Light-initiated oxidative stress

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Light-initiated oxidative stress

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Alfonso Blázquez-Castroa,b, Michael Westberga, Mikkel Bregnhøja, Thomas Breitenbacha, Ditte J. Mogensena, Michael Etzerodtc, Peter R. Ogilbya a

Department of Chemistry, Aarhus University, Aarhus, Denmark Department of Physics of Materials, Faculty of Sciences, Autonomous University of Madrid, Madrid, Spain c Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

b

Abstract The production of reactive oxygen species, ROS, plays an important role in light-mediated oxidative stress. As a tool to study general mechanisms of stress and eustress, and associated cellular signaling pathways, light allows for spatial and temporal control of ROS production in the heterogeneous environment of a living cell. Light is particularly suited for the selective initial production of singlet molecular oxygen at the expense of the superoxide radical anion and vice versa. With fluorescent probes, light is also a valuable tool used to monitor the space- and time-dependent effects of stress on cell physiology, as well as selected reactive intermediates pertinent to oxidative stress. ­Keywords: Singlet oxygen, Superoxide anion, Reactive oxygen species (ROS), Photosensitizer, Optogenetic actuator

­Introduction Light can initiate production of reactive oxygen species (ROS) and, as such, influence oxidative stress. This is important given that we live in a world of oxygen, sunlight, and molecules susceptible to oxidation/oxygenation. Light can also be a mechanistic tool to investigate general ROS-dependent processes. For example, pulsed lasers can probe for and characterize reactive intermediates in both space- and time-resolved subcellular experiments. Although the palette of ROS relevant to oxidative distress and eustress is diverse (Halliwell & Gutteridge, 2015; Sies, Berndt, & Jones, 2017), we focus on two species important in light-induced processes: singlet molecular oxygen, O2(a1Δg), and the superoxide radical anion, O2•− (hereafter denoted as superoxide). However, the reactions of these two species spawn other ROS. Thus, through the selective and Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00019-5 © 2020 Elsevier Inc. All rights reserved.

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spatially localized production of O2(a1Δg) or superoxide, one can provide insight on ROS action in general. Even within the confines of light-induced oxidative stress, an extensive amount of information has accumulated. As such, we cannot provide a comprehensive review. Rather, biased by our own experiments, we summarize recent work.

­Why is light important? The word “light” is often associated only with radiation over the range ~400–700 nm (i.e., visible to the human eye). However, an appreciable amount of nonvisible UV and near-IR light is present to influence living organisms. Although high-energy light (i.e., ionizing gamma and x-ray radiation) is not routinely encountered in our ambient environment, such exposure is relevant for selected medical treatments (Kirakci et al., 2018; Larue et al., 2018). Light can initiate ROS production. The processes involved vary according to the wavelength and the molecules that absorb this light. For many molecules, the excited electronic state produced may initiate electron transfer reactions that involve oxygen. Alternatively, energy transfer from the light-absorbing molecule to oxygen may kinetically compete to produce O2(a1Δg) and, ultimately, a different set of ROS. These two processes are the foundation for the often-used monikers of Type I and Type II photooxidation reactions, respectively (Foote, 1991). The literature is replete with examples of light-initiated ROS-dependent oxidative distress and eustress. One example is photodynamic therapy, PDT, commonly used in cancer treatments, in which ROS initiate cell death (Agostinis et al., 2011). Another is the phenomenon of Low-Level Laser (Light) therapy, LLLT, in which it is inferred that ROS initiate proliferative events in cells (Chung et al., 2012). However, for many such processes, the mechanisms of ROS-based action are still far from being resolved. Light is also a useful mechanistic tool to study ROS-dependent stress. The combination of pulsed lasers, fluorescent probes, and optical microscopes enables subcellular time-resolved spectroscopic studies. Indeed, when combined with optogenetic actuators, as discussed further in the succeeding text, the use of light in this way defines one of the frontiers in the field (Trewin et al., 2018; Westberg, Etzerodt, & Ogilby, 2019).

­Some specifics about light A desirable feature of light, certainly as a source to initiate reactions (i.e., so-called actinic light), is that one can accurately control the dose delivered to the sample. With control over the wavelength, duration of exposure, and incident intensity, one controls the concentration of reactive species initially produced. This alone justifies the use of light as a mechanistic tool; the response to ROS (distress or eustress) depends strongly on dose (Westberg, Bregnhøj, Blázquez-Castro et al., 2016).

­Some specifics about light

With light, one also has the opportunity to control the subcellular location of ROS production. Even with the limiting laws of light diffraction, the volume of excited states obtained simply by focusing the light can be useful (e.g., ~500–700 nm diameter at the beam waist for visible light) (Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). However, the excitation volume can be decreased appreciably, and the effects of scattered light precluded, if excitation occurs via a two-photon process in which light is only absorbed where the photon flux is sufficiently high (Fig. 1) (Gollmer, Besostri, Breitenbach, & Ogilby, 2013; Pimenta et  al., 2012; Westberg, Bregnhøj, Banerjee, et al., 2016). Control over the site of excitation can also be exerted by localizing the lightabsorbing molecule to a specific intracellular organelle. This can be achieved by exploiting (a) solubility parameters and (b) molecular targeting mechanisms (e.g., organelle-specific peptide conjugates) (Celli et al., 2010; Mahon et al., 2007). At the limit, one can use optogenetics to localize the light-activated ROS source on a specific protein (Rodriguez et al., 2017; Trewin et al., 2018; Westberg et al., 2019). For some optogenetic systems, photoinitiated electron transfer reactions quantitatively result in the production of superoxide (Lee, Kim, & Rhee, 2018; Pletnev et al., 2009).

FIG. 1 Fluorescence image of HeLa cells based on a mitochondrial localized dye (green) that illustrates the extent to which actinic light can be controlled. The white dots in the image illustrate the location specificity and spatial resolution accessible through two-photon excitation of a photosensitizer. Reprinted with permission Westberg, M., Bregnhøj, M., Banerjee, C., Blázquez-Castro, A., Breitenbach, T., & Ogilby, P. R. (2016). Exerting better control and specificity with singlet oxygen experiments in live mammalian cells. Methods, 109, 81–91; Westberg, M., Bregnhøj, M., Blázquez-Castro, A., Breitenbach, T., Etzerodt, M., & Ogilby, P. R. (2016). Control of singlet oxygen production in experiments performed on single mammalian cells. Journal of Photochemistry and Photobiology A: Chemistry, 321, 297–308.

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In contrast, different optogenetic actuators can be used to efficiently and selectively produce O2(a1Δg) via energy transfer to ground state oxygen (Westberg et al., 2019; Westberg, Bregnhøj, Etzerodt, & Ogilby, 2017a). Although one can now achieve remarkable control over light-initiated processes, this may not have been the case for experiments performed in the past. As such, given the stated importance of dose, location, and selectivity, many published studies of photoinitiated processes may be “less than conclusive.”

­Light-initiated production of ROS Although light can generate different ROS, the processes most common to living organisms result in the nascent production of O2(a1Δg) and/or superoxide. For example, although irradiation of hydrogen peroxide results in the production of the hydroxyl radical (Okabe, 1978), this occurs only at wavelengths shorter than 300 nm where, in the least, other molecules in a cell will effectively compete for the incident light. In contrast, many molecules, including oxygen itself, absorb light over the range 400–800 nm to produce O2(a1Δg) and/or superoxide. The selective initial production of either O2(a1Δg) or superoxide does not preclude the subsequent formation of other ROS. For example, O2(a1Δg) oxygenates lipids through the “ene” reaction to make an allylic hydroperoxide (Clennan & Pace, 2005). In turn, hydroperoxides readily cleave to yield the hydroxyl radical and an alkoxyl radical (Foote, Valentine, Greenberg, & Liebman, 1995), which propagate to form a plethora of reactive species that can influence the system (Walling, 1995). Similarly, superoxide can be protonated to yield the hydroperoxyl radical that can likewise propagate to yield other ROS. The dismutation reaction of superoxide forms hydrogen peroxide, which, because it is not as reactive as other ROS, can diffuse over greater distances and act as a unique signaling agent (Halliwell & Gutteridge, 2015; Redmond & Kochevar, 2006). Despite the complexity of these reactions, indeed because of the complexity of these reactions, it is mechanistically useful to selectively produce only one ROS at a given initial time.

­Singlet oxygen Molecular oxygen has three low-energy electronic states (Fig. 2). These states are most properly denoted by their term symbols, O2(X3Σg−), O2(a1Δg), and O2(b1Σg+), which also clearly distinguish one state from the other (Atkins, De Paula, & Keeler, 2018; Paterson, Christiansen, Jensen, & Ogilby, 2006). In the past, it has been sufficient to use the moniker “singlet oxygen” with the understanding that this refers to the O2(a1Δg) state. However, recent experiments establish the need to distinguish between the two singlet states of oxygen, O2(a1Δg) and O2(b1Σg+), and we refer to both of these states when using the moniker “singlet oxygen.”

­Singlet oxygen

FIG. 2 Selected features of the three lowest energy electronic states of molecular oxygen: ground state, O2(X3Σg−), first excited state, O2(a1Δg), and second excited state, O2(b1Σg+). (Right side) Crude illustration of the orbital occupancy of the respective states. We only consider the highest occupied orbitals: the degenerate πg orbitals. (Left side) Light can be absorbed by O2(X3Σg−) to directly produce O2(a1Δg) and, independently, O2(b1Σg+). The “wavy” arrow indicates that, once formed, O2(b1Σg+) will decay to produce O2(a1Δg).

­What is ground state oxygen? The ground electronic state of oxygen, O2(X3Σg−), is a spin triplet, as noted by the superscript 3 in the term symbol. This state is a classic example of Hund’s rule of maximum spin multiplicity. When distributing the 16 electrons of O2 into the available molecular orbitals, the last two electrons are placed in degenerate (i.e., equal energy) π antibonding orbitals. The configuration of lowest energy is such that one electron goes into each orbital (Fig. 2), and the spin moments of these electrons are the same yielding a triplet state (Atkins et al., 2018; Paterson et al., 2006). The X in the term symbol indicates that this is the ground state. Of arguably less importance in the present context is the remaining information in the term symbol that characterizes the orbital angular momentum (Σ) and the symmetry (−) and parity (g) of the molecule (i.e., how the wavefunction responds to selected symmetry operations). Although O2(X3Σg−) is not normally considered a ROS, we will deviate somewhat from tradition. As a spin triplet, O2(X3Σg−) is effectively a biradical. In this way, it is a wonderful “radical trap” (i.e., a molecule that will readily react with other free radicals). Thus, if a carbon-based free radical is present, O2(X3Σg−) can trap this radical to form an alkyl peroxide. This now starts well-established propagating processes that result in the formation of other ROS (Walling, 1995). The trapping of free radicals by O2(X3Σg−) can thus be important in oxidative stress.

­What is singlet oxygen? ­The lowest energy singlet state, O2(a1Δg)

The lowest excited electronic state of oxygen, O2(a1Δg), is a spin singlet and results from a change in orbital occupancy relative to O2(X3Σg−). The letter “a” in the term symbol indicates that this is the state next in energy above the ground state, and

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because the spin is different, this letter is written in lower case. Following our description of how the electrons in O2(X3Σg−) are distributed, O2(a1Δg) is often represented as a state in which the final two electrons occupy only one of the degenerate π antibonding orbitals (Fig.  2) (Halliwell & Gutteridge, 2015; Krumova & Cosa, 2016). This can only occur when the spin moments of the respective electrons are opposed (i.e., the Pauli exclusion principle) and, thus, yield a state of net singlet spin. Although this way of illustrating the difference between the O2(X3Σg−) and O2(a1Δg) states is easy to comprehend, it is inaccurate. The proper expression of the more general Pauli Principle is that the total wavefunction for the molecule must change sign (i.e., be antisymmetric) upon interchanging electrons (Atkins et al., 2018). The illustration of orbital occupancy that accurately represents this becomes more complicated (Paterson et al., 2006) and thus is rarely used. The energy difference between the O2(X3Σg−) and O2(a1Δg) states, ~94.3 kJ/mol, depends slightly on the solvent, with the spectroscopic transition falling in the wavelength range ~1270–1280 nm (Bregnhøj, Westberg, Minaev, & Ogilby, 2017; Ogilby, 1999). With few exceptions, light-absorbing molecules of biological importance have excited state energies that exceed the ~94 kJ/mol excitation energy of O2(a1Δg). This is important for one method to produce O2(a1Δg): photosensitization. Although the study of O2(a1Δg) has long been important in quantum mechanics and spectroscopy (Herzberg, 1950; Mulliken, 1928), the reactions of O2(a1Δg) have arguably thrust this molecule into the limelight (Clennan & Pace, 2005; Foote, 1968). Many of these reactions occur with molecules of biological importance (DiMascio et al., 2019) and, as such, are pertinent to oxidative stress.

­The other singlet state, O2(b1Σg+)

The second excited electronic state of oxygen, O2(b1Σg+), is also a spin singlet. The crude way to represent the orbital occupancy in this case is to put the last two of oxygen’s 16 electrons in each of the degenerate antibonding π orbitals and to have their spin moments opposed (Fig. 2) (Paterson et al., 2006). The O2(b1Σg+) - O2(X3Σg−) spectroscopic transition occurs at 765 nm (Fig.  2). Once formed in solution-phase systems, O2(b1Σg+) rapidly decays to produce the O2(a1Δg) state with almost unit efficiency (Schweitzer & Schmidt, 2003). In particular, there is no evidence to indicate that O2(b1Σg+) undergoes chemical reactions in the same way as O2(a1Δg) (Scurlock, Wang, & Ogilby, 1996). Nevertheless, O2(b1Σg+) is important because it is a convenient precursor to O2(a1Δg).

­Transitions between states When discussing transitions between electronic states of a given molecule, the term symbols are important in ascertaining whether the transition is probable (i.e., “allowed”) or improbable (i.e., “forbidden” or weak). Thus, for the aforementioned states of oxygen, we say that the transition between the O2(X3Σg−) and O2(a1Δg) states is “spin forbidden,” among other things; a transition involving a change from triplet to singlet spin is generally not probable.

­Singlet oxygen

­How can singlet oxygen be produced by light? ­Energy transfer from a photosensitizer

The most common and efficient way to produce singlet oxygen using light is via a photosensitized process. In this case, the light is absorbed by a separate molecule, the photosensitizer, and the energy of excitation is transferred to O2(X3Σg−) to produce either O2(a1Δg) or O2(b1Σg+) (Fig. 3A). This process of energy transfer requires a collision between oxygen and the sensitizer and generally occurs with the longer-lived sensitizer triplet state via the reaction 3Sens1 + O2(X3Σg−) → 1Sens0 + O2(a1Δg)/O2(b1Σg+)

FIG. 3 Relevant processes that can occur upon irradiation of a photosensitizer, xSensy, where x denotes the electronic spin and y distinguishes one state from another. Green arrows represent one-photon excitation, whereas red arrows represent two-photon excitation. Depending on the sensitizer used, these transitions can initially populate the same or different states. The “wavy” lines represent nonradiative transitions between states. (A) The sensitized production of O2(a1Δg). (B) A sensitized electron transfer reaction in which an amino acid, AA, donates an electron to the excited state sensitizer to form a radical anion. The latter then reduces O2(X3Σg−) to superoxide in a second electron transfer reaction.

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(Schweitzer & Schmidt, 2003). Although not immediately apparent, a ­collision complex between two triplet states has a component of net singlet spin. Thus, this reaction is spin allowed. A process that kinetically competes with energy transfer to produce singlet oxygen is electron transfer from or to the excited state sensitizer (Fig. 3B). This process also plays an important role in ROS production. The production of the excited state sensitizer can occur through both one- and two-photon processes (Fig. 3). One-photon excitation is most commonly employed and encountered, and absorption can occur with low light intensities. Two-photon excitation is a nonlinear process and occurs only with high light intensities (i.e., lasers) (Ogilby, 2010; Ogilby, 2016). The two-photon process has several advantages: (a) the incident photons are of longer wavelength and generally occur in the so-called “biological window” where many endogenous biological molecules do not absorb, and (b) the required high photon flux can be achieved by focusing the incident light, thereby facilitating spatial control and localization (Fig. 1). Many molecules can act as a singlet oxygen sensitizer, including molecules endogenous to mammalian cells (Redmond & Gamlin, 1999). Exogenous sensitizers can be added, such as the light-absorbing compound used in PDT (Agostinis et al., 2011). Although most molecules of relevance are energetically capable of generating singlet oxygen (vide supra), energy transfer does not always occur with unit efficiency. The site of singlet oxygen production in, on, or near a cell can have significant stress-related consequences (Blázquez-Castro, Breitenbach, & Ogilby, 2018; Gollmer et al., 2013; Kessel, 2004; Redmond & Kochevar, 2006; Rubio, Fleury, & Redmond, 2009). The localization of exogenous sensitizers is often achieved based solely on solubility parameters (e.g., hydrophobic vs hydrophilic domains). This can be undesirable because the stress response may result from a small amount of sensitizer in site A rather than the larger amount in site B, and data can thus be misinterpreted. To address these issues, there is a range of options by which (a) targeting schemes are used to better localize the sensitizer (Celli et al., 2010; Mahon et al., 2007), and (b) location-specific molecular processes are used to activate the sensitizer (e.g., local pH or protein binding) (Callaghan & Senge, 2018). One recent approach is the use of genetically encoded proteins that encapsulate the sensitizer (Trewin et al., 2018; Westberg et al., 2019). A distinct advantage of the latter is that one can achieve molecular level specificity for the site of singlet oxygen production. The design and characterization of photosensitizers that selectively make singlet oxygen at the expense of superoxide, and vice versa, has been extensive (e.g., the kinetic competition illustrated in Fig. 3). Indeed, this issue has been the focus of recent work on genetically encoded sensitizers (Trewin et al., 2018; Westberg et al., 2019).

­Direct irradiation of ground state oxygen

Selective O2(a1Δg) production has also been achieved by directly irradiating O2(X3Σg−) (Fig.  2) (Blázquez-Castro, 2017). Both the O2(X3Σg−)-O2(a1Δg) transition at 1275 nm (Anquez, Yazidi-Belkoura, Randoux, Suret, & Courtade, 2012; Krasnovsky, Roumbal, & Strizhakov, 2008) and the O2(X3Σg−)-O2(b1Σg+) ­transition

­Singlet oxygen

at 765 nm (Blázquez-Castro et  al., 2018; Bregnhøj, Blázquez-Castro, Westberg, Breitenbach, & Ogilby, 2015) have been exploited in this regard, including in live cells. Although these transitions are extremely weak (e.g., they are spin forbidden), one can nevertheless control the O2(a1Δg) dose such as to cover the range from distress to eustress (Blázquez-Castro et al., 2018; Bregnhøj et al., 2015; Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). The O2(X3Σg−)-O2(b1Σg+) transition at 765 nm has favorable attributes in that other compounds common to biological systems, particularly water, do not absorb at this wavelength (i.e., this transition occurs in the “biological window”). Although this is a one-photon experiment, the light can still be sufficiently localized for subcellular irradiation (Fig. 4). In this way, progress through the cell cycle can be accelerated upon low-dose irradiation into the cytoplasm, whereas proliferation is delayed upon analogous irradiation into the nucleus (Blázquez-Castro et al., 2018).

­Dependence on the concentration of ground state oxygen

Both methods described earlier for producing O2(a1Δg) depend on the concentration of O2(X3Σg−) in the system. This can be an important variable for experiments with cells, where there are appreciable location-dependent differences in the O2(X3Σg−) concentration. Specifically, oxygen is more soluble in hydrocarbons than in aqueous media; the concentration of O2(X3Σg−) in air-saturated water is ~10 times smaller than in an air-saturated hydrocarbon solvent (Battino, Rettich, & Tominaga, 1983). Thus, there is less dissolved O2(X3Σg−) in the cytosol than in a membrane.

FIG. 4 Focused 765 nm light can be used to primarily create O2(a1Δg) in the cytoplasm and, independently, the nucleus of a cell. The O2(X3Σg−) → O2(b1Σg+) absorption profile is sufficiently narrow that irradiation with 775 nm light will not produce an appreciable amount of O2(a1Δg) (Bregnhøj et al., 2015). The green fluorescence from a dye localized in the mitochondria of a HeLa cell was used for this image. Reprinted with permission Blázquez-Castro, A., Breitenbach, T., & Ogilby, P. R., 2018. Cell cycle modulation through subcellular spatially resolved production of singlet oxygen via direct 765 nm irradiation: Manipulating the onset of mitosis. Photochemical & Photobiological Sciences, 17, 1310–1318.

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To illustrate salient features of this point, we modeled the O2(X3Σg−) concentration dependence of O2(a1Δg) production for three cases (Fig. 5). Production of O2(a1Δg) upon one-photon excitation of oxygen at 765 nm should depend linearly on the O2(X3Σg−) concentration, as expected from the Lambert–Beer law, and this has been confirmed (Bregnhøj et al., 2015). Given the extended focal volume used thus far in cell-based 765 nm experiments (i.e., beam waist diameter of ~2 μm) (Blázquez-Castro et al., 2018), it is clear that O2(X3Σg−) in both hydrocarbon and aqueous domains is irradiated. Despite the small absorption coefficient for this transition, appreciable and controllable amounts of O2(a1Δg) can still be made using this technique with air-equilibrated cells (Blázquez-Castro et  al., 2018; Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). A key parameter in a photosensitized O2(a1Δg) experiment is the lifetime of the sensitizer triplet state, τT, as measured in a deoxygenated sample. The magnitude of τT is a measure of the deactivation pathways against which quenching by O2(X3Σg−) must kinetically compete. For a freely dissolved sensitizer, a rough guideline is that if τT is longer than ~5–10 μs, then most of the sensitizer triplet states will be quenched by O2(X3Σg−) in an air-saturated aqueous solution. In short, one sees a plateau effect in the O2(a1Δg) yield as [O2(X3Σg−)] is increased beyond an air-saturated solution (Fig. 5) (Hatz, Poulsen, & Ogilby, 2008; Kristiansen, Scurlock, Iu, & Ogilby, 1991). This is a desirable feature if one is concerned that differences in the intracellular O2(X3Σg−)

FIG. 5 A model of how oxygen concentration affects the yield of O2(a1Δg) for three cases: direct irradiation at 765 nm (bottom, green line), and sensitized formation with a shortlived sensitizer triplet state, τT = 1 μs (middle, red line) and with a long-lived sensitizer triplet state, τT = 50 μs (top, blue line). As noted in the text, τT refers to the lifetime in a deoxygenated sample. For the quenching of the sensitizer triplet state by O2(X3Σg−), we used the typical rate constant of 4 × 109 s−1 M−1. The point is to illustrate how, for a given case, the O2(a1Δg) yield responds to a change in the oxygen concentration. Thus, emphasis should not be placed on the yields between cases.

­Singlet oxygen

concentration will influence the dose of O2(a1Δg); the plateau effect ­precludes this problem. In contrast, if τT is comparatively short, then unimolecular triplet state deactivation channels will still compete with quenching by O2(X3Σg−), and the plateau may not be reached even in an oxygen-saturated system (Fig. 5). It is appropriate to carry this discussion over to the photosensitized production of superoxide (Fig. 3B). Superoxide formation is often preceded by electron transfer reactions involving the sensitizer and other molecules (e.g., proteins). As such, the final electron transfer reaction from a radical anion of an organic molecule to O2(X3Σg−) can depend on the O2(X3Σg−) concentration in a convoluted way. Thus, when a sensitizer can produce both O2(a1Δg) and superoxide, the ratio of the amounts of O2(a1Δg) to superoxide may change as a function of the local oxygen concentration.

­ hat reactions of O2(a1Δg) are potentially pertinent to oxidative W stress? The reactions of O2(a1Δg) with organic molecules of biological relevance have been studied for decades (Clennan & Pace, 2005; Davies, 2003; DiMascio et al., 2019; Foote, 1968; Girotti, 2001; Wilkinson, Helman, & Ross, 1995). Classic reactions include the formation of hydroperoxides (e.g., the “ene” reaction with olefins in lipids, Fig. 6), endoperoxides (e.g., 2 + 4 cycloaddition reaction with the dienes in histidine and guanine), dioxetanes (e.g., 2 + 2 cycloaddition with tryptophan), and sulfoxides from sulfides such as methionine. Most importantly, the product initially formed in these reactions is generally just a precursor to other oxidation products, often formed by thermal reactions in the absence of light. Hydroperoxide decomposition to form

FIG. 6 Two O2(a1Δg) reactions of biological importance. Lipids such as oleic acid react via the “ene” reaction to produce a hydroperoxide. The hydroperoxide OO bond is readily cleaved, thermally and photolytically, to yield the hydroxyl radical and an alkoxyl radical, both of which are reactive species. GSH reacts to yield GSSG and other oxidized products.

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alkoxyl and hydroxyl radicals is a good example (Fig. 6) (Walling, 1995). Thus, the O2(a1Δg)-mediated oxidation of a specific amino acid in a protein can have appreciable consequences, particularly if that residue is part of an enzyme’s active site or a site for posttranslational modification (Davies, 2003). Glutathione, GSH, reacts with O2(a1Δg) to predominantly form a dimer, GSSG, linked by a disulfide bridge (Fig. 6) (Devasagayam, Sundquist, DiMascio, Kaiser, & Sies, 1991). It is likely that GSSG is formed via a O2(a1Δg)-mediated electron transfer reaction. Because O2(a1Δg) can influence the GSSG/GSH ratio, it follows that this is one reaction of O2(a1Δg) that can certainly be pertinent to oxidative stress. In this reaction with O2(a1Δg), it is the thiolate, RS−, rather than the thiol, RSH, that is more reactive. This has broad environment-dependent intracellular ramifications for a number of thiol-containing molecules, not just GSH, and is consistent with the fact that O2(a1Δg) is an electrophile. The O2(a1Δg)-influenced redox state of a cell can also be modulated by other antioxidants. It is known from solution-phase experiments that members of the carotenoid family efficiently deactivate O2(a1Δg) to O2(X3Σg−) with rate constants close to the diffusion-controlled limit (DiMascio, Kaiser, & Sies, 1989; Edge, McGarvey, & Truscott, 1997; Foote & Denny, 1968). This process involves energy transfer from O2(a1Δg) to produce the carotenoid triplet state. Carotenoids are unique in this regard because, unlike almost all other organic molecules, they have a triplet state energy that is slightly lower than the ~94 kJ/mol excitation energy of O2(a1Δg). Most importantly, this interaction requires a collision between the respective molecules. Thus, if the system precludes this collision within the lifetime of O2(a1Δg) (e.g., by compartmentalization or by a viscosity that adversely affects diffusion), then the carotenoid is benign as a O2(a1Δg) quencher. However, the carotenoid may still act as a radical trap for secondary/downstream oxidation products of a O2(a1Δg) reaction. With this in mind, it is relevant to note that, when added to a mammalian cell, β-carotene does not quench O2(a1Δg) produced in a photosensitized reaction, but it still protects against O2(a1Δg)-mediated cell death (Bosio et al., 2013).

­O2(a1Δg) as a diffusible signaling agent

Having established that O2(a1Δg)-mediated oxygenation or oxidation reactions can alter the structure and function of biomolecules, we must consider how these reactions kinetically compete with other processes for O2(a1Δg) removal. This analysis provides an estimate for the diffusion distance of O2(a1Δg) from its point of production in a cell. The dimensions of O2(a1Δg)‘s “sphere of activity” help define its role as a diffusible signaling agent capable of influencing stress. We first revisit the phrase “reactive oxygen species.” In the strict chemical sense, O2(a1Δg) is not a “reactive” intermediate; it is a “selective” intermediate. For most biomolecules of significance, the rate constants, k, for interaction with O2(a1Δg) are no greater than ~5 × 107 s−1 M−1, and many are on the order of ~105–106 s−1 M−1 (Wilkinson et  al., 1995). In short, these reactions occur well below the diffusion-­ controlled limit that characterizes “reactive intermediates” such as the hydroxyl radical (i.e., where k ~1010 s−1 M−1) (Redmond & Kochevar, 2006).

­Singlet oxygen

The abundance of organic material in a cell is dominated by proteins, and only selected amino acids have rate constants for reaction with O2(a1Δg) on the order of ~107 s−1 M−1 (e.g., tryptophan, tyrosine, histidine, cysteine, and methionine). If we consider GSH, 10 mM is a representative intracellular concentration, and the rate constant for reaction with O2(a1Δg) is <2 × 106 s−1 M−1 (Devasagayam et al., 1991; Ogilby & Kuimova, 2016). Thus, the concentration of intracellular targets, [R], that rapidly interact with O2(a1Δg) is generally not that great, and the pertinent product with the rate constant, k[R], is small (Briviba & Sies, 2000; Ogilby & Kuimova, 2016). On this basis, the interaction of O2(a1Δg) with the water in a cell should kinetically compete with, for example, the O2(a1Δg)-protein interaction. The lifetime of O2(a1Δg) in neat H2O is ~3.5 μs, and it is ~69 μs in neat D2O (Bregnhøj et al., 2017). As such, there is a pronounced H2O/D2O solvent effect on the lifetime of O2(a1Δg). Thus, the observation of a pronounced H2O/D2O effect on the response of cells to O2(a1Δg)-initiated processes provides compelling evidence that, in a cell, water-mediated O2(a1Δg) deactivation indeed kinetically competes with protein-mediated and glutathione-mediated O2(a1Δg) removal (Ogilby, 2010; Ogilby & Kuimova, 2016; Redmond & Kochevar, 2006). What is the lifetime of O2(a1Δg) in a cell? Based on indirect experiments, the conventional response has been that this lifetime is short (~10–100 ns) (Agostinis et al., 2011; Moan & Berg, 1991). However, an intracellular O2(a1Δg) lifetime of ~100 ns is inconsistent with the observed H2O/D2O cell response mentioned earlier; a lifetime of ~100 ns implies that reactions with proteins, for example, win the kinetic competition over water. Over the past ~15 years, direct time-resolved O2(a1Δg) phosphorescence measurements have pointed to an average intracellular O2(a1Δg) lifetime of ~2–3 μs (i.e., averaged over both hydrophilic and hydrophobic domains) (Kuimova, Yahioglu, & Ogilby, 2009; Silva et al., 2012; Skovsen, Snyder, Lambert, & Ogilby, 2005). These direct measurements also reveal a O2(a1Δg) lifetime of ~15–20 μs in D2O-incubated cells. Thus, interactions of O2(a1Δg) with water indeed principally define the intracellular lifetime of O2(a1Δg). Note that the O2(a1Δg) lifetime in a wide range of hydrocarbons is ~20–30 μs (Bregnhøj et al., 2017; Wilkinson et al., 1995). With a lifetime of 2 μs, a O2(a1Δg) population will be reduced by a factor of ~150 over a period of 10 μs. Upon considering an average intracellular viscosity and the associated diffusion coefficient of oxygen, the radius of O2(a1Δg)‘s sphere of activity from its point of production over this period of 10 μs is ~150 nm (Ogilby, 2010; Ogilby & Kuimova, 2016). Therefore, O2(a1Δg) is a diffusible signaling agent; it can move from its point of production in a cell to initiate a characteristic reaction (i.e., send a message) at a separate location. With a ~150 nm sphere of activity, O2(a1Δg) will cross membranes and readily sample both hydrophobic and hydrophilic intracellular domains.

­ ow can the reactions of O2(a1Δg) modify/modulate cell redox H states and cell response? Redox regulation of cellular function is general and occurs through a variety of pathways (Jones & Sies, 2015). Through its reactions with components of these pathways, O2(a1Δg) can perturb and arguably modulate the redox state of cells. Control of

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cellular function through changes in the redox state may not be as selective as other mechanisms (e.g., phosphorylation and dephosphorylation of a specific protein) (Parvez, Long, Poganik, & Aye, 2018). Rather, given that O2(a1Δg) reacts with GSH, for example, and the redox state of GSH forms the base of a fundamental signaling network (Kemp, Go, & Jones, 2008), it follows that O2(a1Δg) can influence a host of processes. Selectivity can nevertheless still be imparted based on the concentration and localization of the O2(a1Δg) produced (Blázquez-Castro et al., 2018; Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). The local concentration of O2(a1Δg) (i.e., the “dose”) can elicit cell responses ranging from necrosis (high dose), through apoptosis, to proliferation (low dose) (Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). Although necrosis can reasonably be explained (e.g., direct oxygenation that disrupts the plasma membrane), much remains to be determined about how O2(a1Δg) can mediate signaling pathways that steer a cell from apoptosis to proliferation.

­Examples with selected enzymes

Protein phosphorylation/dephosphorylation is involved in many signaling pathways (Hancock, 2005). Thus, perturbing a given phosphatase, an enzyme that cleaves phosphate bonds, is a key signaling step (Ray, Huang, & Tsuji, 2012). The catalytic pocket of phosphatases often has a cysteine that is central to the dephosphorylating action (Bak, Bechtel, Falco, & Weerapana, 2019), and this thiol-containing residue is susceptible to oxidation by O2(a1Δg) (Davies, 2003; Devasagayam et al., 1991). Indeed, inactivation of tyrosine phosphatase by O2(a1Δg) has been reported (Montfort et al., 2006). Perturbing a kinase, an enzyme that catalyzes protein phosphorylation, is likewise a key signaling step. Src kinase has cysteine residues susceptible to redox cycling between the free thiol and bridged disulfide forms, a process that influences enzymatic activity. It has been shown that mild ROS-mediated oxidation to yield a disulfide bridge enhances Src kinase activity (Giannoni & Chiarugi, 2014). Although O2(a1Δg) was not directly implicated, it makes sense that if O2(a1Δg) can mediate the oxidation of a thiol to a disulfide, then Src kinase activity could indeed be enhanced by O2(a1Δg). The action of mitogen activated protein kinases (MAPKs) can be modulated by O2(a1Δg) (Klotz et al., 1999; Klotz, Kröncke, & Sies, 2003).

­Genetic regulation mediated by O2(a1Δg)

O2(a1Δg) will oxygenate nucleic acid bases, particularly guanine, and thereby adversely influence the genetic code carried by DNA and RNA (DiMascio et al., 2019; Ye et al., 2003). Thus, it is easy to see how O2(a1Δg) could directly introduce mutations. A more complicated problem is to ascertain how O2(a1Δg) stimulates the expression of regulatory proteins that mediate stress responses (Ryter & Tyrrell, 1998). A classic example of a O2(a1Δg)-mediated process is the upregulation of the stress protein heme oxygenase-1 (Ryter & Tyrrell, 1998). A key argument used to support the role played by O2(a1Δg) was the observation of a D2O/H2O effect that enhanced gene expression. It was inferred that this was a consequence of the longer lifetime

­Singlet oxygen

of O2(a1Δg) in D2O-incubated cells to kinetically favor the reaction of O2(a1Δg) with target biomolecules. This interpretation is consistent with the perspective that the intracellular lifetime of O2(a1Δg) is principally determined by interaction with the water in a cell (vide supra). Other stress management proteins have been implicated in processes of O2(a1Δg)-mediated gene expression (Ryter & Tyrrell, 1998). It appears that these regulatory responses are a consequence of general changes in the local redox environment that are detected by redox-sensitive transcription factors (e.g., the Nrf2-Keap1 system) that, in turn, initiate gene expression/repression (Espinosa-Diez et al., 2015). An interesting issue is whether the transcription factor responds to a general indicator of redox balance (e.g., the GSSG–GSH redox couple), or whether O2(a1Δg) could specifically and directly influence the transcription factor. The DNA-binding structural motif known as a “zinc finger,” which is common to many transcription factors, contains cysteines that are susceptible to reaction with O2(a1Δg) (Lebrun et al., 2015). It has been shown that transcriptional control by NF-κB can be modulated by O2(a1Δg) (Piette, 2015).

­ orrelating cell response with the O2(a1Δg) reaction in a given C cellular location Redox signaling, as with other cell signaling processes, is space and time dependent (Go & Jones, 2008; Hancock, 2005; Jones & Sies, 2015; Ushio-Fukai, 2009). Subcellular compartmentalization in this regard can simply reflect the spatial localization of a given organelle, or it can reflect the local pH, viscosity, or oxygen concentration. Given the chemical, morphological, and functional inhomogeneity of a cell, it is reasonable to expect that cell response should depend on the site of O2(a1Δg) production. This has been demonstrated using O2(a1Δg) photosensitizers that localize in different cellular domains (Kessel, 2004; Redmond & Kochevar, 2006). With a focus on cell death, it was shown that O2(a1Δg) produced in the mitochondria can have a greater and faster effect than the production of O2(a1Δg) in the cell nucleus (Rubio et al., 2009). We have complemented such sensitizer localization studies using focused lasers to produce O2(a1Δg) (Gollmer et al., 2013; Pimenta et al., 2012).

­Subcellular spatially dependent O2(a1Δg)-mediated eustress response

As illustrated in Fig.  4, we have used 765 nm irradiation to selectively produce O2(a1Δg) in sensitizer-free experiments in two separate cellular domains: the nucleus and the cytoplasm (Blázquez-Castro et al., 2018). These experiments were carried out in the “low-dose” regime where O2(a1Δg) was not toxic but, rather, stimulated the cell to undergo mitosis (Blázquez-Castro et al., 2018; Blázquez-Castro, Breitenbach, & Ogilby, 2014; Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). The production of O2(a1Δg) in the cytoplasm accelerated progress through the cell cycle relative to the unirradiated control cells, whereas the production of O2(a1Δg) in the nucleus delayed cell proliferation relative to the unirradiated control cells

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(Blázquez-Castro et  al., 2018). A small oxidative change in the redox state of the cytoplasm will engage proliferative signaling (Blázquez-Castro et al., 2018; Burhans & Heintz, 2009). Specifically, the mild oxidizing pulse associated with the low dose of O2(a1Δg) could facilitate the transition from the G1 to S phase of the cell cycle by enhancing the activity of Src kinases (Blázquez-Castro et al., 2012; Chiu & Dawes, 2012). In contrast, the nucleus maintains a reducing environment to inhibit chromatin oxidation and thereby better preserve the genetic information (Go & Jones, 2010). As such, the O2(a1Δg)-mediated oxidizing pulse in this location could engage the genetic damage check response to delay mitosis until an “all clear” signal is received (Caputo, Vegliante, & Ghibelli, 2012; Go & Jones, 2010).

­Superoxide radical anion ­Reactivity of superoxide Despite the moniker “super,” superoxide is not characterized by exceptional reactivity (Sawyer, 1991; Sawyer & Valentine, 1981). Thus, like O2(a1Δg), to call it a “reactive oxygen species” is arguably stretching the point. However, when dissolved in aqueous media, superoxide is readily protonated to yield the hydroperoxyl radical, •OOH, a species that is quite reactive (Walling, 1995). Disproportionation yields hydrogen peroxide, which is likewise a unique oxidant. The pKa of the hydroperoxyl radical is ~ 4.8 (Bielski, Cabelli, Arudi, & Ross, 1985). Thus, at pH 7, the equilibrium between •OOH and O2•− + H+ favors superoxide. Nevertheless, despite its low concentration, •OOH is constantly removed via reactions, and through this equilibrium, superoxide serves as a supplier of more •OOH. The oxidation of superoxide can also produce O2(a1Δg), and a number of relevant oxidants are found in a cell (Nanni, Birge, Hubbard, Morrison, & Sawyer, 1981). Thus, the “super” in superoxide is best interpreted in terms of the role that superoxide plays as a precursor to species that are more reactive. Nevertheless, selected reactions of superoxide are still pertinent. For example, superoxide reacts with thiols and, as such, can directly influence redox regulatory systems (i.e., superoxide will slowly oxidize GSH to GSSG) (Winterbourn, 2016). The reaction of superoxide with GSH also produces O2(a1Δg) (Wefers & Sies, 1983; Winterbourn, 2016).

­Photoinitiated production of superoxide as a complication Although superoxide can be formed through “dark” reactions in a cell (Foote et al., 1995; Lambeth & Neish, 2014), our concern is the photoinitiated production of superoxide. A molecule that sensitizes the production of O2(a1Δg) may also be susceptible to electron transfer reactions that produce a radical anion (Fig. 3). Such electron transfer can be efficient in systems containing good electron donors (e.g., amino acids such as tryptophan) (Westberg et al., 2017a; Westberg, Bregnhøj, Etzerodt, & Ogilby, 2017b). The resultant organic radical anion may then reduce O2(X3Σg−) to form superoxide. A related point is to consider the effect of a general reducing agent

­Where does the field stand today? What does the future hold?

on oxygen; with its excitation energy of ~1 eV, O2(a1Δg) will be more easily reduced to superoxide than will O2(X3Σg−) (Ijeri, Daasbjerg, Ogilby, & Poulsen, 2008; Saito, Matsuura, & Inoue, 1983). The concomitant production of both O2(a1Δg) and superoxide can be an undesired complication, certainly for mechanistic studies of photoinitiated stress.

­ elective photoinitiated production of superoxide S as a mechanistic tool A huge effort has been exerted over the last ~40 years to develop photosensitizers that efficiently make O2(a1Δg) at the expense of electron transfer reactions. Although molecular concepts incorporated to achieve this goal may function admirably in ­solution-phase experiments, problems often arise in the complex environment of a cell (Silva et al., 2016; Takizawa et al., 2015). One approach to achieve intracellular selectivity, spatial specificity, and dose control is through the use of genetically encoded proteins that encapsulate the photosensitizer (Trewin et al., 2018; Westberg et al., 2019). A key feature of these optogenetic actuators is that the local environment of the photosensitizer, in principle, will always be the same, irrespective of where in the cell the protein is localized. In this way, the photophysical properties of selectively making either O2(a1Δg) or superoxide, independently characterized in solution-phase experiments, will be retained in the cell. Proteins in the so-called KillerRed family selectively make superoxide (Pletnev et  al., 2009; Trewin et  al., 2018), and a molecular basis for the pertinent electron transfer reactions has been identified (Lee et al., 2018; Magerl, Stambolic, & Dick, 2017). In contrast, a flavin-binding protein has recently been designed and produced as a selective O2(a1Δg) sensitizer (Westberg et al., 2017a). The central feature of this latter work was to design a protein enclosure that precluded electron transfer to the sensitizing chromophore (Fig. 3B).

­ here does the field stand today? What does the future W hold?

­Selective production of O2(a1Δg) and superoxide in space and time Recent work on genetically encoded protein-encased photosensitizers has reached a stage where one can selectively produce superoxide (Trewin et al., 2018) and, independently, O2(a1Δg) (Westberg et al., 2017a) with visible light. Because intracellular localization of such actuators can be protein specific (Riani, Matsuda, Takemoto, & Nagai, 2018; Rodriguez et al., 2017), pathways important for distress and eustress signaling can be targeted as mechanistic studies evolve. Early studies of optogenetic localization involved the flavoprotein miniSOG (for mini Singlet Oxygen Generator) (Shu et al., 2011). It was shown that this protein was more phototoxic when localized in the mitochondria as opposed to the nucleus (Ryumina et al., 2013), an observation consistent with results using other less-­specific

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methods to localize a sensitizer (vide supra). It has been shown, however, that miniSOG is actually a poor O2(a1Δg) photosensitizer, producing superoxide instead (Pimenta, Jensen, Breitenbach, Etzerodt, & Ogilby, 2013; Ruiz-González et al., 2013). A related flavoprotein has since been produced and has been shown to be a selective and efficient O2(a1Δg) sensitizer (Westberg et al., 2017a; Westberg et al., 2017b; Westberg et  al., 2019). Although the pertinent photophysics have been examined in solution, it remains to fully characterize this tool inside cells. However, this approach still has limitations. Specifically, flavins are endogenous and thus will potentially mitigate the effects of the encasing protein to establish distinct localization of O2(a1Δg) production. A complementary approach is to use an exogenous chromophore that becomes a viable sensitizer only upon binding in the localizing protein (He et al., 2016). More experiments should be performed in which O2(a1Δg) is selectively produced using 765 nm irradiation. Although the spatial localization of O2(a1Δg) produced in this way is not as good as in the optogenetic photosensitized experiments (Blázquez-Castro et  al., 2018), any differences observed in the respective cell responses should be considered in light of the O2(a1Δg) diffusion distance. To this end, it will be informative to examine the effect of adding O2(a1Δg) quenchers that can be spatially localized, particularly through genetic control (Pimenta, Jensen, Etzerodt, & Ogilby, 2015; To et al., 2014). H2O should likewise be considered a “quencher,” and complementary experiments should be performed using cells in which H2O has been replaced by D2O (Hatz, Lambert, & Ogilby, 2007; Ogilby, 2010).

E­ xploit the opportunity for better control of O2(a1Δg) and superoxide dose As shown in 765 nm laser-based experiments, the correlation between O2(a1Δg) dose and cell response can range from distress to eustress (Westberg, Bregnhøj, BlázquezCastro, et  al., 2016). It remains to be determined, however, how and why such a change in dose is manifested in this way. For apoptosis and proliferation, where complicated signaling pathways are involved, does O2(a1Δg) interact with different initial targets? Or does the cell response scale according to the concentration of a downstream product from the same initial O2(a1Δg) reaction? Using a system such as KillerRed, the localized dose of intracellular superoxide can likewise be controlled, and the cell responses should be compared to those obtained using a O2(a1Δg) sensitizer.

­ plethora of new ways to monitor cells and cell response A in space and time The continued development of imaging technologies and fluorescent probes dramatically influences the toolbox of the cell biologist (Kaur, Kolanowski, & New, 2016; Rodriguez et  al., 2017). However, the ability to create high-resolution images of dynamic processes has yet to be applied in earnest to address distress and

­References

eustress, particularly images that exceed limits defined by light diffraction (i.e., “super-­resolution” techniques (Leung & Chou, 2011; Schermelleh et al., 2019)). The photo-initiated production of O2(a1Δg) and superoxide are ideally suited to these methods of monitoring cell response; actinic light is readily coupled into the microscope used to monitor cell response (Banerjee, Breitenbach, & Ogilby, 2018; Blázquez-Castro et al., 2018; Pimenta et al., 2012). Unlike other ROS, O2(a1Δg) has the unique feature that it can be directly monitored from a single cell in a time-resolved spectroscopic measurement (Ogilby, 2010; Snyder, Skovsen, Lambert, Poulsen, & Ogilby, 2006) and, as such, provides an informative probe of cell response to oxidative stress (Kuimova, Botchway, et al., 2009). Admittedly, these are not easy experiments (Skovsen, Snyder, & Ogilby, 2006). Nevertheless, continued advances in IR detector technology, for example, should facilitate the use of this important tool. Although fluorescent probes to detect ROS are available (Gomes, Fernandes, & Lima, 2005; Pedersen et al., 2014), there is still a need for probes that are selective for a given ROS. Moreover, one must not neglect other approaches. From work with excitable cells (e.g., neurons), it is known that changes in ion transport across the cell membrane can be induced by O2(a1Δg) (Pooler & Valenzeno, 1979). Many signaling pathways are associated with changes in the transmembrane potential and the flow of ions (e.g., Ca2+), and early cell response to O2(a1Δg), in particular, is reflected in the time-dependent profile of action potentials (Breitenbach, Ogilby, & Lambert, 2010). Thus, it will be beneficial to exploit electrophysiological tools to monitor cell response to the selective production of O2(a1Δg).

­Conclusions Light-initiated oxidative distress and eustress, particularly processes mediated by O2(a1Δg), are common in our world of sunlight, oxygen, and photosensitizers. The reactions of O2(a1Δg) with intracellular regulatory molecules can steer a cell toward a particular fate (e.g., survival, growth, or death). Through judicious use and accurate dose control, O2(a1Δg) can thus be used as an agent to modulate cell behavior. In short, O2(a1Δg) is not just a ROS that will indiscriminately damage a cell. Many of the signaling pathways relevant to these light-initiated events are likewise involved in redox-based stress responses initiated by other ROS. Thus, given that light is accurately controlled and readily used in conjunction with methods to assess cell response, studies of light-initiated O2(a1Δg)-mediated and superoxide-­mediated processes should be useful to a broad cross section of the scientific community.

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