Techniques for Studying Iron in Health and Disease EMBL COURSE EMBL-ATC May 2-4, 2019 coordinated by D Swinkels and I Cabantchik Introduction to Practicals and experimental Protocols General introduction 1 Program DAY 1 2 I. Generation and interpretation of assay test results for biomarkers of iron homeostasis and dyshomeostasis. Rian Roelofs, Rachel van Swelm Dorine Swinkels. Radboud University. Nijmegen NL. 3-25 References (pp 25) Program DAY 2 26 II. Labile 2Fe-2S Clusters. Rachel Nechushtai. Institute of Life Sciences. Hebrew University, Jerusalem, Israel 26-43 References (pp 28-29) II. Biochemical and functional characterization of mitochondrial ISC assembly factors. Oliver Stehling. Institut für Zytobiologie, Philipps- Universität Marburg 44-77 References (pp 76-77) Program DAY 79 Real-time monitoring of intracellular labile iron with fluorescent metal- sensors. Maya Shvartsman. EMBL HD. Rome. IT 80-87 Labile Iron IN BIOLOGICAL FLUIDS. Breno Pannia Espósito, Institute of Chemistry University of São Paulo, Brazil 88-94 Mitochondrial Iron probing. Charareh Pourzand. Department of Pharmacy & Pharmacology. Centre for Therapeutic Innovation University of Bath, UK 95-103 References (pp 98) Techniques for studying iron in health and disease EMBL EMBL, Heidelberg, May 2-4, 2019 1st International laboratory course dedicated to analytical methods for assessing iron in living entities in physiological and pathological states Directors: Dorine Swinkels(NL) and Ioav Cabantchik (IL) https://www.embl.de/training/events/2019/BIR19-02/index.html The workshop is designed to expose the various experimental tools used for: a. tracing iron dynamics in biological systems, from solution to cells to organs, with major emphasis on fluorescence techniques available in most laboratories b. assessing iron status in humans in health and disease and c. exploring methods to study iron sulphur cluster (ISC) proteins (ISP). The 3-day workshop is planned for up to 18 students. Morning sessions comprise lectures and experimental planning and afternoon-evening data analysis and discussions. Bursaries covering the course registration fees are available for selected applicants. TOPICS DAY I. Thursday May 2 2019. GENERATION AND INTERPRETATION OF ASSAY TEST RESULTS FOR BIOMARKERS OF IRON HOMEOSTASIS AND DYSHOMEOSTASIS Coordinator: Dorine W. Swinkels The aim is to reach a level of understanding for adequately generating and interpreting the results of assays for biomarkers of iron homeostasis and dys-homeostasis in both research and clinical setting. Specifically, the students should acquire a level of understanding to be able to: a. Clinically interpret test results of a broad range of conventional and novel iron biomarkers b. Apply fundamentals of implementing an assay and generating a reliable test result in both the research and clinical setting. In the workshop we will: - Discuss interpretation of test results generated by a fully validated assay for the diagnosis of iron disorders. Students will be challenged to solve case –puzzles in small groups, and present their “diagnosis” for the whole group for discussion. - Discuss design of a validation plan for an assay of an analyte. In the afternoon, students will apply this knowledge by performing a validation of a commercial hepcidin ELISA kit in the lab in small groups. By the end of the day each group will present their analytical findings for the whole group for discussion. 1 Techniques for studying iron in health and disease EMBL DAY II. May 3 2019. DYNAMICS OF IRON SULFUR CLUSTER (ISC) PROTEINS (ISP): FROM SOLUTION TO CELLS Coordinator: Coordinators: Stehling, Lill & Nechushtai The aim is to get acquainted with the application of spectroscopic methods for assessing mitochondrial ISP maturation within the cellular environment. I. Biophysical and biochemical analysis of recombinant ISP a. Determination of iron and sulphide content of holo-ISP: ISCA1 and FDX2 as paradigms. b. Assessment of ISC cofactor stability under reducing and oxidizing conditions (UV/Vis spectroscopy). c. Tracing ISC transfer between proteins by native gel chromatography and UV-VIS spectroscopy-CISD2-gene product NAF-1 (wild type and mutants) as paradigm. II. Assessing the consequences of ISP assembly defects of cells as reflected in: a. morphological and metabolic changes of mitochondria; b. Activity measurements of key mitochondrial ISC enzymes by spectroscopic assays; c. ISC-dependent protein modification (lipoylation of DLAT and DLST). b. ISC transfer between proteins by native gel chromatography and UV-VIS spectroscopy: CISD2-gene product NAF-1 (wild type and mutants) as paradigm. d. the roles of amino acid residues in the ISC domain of NAF-1 on cell properties by fluorescence spectroscopy: a. mitochondrial membrane potential; mitochondrial labile (chelatable) iron; mitochondrial ROS susceptible to iron chelators. Techniques: fluorescence plate reader and microscope imaging, UV-VIS spectroscopy, affinity chromatography and native protein gel chromatography DAY III. Thursday May 4 2019. FLUORESCENCE MONITORING OF LABILE IRON TRAFFICKING IN CELLS AND IRON SPECIATION IN ANIMAL FLUIDS AND ORGANS. Coordinators: Pourzand, Hider and Cabantchik We will explore how can fluorescence probes be designed and used for sensing iron dynamically in solution (fluorescence plate reader) or in cell compartments. Theoretical aspects of labile iron sensing with the aid of fluorescence probes will be provided and application in solution and in cells demonstrated by: I. Monitoring on line iron ingress (influx) into cytosol and mitochondria compartments with organelle targeted probes using both transferrin-iron and non-transferrin iron as iron substrates and. K562 as model cells. II. Assessing the level of labile iron in biological fluids of clinical relevance: simulants of labile plasma iron as direct target of chelation. Performance of assays and data evaluation. III. The application of a novel mitochondrial targeted iron sensor as a biochemical and pharmacological tool in iron research- applied to cell model. Techniques: fluorescence plate reader, fluorescence microscope imaging and FACS. 2 Techniques for studying iron in health and disease EMBL Program DAY 1 (May 2nd) GENERATION AND INTERPRETATION OF ASSAY TEST RESULTS FOR BIOMARKERS OF IRON HOMEOSTASIS AND DYSHOMEOSTASIS. Lecturer Topics Allocated time (min) All Organizers Welcome 9:00-9:15 (15´) Yvonne Yeboah& House notes/ ice breaking activity 9:15- 9:45 (30´) Elisabeth Zielonka Course introduction 9:45- 9:55 (10´) Ioav Cabantchik 9:55-10:15 (20´) COFFEE BRAKE Generation and interpretation of assay test results for biomarkers of iron homeostasis and dyshomeostasis 10:15-10:20 (05´) 1) Goal and daily schedule 2) Clinical interpretation of test results 10:15-10:20 (05´) • Presentation 10:35-11:05 (30´) Dorin Swinkels • Case study (in groups) 11:05-11:25 (20´) • Discussion (plenair) 3) Test validation 11:25-11:55 (30´) • Presentation (30´) • Design practical plan (in groups) 11:55-12:25 • Provide groups with final 12:25-12:30 (35´) practical plan Lunch 12:30-13:15 (45´) Rachel van Swelm Practical in Groups 13:30-18:00 (4,5´) Rian Reolofs Rachel van Swelm Calculation of results and prepare 18:00-19:00 (60´) Rian Reolofs presentation Dinner 19:00- 20:00 (60´) Dorin Swinkels Presentation and Discussion 20:00- 21:00 (60´) 3 Techniques for studying iron in health and disease EMBL Day 1 Generation and interpretation of assay test results for biomarkers of iron homeostasis and dyshomeostasis. Day coordinator: Dorine W. Swinkels Audience This course is aimed at PhD students and postdocs in all basic and clinical biomedical specialities including biochemistry, physiology, pharmacology, immunology and molecular biology. There will be a maximum of 18 participants, who will be divided into 6 groups for the practical part. The morning will be devoted to lectures and experimental planning. The experiments will be carried out in the afternoon and analyzed and discussed in the evening. Aim By the end of this day students will be able to adequately generate and interpret results of an assay for biomarkers of iron homeostasis and dyshomeostasis within both a research and clinical setting. More specifically the students will be able to: c. Clinically interpret test results of a broad range of conventional and more novel iron biomarkers d. Apply fundamentals of implementing an assay and generating a reliable test result in both the research and clinical setting. During the workshop we will: - Discuss interpretation of test results generated by a fully validated assay for the diagnosis of iron disorders. Students will be challenged to solve case –puzzles in small groups, and present their “diagnosis” for the whole group for discussion. - Discuss design of a validation plan for an assay of an analyte. In the afternoon, students will apply this knowledge by performing a validation of a commercial hepcidin ELISA kit in the lab in small groups. By the end of the day each group will present their analytical findings for the whole group for discussion. Teachers Dorine Swinkels, Radboudumc Center for Iron Disorders, The Netherlands. [email protected] Rachel van Swelm, Radoudumc Center for Iron Disorders, The Netherlands. [email protected] Rian Roelofs, Radboudumc Center for Iron Disorders, The Netherlands. [email protected] 4 Techniques for studying iron in health and disease EMBL Recommended Literature: 1. Andreasson et al (2015) A Practical Guide to Immunoassay method validation. Frontiers