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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
Simultaneous Monitoring of Mitochondrial Temperature and ATP Fluctuation Using Fluorescent Probes in Living Cells Juan Qiao,†,⊥,# Chuanfang Chen,‡ Dihua Shangguan,†,⊥,# Xiaoyu Mu,§ Shutao Wang,∥,⊥,○ Lei Jiang,*,∥,⊥,○ and Li Qi*,†,⊥,#
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Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P.R. China ‡ Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China § College of Polymer Science & Engineering, Sichuan University, Sichuan 610065, P.R. China ∥ CAS Key Laboratory of Bio-inspired Materials and Interfacial Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, P.R. China ⊥ School of Chemical Sciences, University of Chinese Academy of Sciences, Beijing 100049, P.R. China S Supporting Information *
ABSTRACT: Real-time monitoring of the distribution of energy released during oxidative phosphorylation (OXPHOS) in living cells would advance the understanding of metabolic pathways and cell biology. However, the relationship between intracellular temperature and ATP fluctuation during the OXPHOS process is rarely studied due to the limitation of the sensing approach. Novel fluorescent polymer probes were developed for accurate simultaneous measurements of intracellular temperature and ATP. Utilizing the fluorescence imaging techniques, it was demonstrated for the first time that the temperature in mitochondria increased 2.4 °C and the ATP fluctuation level simultaneously decreased 75% within 2 min during the OXPHOS process. Moreover, the resultant fluorescent polymer probes had good performance and properties for mitochondrial targeting, providing an effective way for investigating mechanisms by which energy is released during the OXPHOS process.
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ntracellular energy release supports the function of many organelles and subcellular organelles. In mitochondria, oxidative phosphorylation (OXPHOS) is an important metabolic pathway by which enzymes oxidize metabolites, producing energy in the form of ATP.1−5 During the OXPHOS reaction, the ATP synthase (complex V) is the final enzyme in the OXPHOS pathway, and the reaction is shown below:
However, it is not known the degree to which temperature and ATP change during the OXPHOS process. Simultaneously measuring intracellular temperature and ATP production would further the understanding of cell biology and OXPHOS process. Efforts have been made to develop different fluorescent probes and to obtain information about energy released during OXPHOS. To address the requirements needed for measuring intracellular temperature and ATP, several fluorescent nanomaterials have been developed, including quantum dot,8,9 fluorescent polymer,10−13 gold nanoclusters,14−16 and fluorescent chemosensors.17−19 However, these probes can only report the temperature or ATP in living cells but not report the entire energy release maps or the relationship between the temperature and ATP in the OXPHOS process. This could be due to the unsatisfactory targeting and distribution properties of the reported materials.
ADP + Pi + 4H+intermembrane ↔ ATP + H 2O + 4H+matrix
The synthesis of ATP is an endergonic process, which requires an input of energy; the flow of electrons from electron donors to electron acceptors through the electron transport chain is an exergonic process, which releases energy.5 In simple terms, if the ADP is converted to ATP, the energy could be consumed; if the reaction is blocked and the ADP could not be converted to ATP, the energy could be released as heat, which would make the temperature increase in the mitochondria. Therefore, the synthesis of ATP is significantly affected by the temperature at which oxidative phosphorylation occurs.6,7 © XXXX American Chemical Society
Received: June 4, 2018 Accepted: September 28, 2018
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DOI: 10.1021/acs.analchem.8b02496 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry Although polymer based fluorescent nanomaterials have been constructed in previous works,20−23 simultaneous monitoring of temperature and ATP during the OXPHOS process has been technically challenging. Therefore, new multifunctional polymer based fluorescent probes which can target mitochondrial organelle and sense temperature and ATP fluctuations simultaneously are highly desirable and necessary. In this study, new fluorescent probes composed of a thermal sensitive polymer containing an intramolecular charge transfer dye (7-(diethylamino) coumarin-3-carbaldehyde) for temperature sensing (T sensing probe) and Rhodamine B derivative for ATP sensing (ATP sensing probe) were developed. The potential of the fluorescent probes for taking simultaneous temperature and ATP measurements during the OXPHOS process in living cells was demonstrated taking advantage of the high quantum yield and intramolecular charge transfer properties and the mitochondrial targeting properties of (4carboxybutyl) triphenylphosphonium bromide (CTPP).
Dynamic light scattering (DLS) was applied to the hydrodynamic diameter of PNIPAm-VBC-DACC changing with temperature, which was determined by a Zetasizer Nano ZS (Malvern Instruments). The PNIPAm-VBC-DACC (1.0 mg/mL) was equilibrated at each temperature for 2 min. The fluorescence intensity analysis of the samples was carried out on a fluorescent spectrophotometer (F-4500, Hitachi, Japan) equipped with a temperature controller. The fluorescence emission at 455 and 477 nm of the HeLa cells was measured by using a microplate reader (Model SpectraMax M5) equipped with a temperature controller. The LCST of PNIPAm-VBC-DACC was detected by the turbidity method using a UV−visible spectrophotometer (UV2450, Shimadzu, Japan) with a temperature controller (Shimadzu, Japan). The phase transition was evaluated by monitoring the transmittance at 500 nm with the temperature ranging from 28.0 to 42.0 °C. The LCST was defined as the temperature with 50% transmittance. Intracellular Temperature and ATP Sensing. The T and ATP sensing probes were utilized in HeLa cells for temperature and ATP monitoring. All of the fluorescence intensity measurement experiments of the cells were carried out on a microplate reader (Model SpectraMax M5). First, a line of HeLa cells formed confluent monolayers in the 96 well plate and incubated with the T sensing probe (0.5 mg mL−1, 20 min) and the ATP sensing probe (5.0 μM, 20 min). Then, the cells were washed 3 times using PBS buffer solution to remove all extracellular probes. The fluorescent emission ratio (454/477 nm) of the T sensing probe and fluorescence emission of the ATP sensing probe at 580 nm at different temperatures were obtained. For the FCCP and Ca2+ shock induced temperature changes, first, the T and ATP sensing probes were incubated with the HeLa cells. Then, FCCP (0.2 mM) or Ca2+ solution (10 nM) was incubated with the HeLa cells for 30 min before the ionomycin calcium complex solution (1.0 μM) was added. Finally, 2 min later, the time course of the relationship between the fluorescence intensity ratio (454 nm/477 nm) of the T sensing probe and the ATP sensing probe at 580 nm was determined.
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EXPERIMENTAL SECTION Chemicals and Materials. The chemical 4-vinylbenzyl chloride (4-VBC) which was applied for the synthesis of the thermosensitive polymer was purchased from Aladdin Reagents Industrial Co., Ltd. (Shanghai, China). 7-(Diethylamino) coumarin-3-carbaldehyde (DACC) was bought from Tianjin Heowns Biochem LLC (Tianjian, China). Dodecyl isobutyric acid trithiocarbonate (DDAT) was bought from Sigma (St. Louis, USA). Azo-bis-isobutyronitrile (AIBN) was obtained from Beijing Chemical Corporation (Beijing, China) and purified before using. N-Isopropylacrylamide (NIPAm) and Rhodamine B were obtained from Aladdin Reagents Industrial Co., Ltd. (Shanghai, China). 2-Aminobenzeneboronic acid (2-ABA) was supplied by Alfa Aesar (MA, USA). ATP was purchased from Ark Pharm Inc. (Illinois, USA). AMP, ADP, UMP, GMP, thymine, glucose, and sucrose were supplied by Sigma-Aldrich (St. Louis, MO). Pyridine, tetrahydrofuran (THF), dichloromethane (DCM), diethyl ether, and other chemicals were of analytical reagent grade purity and were supplied by Beijing Chemical Corporation (Beijing, China). (4-Carboxybutyl)triphenylphosphonium bromide (CTPP), phosphorus oxychloride (POCl3) 2-aminophenylboronic acid (ABA), and triethylamine were purchased from Beijing InnoChem Science &Technology Co., Ltd. (Beijing, China). Throughout the experiments, Milli Q water was used. Instruments and Measurements. The synthesis of T sensing and ATP sensing probes was introduced in the Supporting Information. The 1H NMR detection of PNIPAmVBC and PNIPAm-VBC-DACC was done using a Bruker DMX-400 spectrometer in DMSO. The molecular weight and polydispersity index (PDI) of the PNIPAm-VBC were measured by the gel permeation chromatography (GPC) method. The organic solution THF was used as the eluent for molecular determination, and the flow rate was 1.0 mL/min. A model L-2130 HPLC pump from Hitachi Co., a model 2410 refractive index detector from Waters Co., and a model 2487 ultraviolet detector from Waters Co. were used for the GPC experiment. The columns used for the molecular determination were a combination of MZ-Gel SDplus columns (5 μm, porosity of 103, 104, and 105 Å) from MZ-Analysentechnik GmbH (Mainz, Germany), and the polystyrene standards (MW of 1.3, 3.3 5.2, 13.0, and 25.0 K) were used for calibration.
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RESULTS AND DISCUSSION The fluorescent polymer based T sensing probe was synthesized (Figures S1−S3, Supporting Information). The molecule weight (1.3 kDa) and the distribution index (1.2) of the polymer were investigated using gel permeation chromatography, which demonstrated the successful synthesis of the polymer. The composition of the synthesized polymer was then determined using 1H NMR (Figures S4A and S4B, Supporting Information). To determine the temperature in organelles, CTPP (a mitochondrial targeting agent) was immobilized on the fluorescent polymer chains (Figure S3, Supporting Information).24−27 The lower critical solution temperature (LCST) of the T sensing probe was detected using a turbidity method, with temperatures ranging from 28.0 to 42.0 °C (Figure 1A). The LCST was calculated to be 32.2 °C, which indicated that the polymer had a suitable temperature range for measuring the variations in temperature in living cells. The diameter of the T sensing probe increased from 100.0 to 350.0 nm when the temperature changed from 30.0 to 42.0 °C (Figure 1B). Reversibility results demonstrated that the average fluorescence intensity ratio (I454 nm/I477 nm) of the T B
DOI: 10.1021/acs.analchem.8b02496 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
sensing probe changed linearly with temperature between 30.8 and 38.8 °C, and the correlation coefficient was 0.984 (Figure 2B). To test the thermal responsive performance of the T sensing probe in an intracellular environment, the effect of electrolytes, pH, and proteins was investigated. The results demonstrated that under physiological intracellular conditions, the thermal responsive performance of the T sensing probe was less affected by the environment in living cells (Figures S5A-S5C, Supporting Information). Moreover, the effect of different buffer solutions and FCCP on T and ATP sensing probes has been investigated in detail (Figures S5D and S5E). To determine the amount of ATP in mitochondria of living cells, a derivative of Rhodamine B was used as an ATP sensing probe.28 The synthesis and characterization of the ATP sensing probe are shown in Figure S6 (Supporting Information). Phenylboronic acid was used as the affinity site and introduced into Rhodamine B linked at the ortho-site, to produce an ATP sensing probe. The formation of the closed ring structure makes it nonfluorescent. In the presence of ATP, the ring structure opens and produces fluorescence. It was hypothesized that the covalent bond between boronic acid and ribose, p-p interaction between xanthene and adenine, and electrostatic interactions between amino and phosphate groups could contribute to the opening of the ring in the ATP sensing probe, which generated strong fluorescence. The composition of the synthesized ATP sensing probe was determined using 1H NMR (Figure S7, Supporting Information). Initially, the fluorescence change of the ATP sensing probe with ATP fluctuation was tested (Figure 3). The ATP sensing
Figure 1. Change of transmittance (A), diameter (B), cycling capability (C), and contact angle (D) with temperature change suggested the thermosensitive properties of the T sensing probe within a biological temperature range. The T sensing probe was 5.0 mg mL−1.
sensing probe varied with temperature for six cycles, indicating good cycling capability between 28.0 and 42.0 °C (Figure 1C). Moreover, the thermal sensitive property of the T sensing probe was also demonstrated using the water contact angle which changed with the temperature. The contact angle of the T sensing probe at room temperature was 33.7 ± 2.1° and 56.1 ± 4.4° at 42.0 °C (Figure 1D), respectively. These results suggested that the T sensing probe could change with a variation in temperature. The change in fluorescent properties of the T sensing probe with temperature was investigated to confirm the suitability of the polymer for intracellular nanothermometry. At an excitation wavelength of 365 nm, a fluorescence emission spectrum of the T sensing probe at different temperatures ranging from 30.8 to 40.9 °C was detected (Figure 2A). The
Figure 3. Fluorescence spectra (A) and intensity ratio (B) of the ATP sensing probe responding to ATP at intracellular concentrations (0.0−2.0 mM). F/F0 represents the fluorescence intensity ratio (580 nm), and F0 is the initial fluorescence intensity (580 nm) of the ATP sensing probe. Conditions: λex = 488 nm, ATP sensing probe = 10 μM, Krebs buffer solution (pH 7.8)/ glycerol (40:60, v/v). The ATP sensing probe was 5.0 μM.
Figure 2. (A) Fluorescence spectra of the T sensing probe at different temperatures ranging from 30.8 to 40.9 °C. (B) The fluorescence intensity ratio of R (454/477 nm) changed with temperature. The insert one is the responsive calibration plot for the quantitative determination of temperature. The T sensing probe was 5.0 mg mL−1 in Krebs buffer solution (pH 7.8)/glycerol (40:60, v/v).
probe was dissolved in Krebs buffer solution (pH 7.8)/glycerol (40:60, v/v), to mimic the pH and viscosity inside mitochondria.29 The ATP sensing probe (10.0 μM) had a weak fluorescence, and the addition of different concentrations of ATP resulted in increased fluorescence (Figure 3A). The quantum yields of the ATP probe was 39.6% using Rhodamine B as the reference standard. After the ATP binding, quantum yields of the ATP probe changed to 66.2%. In a range of 10.0 μM to 2.0 mM ATP, the fluorescence intensity increased linearly. The linear relationship was determined, and the correlation coefficient was 0.973 (Figure 3B). Owing to the larger steric hindrance of phenylboronic acid at the ortho position, the fluorescent open ring structure of the ATP sensing probe easily formed, and the fluorescence intensity increased significantly.
results revealed that with an increase in temperature, the fluorescence intensity of the T sensing probe at 477 nm decreased initially, and the wavelength blue-shifted to 454 nm. After which, the fluorescence intensity at 454 nm increased with a temperature increase. This wavelength blue-shift property of the fluorescent dye may be a result of extended p-conjugation and strong intramolecular charge transfer from the donor benzyl group. With an increase in temperature, the fluorescence intensity ratiometric signal of the T sensing probe at 454 to 477 nm increased, as shown in Figure 2A. The average fluorescence intensity ratio (I454 nm/I477 nm) of the T C
DOI: 10.1021/acs.analchem.8b02496 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry The selectivity of the ATP sensing probe for ATP compared to other ATP homologues, metal ions, and some biomolecules was investigated (Figure S8, Supporting Information). The fluorescence intensity of the ATP sensing probe increased 5.0fold in the presence of 2.0 mM ATP, and no significant increase in fluorescence was observed after addition of biomolecules, metal ions, and carbohydrates. Notably, ADP and AMP, which usually compete with ATP in detection methodologies, resulted in a slight fluorescence intensity increase. The ATP sensing probe had good selectivity for ATP compared to other biomolecules. As shown in Figure 4, the composite T sensing probe and the ATP sensing probe could simultaneously determine
Figure 5. Intracellular localization of the ATP sensing probe and the T sensing probe in HeLa cells which indicated the successful mitochondria targeting. Live HeLa cells were prestained with the T sensing probe (0.5 mg mL−1, 20 min) and the ATP sensing probe (5.0 μM, 20 min), respectively, and further treated with Mito-Tracker Green (0.7 μM, 20 min). Green: Mito-Tracker green; blue: T sensing probe; red: ATP sensing probe. Scale bar: 100 μm.
sensing probe (5.0 μM), termed Mix-1, Mix-2, and Mix-3, were tested, and the results suggested that the probes affected the viability of the cells only slightly over a 3 h incubation when compared to untreated cells, suggesting good biocompatibility (Figure S9A, Supporting Information). A Resazurin assay to assess mitochondrial viability at different temperatures in the presence of the T probe has been studied. The results revealed that the toxcicity of the T probe at different temperatures was not obvious, which demonstrated that the T probe was suitable for temperature sensing (Figure S9B, Supporting Information). To demonstrate the suitability of the probes for intracellular temperature and ATP determination during OXPHOS, a calibration experiment was performed with HeLa cells. After incubation with HeLa cells in serum-free cell culture medium at 28.0 °C for 20 min, the probes were efficiently taken up by the HeLa cells via endocytosis (Figure 5). For temperature sensing, the ratios of the fluorescence intensity of the T sensing probe changed with temperatures ranging from 32.0 to 42.0 °C. The results (Figure S10, Supporting Information) showed that the emission ratios of the fluorescence intensity (454/477 nm) changed with temperature linearly. The linear relationship was calculated to be Y = 0.08X − 1.73 with the temperature ranging from 32.0 to 42.0 °C (correlation coefficient R2 =0.996). The positive charge of the T sensing probe and the ATP sensing probe could result in the probes aggregating. The temperature resolution of the T sensing probe in the HeLa cells was calculated as previously described6 (Figure S11). The temperature resolution was smaller than 0.98 ± 0.08 °C over the temperature range 32.0 to 42.0 °C. For intracellular ATP sensing, the applicability of the ATP sensing probe to monitor mitochondrial ATP fluctuation in living cells was investigated. The HeLa cells which were incubated with the ATP sensing probe produced a strong fluorescence in the red channel (559 nm−590 nm). The fluctuation in the ATP level was demonstrated by a change in fluorescence intensity at 580 nm (Figure 3B and Figure 6B). The application of the proposed probes for monitoring mitochondrial temperature and ATP simultaneously changing in living cells was tested. After the cells were incubated with T and ATP sensing probes, the fluorescence changed in the HeLa cells, indicating a change in temperature and ATP. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an
Figure 4. Schematic illustration of the temperature and ATP sensing mechanism of the T and ATP sensing probes.
temperature and ATP fluctuation. As the temperature increased, the thermosensitive moiety in the T sensing probe changed from hydrophilic to hydrophobic, and the fluorescent properties of the T sensing probe changed with the surrounding polarity. Simultaneously, a fluctuation in ATP was reported by the ATP sensing probe. Moreover, due to the CTPP moiety of the T sensing probe and the positive charge of the ATP sensing probe, the composite could successfully realize the mitochondria targeted in living cells. The ability of the T sensing probe and the ATP sensing probe to target mitochondria was investigated in HeLa cells. The HeLa cells stained with the T sensing probe (0.5 mg mL−1, 20 min) and the ATP sensing probe (5.0 μM, 20 min) were costained with the commercially available mitochondrial dye Mito-Tracker Green (0.7 μM, 20 min) in serum-free culture medium. The imaging results (Figure 5) demonstrated that the blue image for the T sensing probe channel (λex = 400 nm) and the red image for the ATP sensing probe channel (λex = 488 nm) were similar to that of the green image for the Mito-Tracker green channel (λex = 514 nm). The strong fluorescence signal of the T sensing probe and the ATP sensing probe colocalized with the Mito-Tracker Green in mitochondria (Pearson’s correction coefficient of 0.95 and 0.94, respectively), suggesting the suitability of the T sensing probe and the ATP sensing probe to target mitochondria. The specific mitochondrial distribution of temperature and the ATP sensing probe could be the result of cooperation of the negative charge in the mitochondrial membrane and the positive charge in the probes. To test the biocompatibility of the T sensing probe and the ATP sensing probe, their cellular toxicity in HeLa cells was investigated using a CCK-8 assay. Differing concentrations of the T sensing probe (0.3 to 0.8 mg mL−1) and the ATP D
DOI: 10.1021/acs.analchem.8b02496 Anal. Chem. XXXX, XXX, XXX−XXX
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increased and ATP decreased and then remained slightly changed across 30 min, which was similar to the FCCP induced temperature and ATP experiments. Therefore, these results provide evidence that temperature and ATP variations during the OXPHOS process can be inhibited by drugs and toxins.
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CONCLUSIONS In this work, ratiometric fluorescent polymer probes were developed for simultaneous temperature and ATP sensing during the OXPHOS process. Cell imaging studies were performed to characterize the organelles-target property of the prepared probes. The measurements of mitochondrial temperature and ATP fluctuation were performed with the probes. The energy release simultaneously created an increase in temperature and a decrease in the ATP level in mitochondria during the OXPHOS process. The probes could be powerful tools for mapping the distribution of energy release and understanding mitochondrial OXPHOS processes that involve thermogenesis in cells.
Figure 6. Mitochondrial temperature (A) and ATP (B) fluctuation response to FCCP inhibition in HeLa cells. The temperature was calculated by the calibration plot in Figure S10 (Supporting Information). The ATP fluctuation was estimated by the fluorescence intensity changing. Live HeLa cells were prestained with the T sensing probe (0.5 mg mL−1, 20 min) and the ATP sensing probe (5.0 μM, 20 min). The intensity data was obtained from 25 live HeLa cells.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b02496.
inhibitor of OXPHOS under glucose starvation conditions, was incubated with HeLa cells. Initially, the prepared T and ATP sensing probes were incubated with the HeLa cells, and the fluorescence signal was measured 2 min after FCCP was added for 30 min. A time course of the relationship between the fluorescence intensity ratios (454/477 nm) and temperature was performed (Figure 6A), and the fluorescent emission of the ATP sensing probe at 580 nm was recorded (Figure 6B). The temperature calculated from the fluorescence ratio of the T sensing probe increased, and the fluorescence intensity of the ATP sensing probe at 580 nm decreased significantly (Figure 6), which indicated a simultaneous decrease in ATP with an increase in temperature. The intracellular temperature was approximately 32.6 °C before the addition of FCCP and increased to approximately 35.0 °C within 2 min of FCCP treatment. Then the temperature gradually decreased as the cells shrank. Simultaneously, there was an approximate 75% decrease in ATP during the first 2 min, and then the level of ATP remained unchanged in the following period. Figure 6 also shows that the T and ATP sensing probes did not affect each other and compared well to other reported fluorescent polymer-based thermosensitive materials (Table S1 in the Supporting Information) and ATP sensing probes (Table S2 in the Supporting Information). To investigate the effect of the FCCP incubation time on temperature and ATP, cell culture containing FCCP was removed after 30 min incubation with the HeLa cells, and then the temperature and ATP were measured. The results (Figure S12) showed that the intracellular temperature approximately returned to the normal temperature before FCCP induced, and ATP remained at a lower level across 30 min. Furthermore, the reagents which cause a change in intracellular temperature were also investigated. Calcium ion shock was used for heat production in living cells. After the T sensing probe and the ATP sensing probe had prestained the HeLa cells, an ionomycin calcium complex was incubated with the cells for 2 min. The results (Figure S12) exhibited that the intracellular temperature
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Synthesis of T and ATP sensing probes, 1H NMR spectroscopy data, and other characterization details (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (L.Q.). *E-mail:
[email protected] (L.J.). ORCID
Dihua Shangguan: 0000-0002-5746-803X Shutao Wang: 0000-0002-2559-5181 Li Qi: 0000-0001-8549-7287 Present Addresses #
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P.R. China. ○ CAS Key Laboratory of Bioinspired Materials and Interfacial Science, Technical Institute of Physics and Chemistry, Chinese Academy of Science, Beijing 100190, P.R. China. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 91732103, 21475137, 21575144, 21375132, 21635008, 21621062) and Chinese Academy of Sciences (QYZDJ-SSW-SLH034).
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DOI: 10.1021/acs.analchem.8b02496 Anal. Chem. XXXX, XXX, XXX−XXX
