A mitochondria-targeted ratiometric probe for the fluorescent and colorimetric detection of SO2 derivatives in live cells

Journal of Luminescence 192 (2017) 297–302

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A mitochondria-targeted ratiometric probe for the fluorescent and colorimetric detection of SO2 derivatives in live cells Yongfei Wanga, Qingtao Menga, Run Zhanga,b, Hongmin Jiaa, Cuiping Wanga, Zhiqiang Zhanga, a b

MARK ⁎

School of Chemical Engineering, University of Science and Technology Liaoning, 185#, Qianshan Zhong Road, Anshan 114044, PR China Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane 4072, Australia

A B S T R A C T An abnormal endogenous SO2 level can induce toxicological effects leading to cancer, cardiovascular diseases, and neurological disorders, as they involve in various physiology and pathological processes in mitochondria cell apparatus. In this study, we report a ratiometric fluorescent probe (NAB-Id) based on the cyanine dye for the detection of sulfur dioxide (SO2) derivatives in aqueous media and live cells. Upon the addition of SO2 derivatives (SO32−/HSO3−) to the solution of NAB-Id, led to the nucleophilic addition to the polymethine, resulting in dramatic colorimetric and fluorescence dual responses. NAB-Id responses to HSO3− with low detection limit (58.6 nM), outstanding specificity (without the interfering of H2S), fast response (t1/2 = 30 s) and working well at physiological pH. Fluorescence co-localization studies demonstrated that NAB-Id was a mitochondria-targeted fluorescent probe for the real-time sensing and bioimaging of SO2 derivatives in living cells.

1. Introduction Sulfur dioxide (SO2) as an industrial waste, has been considered to be a toxic environmental pollutant over the past few decades. [1,2] For example, SO2 derivatives were widely used in pharmaceutical industries as enzyme inhibitor, antimicrobial agent, and can be also used in food industries as anti-oxidant and antibacterial agents. [3–5] Long term exposure to SO2 not only causes some respiratory responses, but also induces cancers, cardiovascular diseases and neurological disorders. [1–2,6] Accordingly, the Joint FAO/WHO Expert Committee on Food Additives has issued that an acceptable daily intake of sulfites should be lower than 0.7 mg kg−1 of body weight. [7] Furthermore, endogenous SO2 can regulate cardiovascular structure and function such as lowering blood pressure, relaxing blood vessels and a negative inotropic effect in the heart. [8–10] Mitochondria is a vital organelle, which is the main source of sulfur-containing species (SCS). Endogenous SO2 can be generated in mitochondria of cells during oxidation of hydrogen sulfide (H2S), sulfur-containing amino acids and decomposition of sulfinylpyruvate to pyruvate in mammals. [11–15] Therefore, monitoring SO2 derivatives in mitochondria environments is highly important for better understanding of its biological functions. Fluorescence probe can be recognized powerful tools for the realtime sensing and imaging in life science and materials science, providing useful information in a short times. [16–21] Among several types of fluorescence probe, ratiometric ones, which can provide built-in self-

⁎

Corresponding author. E-mail addresses: [email protected] (R. Zhang), [email protected] (Z. Zhang).

http://dx.doi.org/10.1016/j.jlumin.2017.06.033 Received 18 January 2017; Received in revised form 22 May 2017; Accepted 16 June 2017 Available online 19 June 2017 0022-2313/ © 2017 Elsevier B.V. All rights reserved.

calibration for correction of a variety of analyte-independent factors, have attracted particular attention for analytical sensing and optical imaging with the potential to provide a precise and quantitative analysis. [22] In the past several years, many novel ratiometric fluorescent probes have been developed for the detection of SO2 derivatives in living cells. [23–44] Although these probes display good responses to SO2 derivatives, the specifically detect mitochondrial SO2 and its derivatives in living cells is still scarcely reported. [45–50] In addition, although some of the SO2 derivatives probes appeared a fast response time toward sulphite, but they suffered great interference by another reactive sulfur, such as H2S. [38,46] Herein, three α,β-unsaturated compounds (NAB-Id, NJB-ID and NHB-ID) based on cyanine dyes have been designed as fluorescence probes for the detection of SO3 2−/HSO3− on the basis of nucleophilic addition reaction (Scheme 1). It has been found that a strong electronwithdrawing group at the o-position of a polymethine promotes the Michael addition reaction significantly. [45,46] Accordingly, amino groups were introduced in the probes to improve their sensitivity and shorten the response time. It was envisioned that the nucleophilic attack of SO3 2−/HSO3− toward the probes will interrupt the π-conjugation and block the ICT process, and, as a result, distinct colorimetric and fluorescence dual responses could be obtained.

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NJB-Id in 45% yield (189.45 mg). 1H NMR (DMSO-d6, 600 MHz) δ 8.32 (d, J = 8.5 Hz, 1H), 8.25 (d, J = 15.3 Hz, 1H), 8.16 (d, 8.5 Hz, 2H), 7.92 (d, J = 8.8 Hz, 1H), 7.73 (t, J = 7.7 Hz, 3H), 7.61 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 15.3 Hz, 1H), 4.60 (m, 2H), 3.44 (t, J = 6.0 Hz, 4H), 2.76 (t, J = 6.2 Hz, 4H), 1.93 (m, 10H), 1.41 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 179.19, 155.17, 139.08, 135.92, 132.57, 131.14, 130.43, 128.48, 127.65, 126.30, 122.99, 112.64, 112.44, 107.52, 96.67, 52.35, 45.16, 27.45, 13.17. ESI-HRMS (m/z): 421.2638 for [C30H33N2]+, found 421.2640. Elemental analysis: calculated: C, 85.47; H, 7.89; N, 6.64. Found: C, 85.48; H, 7.82; N, 6.62.

Scheme 1. Structure of the probe NAB-Id, NJB-ID, and NHB-ID.

2. Experimental 2.1. Reagents and instruments

2.4. Synthesis of NHB-ID 1,1,2-Trimethylbenz[e]indole, iodoethane, piperidine, 6-hydroxy-2naphthaldehyde were purchased from Sinopharm Chemical Reagent Co., Ltd. (China); MitoTracker® Green, 3-(4,5-dimethylthiazol-2yl)−2,5-diphenyltetrazolium bromide (MTT), Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin, and streptomycin sulfate were purchased from Life Technologies. 3-ethyl-1,1,2-trimethyl-1H-benzoeindolium iodide was synthesized by the reported method. Unless otherwise stated, all chemical materials were purchased from commercial sources and used without further purification. Deionized water was used throughout. 1 H NMR and 13C NMR spectra were recorded with an AVANCE500MHZ spectrometer (BRUKER) with chemical shifts reported as ppm (in DMSO, TMS as internal standard). API-ES high resolution mass spectra were recorded on a HP1100LC/MSD spectrometer. The elemental analyses of C, H, N and O were performed on a Vario EL III elemental analyzer. Fluorescence spectra were determined with LS 55 luminescence spectrometer (Perkin Elmer, USA) with excitation and emission slits of 10 nm. The absorption spectra were measured with a Lambda 900 UV/VIS/NIR spectrophotometer (Perkin Elmer, USA). Fluorescent living cell images were acquired on an Olympus Fluoview FV 1000 IX81 inverted confocal laser-scanning microscope equipped with 405, 473, 559 and 635 nm laser diodes. The relative fluorescence intensity and colocalization of images were analysed by using Image J software version 1.44p.

3-Ethyl-1,1,2-trimethyl-1H-benzoeindolium iodide (249 mg, 1.05 mmol) was dissolved in 20 mL EtOH, followed by the addition of 4-(diethylamino)salicylaldehyde (193.2 mg, 1 mmol) and one drop of piperidine. The mixture was then heated to reflux overnight. After cooling the mixture to R. T., the formed precipitate was filtered and rinsed with 20 mL of cooled EtOH, and dried under vacuum to obtain NHB-Id in 50% yield (206.5 mg). 1H NMR (DMSO-d6, 600 MHz) δ 11.05 (s, 1 H), δ 8.53 (d, J = 15 Hz, 1H), 8.35 (d, J = 6 Hz, 1H), 8.19 (d,5 Hz, 1H),8.13 (d, 10 Hz, 1H), 8.04 (s, 1H), 7.92 (t, J = 10 Hz, 1H), 7.71 (t, J = 5 Hz, 1H), 7.60 (d, J = 5 Hz, 1H), 7.23 (s, 1H), 6.53 (d, J = 10 Hz, 1H), 6.25 (s, 1H), 4.52 (m, 2H), 3.49 (m, 4H), 1.96 (s, 6H), 1.41 (t, J = 5 Hz, 3H), 1.18 (t, J = 5 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 178.18, 152.71, 149.07, 138.36, 135.49, 132.01, 130.48, 129.79, 127.85, 127.00, 125.67, 122.36, 121.55, 121.43, 112.01, 101.80, 55.86, 51.90, 49.82, 26.69, 26.23, 20.35, 18.39, 13.09. ESI-HRMS (m/ z): 413.2587 for [C28H33N2O]+, found 413.2640. Elemental analysis: calculated: C, 81.32; H, 8.04; N, 6.77; Found: C, 81.28; H, 8.12; N, 6.72. 2.5. Determination of the detection limit The detection limit was calculated based on the fluorescence titration (Fig. 3d) of NAB-Id in the presence of NaHSO3 (1–5 μM). The fluorescence intensity of NAB-Id was measured by three times and standard deviation of the blank measurement was achieved. The detection limit was calculated with the following equation: [51,52]

2.2. Synthesis of NAB-Id

Detection limit (DL) = 3σ/ k

3-Ethyl-1,1,2-trimethyl-1H-benzoeindolium iodid e46 (249 mg, 1.05 mmol) was dissolved in 20 mL EtOH, followed by the addition of 4-dimethylaminobenzaldehyde (172.2 mg, 1 mmol) and one drop of piperidine. The mixture was then heated to reflux overnight. After cooling the mixture to R. T., the formed precipitate was filtered and rinsed with 20 mL of cooled EtOH, and dried under vacuum to obtain NAB-Id in 55% yield (185 mg). 1H NMR (DMSO-d6, 600 MHz) δ(ppm): 8.45 (s, 1H), 8.42 (s, 1H), 8.22 (d, J = 7.5 Hz, 1H), 8.16 (d, J = 7.0 Hz, 1H), 8.12 (d, J = 7 Hz, 2H), 7.99 (d, J = 7.5 Hz, 1H), 7.75 (t, J = 6.5 Hz, 1H), 7.64 (d, J = 6 Hz, 1H), 7.30 (d, J = 13 Hz, 1H), 6.89 (d, J = 7 Hz, 2H), 4.66 (m, 2H), 3.18 (s, 6H), 2.00 (s, 6H); 1.44 (t, J = 6 Hz, 3H) 13C NMR (DMSO-d6, 150 MHz) δ (ppm): 179.31, 153.81, 153.90, 138.87, 138.62, 133.1, 137.90, 135.54, 133.61, 132.03, 129.82, 128.92, 152.97 137.77, 135.95, 131.90, 130.18, 129.40, 127.58, 126.42, 125.69,122.10, 111.92, 111.58, 103.49, 55.41, 52.04, 43.16, 40.12, 25.58, 12.84. ESI-HRMS (m/z): 369.2325 for [C28H26NO]+, found 369.2333. Elemental analysis: calculated: C, 84.51; H, 7.91; N, 7.58. Found: C, 84.58; H, 7.85; N, 7.51.

where σ is the standard deviation of the blank measurement, k is the slope between the fluorescence ratios (F467 nm/F611 nm) versus NaHSO3 concentration. 2.6. General procedures of spectra detection Stock solutions of NAB-Id was prepared in PBS aqueous buffer (DMSO: H2O = 3:7, 50 mM, pH = 7.4). Excitation wavelength for NAB-Id, NJB-Id and NHB-ID were 405 nm, 540 nm. Before spectroscopic measurements, the solution was freshly prepared by diluting the high concentration stock solution to corresponding solution (10 μM). Solution of sulphite and bisulfate were prepared by dissolving of Na2SO3 or NaHSO3 in deionized water. The mixture was then stirred at R. T for 5 min. A 3 mL solution of NAB-Id ( NJB-Id and NHB-ID) and analytes were filled in a 1 cm quartz cell and applied for each of the spectroscopic test. 2.7. Living cell imaging experiments

2.3. Synthesis of NJB-ID Human breast cancer cells (MCF-7) were purchased from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). The cells were seeded in 24-well flat-bottomed plates and then incubated for 24 h at 37 °C under 5% CO2. Before imaging, the living cells

3-Ethyl-1,1,2-trimethyl-1H-benzoeindolium iodide (249 mg, 1.05 mmol) was dissolved in 20 mL EtOH, followed by the addition of 9-Julolidinecarboxaldehyde (201.2 mg, 1 mmol) and one drop of piperidine. The mixture was then heated to reflux overnight. After cooling the mixture to R. T., the formed precipitate was filtered and rinsed with 20 mL of cooled EtOH, and dried under vacuum to obtain 298

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were incubated with NAB-Id (5 μM) for another 30 min and then washed with PBS aqueous buffer (DMSO: H2O = 3:7, pH = 7.4) three times. Fluorescence imaging was performed using an OLYMPUSFV1000 inverted fluorescence microscope with a 60 × objective lens. Under the confocal fluorescence microscope, NAB-Id was excited at 405 nm and emission was collected at 450–510 nm (green channel) and 585–645 nm (red channel). For the detection of NaHSO3, MCF-7 cells were incubated with NAB-Id (5 μM) at 37 °C for 30 min and then NaHSO3 (20 equiv.) was added at 37 °C for 15 min.

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3. Results and discussion

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3.1. Synthesis and characterization of Cyanine derivatives

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Cyanine dyes were chosen as the fluorophore due to their excellent photochemical and photophysical properties, such as long-wavelength absorption and emission, high fluorescence quantum yield, high stability against light and large Stokes shift. [53] Cyanine derivatives were facilely synthesized by coupling of 3-ethyl-1,1,2-trimethyl-1H-benzoeindolium iodide with benzaldehydes derivatives in ethanol. The structure of intermediates and target products were confirmed by 1H NMR, 13C NMR and HRMS (Fig. S1-9, ESI†).

ab c de f g h i j k l mno pq r s t u Fig. 2. Ratiometric fluorescence enhancement (F467 nm/F611 nm) of NAB-Id (10 µM) towards various anions and biothiols (1 mM) in PBS aqueous buffer (DMSO: H2O = 3:7, 50 mM, pH = 7.4). The anions and molecules are: a, blank; b, SO4 2–; c, HCO3–; d, CH3COO–; e, SCN–; f, HPO4 2–; g, NO2–; h, F–; i, Cl–; j, Br–; k, I–; l, NO3–;m, HSO4–; n, CO3 2– ;o, ClO–; and reactive sulfur( s, S 2– (300 µM); p, Cys (1 mM); q, Hcy (1 mM); r, GSH (1 mM); t, SO3 2– (50 µM); u, HSO3– (50 µM). Insert is the image under natural light and 365 nm UV lamp of NAB-Id in the presence of different anions and molecules. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. Spectroscopic studies of NAB-Id toward SO2 derivatives in aqueous medium

; HCO3–; AcO–; SCN–; HPO4 2–; NO2–; F–; Cl–; Br–; I–; NO3–; HSO4–; CO3 ; ClO–. In addition, treatment of NAB-Id with 300 μM S2- and 1 mM of Cys, Hcy, and GSH showed negligible changes in fluorescence emission of NAB-Id. The Insert picture in Fig. 4 showed the changes in color and fluorescence color (under 365 nm UV lamp) of NAB-Id in the presence of various anions and molecules. No obvious difference in color and fluorescence color was noticed except the addition of HSO3– or SO32−, which indicated that the specific response of NAB-Id towards SO2 derivatives can be perceived even by the naked eyes. These results revealed that the ratiometric fluorescence response of NAB-Id towards SO2 derivatives is highly selective without interference of other species, which enable it to be used as the specific fluorescence probes for SO2 derivatives detection in complicated biological systems. To further investigate the sensitivity of NAB-Id towards HSO3−, the absorption and emission spectra were investigated in stimulated physiological media (10 µM DMSO:PBS buffer = 3:7, pH = 7.4). The free NAB-Id displayed a broad absorption band centered at 550 nm and a corresponding emission maximum at 611 nm with a moderate fluorescent intensity as shown in Fig. 3a, b, c. Upon the addition of an increasing amount of NaHSO3, the absorption intensity at 550 nm was gradually decreased accompanied by a new absorption band at 323 nm. Furthermore, the free NAB-Id probe showed intrinsic emission band at 611 nm, Treatment of HSO3− induces a marked emission decrease at 611 nm and concurrently a dramatic enhancement of the emission intensity at 467 nm (Fig. 3b, c). The results demonstrated that the emission intensity ratios of the NAB-Id could be amplified evidently upon the addition of HSO3−. Furthermore, the emission ratios (I467 nm /I611 nm) are linearly proportional to the amount of sulphite (1–5 μM) (Fig. 3d). According to the titration profiles, the detection limits (3σ/k) toward HSO3− was calculated to be 56.8 nM for NAB-Id (R 2 = 0.9920), which is enough for the mitochondrio-targeted work in the living cells. 2–

2–

The fluorescence responses of cyanine derivatives (NAB-Id, NJB-ID and NHB-ID) toward SO2 derivatives (SO3 2−/HSO3−) were firstly investigated. Fig. 1 shows the time-dependent (0−600 s) maximum emission changes in the presence of HSO3−. NAB-Id could promptly response to NaHSO3 within 90 s, which is favorable for real-time monitoring of HSO3− levels in living samples. Nevertheless, no obvious fluorescence changes were found for NJB-ID and NHB-ID within 10 min under the same conditions, demonstrating that NJB-ID and NHB-ID could not respond to SO2 derivatives. We suspected that the different distribution of the electron cloud in the two probes resulted in the different reactivity towards SO2 derivatives. Accordingly, only NAB-Id was investigated by fluorescence and absorption spectra in detail. The fluorescence selectivity of NAB-Id towards SO2 derivatives in PBS aqueous buffer (DMSO: H2O = 3:7, 50 mM, pH = 7.4) was investigated. The ratiometric fluorescence enhancement (F467 nm/F611 nm) of NAB-Id (10 µM) towards various anions and biothiols were shown in Fig. 2, no obvious changes in fluorescence intensity ratio (F467 nm /F611 nm) were noticed in the presence of 1 mM anions, including SO4

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Time (second) Fig. 1. Time course fluorescence responses of NAB-Id (■, 10 µM), NJB-Id (■, 10 µM) and NHB-Id (■, 10 µM) towards HSO3– (10.0 equiv.) in 50 mM PBS solution of pH = 7.40.

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Fig. 3. Changes in absorption spectra (a) and fluorescence spectra (b, c) of NAB-Id (10 M) in the presence of difference concentration of HSO3– (0–120 μM) in PBS aqueous buffer (DMSO: H2O = 3:7, 50 mM, pH = 7.4); (b) Excitation at 404 nm, slit: 10/10 nm; (c) excitation at 550 nm, slit: 10/20 nm. (D) Ratiometric fluorescence enhancement (F467 nm/F611 nm) of NAB-Id (10 M) as a function of HSO3– concentration (1–5 μM) in PBS buffer of pH 7.4.

fluorescence imaging HSO3− in live cells. The fluorescence imaging HSO3− was conducted by confocal laser-scanning microscopy measurement. The MCF-7 cells were incubated with a 5 μM solution of NAB-Id for 30 min at 37 °C in a CO2 incubator (95% relative humidity, 5% CO2). Then washed with PBS three times and mounted on a microscope stage. As shown in Fig. 4a, no obvious intracellular fluorescence signals in the green channel (450–510 nm) were collected by the confocal microscope FV1000. However, obvious fluorescence signals at the red channel of 585–645 nm were also gathered (Fig. 4b), which indicated that the probe NAB-Id had remarkable member permeability. Furthermore, when the MCF-7 cells pre-treated with NAB-Id and further incubated with 100 μM NaHSO3 at 37 °C for 15 min (Fig. 4f–j), the green fluorescence channel gradually became brightened and the red fluorescence channel became darkened, respectively. The results indicated that NAB-Id has fine resolution in bioimaging and can be used for monitoring HSO3− through the ratiometric fluorescence imaging in live cells.

3.4. Recognition mechanism We speculated that a 1,4-addition reaction occurred between NABId and HSO3– rather than a 1,2-addition reaction. The proposed reaction mechanism was firstly confirmed by the 1H NMR titration with different amounts of NaHSO3 in DMSO-d6-D2O solution (Fig. S12, ESI†), After treatment of NAB-Id with NaHSO3, all of the 1H NMR signals shifted to upfield due to the nucleophilic attack of HSO3– toward C4 interrupting ▯-▯ conjugation and weakening its electron-withdrawing characteristic. High-resolution mass spectroscopy was also performed to confirm the Michael addition of HSO3− to probe NAB-Id. As shown in the Fig. S13, the addition of NaHSO3 into the aqueous solution of NAB-Id, the peak of [NAB-Id]+ at m/z 369.2333 disappeared, where a new peak at m/z 449.1895 assigned to [NAB-Id-S-H]– was emerged. The results were in agreement with proposed sensing mechanism of NAB-Id depicted in Scheme 2.

3.5. Intracellular imaging 3.6. Mitochondrial-localization The excellent fluorescence properties of NAB-Id in stimulated physiological media encouraged us to study its application in

As reported in the literature, the indolium moiety not only can improve the water solubility of the probe but act as a mitochondriatargeted carrier. To prove whether NAB-Id could specifically stain in the mitochondria, intracellular co-localization experiment of NAB-Id with commercial mitochondrial tracker, Mito Tracker Green FM was conducted in MCF-7 cells. As shown in Fig. 5, obvious overlap between the red fluorescence of NAB-Id and the green fluorescence of Mito Tracker Green FM was observed in the cell line. Moreover, the Pearson's co-localization coefficient, used to describe correlation of the intensity

Scheme 2. The proposed sensing mechanism of the probe NAB-Id for HSO3−.

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Fig. 4. Confocal fluorescence imaging of MCF-7 cells incubated with NAB-Id (5 μM) for 30 min at 37 °C (Top) and then treated with 100 μM NaHSO3 at 37 °C for 15 min (Bottom): (a) Confocal image of NAB-Id (5 μM) on green channel (450−510 nm). (b) Confocal image of NAB-Id (5 μM) on red channel (585−645 nm). (c) Bright image. (d) Merged image of (a), (b), and (c). (e) Ratio image (green channel/red channel). (f) the emission was collected at 450–510 nm with green pseudocolor. (g) the emission was collected at 585–645 nm with red pseudocolor. (h) Bright image. (i) Merged image of (f), (g), and (h). (j) Ratio image (green channel/red channel). Excitation wavelength at 405 nm, scale bar = 20 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Conclusions

distribution between two channels, was calculated to be 0.95, indicating that the probe NAB-Id could localize in the mitochondria, where is one of the main organelle for generation of the endogenous SO2 in vivo, The results indicated that the potential application of NABId in monitoring endogenous SO2 in living cells.

In summary, a ratiometric fluorescent probe NAB-Id base on cyanine dye was developed for HSO3− sensing and imaging. NAB-Id exhibits colorimetric and ratiometric responses to HSO3− based on the nucleophilic addition mechanism. The sensing mechanism of NAB-Id towards SO2 derivatives were confirmed by 1H NMR titration and high resolving mass spectrum. The probe NAB-Id shows fast response, high

Fig. 5. Colocalization fluorescence imaging of MCF-7 cells incubated with NAB-Id (5 μM) for 30 min and Mito Tracker Green FM (1.0 μM) for another 10 min at 37 °C. (a) Confocal image from Mito Tracker Green FM on green channel (λex = 488 nm). (b) Confocal image from NAB-Id on red channel (λex = 405 nm). (c) Bright image. (d) Merged image of (a), (b) and (c). (e) Correlation plot of the intensities of NAB-Id and Mito Tracker Green FM (Rr = 0.95). (f), (g) Normalized intensity profile of regions of interest (ROIs) across MCF-7 cells. Scale bar = 20 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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