Welcome to the Symposium on Biophysics for Biology and Medicine
Dear Colleagues,
Welcome to the Symposium, the International Conference on Biophysics for Biology and Medicine.
Outstanding scientists will review progress in redox biology, cancer, neurodegenerative diseases and cover
selected topics of biomedical applications of advanced EPR spectroscopy and imaging, such as metal proteins
and macromolecules, development and use of new spin labels, spin traps and spin probes for EPR and dynamic
nuclear polarization, oxidative damage, oximetry, biophysics and clinical applications.
The meeting should provide great opportunities for sharing new ideas and research experience. The sessions
will include lectures, plenary conferences and talks.
The conference is organized at the Hotel Dieu intercontinental located in the heart of Marseille. In addition to
exciting sessions and lively discussions, do not miss the gala dinner organized on April 10
th
and Raman’s
anniversary on April 12
th
to enjoy together French cooking, Vieux Port view and champagne.
We are grateful to the sponsors who have provided financial support to help us in the organization of this
international conference.
We wish you a very stimulating meeting. Enjoy the conference and Marseille!
The organizers
Direction to the symposium
The conference is held at the hotel Dieu Intercontinental, 1 Pl. Daviel, 13002 Marseille 45, rue des Saints Pères
(cf. map). Access with public transportations:
At your arrival at Marseille airport (Marseille Provence Airport)
-The fastest way to reach your hotel (Intercontinental or other hotel located in the downtown area) is to
take a taxi.
-There is a possibility to take a Bus which goes from the Airport to Marseille St Charles Station and Marseille
city center (motorway). From the airport, there is a bus every 20 min.
https://www.marseille-airport.com/access-car-parks/access/bus/marseille-st-charles-station
At Saint Charles train station, there is a direct access to the subway station. (https://www.rtm.fr/en). Take
the M1 metro (direction La Fourragère) and get out to VIEUX PORT station. Take the QUAI DES BELGES exit
and you will be on the Vieux Port big place.
Hôtel Dieu Intercontinental
Subway exit
VIEUX PORT
MUCEM with a rooftop
restaurant
Direction to the Gala dinner (Wednesday, April 10
nd
)
The gala dinner will take place at the Intercontinental Hotel at the “escaliers Monumentaux”
Direction to Atelier Ferroni dinner (Friday, April 12
nd
)
We have privatized a “rum workshop” from the company Ferroni. https://carrynation.fr/bar/atelier
The place is located at 8, rue Neuve Sainte Catherine - 13007 Marseille (10 min walk from the hotel).
Sponsors
Scientific Program
Wednesday, April 10
nd
8.30 – 8.50
Registration
8.50 – 9.00
Welcome words
9.00 – 11.00
Session 1: Redox biology, cancer and neurodegeneration
Chair: Olivier Ouari
9.00 – 9.45
Plenary - Nitrogen oxide-derived anti-inflammatory lipid signaling mediators
New drug candidates?
Bruce Freeman (Pittsburgh, USA)
10.00 – 10.30
Implications of SPR mimetics to BH4-deficiency and pain management
Jeannette Vasquez-Vivar (Milwaukee, USA)
10.30 – 11.00
Coffee break
11.00 – 11.30
Mitigating gut microbial degradation of levodopa and enhancing dopamine in the
brain: Implications in Parkinson’s disease
Gang Cheng (Milwaukee, USA)
11.30 – 12.30
Session 2: EPR spectroscopy for structural biology
Chair: : Valérie Belle
11.30 – 12.00
Electroformation of giant unilamellar vesicles from moist lipid films formed by vesicle
fusion on the surface of an electrode with emphasis on membranes with high
cholesterol content
Marija Raguz (Split, Croatia)
12.00-12.30
In-cell EPR: exploring protein structural dynamics inside cells by SDSL-EPR
Elisabetta Mileo (Marseille, France)
12.30-14h30
Lunch
14.30 – 16.30
Session 3: Structure / function by DNP MRI and NMR
Chair: Elisabetta Mileo
14.30 – 15.00
Structural insights from NMR of protein-lipid assemblies in degenerative diseases
Francesca M. Marassi (Milwaukee, USA)
15.00 – 15.30
Hyperpolarized
13
C NMR probes to assess the activity of the mitochondrial TCA cycle
Zoltan Kovacs (Dallas, USA)
15.30 – 16.00
Coffee break
16.00 – 17.00
Session 4: Photoreactivity, phototoxicity in Krakow city !
Chair: Jacek Zielonka
16.00 – 16.30
Photoreactivity and phototoxicity of fine particulate matter from air pollutants
Tadeusz Sarna (Krakow, Poland)
16.30 – 17.00
Ravinder J. Singh (Rochester, USA)
19h30
Gala dinner
Thursday, April 11
nd
9.00 – 10.00
Session 5: in vivo Imaging and Oximetry
Chair: Periannan Kuppusamy
9.00 – 9.30
EPR Oximetry: Capillary to Clinic
Periannan Kuppusamy (Dartmouth, USA)
9h.30 – 10.00
An autocatalytic mechanism for the reaction between S-nitrosoglutathione and
hydrogen sulfide
Neil Hogg (Milwaukee, USA)
10.00 – 10.30
Coffee break
10.30 – 12.00
Session 6: Spin labels, spin traps, spin probes for EPR
Chair: Micael Hardy
10.30 – 11.00
Fluorogenic cyclization of phenyl-type radicals for specific detection of peroxynitrite
Jacek Zielonka (Milwaukee, USA)
11.00 – 11.30
The reaction of glutathione-derived dinitrosyl iron complexes and superoxide radical
anion - the detection and quantitation of peroxynitrite
Adam Sikora (Lodz, Poland)
11.30 – 12.00
Unveiling the Therapeutic Frontiers of Targeted Nitroxides
Marcos Lopez (Puerto Rico, USA)
12.00
Lunch
Oral presentations
Redox biology, cancer and neurodegeneration
Nitrogen oxide-derived anti-inflammatory lipid signaling mediators
New drug candidates?
Bruce Freeman.
1
1
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh
Pennsylvania, USA.
A broad array of nitrogen oxide (NOx) and oxygen-derived oxidizing, nitrosating and nitrating species
are generated endogenously during metabolism and inflammation. Nitric oxide (
•
NO), nitrite (NO
2
-
) and nitrate
(NO
3
-
) are readily transformed by metabolic and inflammatory reduction-oxidation (redox) reactions to yield
an array of reactive species that can mediate toxic and adaptive signaling responses, depending on
concentration. For example, unsaturated fatty acid reaction with
•
NO
2
produces fatty acid nitroalkene and
nitro-nitrate derivatives (NO
2
-FA). These species are conferred with an electrophilic character that supports
kinetically rapid and reversible adduction of nucleophilic amino acids, predominantly cysteine (Cys). Preclinical
and clinical data affirms that NO
2
-FA at endogenous and therapeutic concentrations react with the “hyper-
reactive Cys proteome”. These covalent reactions broadly impact cell and organ function by altering the
structure/function of ~75-150 hyper-reactive protein targets that evolved to mediate adaptive signaling
responses. This includes redox-sensitive transcription factors, protein chaperones and enzymes regulating
stress responses and tissue repair.
Downstream responses include inhibition of cytokine expression, improved oxidant-antioxidant “balance”,
enhanced mitochondrial and metabolic function. These concerted actions will suppress inflammation and limit
pathologic cell proliferation, thus potentially providing clinical benefit. This presentation will also summarize
a) Phase 1 and 2 clinical trial results for orally-administered electrophilic 10-nitro-octadec-9-enoic acid (nitro-
oleate, CP-6) in renal and pulmonary disease patients and b) recent discoveries of how small molecule
nitroalkenes can limit other pathologic cell proliferation responses.
Implications of SPR mimetics to BH4-deficiency and pain management
Jeannette Vasquez-Vivar,
1
James Woodcock,
1
Steven Traeger,
1
Zhongjie Shi,
2
Sidhartha Tan.
2
1
Department of Biophysics, Redox Biology Program, Medical College of Wisconsin, Milwaukee, Wisconsin
53226.
2
Department of Pediatrics Wayne State University, Detroit, Michigan 48201.
Sepiapterin reductase (SPR) is a NADPH-dependent enzyme in the tetrahydrobiopterin synthetic
pathway. Biallelic SPR deficiency (SPRD) leads to intellectual disabilities and motor disorders. Still, it is
distinctive because it does not cause hyperphenylalaninemia, which is generally present in other deficiencies.
It is thought that carbonyl and aldo-keto reductases (CR, AKR) mimicking SPR activity explain this finding,
although their efficiencies in vivo remain unclear. Recent human genome-wide association studies have
indicated a role for the BH4 pathway in neuropathic chronic pain, a condition caused by injury to the
somatosensory system. Since both CR and AKRs could represent a therapeutic approach for SPRD and could
be an unrealized target of pain therapies focused on inhibiting SPR, we sought to better characterize the role
of AKRs in the BH4 pathway. We generated homozygous knockout (SPR-KO) HEK cells targeting SPR exon 2, a
frequent mutation locus of human SPRD, using CRISPR/Cas9. The biallelic SPR mutation was confirmed by
Sanger sequencing, and the absence of SPR protein expression was confirmed by Western blot. Activity assays
showed the total loss of SPR activity, yet 18% of the basal wild-type BH4 persisted. The pattern of BH2
depletion in SPR-KO indicated that at least some of the cellular BH2 arises from the biosynthetic pathway. The
overexpression of the human AKR1C3 isoform in SPR-KO cells, in direct contradiction to previous in vitro
studies, failed to bring BH4 to wild-type levels. A 21% increase in BH4 was found in clones expressing AKR1C3
SPR-KO cells. Also, no significant BH4 changes were found in wild-type cells overexpressing AKR1C3. We
confirmed that AKR1B1 and CR are expressed in HEK cells, although CR is expressed at exceedingly low levels.
The impact of SPR/BH4 insufficiency and SPR mimetics on cellular functions and their possible role in
inflammatory conditions will be discussed.
Research reported in this work was supported by the National Institute of Neurological Disorders and Stroke of the
National Institutes of Health under award numbers R01NS1149972 to ST, R01NS117146 to ST, JVV, XJ.
Mitigating gut microbial degradation of levodopa and enhancing dopamine in the
brain: Implications in Parkinson’s disease
Gang Cheng,
1
Micael Hardy,
2
Cecilia J. Hillard,
3
Jimmy B. Feix,
1
Balaraman Kalyanaraman.
1*
1
Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
2
Aix-Marseille Univ, CNRS, ICR, UMR 7273, Marseille 13013, France.
3
Department of Pharmacology and Toxicology and Neuroscience Research Center, Medical College of
Wisconsin, Milwaukee, WI 53226, United States.
Levodopa is used to manage Parkinson’s disease symptoms. As Parkinson’s disease progresses, patients
require increased doses of levodopa, causing undesirable side effects. Additionally, the oral bioavailability of
levodopa decreases in Parkinson’s disease patients due to the increased metabolism of levodopa to dopamine
by gut bacteria, Enterococcus faecalis, resulting in decreased neuronal uptake and dopamine formation.
Parkinson’s disease patients have varying levels of these bacteria. Thus, decreasing bacterial metabolism is a
promising therapeutic approach to enhance the bioavailability of levodopa in the brain. Mito-ortho-HNK,
formed by modification of a naturally occurring molecule such as honokiol conjugated to a
triphenylphosphonium moiety, mitigated metabolism of levodopa—alone or combined with carbidopa—to
dopamine. Mito-ortho-HNK suppressed the growth of E. faecalis, decreased dopamine levels in the gut, and
increased dopamine levels in the brain. One way to enhance the bioavailability of levodopa is to mitigate its
gut metabolism to dopamine using Mito-ortho-HNK and analogs.
Keywords: microbiome, mitochondria-targeted drugs, bioenergetics, levodopa, TPP
+
-based drugs, Parkinson’s
disease.
EPR Spectroscopy for structural biology
Electroformation of giant unilamellar vesicles from moist lipid films formed by
vesicle fusion on the surface of an electrode with emphasis on membranes with
high cholesterol content
M. Raguz,
1
I. Mardešić
1
Z. Boban.
1
1
University of Split School of Medicine, Department of medical physics and biophysics, Split, Croatia.
Artificial models that are often used to mimic cell membranes are giant unilamellar vesicles (GUVs). They are
most commonly produced using the electroformation method. The traditional protocol involves a step in
which the organic solvent is completely evaporated, forming a dry lipid film. This leads to an artifactual
demixing of cholesterol (Chol) in the form of anhydrous crystals. These crystals do not participate in the
formation of the lipid bilayer, which leads to a decrease in the Chol concentration in the bilayer compared to
the initial lipid solution. We propose a novel protocol that bypasses the dry lipid film phase by combining of
rapid solvent exchange (RSE), extrusion, plasma cleaning and spin-coating to produce GUVs from moist lipid
films. We tested the efficiency of the protocol with Chol/phosphatidylcholine (POPC) lipid mixtures with a
mixing ratio between 0 and 2.5. The most reproducible results were obtained when the duration of vesicle
spin-coating was 30 s and large unilamellar vesicles (LUVs) were extruded through 100 nm membrane pores.
The reduction in vesicle size was about 40% for all vesicles with a Chol/POPC mixing ratio above 1.5 compared
to the pure POPC bilayer (Figure 1). We believe that this new, improved electroformation protocol will allow
us to successfully study the physical properties, lateral organization, and domain formation of membranes
with very high Chol content such as the plasma membranes of the eye lens fiber cells. The elimination of
organic solutions by the RSE method and the plasma cleaning of the electrode have proven to be advantageous
for the preparation of GUVs with charged lipids and buffer solutions.
Figure 1. GUVs sizes for different Chol/POPC mixing ratios.
In-cell EPR:
exploring protein structural dynamics inside cells by SDSL-EPR
A. Pierro,
1
A. Bonucci,
2
B. Zambelli,
3
O. Ouari,
4
A. Magalon
5
, V. Belle
2
, E. Mileo.
2
1
Department of Chemistry, University of Konstanz, Konstanz, Germany.
2
Aix Marseille Univ, CNRS, BIP, Marseille, France.
3
Laboratory of Bio-Inorganic Chemistry, University of Bologna, Italy.
4
Aix Marseille Univ, CNRS, ICR, Marseille, France.
5,6
Aix Marseille Univ, CNRS, LCB UMR7283, Marseille, France.
Understanding how the intracellular medium modulates protein structural dynamics and protein-protein
interactions is an intriguing but required topic scientists search to address by studying biomolecules in their
native environment. As the cellular environment cannot be reproduced in vitro, investigation of biomolecules
directly inside cells has attracted a growing interest in the past decade. Indeed, efforts in magnetic resonance
spectroscopies have enabled important improvements in the study of structural dynamics directly in the
cellular context.
Among magnetic resonances approaches, site-directed spin labeling coupled to electron paramagnetic
resonance spectroscopy (SDSL-EPR) has demonstrated to be one of the powerful approaches to study
structural properties of biomolecules [1]. In particular, nitroxide-based SDSL-EPR couples the benefits of high
sensitivity and the lack of size constraints for the biomolecule of interest with the ability to study protein
structural transitions and interactions at physiological temperature. [2-3]
In this lecture, I will introduce the basic principles of in-cell EPR spectroscopy of proteins. I will discuss the
main technical problems limiting the application of the method. In parallel, I will overview recent
developments and highlight the main future research directions in the field. [3-4-5]
References:
[1] - A. Bonucci, O. Ouari, B. Guigliarelli, V. Belle, E. Mileo, ChemBioChem 2020, 21, 451-460.
[2] - G. Karthikeyan, A. Bonucci, G. Casano, G. Gerbaud, S. Abel, V. Thomé, L. Kodjabachian, A. Magalon, B. Guigliarelli, V.
Belle, O. Ouari, E. Mileo, Angew. Chem. Int. Ed. 2018, 130, 1380-1384.
[3] – A. Pierro, A. Bonucci, D. Normanno, M. Ansaldi, E. Etienne, G. Gerbaud, E. Pilet, B. Guigliarelli, O. Ouari, A. Magalon,
V. Belle, E. Mileo, Chem. – Eur. J. 2022, 28, e202202249.
[4] – A. Pierro, K. C. Tamburrini, H. Leguenno, E. Etienne, G. Gerbaud, B. Guigliarelli, V. Belle, B. Zambelli, E. Mileo, iScience,
2023, 26 (10), 107855.
[5]- Y. Ben-Ishay, Y. Barak, O. Ouari, A. Pierro, E. Mileo, X.-C.-Su and D. Goldfarb, Protein Science, 2024, in press.
Structure / function by DNP MRI and NMR
Structural insights from NMR of protein-lipid assemblies in degenerative diseases
Francesca M. Marassi, Rajlaxmi Panigrahi, Kyungsoo Shin, Gopinath Tata, Nicholas Wood.
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Medical College of
Wisconsin, Milwaukee, WI 53226, United States.
Elucidating the structure and activity of proteins in their native environment is a fundamental goal of
structural biology. Here we show that nuclear magnetic resonance (NMR) spectroscopy is ideally suited for
this task. Using NMR experiments with both soluble and solid-state samples we explore proteins in complex
biomolecular assemblies, including microbial membranes and calcified protein-lipid deposits. NMR studies of
bacterial samples yield insights about the native structure and function of virulence proteins and set the stage
for probing their interactions with the complex milieu of immune response proteins present in human serum.
Similarly, the ability to focus on calcified protein-lipid assemblies with NMR sheds light on the calcified
depositions that accumulate in the aging eye and brain in degenerative diseases such as agerelated macular
degeneration and Alzheimer's disease. These deposits are rich in blood proteins, lipids and hydroxyapatite,
the stable, mineralized form of hydroxylated calcium phosphate that makes up the bulk of bone and teeth,
but the process of deposit formation is poorly understood. We show that detailed structural and functional
information can be obtained from solid-state NMR and solution NMR with complex native or native-like
samples.
Hyperpolarized
13
C NMR probes to assess the activity of the mitochondrial TCA
cycle
M. Huynh,
1
J. Singh,
1
Z. Erfani,
1
E. H. Suh,
1,2
J. Chen,
1
J. M. Park,
1
and Z. Kovacs.
1
1
Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA.
2
Department of Pharmaceutical Sciences, HSC College of Pharmacy, The University of North Texas Health
Science Center, Fort Worth, TX USA.
The mitochondrial TCA cycle is a central metabolic pathway for energy production and biosynthesis. The goal
of this project was to develop hyperpolarized (HP)-
13
C labeled probes that can report TCA cycle activity in vivo
in real-time. We synthesized [2-
13
C, 3-
2
H
3
]pyruvate and diethyl [1,2-
13
C
2
]-2-ketoglutarate as HP-
13
C MRS probes
for the TCA cycle. The C2 carbon of pyruvate enters the TCA cycle as acetyl-CoA and appears in downstream
metabolites such as [5-
13
C]glutamate. 2-Ketoglutarate in its cell permeable ester form is taken up by cells,
hydrolyzed by esterases and enters the cycle as ketoglutarate and is decarboxylated to [
13
C]bicarbonate by
ketoglutarate dehydrogenase. The probes were hyperpolarized by dynamic nuclear polarization and
administered into Wistar rats via tail vein injection. In vivo
13
C MRS was performed at 3 T with a
13
C surface
coil placed over the liver. As expected, HP-[2-
13
C, 3-
2
H
3
]pyruvate produced large amounts of lactate and
alanine and several metabolites of the glycolysis / gluconeogenesis pathway via pyruvate carboxylation.
However, only a weak signal [5-
13
C]glutamate was observed. On the other hand, diethyl [1,2-
13
C
2
]-2-
ketoglutarate produced easily detectable HP-[
13
C]bicarbonate via ketoglutarate dehydrogenase in the liver
reflecting flux through the TCA cycle. Thus, diethyl [1,2-
13
C
2
]-2-ketoglutarate is a better probe for monitoring
TCA cycle activity than [2-
13
C, 3-
2
H
3
]pyruvate. This is likely due to substrate competition as other sources of
acetyl-CoA can contribute to TCA flux competing with the probe.
Figure 1. The TCA cycle (left), the downstream metabolites of HP-[2-
13
C, 3-
2
H
3
]pyruvate (middle) and the formation of
HP-[
13
C]bicarbonate from HP diethyl [1,2-
13
C
2
]-2-ketoglutarate in vivo (right).
HP-[
13
C]HCO
3
-
Oxaloacetate
Citrate
Isocitrate
Ketoglutarate
Succinyl-CoA
Succinate
Malate
Fumarate
Acetyl-CoA
Glutamate
Pyruvate
CO
2
CO
2
TCA
cycle
Glycolysis
-Oxidation
CO
2
LactateAlanine
CO
2
Photoreactivity, phototoxicity in Krakow city !
Photoreactivity and phototoxicity of fine particulate matter from air pollutants
K. Mokrzynski,
1
O. Krzysztynska-Kuleta,
1
M. Zawrotniak,
2
M. Sarna,
1
M. Wojtala,
1
D. Wnuk
3
and T. Sarna.
1
1
Jagiellonian University,
1
Department of Biophysics.
2
Department of Comparative Biochemistry and Bioanalytics.
3
Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Krakow, Poland.
Although human skin efficiently protects the body against harmful effects of environmental factors,
excessive exposure of the skin to certain air pollutants, such as fine particulate matter (PM), could lead to
oxidative stress and result in pathological conditions. It has been reported that PM
2.5
containing particles with
aerodynamic diameter smaller than 2.5 m could not only penetrate through barrier-disrupted skin, but also
induce skin barrier dysfunction (1). Because typical constituents of PM exhibit photochemical activity, their
harmful effects could be intensified by solar radiation.
This paper will review a recent study aimed at determining phototoxicity of PM
2.5
collected in the
Krakow area in the four seasons (2). The effect of different PM
2.5
on selected parameters of cultured human
keratinocytes, subjected to irradiation with light derived from a solar simulator, were measured by standard
cell and molecular biology techniques. Photoreactivity of the studied PM
2.5
, particularly their efficiency to
photogenerate singlet oxygen and free radicals were determined by time-resolved singlet oxygen
phosphorescence and EPR-spin trapping, respectively.
Our data showed that the highest phototoxicity and photoreactivity exhibited PM
2.5
, collected from
air pollutants in the winter season. This, in part, could be due to the fact that increased coal burning occurs in
the Krakow area in the winter season. As a result, the winter season ambient particles are likely to contain
substantial amount of photoreactive aromatic hydrocarbons. In a follow up study, we also examined if selected
antioxidants could protect HaCaT cells from phototoxicity mediated by PM
2.5
(3). The obtained data revealed
that L-ascorbic acid and resveratrol, especially when used in combination, could serve as protective agents
against adverse effects of PM
2.5
both dark and light in an in vitro model of human skin.
Literature
1. Liao, Z. et al., Toxicol Rep 2020, 7, 1-9
2. Mokrzynski, K. et al., Int J Mol Sci 2021, 22, 10645, 1-20
3. Mokrzynski, K. et al., Photochem Photobiol 2023, 99: 983–992
In vivo Imaging and Oximetry
EPR Oximetry: Capillary to Clinic
P. Kuppusamy.
1
1
Geisel School of Medicine, Dartmouth College, Lebanon, NH 03756, USA.
Knowledge of tissue oxygen levels holds paramount importance to understand the mechanism of
several pathophysiological disorders and to develop treatment strategies to mitigate disease progression. This
would require methods capable of quantifying the level of tissue oxygen with good reliability and accuracy in
the clinical setting. Electron paramagnetic resonance (EPR) spectroscopy is capable of measuring tissue
oxygen levels using injected (soluble) or implanted (solid) paramagnetic probes as oxygen-sensors. The
principle of EPR oximetry is based on the paramagnetic property of molecular oxygen, which can shorten spin-
spin relaxation time (T2) of EPR spin-probes leading to EPR line-broadening. The effect of oxygen on EPR
spectrum was first observed in DPPH solutions by Deguchi in 1960 and later in nitroxide solutions by others
(Edelstein, et. al., 1964; Povich, 1975; Backer, et. al., 1977). The practical application of this phenomenon,
termed as ‘spin label oximetry’, was developed by Hyde and others in the 1980s for studying oxygen transport
and metabolism in cell systems. The developments of low-frequency EPR instrumentation and probes during
1990s and later, have enabled the use EPR oximetry for a variety of in vivo applications such as isolated
functioning organs (e.g., heart), intact animals, and humans. Over the years, the EPR oximetry technology has
continuously evolved, with respect to both the probes and methods, to make reliable, accurate, and repeated
measurements in living systems under minimally invasive conditions. EPR oximetry applications have been
rapidly expanding to include measuring myocardial tissue oxygen for assessing the extent of myocardial injury,
chronic wound healing, ischemic stroke, and monitoring tumor oxygenation levels for enhancing cancer-
treatment efficacy in animal models and cancer patients. This presentation will provide an overview of the
principle of EPR oximetry, available methods/probes, and applications from in vitro studies in capillaries to in
vivo measurements in cancer patients in the clinic.
Acknowledgments: The work was supported by NIH grant funding R01 EB004031 and R01 CA269234.
An Autocatalytic Mechanism for the Reaction between
S-Nitrosoglutathione and Hydrogen Sulfide
Neil Hogg
Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI.
Small-molecule gaseous signaling agents, that have been collectively referred to as gasotransmitters,
include nitric oxide (NO), hydrogen sulfide (H
2
S) and carbon monoxide. There is evidence that these molecules
are able to work together and, in some circumstances, may act synergistically. For this reason, there has been
significant interest in understanding the biological chemistry of interactions between gasotransmitters.
Studies of the reaction between NO and H
2
S uncovered the synthesis of a yellow-colored molecule with an
absorbance maximum at 402 nm. While there has been some controversy regarding the chemical nature of
this molecule, it has now been well established to be nitrosopersulfide (ONSS
-
). Nitrosopersulfide can be
generated in good yield from the reaction between H
2
S and nitrosothiols such as S-nitrosoglutathione (GSNO).
Although this reaction has been know for several decades, no viable mechanism has been established. The
reaction kinetics indicate significant sigmoidal behavior indicating possible autocatalysis. We will present a
mechanism (see scheme) that contains all the salient features of the reaction kinetics and product formation,
and that fits well to experimental kinetic data, and will also discuss possible biological significance of this
mechanism.
Spin labels, spin traps, spin probes for EPR
Fluorogenic cyclization of phenyl-type radicals for specific detection of
peroxynitrite
A. Grzelakowska,
1,2
R. Podsiadly,
1
J. Zielonka.
2
1
Institute of Polymer and Dye Technology, Lodz University of Technology, Poland.
2
Department of Biophysics, Medical College of Wisconsin, Milwaukee, United States.
Peroxynitrite (ONOO
–
), a biological oxidizing and nitrating species responsible for posttranslational
modification of cellular proteins, has been implicated in numerous pathologies carrying an inflammatory
component. Specific detection of ONOO
–
in biological systems remains a challenge and boronates are regarded
as a most promising class of probes for the detection and quantitation of ONOO
–
. However, boronates can be
oxidized not only by ONOO
–
, but also by several other biologically relevant oxidants, including hydrogen
peroxide (H
2
O
2
) and hypochlorous acid (HOCl). Oxidation of boronate probes by ONOO
–
results in the
formation of minor, ONOO
–
-specific products via a reaction pathway involving a phenyl radical-type
intermediate, in addition to the major, phenolic product.
1
Here, we report a new approach for specific detection of ONOO
–
, based on fluorogenic cyclization of the
phenyl type radical formed during oxidation of a boronate probe by ONOO
–
, with the production of a
fluorescent product. We characterized the kinetics and stoichiometry of the reaction of benzophenone-2-
boronic acid (2-BP-BA) with ONOO
–
and identified 2-hydroxybenzophenone (2-HBP) as the major product and
fluorescent fluorenone (FLN) and 2-nitrobenzophenone (2-NBP) as the minor, ONOO
–
-specific products
(Scheme 1). Neither hydrogen peroxide alone nor in the presence of myeloperoxidase and nitrite produce FLN
or 2-NBP. Moreover, FLN is formed both from bolus ONOO
–
and from in situ generated ONOO
–
, produced
during the decomposition of SIN-1, a thermal source of O
2
•–
and
•
NO.
Scheme 1. Products detected upon oxidation of 2-BP-BA by H
2
O
2
vs. ONOO
–
As fluorenone can be selectively detected using fluorescence spectroscopy, the observed reaction provides a
model for the development of next generation probes for ONOO
–
, for non-invasive, fluorescence-based
specific detection of ONOO
–
.
1
Sikora A. et al. Boronate-based probes for biological oxidants: A novel class of molecular tools for redox biology. Front.
Chem. 2020; 8:580899. doi: 10.3389/fchem.2020.580899
The reaction of glutathione-derived dinitrosyl iron complexes
and superoxide radical anion - the detection and quantitation
of peroxynitrite
M. Rola,
1
A. Artelska,
1
J. Zielonka,
2
R. Michalski,
1
A. Sikora.
1
1
Institute of Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland.
2
Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, United States.
Nitric oxide produced in cells reacts with iron ions and thiols to form the so-called dinitrosyl iron complexes
(DNIC). It is assumed that DNICs are important products of the cellular metabolism of nitric oxide, constituting
its reservoir. Despite this, the biological chemistry of DNICs is not well understood, and little is known about
the mechanism of their formation and degradation. An important reaction of nitric oxide is its interaction with
the superoxide radical anion (O
2
•
−
), leading to the formation of peroxynitrite (ONOO
−
). Here, we present the
results of our study on the detection and quantification of peroxynitrite formed in the reaction of glutathione-
derived DNIC and superoxide radical anion. Boronate probes FlBA and o-MitoPBA were employed for the
detection of peroxynitrite and its quantitative determination. We compared the yield of peroxynitrite
generation in the DNIC/O
2
•
−
system with the efficiency of its production in the GSNO/O
2
•
−
system. The effects
of SOD and catalase were also investigated.
Unveiling the Therapeutic Frontiers of Targeted Nitroxides
KM Vega-Escobar,
2
K. Valentín-Meléndez,
2, 3
S. Sanabria-Barrera,
4
O. Ouari,
5
M. Hardy,
5
and M. Lopez.
1, 2
1
Research Institute, Puerto Rico Science, Technology, and Research Trust, San Juan, Puerto Rico.
2
Department of Chemistry and NIH-URISE Program, University of Puerto Rico at Humacao, Humacao, Puerto
Rico.
3
Department of Biology, University of Puerto Rico at Humacao, Humacao, Puerto Rico.
4
Innovation and Technology Development, Fundación Cardiovascular de Colombia, Floridablanca, Colombia.
5
Aix Marseille Univ, CNRS, ICR, Equipe SREP, Marseille, France.
Nitroxides have evolved from their well-established role as potent antioxidants and superoxide
dismutase (SOD) mimetics to become critical players in experimental therapeutics. Here, we highlight the
delves into the strategic targeting of nitroxides to mitochondria, a critical evolution that has significantly
broadened their therapeutic horizons beyond their antioxidant activity. Once celebrated primarily for their
capacity to neutralize reactive oxygen species, nitroxides have revealed their prowess as versatile therapeutic
agents.
Mitochondria-targeted nitroxides like Mito-CP and Mito-SG1 have emerged as a groundbreaking
strategy, addressing the intricate challenges of mitochondrial-derived free radicals. This targeting endeavor
has reinforced the traditional antioxidant paradigm and unveiled a spectrum of novel therapeutic potentials
in cancer, neuroscience, and cardiometabolic diseases. These include modulation of cell signaling pathways,
selective inhibition of mitochondrial functions, and initiating protective mechanisms against cellular stress and
damage.
The transition from general antioxidants to mitochondria-specific interventions marks a significant
milestone, reflecting a deeper understanding of cellular oxidative processes and the critical role of
mitochondria in health and disease. Drawing from the contributions of researchers like our mentor Balaraman
Kalyanaraman, here we showcase the multifaceted therapeutic applications of targeted nitroxides, from
cancer and neuroprotection to cardiovascular diseases, positioning them at the forefront of therapeutic
innovation. The strategic targeting of nitroxides represents a paradigm shift from conventional antioxidant
applications to a multifaceted therapeutic approach. This evolution highlights the potential of targeted
nitroxides to navigate the complex interplay between oxidative stress and mitochondrial dysfunction, offering
promising avenues for developing targeted therapeutic interventions.
Figure 1 – Structures of Mito-CP and Mito-SG1 nitroxides
Enjoying Marseille
Restaurants
Around the Intercontinental Hotel, there is the Panier neighborhood
You will find places for lunch and dinner.
“La VIEILLE PELLE” Pizza, Italian cuisine. One of the oldest restaurants in this district
Le Tribeca Pizza and Mediterranean food 200 quai du Vieux Port (5 min walking distance from
Hotel Intercontinental)
“Entre Ciel et Mer” seafood, cheeses, charcuteries
https://www.entre-terre-et-mer-marseille.com/
And the menu
https://www.entre-terre-et-mer-marseille.com/carte-entre-terre-et-mer-restaurant-fruits-de-mer-marseille
« Le Barrio »
Peruvian fusion cuisine
https://www.barrio28.fr/
And the menu
https://www.barrio28.fr/carte
If you on the other side of the Vieux Port, you will find an Italian-style piazza, the COURS ESTIENNE D’ORVES
with places to eat.
“Les ARCENAULX” French cuisine, great wines
https://www.les-arcenaulx.com/
And example of menus
https://www.les-arcenaulx.com/le-restaurant/la-carte/
For the meat lovers, “La Côte de Boeuf”, great meat, outstanding wine selection
https://www.lacotedeboeuf.net/la-carte-la-cote-de-boeuf-restaurant-marseille
Wine
https://www.lacotedeboeuf.net/cave-la-cote-de-boeuf-restaurant-marseille
To visit (nearby)
MUCEM (museum) and the Fort Saint Jean (bridge)
Grotte Cosquer
https://www.grotte-cosquer.com/
La Vieille Charité (church and museum) dans le Panier
Cathédrale La Major
Le Pharo (on the other side of the Vieux Port)
Further
La Corniche Kennedy
