TORC1 (Target of rapamycin complex 1), as important for magnetization; loss of TCO89 and treatment with rapamycin reduced magnetization in a TCO89-dependent manner. The TCO89 expression level positively correlated with magnetization, enabling inducible magnetization. Several carbon metabolism genes were also shown to affect magnetization. Redox mediators indicated that TCO89 alters the intracellular redox to an oxidized state in a dose-dependent manner. Taken together, we demonstrated that synthetic induction of magnetization is possible and that the key factors are local redox control through carbon metabolism and iron supply

Standardising Clinical Caremaps: Model, Method and Graphical Notation for Caremap Specification

Standardising care can improve patient safety and outcomes, and reduce the cost of providing healthcare services. Caremaps were developed to standardise care, but contemporary caremaps are not standardised. Confusion persists in terms of terminology, structure, content and development process. Unlike existing methods in the literature, the approach, model and notation presented in this chapter pays special attention to incorporation of clinical decision points as first-class citizens within the modelling process. The resulting caremap with decision points is evaluated through creation of a caremap for women with gestational diabetes mellitus. The proposed method was found to be an effective way for comprehensively specifying all features of caremaps in a standardised way that can be easily understood by clinicians. This chapter contributes a new standardised method, model and notation for caremap content, structure and development

Investigating infection management and antimicrobial stewardship in surgery: a qualitative study from India and South Africa

Appendix A. Supplementary data: The following is the Supplementary data to this article: Multimedia component 1. Avalable online at https://www.sciencedirect.com/science/article/pii/S1198743X20307734#appsec1 .This work was supported by the Economic and Social Science Research Council (ESRC) and the National Institute for Health Research, UK Department of Health (HPRU-2012-10047) in partnership with Public Health England. This study is part of the ASPIRES project (Antibiotic use across Surgical Pathways—Investigating, Redesigning and Evaluating Systems) (https://www.imperial.ac.uk/arc/aspires/). ASPIRES aims to address antimicrobial resistance and improve clinical outcomes optimizing antibiotic usage along surgical pathways. The support of ESRC as part of the Antimicrobial Cross Council initiative supported by the seven UK research councils, and also the support of the Global Challenges Research Fund, is gratefully acknowledged