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1.
Phys Chem Chem Phys ; 25(33): 22336-22344, 2023 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-37580966

RESUMEN

The excited states of carotenoids have been a subject of numerous studies. While a majority of these reports target the excited state dynamics initiated by the excitation of the S2 state, the upper excited state(s) absorbing in the UV spectral region (denoted as SUV) has been only scarcely studied. Moreover, the relation between the SUV and Sn, the final state of the well-known S1-Sn transition of carotenoids, remains unknown. To address this yet-unresolved issue, we compared the excited state dynamics of two carotenoids, namely, ß-carotene and astaxanthin, after excitation of either the SUV or Sn state. The SUV state was excited directly by UV light, and the excitation of the Sn state was achieved via re-pumping the S1-Sn transition. The results indicated that direct SUV excitation produces an S1-Sn band that is significantly broader than that obtained after S2 excitation, most probably due to the generation of multiple S1 conformations produced by excess energy. No such broadening is observed if the Sn state is excited by the re-pump pulse. This shows that the Sn and SUV states are different, each initializing a specific relaxation pathway. We propose that the Sn state retains the coupled triplet pair character of the S1 state, while the SUV state is the higher state of Bu+ symmetry accessible by one-photon transition.

2.
Photosynth Res ; 154(1): 75-87, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-36066816

RESUMEN

The functions of both (bacterio) chlorophylls and carotenoids in light-harvesting complexes have been extensively studied during the past decade, yet, the involvement of BChl a high-energy Soret band in the cascade of light-harvesting processes still remains a relatively unexplored topic. Here, we present transient absorption data recorded after excitation of the Soret band in the LH2 complex from Rhodoblastus acidophilus. Comparison of obtained data to those recorded after excitation of rhodopin glucoside and B800 BChl a suggests that no Soret-to-Car energy transfer pathway is active in LH2 complex. Furthermore, a spectrally rich pattern observed in the spectral region of rhodopin glucoside ground state bleaching (420-550 nm) has been assigned to an electrochromic shift. The results of global fitting analysis demonstrate two more features. A 6 ps component obtained exclusively after excitation of the Soret band has been assigned to the response of rhodopin glucoside to excess energy dissipation in LH2. Another time component, ~ 450 ps, appearing independently of the excitation wavelength was assigned to BChl a-to-Car triplet-triplet transfer. Presented data demonstrate several new features of LH2 complex and its behavior following the excitation of the Soret band.


Asunto(s)
Carotenoides , Complejos de Proteína Captadores de Luz , Bacterioclorofilas/metabolismo , Beijerinckiaceae , Carotenoides/metabolismo , Glucósidos , Complejos de Proteína Captadores de Luz/metabolismo
3.
Photosynth Res ; 144(2): 127-135, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-31802367

RESUMEN

We used ultrafast transient absorption spectroscopy to study excited-state dynamics of two keto-carotenoids, siphonaxanthin and siphonein. These two carotenoids differ in the presence of dodecanoyl-oxy group in siphonein, which is attached to the C19 carbon on the same side of the molecule as the conjugated keto group. We show that this dodecanoyl-oxy group, though not in conjugation, is still capable of modifying excited state properties. While spectroscopic properties of siphonein and siphonaxanthin are nearly identical in a non-polar solvent, they become markedly different in polar solvents. In a polar solvent, siphonein, having the dodecanoyl-oxy moiety, exhibits less pronounced vibrational bands in the absorption spectrum and has significantly enhanced characteristic features of an intramolecular charge-transfer (ICT) state in transient absorption spectra compared to siphonaxanthin. The presence of the dodecanoyl-oxy moiety also alters the lifetimes of the S1/ICT state. For siphonaxanthin, the lifetimes are 60, 20, and 14 ps in n-hexane, acetonitrile, and methanol, whereas for siphonein these lifetimes yield 60, 11, and 10 ps. Thus, we show that even a non-conjugated functional group can affect the charge-transfer character of the S1/ICT state. By comparison with fucoxanthin acyl-oxy derivatives, we show that position of the acyl-oxy group in respect to the conjugated keto group is the key feature determining whether the polarity-dependent behavior is enhanced or suppressed.


Asunto(s)
Carotenoides/química , Xantófilas/química , Acetonitrilos/química , Hexanos/química , Enlace de Hidrógeno , Estructura Molecular , Solventes/química , Espectroscopía de Absorción de Rayos X/instrumentación , Espectroscopía de Absorción de Rayos X/métodos
4.
Phys Chem Chem Phys ; 22(17): 9195-9203, 2020 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-32149285

RESUMEN

Phytochromes are photosensory proteins in plants, fungi, and bacteria, which detect red- and far-red light. They undergo a transition between the resting (Pr) and photoactivated (Pfr) states. In bacterial phytochromes, the Pr-to-Pfr transition is facilitated by two intermediate states, called Lumi-R and Meta-R. The molecular structures of the protein in these states are not known and the molecular mechanism of photoconversion is not understood. Here, we apply transient infrared absorption spectroscopy to study the photocycle of the wild-type and Y263F mutant of the phytochrome from Deinococcus radiodurans (DrBphP) from nano- to milliseconds. We identify two sequentially forming Lumi-R states which differ in the local structure surrounding the carbonyl group of the biliverdin D-ring. We also find that the tyrosine at position 263 alters local structure and dynamics around the D-ring and causes an increased rate of Pfr formation. The results shed new light on the mechanism of light-signalling in phytochrome proteins.


Asunto(s)
Deinococcus/química , Deinococcus/genética , Modelos Moleculares , Fitocromo/química , Espectrofotometría Infrarroja , Proteínas Bacterianas/química , Fototransducción/genética , Mutación , Estructura Terciaria de Proteína
5.
Biochim Biophys Acta Bioenerg ; 1859(5): 357-365, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29499185

RESUMEN

We have applied femtosecond transient absorption spectroscopy in pump-probe and pump-dump-probe regimes to study energy transfer between fucoxanthin and Chl a in fucoxanthin-Chl a complex from the pennate diatom Phaeodactylum tricornutum. Experiments were carried out at room temperature and 77 K to reveal temperature dependence of energy transfer. At both temperatures, the ultrafast (<100 fs) energy transfer channel from the fucoxanthin S2 state is active and is complemented by the second pathway via the combined S1/ICT state. The S1/ICT-Chl a pathway has two channels, the fast one characterized by sub-picosecond energy transfer, and slow having time constants of 4.5 ps at room temperature and 6.6 ps at 77 K. The overall energy transfer via the S1/ICT is faster at 77 K, because the fast component gains amplitude upon lowering the temperature. The pump-dump-probe regime, with the dump pulse centered in the spectral region of ICT stimulated emission at 950 nm and applied at 2 ps after excitation, proved that the S1 and ICT states of fucoxanthin in FCP are individual, yet coupled entities. Analysis of the pump-dump-probe data suggested that the main energy donor in the slow S1/ICT-Chl a route is the S1 part of the S1/ICT potential surface.


Asunto(s)
Clorofila/química , Diatomeas/química , Espectrofotometría Atómica , Xantófilas/química , Clorofila A
6.
Photosynth Res ; 131(1): 105-117, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27612863

RESUMEN

A quenching mechanism mediated by the orange carotenoid protein (OCP) is one of the ways cyanobacteria protect themselves against photooxidative stress. Here, we present a femtosecond spectroscopic study comparing OCP and RCP (red carotenoid protein) samples binding different carotenoids. We confirmed significant changes in carotenoid configuration upon OCP activation reported by Leverenz et al. (Science 348:1463-1466. doi: 10.1126/science.aaa7234 , 2015) by comparing the transient spectra of OCP and RCP. The most important marker of these changes was the magnitude of the transient signal associated with the carotenoid intramolecular charge-transfer (ICT) state. While OCP with canthaxanthin exhibited a weak ICT signal, it increased significantly for canthaxanthin bound to RCP. On the contrary, a strong ICT signal was recorded in OCP binding echinenone excited at the red edge of the absorption spectrum. Because the carbonyl oxygen responsible for the appearance of the ICT signal is located at the end rings of both carotenoids, the magnitude of the ICT signal can be used to estimate the torsion angles of the end rings. Application of two different excitation wavelengths to study OCP demonstrated that the OCP sample contains two spectroscopically distinct populations, none of which is corresponding to the photoactivated product of OCP.


Asunto(s)
Carotenoides/análisis , Cianobacterias/química , Análisis Espectral/métodos
7.
J Phys Chem Lett ; 12(12): 3176-3181, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33755477

RESUMEN

Femtosecond transient absorption spectroscopy following two-photon excitation (2PE) is used to determine the contributions of carotenoids and chlorophylls to the 2PE signals in the main plant light-harvesting complex (LHCII). For 2PE, excitation at 1210 and 1300 nm was used, being within the known 2PE profile of LHCII. At both excitation wavelengths, the transient absorption spectra exhibit a shape characteristic of excited chlorophylls with only a minor contribution from carotenoids. We compare the 2PE data measured for LHCII with those obtained from 2PE of a lutein/chlorophyll a mixture in acetone. We estimate that although the 2PE cross section of a single carotenoid in acetone is ∼1.7 times larger than that of a Chl a, due to the 1:3.5 carotenoid/Chl ratio in LHCII, only one-third of the absorbed 2PE photons excite carotenoids in LHCII in the 1200-1300 nm range.

8.
Nat Commun ; 12(1): 6890, 2021 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-34824207

RESUMEN

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.


Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/metabolismo , Proteínas de Arabidopsis/genética , Carotenoides/metabolismo , Clorofila/metabolismo , Proteínas de Cloroplastos/genética , Transferencia de Energía , Mutación , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Synechocystis/genética , Synechocystis/metabolismo , Xantófilas/metabolismo , Zeaxantinas/genética , Zeaxantinas/metabolismo
9.
Acta Crystallogr D Struct Biol ; 77(Pt 8): 1001-1009, 2021 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-34342273

RESUMEN

(6-4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6-4) photolyase [Dm(6-4)photolyase] at room and cryogenic temperatures are reported. The room-temperature structure was solved to 2.27 Šresolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79 Šresolution using conventional X-ray crystallography. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV-Vis absorption spectroscopy confirms that Dm(6-4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.


Asunto(s)
Flavoproteínas/química , Animales , Cristalografía , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Drosophila melanogaster , Flavoproteínas/metabolismo , Temperatura
10.
Biochim Biophys Acta Bioenerg ; 1861(2): 148120, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31734194

RESUMEN

The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and back-conversion, and lower fluorescence quenching of phycobilisomes relative to the well-characterized OCP1. Moreover, OCP2 is not regulated by the fluorescence recovery protein (FRP). In this work, we present a comprehensive study combining ultrafast spectroscopy and structural analysis to compare the photoactivation mechanisms of OCP1 and OCP2 from Tolypothrix PCC 7601. We show that despite significant differences in their functional characteristics, the spectroscopic properties of OCP1 and OCP2 are comparable. This indicates that the OCP functionality is not directly related to the spectroscopic properties of the bound carotenoid. In addition, the structural analysis by X-ray footprinting reveals that, overall, OCP1 and OCP2 have grossly the same photoactivation mechanism. However, the OCP2 is less reactive to radiolytic labeling, suggesting that the protein is less flexible than OCP1. This observation could explain fast photoconversion of OCP2.


Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/química , Ficobilisomas/química , Espectrometría de Fluorescencia
11.
Elife ; 92020 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-32228856

RESUMEN

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.


Plants adapt to the availability of light throughout their lives because it regulates so many aspects of their growth and reproduction. To detect the level of light, plant cells use proteins called phytochromes, which are also found in some bacteria and fungi. Phytochrome proteins change shape when they are exposed to red light, and this change alters the behaviour of the cell. The red light is absorbed by a molecule known as chromophore, which is connected to a region of the phytochrome called the PHY-tongue. This region undergoes one of the key structural changes that occur when the phytochrome protein absorbs light, turning from a flat sheet into a helix. Claesson, Wahlgren, Takala et al. studied the structure of a bacterial phytochrome protein almost immediately after shining a very brief flash of red light using a laser. The experiments revealed that the structure of the protein begins to change within a trillionth of a second: specifically, the chromophore twists, which disrupts its attachment to the protein, freeing the protein to change shape. Claesson, Wahlgren, Takala et al. note that this structure is likely a very short-lived intermediate state, which however triggers more changes in the overall shape change of the protein. One feature of the rearrangement is the disappearance of a particular water molecule. This molecule can be found at the core of many different phytochrome structures and interacts with several parts of the chromophore and the phytochrome protein. It is unclear why the water molecule is lost, but given how quickly this happens after the red light is applied it is likely that this disappearance is an integral part of the reshaping process. Together these events disrupt the interactions between the chromophore and the PHY-tongue, enabling the PHY-tongue to change shape and alter the structure of the phytochrome protein. Understanding and controlling this process could allow scientists to alter growth patterns in plants, such as crops or weeds.


Asunto(s)
Proteínas Bacterianas/química , Cristalografía por Rayos X , Luz , Fitocromo/química , Sitios de Unión , Deinococcus/química , Rayos Láser , Modelos Moleculares , Procesos Fotoquímicos , Conformación Proteica
12.
Biochim Biophys Acta Bioenerg ; 1860(2): 111-120, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30414929

RESUMEN

Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690 nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S2 and hot S1 states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed.


Asunto(s)
Clorofila A , Transferencia de Energía/fisiología , Complejos de Proteína Captadores de Luz/química , Estramenopilos/fisiología , Chlorophyta/fisiología , Plastidios , Rhodophyta/fisiología , Espectrometría de Fluorescencia/métodos , Xantófilas
13.
J Phys Chem B ; 122(29): 7264-7276, 2018 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-29963865

RESUMEN

To demonstrate the value of the multipulse method in revealing the nature of coupling between excited states and explore the environmental dependencies of lowest excited singlet state (S1) and intramolecular charge transfer (ICT) state equilibration, we performed ultrafast transient absorption pump-dump-probe and pump-repump-probe spectroscopies on fucoxanthin in various solvent conditions. The effects of polarity, proticity, and temperature were tested in solvents methanol at 293 and 190 K, acetonitrile, and isopropanol. We show that manipulation of the kinetic traces can produce one trace reflecting the equilibration kinetics of the states, which reveals that lower polarity, proticity, and temperature delay S1/ICT equilibration. On the basis of a two-state model representing the S1 and ICT states on the same S1/ICT potential energy surface, we were able to show that the kinetics are strictly dependent on the initial relative populations of the states as well as the decay of the ICT state to the ground state. Informed by global analysis, a systematic method for target analysis based on this model allowed us to quantify the population transfer rates throughout the life of the S1/ICT state as well as separate the S1 and ICT spectral signatures. The results are consistent with the concept that the S1 and ICT states are part of one potential energy surface.

14.
J Phys Chem B ; 122(11): 2922-2930, 2018 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-29469573

RESUMEN

We used ultrafast transient absorption spectroscopy to study excited-state dynamics of the keto-carotenoid fucoxanthin (Fx) and its two derivatives: 19'-butanoyloxyfucoxanthin (bFx) and 19'-hexanoyloxyfucoxanthin (hFx). These derivatives occur in some light-harvesting systems of photosynthetic microorganisms, and their presence is typically related to stress conditions. Even though the hexanoyl (butanoyl) moiety is not a part of the conjugated system of hFx (bFx), their absorption spectra in polar solvents exhibit more pronounced vibrational bands of the S2 state than for Fx. The effect of the nonconjugated acyloxy moiety is further observed in transient absorption spectra, which for Fx exhibit characteristic features of an intramolecular charge transfer (ICT) state in all polar solvents. For bFx and hFx, however, much weaker ICT features are detected in methanol, and the spectral markers of the ICT state disappear completely in polar, but aprotic acetonitrile. The presence of the acyloxy moiety also alters the lifetimes of the S1/ICT state. For Fx, the lifetimes are 60, 30, and 20 ps in n-hexane, acetonitrile, and methanol, whereas for bFx and hFx, these lifetimes yield 60, 60, and 40 ps, respectively. Testing the S1/ICT state lifetimes of hFx in other solvents revealed that some ICT features can be induced only in polar, protic solvents (methanol, ethanol, and ethylene glycol). Thus, bFx and hFx represent a rather rare example of a system in which a nonconjugated functional group significantly alters excited-state dynamics. By comparison with other carotenoids, we show that a keto group at the acyloxy tail, even though it is not in conjugation, affects the electron distribution along the conjugated backbone, resulting in the observed decrease of the ICT character of the S1/ICT state of bFx and hFx.


Asunto(s)
Simulación de Dinámica Molecular , Xantófilas/química , Conformación Molecular
15.
J Phys Chem B ; 121(17): 4438-4447, 2017 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-28382818

RESUMEN

Ultrafast transient absorption spectroscopy and single-wavelength anisotropy measurements were used to study the effect of isomerization on the excited-state properties of fucoxanthin in polar and nonpolar solvents. The excitation wavelengths were 477 nm for all-trans-fucoxanthin, and 333 and 477 nm for cis-fucoxanthin. All transient absorption spectra of the fucoxanthin isomers in polar solvents show intramolecular charge transfer (ICT) state features, typical for carbonyl carotenoids. Global analysis of the data requires an additional fitting component, originated from the presence of blue and red forms of fucoxanthin in a polar protic solvent. Here we demonstrate that the ICT state decays faster than the S1 state, due to the significant contribution of the red form to the ICT state dynamics. The isomerization does not affect the S1 lifetime, but induces a larger difference between the S1- and ICT-state lifetimes in cis-fucoxanthin, which is likely caused by alterations of ICT coupling to either the S1 or S0 states; the S*-state signal is more pronounced for cis-isomers in a nonpolar solvent.


Asunto(s)
Teoría Cuántica , Xantófilas/química , Estructura Molecular , Estereoisomerismo
17.
J Phys Chem B ; 119(39): 12653-63, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26362118

RESUMEN

In order to estimate the possible structure of the unknown carbonyl carotenoid related to isofucoxanthin from Chromera velia denoted as isofucoxanthin-like carotenoid (Ifx-l), we employed steady-state and ultrafast time-resolved spectroscopic techniques to investigate spectroscopic properties of Ifx-l in various solvents. The results were compared with those measured for related carotenoids with known structure: fucoxanthin (Fx) and isofucoxanthin (Ifx). The experimental data were complemented by quantum chemistry calculations and molecular modeling. The data show that Ifx-l must have longer effective conjugation length than Ifx. Yet, the magnitude of polarity-dependent changes in Ifx-l is larger than for Ifx, suggesting significant differences in structure of these two carotenoids. The most interesting spectroscopic feature of Ifx-l is its response to solvent proticity. The transient absorption data show that (1) the magnitude of the ICT-like band of Ifx-l in acetonitrile is larger than in methanol and (2) the S1/ICT lifetime of Ifx-l in acetonitrile, 4 ps, is markedly shorter than in methanol (10 ps). This is opposite behavior than for Fx and Ifx whose S1/ICT lifetimes are always shorter in protic solvent methanol (20 and 13 ps) than in aprotic acetonitrile (30 and 17 ps). Comparison with other carbonyl carotenoids reported earlier showed that proticity response of Ifx-l is consistent with presence of a conjugated lactone ring. Combining the experimental data and quantum chemistry calculations, we estimated a possible structure of Ifx-l.


Asunto(s)
Alveolados/química , Carotenoides/química , Solventes/química
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