XIth EPR Workshop on Applications of EPR in Biology and Medicine.

We are pleased and honored to present this special issue for CBBI on the broad topic of biomedical EPR. The papers herein resulted from the most recent October 2019 EPR Workshop in Kraków that encompasses work from outstanding researchers in the field. Before describing the range of articles, we have briefly summarized the history of these workshops and the publications that resulted.

What are the methodological and theoretical prospects for paramagnetic NMR in structural biology? A glimpse into the crystal ball.

NMR spectroscopy is very sensitive to the presence of unpaired electrons, which perturb the NMR chemical shifts, J splittings and nuclear relaxation rates. These paramagnetic effects have attracted increasing attention over the last decades, and their use is expected to increase further in the future because they can provide structural information not easily achievable with other techniques. In fact, paramagnetic data provide long range structural restraints that can be used to assess the accuracy of crystal structures in solution and to improve them by simultaneous refinements with the X-ray data. They are also precious for obtaining information on the conformational variability of biomolecular systems, possibly in conjunction with SAXS and/or DEER data. We foresee that new tools will be developed in the next years for the simultaneous analysis of the paramagnetic data with data obtained from different techniques, in order to take advantage synergistically of the information content of all of them. Of course, the use of the paramagnetic data for structural purposes requires the knowledge of the relationship between these data and the molecular coordinates. Recently, the equations commonly used, dating back to half a century ago, have been questioned by first principle quantum chemistry calculations. Our prediction is that further theoretical/computational improvements will essentially confirm the validity of the old semi-empirical equations for the analysis of the experimental paramagnetic data.

Pulse EPR in biological systems - Beyond the expert's courtyard.

Application of EPR to biological systems includes many techniques and applications. In this short perspective, which dares to look into the future, I focus on pulse EPR, which is my field of expertise. Generally, pulse EPR techniques can be divided into two main groups: (1) hyperfine spectroscopy, which explores electron-nuclear interactions, and (2) pulse-dipolar (PD) EPR spectroscopy, which is based on electron-electron spin interactions. Here I focus on PD-EPR because it has a better chance of becoming a widely applied, easy-to-use table-top method to study the structural and dynamic aspects of bio-molecules. I will briefly introduce this technique, its current state of the art, the challenges it is facing, and finally I will describe futuristic scenarios of low-cost PD-EPR approaches that can cross the diffusion barrier from the core of experts to the bulk of the scientific community.

Advanced paramagnetic resonance spectroscopies of iron-sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM).

The advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of nature's widely distributed iron-sulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

High-frequency and high-field electron paramagnetic resonance (HFEPR): a new spectroscopic tool for bioinorganic chemistry.

This minireview describes high-frequency and high-field electron paramagnetic resonance (HFEPR) spectroscopy in the context of its application to bioinorganic chemistry, specifically to metalloproteins and model compounds. HFEPR is defined as frequencies above ~100 GHz (i.e., above W-band) and a resonant field reaching 25 T and above. The ability of HFEPR to provide high-resolution determination of g values of S = 1/2 is shown; however, the main aim of the minireview is to demonstrate how HFEPR can extract spin Hamiltonian parameters [zero-field splitting (zfs) and g values] for species with S > 1/2 with an accuracy and precision unrivalled by other physical methods. Background theory on the nature of zfs in S = 1, 3/2, 2, and 5/2 systems is presented, along with selected examples of HFEPR spectroscopy of each that are relevant to bioinorganic chemistry. The minireview also provides some suggestions of specific systems in bioinorganic chemistry where HFEPR could be rewardingly applied, in the hope of inspiring workers in this area.

The world as viewed by and with unpaired electrons.

Recent advances in electron paramagnetic resonance (EPR) include capabilities for applications to areas as diverse as archeology, beer shelf life, biological structure, dosimetry, in vivo imaging, molecular magnets, and quantum computing. Enabling technologies include multifrequency continuous wave, pulsed, and rapid scan EPR. Interpretation is enhanced by increasingly powerful computational models.

In vivo electron spin resonance: An effective new tool for reactive oxygen species/reactive nitrogen species measurement.

Reactive oxygen species are regarded as important factors in the initiation and progression of many diseases. Therefore, measurement of redox status would be helpful in understanding the "Redox Navigation" of such diseases. Because electron spin resonance (ESR) shows good signal responses to nitroxyl radical and various redox-related species, such as oxygen radicals and antioxidants, the in vivo ESR/nitroxyl probe technique can provide useful information on real-time redox status in a living body. ESR spectrometers for in vivo measurements can be operated at lower frequencies (approximately 3.5 GHz, 1 GHz, 700 MHz, and 300 MHz) than usual (9-10 GHz). Several types of resonators were also designed to minimize the dielectric loss of electromagnetic waves caused by water in animal bodies. In vivo ESR spectroscopy and its imaging have been used to analyze radical generation, redox status, partial pressure of oxygen and other conditions in various diseases. In addition, ESR has been used to analyze injury models related to oxidative stresses, such as nitroxyl radicals. The application of in vivo ESR for diseases related to oxidative injuries currently being investigated and the accumulation of basic data for therapy is ongoing. Recent progress in the instrumentation for in vivo ESR spectroscopy and its application to the life sciences are reviewed, because measurement of redox status in vivo is considered necessary to understand the initiation and progression of diseases.

Fingernail dosimetry: current status and perspectives.

A summary of recent developments in fingernail EPR dosimetry is presented in this paper. Until 2007, there had been a very limited number of studies of radiation-induced signals in fingernails. Although these studies showed some promising results, they were not complete with regard to the nature of non-radiation signals and the variability of dose dependence in fingernails. Recent study has shown that the two non-radiation components of the EPR spectrum of fingernails are originated from mechanical stress induced in the samples at their cut. The mechanical properties of fingernails were found to be very similar to those of a sponge; therefore, an effective way to eliminate their mechanical deformation is by soaking them in water. Stress caused by deformation can also significantly modify the dose response and radiation sensitivity. Consequently, it is critically important to take into account the mechanical stress in fingernail samples under EPR dose measurements. Obtained results have allowed formulating a prototype of a protocol for dose measurements in human fingernails.

From spin-labeled proteins to in vivo EPR applications.

This is a historical overview of the advent of applications of spin labeling to biological systems and the subsequent developments from the perspective of a scientist who was working as a Ph.D. student when the technique was conceived and was fortunate enough to participate in its development. In addition, the historical development of in vivo applications of EPR on animals and other living systems is described from a personal perspective.

Applications of electron paramagnetic resonance to studies of neurological disease.

Electron paramagnetic resonance spectroscopy (EPR) has the potential to give much detail on the structure of the paramagnetic transition ion coordination sites, principally of Cu2+, in a number of proteins associated with central nervous system diseases. Since these sites have been implicated in misfolding/mis-oligomerisation events associated with neurotoxic molecular species and/or the catalysis of damaging redox reactions in neurodegeneration, an understanding of their structure is important to the development of therapeutic agents. Nevertheless EPR, by its nature an in vitro technique, has its limitations in the study of such complex biochemical systems involving self-associating proteins that are sensitive to their chemical environment. These limitations are at the instrumental and theoretical level, which must be understood and the EPR data interpreted in the light of other biophysical and biochemical studies if useful conclusions are to be drawn.

Recent advances and applications of site-directed spin labeling.

Site-directed spin labeling has become a popular biophysical tool for the characterization of protein structure, dynamics and conformational change. This method is well suited and widely used to study small soluble proteins, membrane proteins and large protein complexes. Recent advances in site-directed spin labeling methodology have occurred in two areas. The first involves an understanding of the conformations and local dynamics of the spin-labeled sidechain, including the features of proteins that influence electron paramagnetic resonance lineshape. The second advance is the application of pulse techniques to determine long-range distances and distance distributions in proteins. During the past two years, these technical developments have been used to address several important problems concerning the molecular function of proteins.

Joint International Conference on EPR Spectroscopy and wound healing.

The International Conference on Electron Paramagnetic Resonance Spectroscopy and Imaging of Biological Systems (EPR 2005) was held September 4 through 9, 2005, at Columbus, Ohio, U.S.A. Nearly 200 participants from 16 countries presented recent advances on the use of EPR technology to study biologic processes, with an emphasis on human health. For the first time, the EPR conference allied with the Wound Healing Conference, and the alliance opened an avenue for successful amalgamation of the basic biomedical and clinical aspects of wound healing with EPR technology and vice versa. This should lead to emerging applications of EPR technology in biomedical research and clinical practice.

The Third International Intercomparison on EPR Tooth Dosimetry: part 2, final analysis.

The objective of the Third International Intercomparison on EPR Tooth Dosimetry was to evaluate laboratories performing tooth enamel dosimetry <300 mGy. Final analysis of results included a correlation analysis between features of laboratory dose reconstruction protocols and dosimetry performance. Applicability of electron paramagnetic resonance (EPR) tooth dosimetry at low dose was shown at two applied dose levels of 79 and 176 mGy. Most (9 of 12) laboratories reported the dose to be within 50 mGy of the delivered dose of 79 mGy, and 10 of 12 laboratories reported the dose to be within 100 mGy of the delivered dose of 176 mGy. At the high-dose tested (704 mGy) agreement within 25% of the delivered dose was found in 10 laboratories. Features of EPR dose reconstruction protocols that affect dosimetry performance were found to be magnetic field modulation amplitude in EPR spectrum recording, EPR signal model in spectrum deconvolution and duration of latency period for tooth enamel samples after preparation.

ESR spectrometry: a future-oriented tool for dosimetry and dating.

ESR spectroscopy is currently taking root as a key technology in dosimetry, dating and imaging. In dosimetry, it competes with cytometry in the fields of biological dosimetry and retrospective dosimetry, leads in high-level reference and routine dosimetry, is high-ranking among the methods to identify radiation preserved foods, represents a method of choice to date geological, archaeological and paleontological materials back millions of years, and has demonstrated capacity for imaging. Further scientific and technological progress as predicted in the recent past (Appl. Radiat. Isot. 52 (2000) 1023) is reviewed here. Additionally, the review is expanded to include international reports and recommendations on ESR dosimetry and dose reconstruction, under way at the American Society for Testing and Materials (ASTM), the International Organisation of Standards (ISO), the International Atomic Energy Agency (IAEA) and the International Commission on Radiation Units and Measurements (ICRU). Emphasis is placed on interpretation of tooth enamel doses in terms of organ and effective doses, using CT-based virtual humans. The future of EPR spectroscopy for in situ dose measurements is noted, depicting a non-destructive in vivo dosimetry applicable directly to individuals, but also to hominid and animal fossils for direct dating.

Recent progress in in vivo ESR spectroscopy.

The generation of free radicals and redox status is related to various diseases and injuries that are related to radiation, aging, ischemia-reperfusion, and other oxidative factors. In vivo electron spin resonance (ESR) spectroscopy is noninvasive and detects durable free radicals in live animals. ESR spectrometers for in vivo measurements operate at a lower frequency (approximately 3.5 GHz, approximately 1 GHz, 700 MHz, and approximately 300 MHz) than usual (9-10 GHz). Several types of resonators have been designed to minimize the dielectric loss of electromagnetic waves caused by water in animal bodies. In vivo ESR spectroscopy and its imaging have been used to analyze radical generation, redox status, partial pressure of oxygen and other conditions in various disease and injury models related to oxidative stress with probes, such as nitroxyl radicals. Through these applications, the clarification of the mechanisms related to oxidative diseases (injuries) and the accumulation of basic data for radiological cancer therapy are now ongoing. In vivo ESR measurement is performed in about 10 laboratories worldwide, including ours. To introduce in vivo ESR spectroscopy to life scientists, this article reviews the recent progress of in vivo ESR spectroscopy in instrumentation and its application to the life sciences.

In vivo EPR: when, how and why?

This special issue is aimed at providing the readers of this journal with an indication of the exciting and important areas in which in vivo electron paramagnetic resonance (EPR) [or equivalently electron spin resonance (ESR)] is making contributions to experimental progress and to provide perspectives on future developments, including the potential for in vivo EPR to be an important new clinical tool. There also are many situations where the combination of in vivo EPR with NMR may be very synergistic. EPR (ESR) is a magnetic resonance-based technique that detects species with unpaired electrons. The technique has become a major tool in diverse fields ranging from biology and chemistry to solid-state physics. In the last few years, many publications have demonstrated that EPR measurements in living animals (in vivo EPR) can provide very significant new insights to physiology, pathophysiology and pharmacology. The most successful applications of in vivo EPR have been non-invasive measurements of oxygen, nitric oxide, bioradicals, pH and redox state, with applications in oncology, cardiology, neuroscience and toxicology. EPR also appears to be the method of choice for measuring radiation dose retrospectively, including the potential to do this in vivo in human subjects. While far from comprehensive, the reviews, original contributions and viewpoints provided in this issue by several leaders in the field of in vivo EPR should provide the readers with confirmation that in vivo EPR is an exciting field that is likely to provide very valuable complementary information for many NMR-based studies in experimental animals and, probably, also for clinical studies.

Assessment of tumor oxygenation by electron paramagnetic resonance: principles and applications.

This review paper attempts to provide an overview of the principles and techniques that are often termed electron paramagnetic resonance (EPR) oximetry. The paper discusses the potential of such methods and illustrates they have been successfully applied to measure oxygen tension, an essential parameter of the tumor microenvironment. To help the reader understand the motivation for carrying out these measurements, the importance of tumor hypoxia is first discussed: the basic issues of why a tumor is hypoxic, why these hypoxic microenvironments promote processes driving malignant progression and why hypoxia dramatically influences the response of tumors to cytotoxic treatments will be explained. The different methods that have been used to estimate the oxygenation in tumors will be reviewed. To introduce the basics of EPR oximetry, the specificity of in vivo EPR will be discussed by comparing this technique with NMR and MRI. The different types of paramagnetic oxygen sensors will be presented, as well as the methods for recording the information (EPR spectroscopy, EPR imaging, dynamic nuclear polarization). Several applications of EPR for characterizing tumor oxygenation will be illustrated, with a special emphasis on pharmacological interventions that modulate the tumor microenvironment. Finally, the challenges for transposing the method into the clinic will also be discussed.

Radio frequency continuous-wave and time-domain EPR imaging and Overhauser-enhanced magnetic resonance imaging of small animals: instrumental developments and comparison of relative merits for functional imaging.

Electron paramagnetic resonance (EPR) imaging in the continuous wave (CW) and time-domain modes, as well as Overhauser-enhanced magnetic resonance imaging in vivo is described. The review is based mainly on the CW and time-domain EPR instrumentation at 300 MHz developed in our laboratory, and the relative merits of these methods for functional in vivo imaging of small animals to assess hypoxia and tissue redox status are described. Overhauser imaging of small animals at magnetic fields in the range 10-15 mT that is being carried out in our laboratory for tumor imaging and the evaluation of tumor hypoxia based on quantitative evaluation of Overhauser enhancement is also described. Alternate approaches to spectral-spatial imaging using the transverse decay constants to infer in situ line widths and hence in vivo pO2 using CW and time-domain EPR imaging are also discussed. The nature of the spin probes used, the quality of the images obtained in all the three methods, the achievable resolution, limitations and possible future directions in small animal functional imaging with these modalities are summarized.

Tissue oxygenation in sepsis; new insights from in vivo EPR.

Nitric oxide (NO) is a key mediator in the maldistribution of oxygen by tissue and organ dysfunction observed in sepsis. Despite this, few techniques are capable of measuring these parameters directly in vivo. We describe here several techniques that have been developed by our group to address this directly by in vivo EPR in animal models of sepsis. Oxygen-sensitive materials can be implanted or administered and report on local tissue pO2. Spin trapping of NO can simultaneously report on tissue NO content. Repeat measures of these parameters can be made directly from a defined tissue site, allowing development of new models and experiments to study the defects in tissue and organ function seen in sepsis.