Photooxygenation mechanisms in naproxen-amino acid linked systems

The photooxygenation of model compounds containing the two enantiomers of naproxen (NPX) covalently linked to histidine (His), tryptophan (Trp) and tyrosine (Tyr) has been investigated by steady state irradiation, fluorescence spectroscopy and laser flash photolysis. The NPX–His systems presented the highest oxygen-mediated photoreactivity. Their fluorescence spectra matched that of isolated NPX and showed a clear quenching by oxygen, leading to a diminished production of the NPX triplet excited state ( 3 NPX*–His). Analysis of the NPX–His and NPX–Trp photolysates by UPLC-MS–MS revealed in both cases the formation of two photoproducts, arising from the reaction of singlet oxygen (1 O2) with the amino acid moiety. The most remarkable feature of NPX–Trp systems was a fast and stereoselective intramolecular fluorescence quenching, which prevented the efficient formation of 3 NPX*–Trp, thus explaining their lower reactivity towards photooxygenation. Finally, the NPX–Tyr systems were nearly unreactive and exhibited photophysical properties essentially coincident with those of the parent NPX. Overall, these results point to a type II photooxygenation mechanism, triggered by generation of 1 O2 from the 3 NPX* chromophoreFinancial support from the Spanish Government (CTQ2010-14882, JCI-2011-09926, Miguel Servet CP11/00154), from the EU (PCIG12-GA-2012-334257), from the Universitat Politecnica de Valencia (SP20120757) and from the Conselleria de Educacio, cultura i Esport (PROMETEOII/2013/005, GV/2013/051) is gratefully acknowledged.Vayá Pérez, I.; Andreu Ros, MI.; Jiménez Molero, MC.; Miranda Alonso, MÁ. (2014). Photooxygenation mechanisms in naproxen-amino acid linked systems. Photochemical & Photobiological Sciences Photochemical and Photobiological Sciences. 13:224-230. https://doi.org/10.1039/c3pp50252jS22423013L. I. Grossweiner and K. C.Smith, Photochemistry, in The Science of Photobiology, ed. K. C. Smith, Plenum Press, New York, 2nd edn, 1989, pp. 47–78L. Pretali and A.Albini, in CRC Handbook of Organic Photochemistry and Photobiology, ed. A. Griesbeck, M. Oelgemöller and F. Ghetti, CRC Press, Boca Raton, FL, 3rd edn, 2012, pp. 369–391Foote, C. S. (1991). DEFINITION OF TYPE I and TYPE II PHOTOSENSITIZED OXIDATION. Photochemistry and Photobiology, 54(5), 659-659. doi:10.1111/j.1751-1097.1991.tb02071.xDavies, M. J. (2003). Singlet oxygen-mediated damage to proteins and its consequences. Biochemical and Biophysical Research Communications, 305(3), 761-770. doi:10.1016/s0006-291x(03)00817-9Davies, M. J. (2004). Reactive species formed on proteins exposed to singlet oxygen. Photochemical & Photobiological Sciences, 3(1), 17. doi:10.1039/b307576cGirotti, A. W. (2001). Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms. Journal of Photochemistry and Photobiology B: Biology, 63(1-3), 103-113. doi:10.1016/s1011-1344(01)00207-xAndreu, I., Morera, I. M., Boscá, F., Sanchez, L., Camps, P., & Miranda, M. A. (2008). Cholesterol–diaryl ketone stereoisomeric dyads as models for «clean» type I and type II photooxygenation mechanisms. Organic & Biomolecular Chemistry, 6(5), 860. doi:10.1039/b718068cStadtman, E. R. (1993). Oxidation of Free Amino Acids and Amino Acid Residues in Proteins by Radiolysis and by Metal-Catalyzed Reactions. Annual Review of Biochemistry, 62(1), 797-821. doi:10.1146/annurev.bi.62.070193.004053Garrison, W. M. (1987). Reaction mechanisms in the radiolysis of peptides, polypeptides, and proteins. Chemical Reviews, 87(2), 381-398. doi:10.1021/cr00078a006P. U. Giacomoni , Sun Protection in Man, Comprehensive Series in Photosciences, Elsevier, Amsterdam, 2001, vol. 3R. C. Straight and J. D.Spikes, Photosensitized oxidation of biomolecules. in Polymers and Biopolymers, ed. A. A. Frimer and O. Singlet, CRC Press, Boca Raton, FL, 1985, pp. 91–143Wright, A., Bubb, W. A., Hawkins, C. L., & Davies, M. J. (2002). Singlet Oxygen–mediated Protein Oxidation: Evidence for the Formation of Reactive Side Chain Peroxides on Tyrosine Residues¶. Photochemistry and Photobiology, 76(1), 35. doi:10.1562/0031-8655(2002)0762.0.co;2Agon, V. V., Bubb, W. A., Wright, A., Hawkins, C. L., & Davies, M. J. (2006). Sensitizer-mediated photooxidation of histidine residues: Evidence for the formation of reactive side-chain peroxides. Free Radical Biology and Medicine, 40(4), 698-710. doi:10.1016/j.freeradbiomed.2005.09.039Huyett, J. E., Doan, P. E., Gurbiel, R., Houseman, A. L. P., Sivaraja, M., Goodin, D. B., & Hoffman, B. M. (1995). Compound ES of Cytochrome c Peroxidase Contains a Trp .pi.-Cation Radical: Characterization by Continuous Wave and Pulsed Q-Band External Nuclear Double Resonance Spectroscopy. Journal of the American Chemical Society, 117(35), 9033-9041. doi:10.1021/ja00140a021Redmond, R. W., & Gamlin, J. N. (1999). A Compilation of Singlet Oxygen Yields from Biologically Relevant Molecules. Photochemistry and Photobiology, 70(4), 391-475. doi:10.1111/j.1751-1097.1999.tb08240.xA. J. Lewis and D. E.Furst, Nonsteroidal Anti-Inflammatory Drugs: Mechanisms and Clinical Uses, Marcel Dekker, New York, 2nd edn, 1994Boscá, F., Marín, M. L., & Miranda, M. A. (2001). Photoreactivity of the Nonsteroidal Anti-inflammatory 2-Arylpropionic Acids with Photosensitizing Side Effects¶. Photochemistry and Photobiology, 74(5), 637. doi:10.1562/0031-8655(2001)0742.0.co;2Beijersbergen van Henegouwen, G. M. J. (1991). New trends in photobiology. Journal of Photochemistry and Photobiology B: Biology, 10(3), 183-210. doi:10.1016/1011-1344(91)85002-xMiranda, M. A., Castell, J. V., Hernández, D., Gómez-Lechón, M. J., Bosca, F., Morera, I. M., & Sarabia, Z. (1998). Drug-Photosensitized Protein Modification:  Identification of the Reactive Sites and Elucidation of the Reaction Mechanisms with Tiaprofenic Acid/Albumin as Model System†. Chemical Research in Toxicology, 11(3), 172-177. doi:10.1021/tx970082dJiménez, M. C., Pischel, U., & Miranda, M. A. (2007). Photoinduced processes in naproxen-based chiral dyads. Journal of Photochemistry and Photobiology C: Photochemistry Reviews, 8(3), 128-142. doi:10.1016/j.jphotochemrev.2007.10.001Catalfo, A., Bracchitta, G., & De Guidi, G. (2009). Role of aromatic amino acid tryptophan UVA-photoproducts in the determination of drug photosensitization mechanism: a comparison between methylene blue and naproxen. Photochemical & Photobiological Sciences, 8(10), 1467. doi:10.1039/b9pp00028cVayá, I., Pérez-Ruiz, R., Lhiaubet-Vallet, V., Jiménez, M. C., & Miranda, M. A. (2010). Drug–protein interactions assessed by fluorescence measurements in the real complexes and in model dyads. Chemical Physics Letters, 486(4-6), 147-153. doi:10.1016/j.cplett.2009.12.091Vayá, I., Jiménez, M. C., & Miranda, M. A. (2007). Excited-State Interactions in Flurbiprofen−Tryptophan Dyads. The Journal of Physical Chemistry B, 111(31), 9363-9371. doi:10.1021/jp071301zVayá, I., Bonancía, P., Jiménez, M. C., Markovitsi, D., Gustavsson, T., & Miranda, M. A. (2013). Excited state interactions between flurbiprofen and tryptophan in drug–protein complexes and in model dyads. Fluorescence studies from the femtosecond to the nanosecond time domains. Physical Chemistry Chemical Physics, 15(13), 4727. doi:10.1039/c3cp43847cGriesbeck, A. G., Neudörfl, J., & de Kiff, A. (2011). Photoinduced electron-transfer chemistry of the bielectrophoric N-phthaloyl derivatives of the amino acids tyrosine, histidine and tryptophan. Beilstein Journal of Organic Chemistry, 7, 518-524. doi:10.3762/bjoc.7.60Giese, B., Wang, M., Gao, J., Stoltz, M., Müller, P., & Graber, M. (2009). Electron Relay Race in Peptides. The Journal of Organic Chemistry, 74(10), 3621-3625. doi:10.1021/jo900375fCordes, M., Köttgen, A., Jasper, C., Jacques, O., Boudebous, H., & Giese, B. (2008). Influence of Amino Acid Side Chains on Long-Distance Electron Transfer in Peptides: Electron Hopping via «Stepping Stones». Angewandte Chemie International Edition, 47(18), 3461-3463. doi:10.1002/anie.200705588Abraham, B., & Kelly, L. A. (2003). Photooxidation of Amino Acids and Proteins Mediated by Novel 1,8-Naphthalimide Derivatives. The Journal of Physical Chemistry B, 107(45), 12534-12541. doi:10.1021/jp0358275Cadenas, E. (1989). Biochemistry of Oxygen Toxicity. Annual Review of Biochemistry, 58(1), 79-110. doi:10.1146/annurev.bi.58.070189.000455Peña, D., Martí, C., Noneil, S., Martínez, L. A., & Miranda, M. A. (1997). Time-Resolved Near Infrared Studies on Singlet Oxygen Production by the Photosensitizing 2-Arylpropionic Acids. Photochemistry and Photobiology, 65(5), 828-832. doi:10.1111/j.1751-1097.1997.tb01930.xKerwin, B. A., & Remmele, R. L. (2007). Protect from Light: Photodegradation and Protein Biologics. Journal of Pharmaceutical Sciences, 96(6), 1468-1479. doi:10.1002/jps.20815Kang, P., & Foote, C. S. (2000). Synthesis of a 13C,15N labeled imidazole and characterization of the 2,5-endoperoxide and its decomposition. Tetrahedron Letters, 41(49), 9623-9626. doi:10.1016/s0040-4039(00)01731-7Saito, I., Matsuura, T., Nakagawa, M., & Hino, T. (1977). Peroxidic intermediates in photosensitized oxygenation of tryptophan derivatives. Accounts of Chemical Research, 10(9), 346-352. doi:10.1021/ar50117a00

Impact of climate change environmental conditions on the resilience of different formulations of the biocontrol agent Candida sake CPA‐1 on grapes

Biocontrol agents have become components of integrated crop protection systems for controlling economically important fungal pathogens. Candida sake CPA‐1 is a biocontrol agent of fungal pathogens of fruits, both pre‐ and post‐harvest. While the efficacy of different formulations have been examined previously, few studies have considered the resilience of different formulations under changing climatic conditions of elevated temperature, drought stress and increased atmospheric CO2. This study examined the effect of (a) temperature × RH × elevated CO2 (400 vs 1000 ppm) on the temporal establishment and viability of two dry and one liquid C. sake CPA‐1 formulations on grape berry surfaces; (b) temperature stress (25 vs 35°C); and (c) elevated CO2 levels. Results indicated that temperature, RH and CO2 concentration influenced the establishment and viability of the formulations but there was no significant difference between formulations. For the combined three‐component factors, increased temperature (35°C) and lower RH (40%) reduced the viable populations on grapes. The interaction with elevated CO2 improved the establishment of viable populations of the formulations tested. Viable populations greater than Log 4 CFUs per g were recovered from the grape surfaces suggesting that these had conserved resilience for control of Botrytis rot in grapes

Predicted ecological niches and environmental resilience of different formulations of the biocontrol yeast Candida sake CPA-1 using the Bioscreen C

Environmental resilience of biocontrol microorganisms has been a major bottleneck in the development of effective formulations. Candida sake is an effective biocontrol agent (BCA) against Penicillium expansum, Botrytis cinerea or Rhizopus stolonifer, and different formulations of the BCA have been optimised recently. The objective of this study was to compare the relative tolerance of different dry and liquid formulations of the biocontrol yeast C. sake CPA-1 to interacting environmental conditions using the Bioscreen C. Initially, the use of this automated turbidimetric method was optimised for use with different formulations of the biocontrol yeast. The best growth curves were obtained for the C. sake CPA-1 strain when grown in a synthetic grape juice medium under continuous shaking and with an initial concentration of 105 CFUs ml−1. All the formulations showed a direct relationship between optical density values and yeast concentrations. Temperature (15–30 °C) and water activity (aw; 0.94–0.99) influenced the yeast resilience most profoundly, whereas the effect of pH (3–7) was minimal. In general, the liquid formulation grew faster in more interacting environmental conditions but only the yeast cells in the dry potato starch formulation could grow in some stress conditions. This rapid screening method can be used for effective identification of the resilience of different biocontrol formulations under interacting ecological abiotic conditions

Long-lived fluorescence of homopolymeric guanine–cytosine DNA duplexes

International audienceThe fluorescence spectrum of the homopolymeric double helix poly(dG)·poly(dC) is dominated by emission decaying on the nanosecond time-scale, as previously reported for the alternating homologue poly(dGdC)·poly(dGdC). Thus, energy trapping over long periods of time is a common feature of GC duplexes which contrast with AT duplexes. The impact of such behaviour on DNA photodamage needs to be evaluated

Excited state interactions between flurbiprofen and tryptophan in drug-protein complexes and in model dyads. Fluorescence studies from the femtosecond to the nanosecond time domains

International audienceWe report here on the interaction dynamics between flurbiprofen (FBP) and tryptophan (Trp) covalently linked in model dyads and in a complex of FBP with human serum albumin (HSA) probed by time-resolved fluorescence spectroscopy from the femto- to the nano-second timescales. In the dyads, a rapid (k > 1010 s−1) dynamic quenching of the 1FBP* fluorescence is followed by a slower (k > 109 s−1) quenching of the remaining 1Trp* fluorescence. Both processes display a clear stereoselectivity; the rates are 2-3 times higher for the (R,S)-dyad. In addition, a red-shifted exciplex emission is observed, rising in the range of 100-200 ps. A similar two-step dynamic fluorescence quenching is also observed in the FBP-HSA complex, although the kinetics of the involved processes are slower. The characteristic reorientational times determined for the two enantiomeric forms of FBP in the protein show that the interaction is stronger for the (R)-form. This is, to our knowledge, the first observation of stereo-selective flurbiprofen-tryptophan interaction dynamics with femtosecond time resolution

Stereodifferentiation in the intramolecular singlet excited state quenching of hydroxybiphenyl-tryptophan dyads

The photochemical processes occurring in diastereomeric dyads (S, S)-1 and (S, R)-1, prepared by conjugation of (S)-2-(2-hydroxy-1,1'-biphenyl-4-yl) propanoic acid ((S)-BPOH) with (S)- and (R)-Trp, have been investigated. In acetonitrile, the fluorescence spectra of (S, S)-1 and (S, R)-1 were coincident in shape and position with that of (S)-BPOH, although they revealed a markedly stereoselective quenching. Since singlet energy transfer from BPOH to Trp is forbidden (5 kcal mol(-1) uphill), the quenching was attributed to thermodynamically favoured (according to Rehm-Weller) electron transfer or exciplex formation. Upon addition of 20% water, the fluorescence quantum yield of (S)-BPOH decreased, while only minor changes were observed for the dyads. This can be explained by an enhancement of the excited state acidity of (S)-BPOH, associated with bridging of the carboxy and hydroxy groups by water, in agreement with the presence of water molecules in the X-ray structure of (S)-BPOH. When the carboxy group was not available for coordination with water, as in the methyl ester (S)-BPOHMe or in the dyads, this effect was prevented; accordingly, the fluorescence quantum yields did not depend on the presence or absence of water. The fluorescence lifetimes in dry acetonitrile were 1.67, 0.95 and 0.46 ns for (S)-BPOH, (S, S)-1 and (S, R)-1, respectively, indicating that the observed quenching is indeed dynamic. In line with the steady-state and time-resolved observations, molecular modelling pointed to a more favourable geometric arrangement of the two interacting chromophores in (S, R)-1. Interestingly, this dyad exhibited a folded conformation in the solid state.Financial support from the Spanish Government (CTQ2010-14882, BES-2008-003314, JCI-2011-09926, PR2011-0581), from the Generalitat Valenciana (Prometeo 2008/090) and from the Universitat Politecnica de Valencia (PAID 05-11, 2766) is gratefully acknowledged.Bonancía Roca, P.; Vayá Pérez, I.; Markovitsi, D.; Gustavsson, T.; Jiménez Molero, MC.; Miranda Alonso, MÁ. (2013). Stereodifferentiation in the intramolecular singlet excited state quenching of hydroxybiphenyl-tryptophan dyads. Organic and Biomolecular Chemistry. 11(12):1958-1963. https://doi.org/10.1039/c3ob27278hS195819631112Jiménez, M. C., Pischel, U., & Miranda, M. A. (2007). Photoinduced processes in naproxen-based chiral dyads. Journal of Photochemistry and Photobiology C: Photochemistry Reviews, 8(3), 128-142. doi:10.1016/j.jphotochemrev.2007.10.001Abad, S., Pischel, U., & Miranda, M. A. (2005). Wavelength-Dependent Stereodifferentiation in the Fluorescence Quenching of Asymmetric Naphthalene-Based Dyads by Amines. The Journal of Physical Chemistry A, 109(12), 2711-2717. doi:10.1021/jp047996aAbad, S., Vayá, I., Jiménez, M. C., Pischel, U., & Miranda, M. A. (2006). Diastereodifferentiation of Novel Naphthalene Dyads by Fluorescence Quenching and Excimer Formation. ChemPhysChem, 7(10), 2175-2183. doi:10.1002/cphc.200600337Bonancía, P., Vayá, I., Climent, M. J., Gustavsson, T., Markovitsi, D., Jiménez, M. C., & Miranda, M. A. (2012). Excited-State Interactions in Diastereomeric Flurbiprofen–Thymine Dyads. The Journal of Physical Chemistry A, 116(35), 8807-8814. doi:10.1021/jp3063838Paris, C., Encinas, S., Belmadoui, N., Climent, M. J., & Miranda, M. A. (2008). Photogeneration of 2-Deoxyribonolactone in Benzophenone−Purine Dyads. Formation of Ketyl−C1′ Biradicals. Organic Letters, 10(20), 4409-4412. doi:10.1021/ol801514vBelmadoui, N., Encinas, S., Climent, M. J., Gil, S., & Miranda, M. A. (2006). Intramolecular Interactions in the Triplet Excited States of Benzophenone–Thymine Dyads. Chemistry - A European Journal, 12(2), 553-561. doi:10.1002/chem.200500345Lhiaubet-Vallet, V., Boscá, F., & Miranda, M. A. (2007). Stereodifferentiating Drug−Biomolecule Interactions in the Triplet Excited State:  Studies on Supramolecular Carprofen/Protein Systems and on Carprofen−Tryptophan Model Dyads. The Journal of Physical Chemistry B, 111(2), 423-431. doi:10.1021/jp066968kVayá, I., Pérez-Ruiz, R., Lhiaubet-Vallet, V., Jiménez, M. C., & Miranda, M. A. (2010). Drug–protein interactions assessed by fluorescence measurements in the real complexes and in model dyads. Chemical Physics Letters, 486(4-6), 147-153. doi:10.1016/j.cplett.2009.12.091Seedher, N., & Bhatia, S. (2005). Mechanism of interaction of the non-steroidal antiinflammatory drugs meloxicam and nimesulide with serum albumin. Journal of Pharmaceutical and Biomedical Analysis, 39(1-2), 257-262. doi:10.1016/j.jpba.2005.02.031SEEDHER, N., & BHATIA, S. (2006). Reversible binding of celecoxib and valdecoxib with human serum albumin using fluorescence spectroscopic technique. Pharmacological Research, 54(2), 77-84. doi:10.1016/j.phrs.2006.02.008Nanda, R. K., Sarkar, N., & Banerjee, R. (2007). Probing the interaction of ellagic acid with human serum albumin: A fluorescence spectroscopic study. Journal of Photochemistry and Photobiology A: Chemistry, 192(2-3), 152-158. doi:10.1016/j.jphotochem.2007.05.018Zhou, B., Li, R., Zhang, Y., & Liu, Y. (2008). Kinetic analysis of the interaction between amphotericin B and human serum albumin using surface plasmon resonance and fluorescence spectroscopy. Photochemical & Photobiological Sciences, 7(4), 453. doi:10.1039/b717897bVahedian-Movahed, H., Saberi, M. R., & Chamani, J. (2011). Comparison of Binding Interactions of Lomefloxacin to Serum Albumin and Serum Transferrin by Resonance Light Scattering and Fluorescence Quenching Methods. Journal of Biomolecular Structure and Dynamics, 28(4), 483-502. doi:10.1080/07391102.2011.10508590Katrahalli, U., Kalalbandi, V. K. A., & Jaldappagari, S. (2012). The effect of anti-tubercular drug, ethionamide on the secondary structure of serum albumins: A biophysical study. Journal of Pharmaceutical and Biomedical Analysis, 59, 102-108. doi:10.1016/j.jpba.2011.09.013El-Kemary, M., Gil, M., & Douhal, A. (2007). Relaxation Dynamics of Piroxicam Structures within Human Serum Albumin Protein. Journal of Medicinal Chemistry, 50(12), 2896-2902. doi:10.1021/jm061421fTormo, L., Organero, J. A., Cohen, B., Martin, C., Santos, L., & Douhal, A. (2008). Dynamical and Structural Changes of an Anesthetic Analogue in Chemical and Biological Nanocavities. The Journal of Physical Chemistry B, 112(43), 13641-13647. doi:10.1021/jp803083yTardioli, S., Lammers, I., Hooijschuur, J.-H., Ariese, F., van der Zwan, G., & Gooijer, C. (2012). Complementary Fluorescence and Phosphorescence Study of the Interaction of Brompheniramine with Human Serum Albumin. The Journal of Physical Chemistry B, 116(24), 7033-7039. doi:10.1021/jp300055cVayá, I., Jiménez, M. C., & Miranda, M. A. (2007). Excited-State Interactions in Flurbiprofen−Tryptophan Dyads. The Journal of Physical Chemistry B, 111(31), 9363-9371. doi:10.1021/jp071301zCallis, P. R., & Burgess, B. K. (1997). Tryptophan Fluorescence Shifts in Proteins from Hybrid Simulations:  An Electrostatic Approach. The Journal of Physical Chemistry B, 101(46), 9429-9432. doi:10.1021/jp972436fLakowicz, J. R. (2000). On Spectral Relaxation in Proteins†¶‖. Photochemistry and Photobiology, 72(4), 421. doi:10.1562/0031-8655(2000)0722.0.co;2Schuler, B., & Eaton, W. A. (2008). Protein folding studied by single-molecule FRET. Current Opinion in Structural Biology, 18(1), 16-26. doi:10.1016/j.sbi.2007.12.003Shen, X., & Knutson, J. R. (2001). Subpicosecond Fluorescence Spectra of Tryptophan in Water. The Journal of Physical Chemistry B, 105(26), 6260-6265. doi:10.1021/jp010384vBeechem, J. M., & Brand, L. (1985). Time-Resolved Fluorescence of Proteins. Annual Review of Biochemistry, 54(1), 43-71. doi:10.1146/annurev.bi.54.070185.000355Callis, P. R. (1997). [7] 1La and 1Lb transitions of tryptophan: Applications of theory and experimental observations to fluorescence of proteins. Flourescence Spectroscopy, 113-150. doi:10.1016/s0076-6879(97)78009-1Basarić, N., & Wan, P. (2006). Competing Excited State Intramolecular Proton Transfer Pathways from Phenol to Anthracene Moieties. The Journal of Organic Chemistry, 71(7), 2677-2686. doi:10.1021/jo0524728Lukeman, M., & Wan, P. (2003). Excited-State Intramolecular Proton Transfer ino-Hydroxybiaryls:  A New Route to Dihydroaromatic Compounds. Journal of the American Chemical Society, 125(5), 1164-1165. doi:10.1021/ja029376yKeck, J., Kramer, H. E. A., Port, H., Hirsch, T., Fischer, P., & Rytz, G. (1996). Investigations on Polymeric and Monomeric Intramolecularly Hydrogen-Bridged UV Absorbers of the Benzotriazole and Triazine Class. The Journal of Physical Chemistry, 100(34), 14468-14475. doi:10.1021/jp961081hVollmer, F., & Rettig, W. (1996). Fluorescence loss mechanism due to large-amplitude motions in derivatives of 2,2′-bipyridyl exhibiting excited-state intramolecular proton transfer and perspectives of luminescence solar concentrators. Journal of Photochemistry and Photobiology A: Chemistry, 95(2), 143-155. doi:10.1016/1010-6030(95)04252-0Lukeman, M., & Wan, P. (2002). A New Type of Excited-State Intramolecular Proton Transfer:  Proton Transfer from Phenol OH to a Carbon Atom of an Aromatic Ring Observed for 2-Phenylphenol1. Journal of the American Chemical Society, 124(32), 9458-9464. doi:10.1021/ja0267831Jiménez, M. C., Miranda, M. A., Tormos, R., & Vayá, I. (2004). Characterisation of the lowest singlet and triplet excited states of S-flurbiprofen. Photochem. Photobiol. Sci., 3(11-12), 1038-1041. doi:10.1039/b408530bWeller, A. (1982). Photoinduced Electron Transfer in Solution: Exciplex and Radical Ion Pair Formation Free Enthalpies and their Solvent Dependence. Zeitschrift für Physikalische Chemie, 133(1), 93-98. doi:10.1524/zpch.1982.133.1.093Winget, P., Cramer, C. J., & Truhlar, D. G. (2004). Computation of equilibrium oxidation and reduction potentials for reversible and dissociative electron-transfer reactions in solution. Theoretical Chemistry Accounts, 112(4). doi:10.1007/s00214-004-0577-0ÇAKIR, S., & BÇER, E. (2010). SYNTHESIS, SPECTRAL CHARACTERIZATION AND ELECTROCHEMISTRY OF VANADIUM(V) COMPLEX WITH TRYPTOPHAN. Journal of the Chilean Chemical Society, 55(2). doi:10.4067/s0717-9707201000020002

Automatic Semantic Segmentation of the Lumbar Spine: Clinical Applicability in a Multi-parametric and Multi-centre Study on Magnetic Resonance Images

One of the major difficulties in medical image segmentation is the high variability of these images, which is caused by their origin (multi-centre), the acquisition protocols (multi-parametric), as well as the variability of human anatomy, the severity of the illness, the effect of age and gender, among others. The problem addressed in this work is the automatic semantic segmentation of lumbar spine Magnetic Resonance images using convolutional neural networks. The purpose is to assign a class label to each pixel of an image. Classes were defined by radiologists and correspond to different structural elements like vertebrae, intervertebral discs, nerves, blood vessels, and other tissues. The proposed network topologies are variants of the U-Net architecture. Several complementary blocks were used to define the variants: Three types of convolutional blocks, spatial attention models, deep supervision and multilevel feature extractor. This document describes the topologies and analyses the results of the neural network designs that obtained the most accurate segmentations. Several of the proposed designs outperform the standard U-Net used as baseline, especially when used in ensembles where the output of multiple neural networks is combined according to different strategies.Comment: 19 pages, 9 Figures, 8 Tables; Supplementary Material: 6 pages, 8 Table

Cellular photo(geno)toxicity of gefitinib after biotransformation

Gefitinib (GFT) is a selective epidermal growth factor receptor (EGFR) inhibitor clinically used for the treatment of patients with non-small cell lung cancer. Bioactivation by mainly Phase I hepatic metabolism leads to chemically reactive metabolites such as O-Demethyl gefitinib (DMT-GFT), 4-Defluoro-4-hydroxy gefitinib (DF-GFT), and O-Demorpholinopropyl gefitinib (DMOR-GFT), which display an enhanced UV-light absorption. In this context, the aim of the present study is to investigate the capability of gefitinib metabolites to induce photosensitivity disorders and to elucidate the involved mechanisms. According to the neutral red uptake (NRU) phototoxicity test, only DF-GFT metabolite can be considered non-phototoxic to cells with a photoirritation factor (PIF) close to 1. Moreover, DMOR-GFT is markedly more phototoxic than the parent drug (PIF = 48), whereas DMT-GFT is much less phototoxic (PIF = 7). Using the thiobarbituric acid reactive substances (TBARS) method as an indicator of lipid photoperoxidation, only DMOR-GFT has demonstrated the ability to photosensitize this process, resulting in a significant amount of TBARS (similar to ketoprofen, which was used as the positive control). Protein photooxidation monitored by 2,4-dinitrophenylhydrazine (DNPH) derivatization method is mainly mediated by GFT and, to a lesser extent, by DMOR-GFT; in contrast, protein oxidation associated with DMT-GFT is nearly negligible. Interestingly, the damage to cellular DNA as revealed by the comet assay, indicates that DMT-GFT has the highest photogenotoxic potential; moreover, the DNA damage induced by this metabolite is hardly repaired by the cells after a time recovery of 18 h. This could ultimately result in mutagenic and carcinogenic effects. These results could aid oncologists when prescribing TKIs to cancer patients and, thus, establish the conditions of use and recommend photoprotection guidelines

Modulation of the photobehavior of gefitinib and its phenolic metabolites by human transport proteins

The photobiological damage that certain drugs or their metabolites can photosensitize in proteins is generally associated with the nature of the excited species that are generated upon interaction with UVA light. In this regard, the photoinduced damage of the anticancer drug gefitinib (GFT) and its two main photoactive metabolites GFT-M1 and GFT-M2 in cellular milieu was recently investigated. With this background, the photophysical properties of both the drug and its metabolites have now been studied in the presence of the two main transport proteins of human plasma, i.e., serum albumin (HSA) and α1-acid glycoprotein (HAG) upon UVA light excitation. In general, the observed photobehavior was strongly affected by the confined environment provided by the protein. Thus, GFT-M1 (which exhibits the highest phototoxicity) showed the highest fluorescence yield arising from long-lived HSA-bound phenolate-like excited species. Conversely, locally excited (LE) states were formed within HAG, resulting in lower fluorescence yields. The reserve was true for GFT-M2, which despite being also a phenol, led mainly to formation of LE states within HSA, and phenolate-like species (with a minor contribution of LE) inside HAG. Finally, the parent drug GFT, which is known to form LE states within HSA, exhibited a parallel behavior in the two proteins. In addition, determination of the association constants by both absorption and emission spectroscopy revealed that the two metabolites bind stronger to HSA than the parent drug, whereas smaller differences were observed for HAG. This was further confirmed by studying the competing interactions between GFT or its metabolites with the two proteins using fluorescence measurements. These above experimental findings were satisfactorily correlated with the results obtained by means of molecular dynamics (MD) simulations, which revealed the high affinity binding sites, the strength of interactions and the involved amino acid residues. In general, the differences observed in the photobehavior of the drug and its two photoactive metabolites in protein media are consistent with their relative photosensitizing potentials

Investigation of metabolite-protein interactions by transient absorption spectroscopy and in silico methods

[EN] Transient absorption spectroscopy in combination with in silico methods has been employed to study the interactions between human serum albumin (HSA) and the anti-psychotic agent chlorpromazine (CPZ) as well as its two demethylated metabolites (MCPZ and DCPZ). Thus, solutions containing CPZ, MCPZ or DCPZ and HSA (molar ligand:protein ratios between 1:0 and 1:3) were submitted to laser flash photolysis and the Delta A(max) value at lambda = 470 nm, corresponding to the triplet excited state, was monitored. In all cases, the protein-bound ligand exhibited higher Delta Amax values measured after the laser pulse and were also considerably longer-lived than the non-complexed forms. This is in agreement with an enhanced hydrophilicity of the metabolites, due to the replacement of methyl groups with H that led to a lower extent of protein binding. For the three compounds, laser flash photolysis displacement experiments using warfarin or ibuprofen indicated Sudlow site I as the main binding site. Docking and molecular dynamics simulation studies revealed that the binding mode of the two demethylated ligands with HSA would be remarkable different from CPZ, specially for DCPZ, which appears to come from the different ability of their terminal ammonium groups to stablish hydrogen bonding interactions with the negatively charged residues within the protein pocket (Glu153, Glu292) as well as to allocate the methyl groups in an apolar environment. DCPZ would be rotated 180 degrees in relation to CPZ locating the aromatic ring away from the Sudlow site I of HSA. (C) 2019 Elsevier B.V. All rights reserved.Financial support from Ministerio de Economia, Industria y Competitividad (CTQ2016-78875-P, SAF2016-75638-R, BES-2011-043706), Generalitat Valenciana (Prometeo 2017/075), Xunta de Galicia [Centro Singular de Investigacion de Galicia accreditation 2016-2019 (ED431G/09, ED431B 2018/04) and post-doctoral fellowship to E. L.] and European Union (European Regional Development Fund-ERDF) is gratefully acknowledged. I. A. holds a "Miguel Servet" contract (CP1116/00052) funded by the Carlos III Health Institute. We are grateful to the Centro de Supercomputacion de Galicia (CESGA) for computational facilities.Limones Herrero, D.; Palumbo, F.; Vendrell Criado, V.; Andreu Ros, MI.; Lence, E.; González-Bello, C.; Miranda Alonso, MÁ.... (2020). Investigation of metabolite-protein interactions by transient absorption spectroscopy and in silico methods. 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