Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation

Abstract

The killing effect of nitrogen-doped titanium dioxide (N-TiO2) nanoparticles on human cervical carcinoma (HeLa) cells by visible light photodynamic therapy (PDT) was higher than that of TiO2 nanoparticles. To study the mechanism of the killing effect, the reactive oxygen species produced by the visible-light-activated N-TiO2 and pure-TiO2 were evaluated and compared. The changes of the cellular parameters, such as the mitochondrial membrane potential (MMP), intracellular Ca^sup 2+^, and nitrogen monoxide (NO) concentrations after PDT were measured and compared for N-TiO2- and TiO2-treated HeLa cells. The N-TiO2 resulted in more loss of MMP and higher increase of Ca^sup 2+^ and NO in HeLa cells than pure TiO2. The cell morphology changes with time were also examined by a confocal microscope. The cells incubated with N-TiO2 exhibited serious distortion and membrane breakage at 60 min after the PDT.

Li et al. Nanoscale Research Letters 2013, 8:96 http://www.nanoscalereslett.com/content/8/1/96

NANO EXPRESS Open Access

Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation

Zheng Li1, Xiaobo Pan1, Tianlong Wang1, Pei-Nan Wang1, Ji-Yao Chen2 and Lan Mi1*

Abstract

The killing effect of nitrogen-doped titanium dioxide (N-TiO2) nanoparticles on human cervical carcinoma (HeLa) cells by visible light photodynamic therapy (PDT) was higher than that of TiO2 nanoparticles. To study the mechanism of the killing effect, the reactive oxygen species produced by the visible-light-activated N-TiO2 and pure-TiO2 were evaluated and compared. The changes of the cellular parameters, such as the mitochondrial membrane potential (MMP), intracellular Ca2+, and nitrogen monoxide (NO) concentrations after PDT were measured and compared for N-TiO2- and TiO2-treated HeLa cells. The N-TiO2 resulted in more loss of MMP and higher increase of Ca2+ and NO in

HeLa cells than pure TiO2. The cell morphology changes with time were also examined by a confocal microscope. The cells incubated with N-TiO2 exhibited serious distortion and membrane breakage at 60 min after the PDT.

Keywords: Nitrogen-doped TiO2, Visible-light-activated, Photodynamic therapy, Reactive oxygen species

Background

In recent years, semiconductor titanium dioxide (TiO2)

was noticed as a potential photosensitizer in the field of photodynamic therapy (PDT) due to its low toxicity, high stability, excellent biocompatibility, and photoreactivity [1-4]. The electrons in the valence band of TiO2 can be excited to the conduction band by ultraviolet (UV) radiation with the wavelength shorter than 387 nm (corresponding to 3.2 eV as the band gap energy of anatase TiO2), thus resulting in the photoinduced hole-electron pairs. These photoinduced electrons and holes can interact with surrounding H2O or O2 molecules and generate various reactive oxygen species (ROS, such as superoxide anion radical O2 [5], hydroxyl radical OH [6], singlet oxygen 1O2 [7], and hydrogen peroxide H2O2 [8]), which can react with biological molecules, such as lipids, proteins, and DNA, cause their damages, and eventually kill cancer cells [1,9,10].

However, the pure TiO2 can only be excited by UV light which is harmful and hinders its practical applications

[11]. Fortunately, recent studies have reported that the optical absorption of TiO2 in the visible region could be improved by doping [12-14] or dye-adsorbed methods [15,16], which will facilitate the application of TiO2 as a photosensitizer for PDT. In our previous study [10], we enhanced the visible light absorption of TiO2 by nitrogen doping and found that the nitrogen-doped TiO2 (N-TiO2) showed much higher visible-light-induced photokilling effects on cancer cells than the pure TiO2.

Although great efforts have been made to prepare doped TiO2 with visible light absorption, the underlying mechanism of the killing effects of photoactivated TiO2 on cancer cells has not yet been investigated in details. It is unclear how the TiO2 interacts with the cancer cells, and what are the differences for their photokilling effects between pure and doped TiO2. For possible medical applications of N-TiO2, it is of crucial importance to understand the killing effect of N-TiO2 on cancer cells and the mechanism of cell damages induced by PDT.

As ROS has been claimed to be of major importance for various kinds of PDT [17-20], the time-dependent ROS productions during visible light irradiation were evaluated in this work for both N-TiO2 and TiO2 in aqueous suspensions. The productions of different ROS species, such as O2, H2O2, and OH, were also studied.

* Correspondence: mailto:[email protected]

Web End [email protected]

1Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Department of Optical Science and Engineering, Shanghai Ultra-Precision Optical Manufacturing Engineering Center, Fudan University, 220 Handan Road, Shanghai 200433, ChinaFull list of author information is available at the end of the article

2013 Li et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0

Web End =http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Furthermore, a systematic comparison of the intracellular parameters with N-TiO2 and TiO2 nanoparticles as photosensitizers for PDT was investigated. The changes of mitochondrial membrane potential (MMP), intracellular Ca2+, and nitrogen monoxide (NO) concentrations with time after the PDT were measured. The relationships between these parameters were discussed. The morphological changes of cytoskeletons after irradiation were also examined by a confocal microscope at different times after the PDT. The killing effects between pure and nitrogen-doped TiO2 were compared.

Methods

Preparation and characterization of N-TiO2 samplesThe details of preparation of N-TiO2 nanoparticles were described in our previous paper [10]. Briefly, The anatase TiO2 nanoparticles (particle size <25 nm; Sigma-Aldrich,

St. Louis, MO, USA) were calcined at a flow rate of3.5 L/min in ammonia atmosphere at 550C for 20 min to produce the N-TiO2 nanoparticles. The crystalline phases of the N-TiO2 nanoparticles were determined by Raman spectra to be anatase. The ultraviolet-visible (UV/Vis) diffuse reflectance absorption spectra (Additional file 1: Figure S1) of the N-TiO2 and TiO2 samples were measured with a Jasco V550 UV/Vis spectrophotometer (Jasco, Inc., Tokyo, Japan).

Pure and N-doped TiO2 nanoparticles were autoclaved and dispersed in DMEM-H medium at a concentration of 100 g/ml, respectively. The samples were ultrasonicated for 15 min before using.

Cell culture and PDT treatmentThe human cervical carcinoma cells (HeLa) procured from the Cell Bank of Shanghai Science Academy were grown in Petri dishes in DMEM-H solution supplemented with 10% fetal calf serum in a fully humidified incubator at 37C with 5% CO2 for 24 h.

The cells were incubated with 100 g/ml pure or N-doped TiO2 under light-free conditions for 2 h and were then illuminated with a visible light filtered by a bandpass filter (400 to 440 nm) from a Xe lamp (100-W; Olympus, Center Valley, PA, USA) at a power density of 40 mW/cm2 for 5 min. The transmission spectrum of that bandpass filter was shown in Additional file 2: Figure S2. As shown in the figure, the filter could transmit some light with the wavelength below 400 nm. Therefore, the pure TiO2 could still absorb a small amount of the transmitted light.

Measurement of ROS induced by TiO2 or N-TiO2 in aqueous suspensionsFor the measurement of photo-induced ROS in TiO2 or

N-TiO2 aqueous suspensions, 20,70-dichlorfluorescein (DCFH), was used as a probe. The DCFH was converted

from the diacetate form DCFH (DCFH-DA) (Sigma-Aldrich) by adding 0.5 ml of 1 mM DCFH-DA in methanol into 2 ml of 0.01 N NaOH and keeping the mixture at room temperature in the dark for 30 min. It was then neutralized with 10 ml sodium phosphate buffer (pH = 7.2) [21]. Pure or N-doped TiO2 in phosphate buffered saline (PBS, 100 g/ml) were mixed with DCFH (25 M) before visible light irradiation. The non-fluorescent DCFH can rapidly react with ROS to form fluorescent 20,70-dichlorofluorescein (DCF). By measuring the fluorescent intensity, the production of ROS could be estimated.

To measure the generations of specific ROS, two probes were used respectively. Dihydrorhodamine 123 (DHR) is mainly sensitive to O2 [22] and H2O2 [23], and 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]-benzoic acid (APF) is selectively sensitive to OH [23]. It was already demonstrated that the reactive species H2O2,

O2, and 1O2 did not cause any modification in the fluorescence of the probe APF [24]. Pure or N-doped TiO2 in

PBS (100 g/ml) were mixed with DHR (25 M, Sigma-Aldrich) or APF (10 M, Cayman Chemical, Ann Arbor, MI, USA) before irradiation. Upon oxidation, the non-fluorescent DHR or APF is converted to the highly fluorescent Rhodamine 123 or fluorescein.

After the samples were irradiated by a visible light (400 to 440 nm) with a power density of 40 mW/cm2 for different times ranging from 1 to 5 min, the fluorescence spectra were recorded by a spectrometer (F-2500, Hitachi, Brisbane, CA, USA) and the fluorescent intensities were compared.

MMP assayRhodamine 123 [2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester] (Beyotime, Jiangsu, China), which could bind specifically to the mitochondria, was used to estimate the MMP. When MMP is decreased, the dye could be released from the mitochondria and the fluorescence vanished. The PDT-treated cells were incubated with Rhodamine 123 (5 g/ml) for 30 min in the dark at 37C and then were washed with Dulbeccos PBS (D-PBS) for three times before the visible light illumination.

Measurement of Ca2+ concentrationTo study the intracellular calcium concentration, HeLa cells were loaded with 10 M Fluo-3 AM (Beyotime) for 30 min at 37C and followed by washing with D-PBS for three times. Then the cells were incubated for another 20 min to ensure complete cleavage of Fluo-3 AM by the intracellular ester enzyme that releases Fluo-3 before the illumination.

Measurement of intracellular NOThe intracellular NO level was detected using a NO-sensitive fluorescence probe DAF-FM DA [3-amino, 4-

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aminomethyl-20,70-difluorescein, diacetate] (Beyotime). The cells were loaded with 10 M DAF-FM DA at 37C in the kit buffer for 20 min and were then gently washed with D-PBS for three times and incubated for another 20 min to ensure that the intracellular DAF-FM DA was completely catalyzed to form DAF-FM by ester enzyme before the illumination.

Cell morphology and cytoskeleton observationThe HeLa cells were fixed with 4% paraformaldehyde for 15 min at room temperature with different time intervals after the illumination. Then they were permeabilized with 0.025% Triton X-100 in D-PBS (Sigma-Aldrich Corp., St. Louis, MO, USA) for 2 min. After washing with D-PBS three times, the cells were treated with 1% bovine serum albumin (BSA) for 2 h at 4C. The fixed cells were stained with 5 nM Alexa FluorW 488 phalloidin conjugate (Invitrogen, Eugene, OR, USA) for F-actin labeling for 40 min at 37C. Meanwhile, 1% BSA was added to the staining solution to reduce nonspecific background staining. The cells were washed with 0.05% PBS-Tween20 three times before microscopic observation.

Microscopy and image analysisThe fluorescence images of cells were observed by a laser scanning confocal microscope (FV-300, IX71; Olympus, Tokyo, Japan) using a 488-nm continuous wave Ar+ laser (Melles Griot, Carlsbad, CA, USA) as the excitation source and a 60 water objective to focus the laser beam. A 505- to 550-nm bandpass filter was used for the fluorescence images. Each experiment was repeated three times independently.

The fluorescence intensities of MMP, Ca2+, and NO

probes from the microscopic images were analyzed with the Olympus Fluoview software. The data were expressed in terms of the relative fluorescence intensity of the probes and expressed as mean SD. The fluorescence intensity was averaged from 100 to 150 cells for each experiment.

Results and discussion

Generation of ROS by pure and N-doped TiO2 in aqueous suspensionsThe generations of ROS induced by TiO2 or N-TiO2 nanoparticles in aqueous suspensions under visible light irradiation were studied using the fluorescence probes as described in the Methods section. The fluorescence intensities with the irradiation times ranging from 1 to 5 min were shown in Figure 1a. The fluorescence intensities of both TiO2 (the black line) and N-TiO2 (the red line) samples increased with irradiation time but the fluorescence intensities of N-TiO2 samples were always higher than that of the TiO2 ones. It means that N-TiO2 could generate more ROS than TiO2 under visible light

irradiation, which agrees well with the spectral result that N-TiO2 showed higher visible light absorption than

TiO2 (see Additional file 1: Figure S1, where a shoulder was observed at the edge of the absorption spectra, which extended the absorption of N-TiO2 from 380 to 550 nm).

The major reactions for the formation of ROS upon illumination of TiO2 have been proposed as follows [25]:

TiO2 hv Eg

[C0][notdef]h e 1

H2O hOH H 2 2OHH2O2 3

O2 e O2 4

O2 h1O2 5

H2O2 O2 OH OH O2 6

OH is mainly formed in the reaction of photogenerated holes with surrounding water, while O2 is formed in the reaction of photogenerated electrons with dissolved oxygen molecules. Some O2 can form 1O2 by reacting with the holes. Moreover, some OH can form H2O2, and the

reactions of H2O2 can also result in the formation of OH with a lesser extent.

Since DCFH is a nonspecific ROS probe, it is necessary to further analyze the specific ROS. As described above, DHR and APF were used to evaluate the generation of O2/H2O2 and OH, respectively. The fluorescence measurements in Figure 1b,c shows that all the specific ROS increased with the irradiation time, but the N-TiO2 induced more O2/H2O2 (Figure 1b) while less

OH (Figure 1c) than TiO2. It was reported that the photogenerated holes of N-TiO2 were trapped in the N 2p levels and had a very low mobility [26], thus were barely involved in the photocatalysis when the N-TiO2 was illuminated by visible light [27]. In this study, the lower production of OH from N-TiO2 might result from the same reason. However, the photogenerated electrons in the conduction band can react with oxygen molecules to generate O2, which is thermodynamically favored [28]. Thus,

N-TiO2 could generate more O2 /H2O2 than the pure TiO2 due to the higher visible light absorption efficiency.

When cells were treated with TiO2 or N-TiO2 nano-particles, the nanoparticles were not only found on the cell membrane but also in the cytoplasm, and some of them aggregated around or in Golgi complexes and even in nuclei [10]. As the TiO2 or N-TiO2 nanoparticles can induce

ROS under visible light irradiation, the photokilling effect on cancer cells was observed in our previous work [10]. Considering that the productions of the specific ROS

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Figure 1 Comparison of ROS induced by TiO2 and N-TiO2. Fluorescence measurements as a function of irradiation time to compare the productions of ROS and specific ROS in aqueous suspensions induced by TiO2 and N-TiO2: (a) total ROS, (b) O2/H2O2, and (c) OH .

species generated by TiO2 or N-TiO2 are different and the contributions from the specific ROS to PDT may also be different, the PDT-induced changes of the intracellular parameters, such as MMP, Ca2+, and NO concentrations in HeLa cells treated with TiO2 or N-TiO2 were studied as follows.

MMP changesWhen TiO2- or N-TiO2-treated cells were illuminated by light, the generated ROS may attack the mitochondria [29] or the activated nanoparticles may interact with the mitochondria directly [30], which would affect the function of mitochondria and cause the opening of mitochondrial permeability pores, resulting in the dissipation of MMP [30-32]. In this study, the MMP decreased immediately after the PDT as shown in Figure 2. It seems that the

mitochondrion is a very sensitive cellular organelle during the PDT, and the defects can be detected immediately in our study. For TiO2-treated cells, the MMP level decreased continuously after the PDT with an approximate rate of 1.2% per min within 60 min. The MMP level for N-TiO2 samples dropped much faster (around 4.2%

per min) within the first 10 min after the PDT, then decreased at slower and slower rate within 45 min, and almost kept in a constant rate of 20% after 45 min. However, the MMP levels of control cells and the cells incubated with TiO2 and N-TiO2 under light-free conditions did not show any change during 60 min (data not shown), which confirmed the low cytotoxicity of TiO2 and N-TiO2.

It should be noted that the MMP level of N-TiO2-

treated cells decreased 3.5 times faster than that of the TiO2-treated cells at the beginning after the PDT.

Figure 2 MMP of HeLa cells as a function of the time after the PDT. Cells were incubated with 100 g/ml TiO2 (white square)

or N-TiO2 (black circle) for 2 h and illuminated by the visible light for 5 min. The averaged fluorescence intensity of control cells (white triangle) at 0 min was set as 100%.

Figure 3 Time-dependent changes of the intracellular Ca2+

levels after the PDT. The averaged fluorescence intensity of control cells (white triangle) was set as 100%. TiO2 (white square)- or N-TiO2 (black circle)-treated cells (100 g/ml) were incubated under light-free conditions for 2 h and illuminated by the visible light for 5 min.

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Figure 4 Changes of the intracellular NO levels as a function of the time after the PDT. The averaged fluorescence intensity of control cells (white triangle) was set as 100%. TiO2 (white square)- or

N-TiO2 (black circle)-treated cells were incubated with 100 g/ml under light-free conditions for 2 h before the irradiation.

Compared with Figure 1c that there were considerably more OH induced by TiO2 than N-TiO2 under visible light, it strongly suggested that the hydroxyl radicals with the rather shorter lifetime and lower diffusion length than O2 and H2O2 [33] might contribute less on the damage of mitochondria among a variety of ROS in PDT.

Intracellular Ca2+ concentrationIt has been reported that some signal transduction pathways were activated by PDT [34]. Calcium expression level was one of the concerning principal factor since it is an important link between the pathways. The

activation of Ca2+ was also known as a contributor to the cell morphological and functional changes associated with apoptosis [35]. The raise of intracellular calcium levels would result in various changes of cellular metabolism as well as the cell morphology.

The time-dependent intracellular Ca2+ concentrations after the PDT were measured as shown in Figure 3. The detectable increase of the intracellular Ca2+ levels for TiO2 samples was first observed at 15 min after the

PDT, while that for N-TiO2 samples, it was observed at the first measurement point of 5 min after the PDT. Comparing the data in Figure 3 with that in Figure 2, we can see the elevation of Ca2+ followed by the loss of MMP. To demonstrate the correlativity of Ca2+ and

MMP, the starting times of the detectable increase of Ca2+ were marked as two red squares in Figure 2. It suggests that a certain amount of the MMP loss (about 24% 5%) would cause the detectable increase of Ca2+.

As shown in Figure 3, the Ca2+ levels for both TiO2 and N-TiO2 samples reached the maximum values at about 45 min after the PDT, where N-TiO2 induced release of Ca2+ at around 2.1-fold than TiO2 did. Since there was no calcium ion in the D-PBS solution, the detected Ca2+ might be released from the damaged calcium stores, such as mitochondria and possibly other organelles, and flow into the cytoplasm through ion channels [36].

This result agreed with the data of MMP changes. The MMP levels of N-TiO2 decreased around 3.5 times faster than that of TiO2 at the early time after the PDT, which means the N-TiO2 induced damage of mitochondria was more serious. Therefore, the released Ca2+ could be observed earlier and the Ca2+ levels were higher in N-TiO2 samples as compared to the TiO2 samples.

Figure 5 The morphology and cytoskeleton of HeLa cells at different time points after the PDT. (a) Control cells. (b) TiO2-treated cells. (c) N-TiO2-treated cells (scale bar, 20 m). Cells were incubated with 100-g/ml TiO2 or N-TiO2 under light-free conditions for 2 h before the PDT and then fixed at 15 min and 60 min after the PDT, respectively. The cells were stained with Alexa FluorW 488 phalloidin for F-actin.

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Generation of NOThe cells have defense mechanisms such as the endogenous generation of NO, which can scavenge a certain amount of ROS and protect cells from ROS attack [32,36,37]. The change of the NO level after the PDT was also detected in this work. The intracellular NO levels of N-TiO2 samples increased faster than that of the TiO2 ones (Figure 4), the former increased from 100% (as control cells) to 141% in 60 min after the PDT, while the latter increased to 121% only. It means that more NO was generated to buffer the increased ROS under higher oxidative stress for N-TiO2 samples although TiO2 induced higher amount of OH. This result also suggested that the OH species played a less important role among a variety of ROS in the PDT. Taken the above findings together, it suggested that the ROS overwhelmed the antioxidant defense capacity of NO in the cells, although NO could buffer the ROS to a certain extent. The remaining ROS would become highly harmful and lead to irreversible cellular damage.

Cell morphology and cytoskeleton defectsThe cell morphology images of HeLa cells at different times after the PDT were acquired by a confocal microscope with the labeled F-actin. No morphology and cytoskeleton defects were found at 15 min after the PDT for both TiO2 and N-TiO2 samples (Figure 5b,c, upper images).

At 60 min after the PDT, the organization of actin cyto-skeleton of the cells incubated with TiO2 seemed disrupted (Figure 5b, lower image), while the cells incubated with N-TiO2 exhibited serious distortion and membrane breakage (Figure 5c, lower image).

As ROS can be generated around TiO2 or N-TiO2, the nanoparticles near the cell membranes may directly cause cell membrane damage by biochemical reactions. Additionally, the PDT-induced defect of mitochondria and the release of Ca2+ into the cytoplasm might trigger cell apoptosis or necrosis, which may result in the cell morphology and cytoskeleton defects eventually. As the cytoskeleton is involved in many intracellular signaling pathways, the cytoskeletal distortion and shrinkage need to be further studied for a long observation time in future studies.

Conclusions

A comparison of the killing effects between N-TiO2 and TiO2 on HeLa cells with visible light irradiation was conducted. N-TiO2 produced more ROS and specifically more O2/H2O2 under visible light irradiation. Contrarily, more OH were produced by TiO2. The MMP levels were sensitive in the PDT, and rapid loss of MMP was detected at the very beginning after the PDT as one of the earliest detectable biochemical changes in this study. A certain amount of MMP loss (around 24%) was followed

by the detectable increase of Ca2+ (at 5 and 15 min after the PDT for N-TiO2 and TiO2, respectively). The increase of NO was detected later than the other intracellular parameters, which indicates that the NO generation was caused by the generation of ROS. The N-TiO2 resulted in more loss of MMP and higher increase of Ca2+ and NO in HeLa cells and, finally, induced more cell damages than pure TiO2. At 60 min after irradiation, significant cytoskeletal shrinkage and breakage were observed for N-TiO2-

treated cells, whereas for TiO2-treated cells, only slight damage was demonstrated. Overall, N-TiO2 can induce more cell damages than pure TiO2. The hydroxyl radicals might contribute less to the cell damages among a variety of ROS.

Additional files

http://www.biomedcentral.com/content/supplementary/1556-276X-8-96-S1.tiff

Web End =Additional file 1: Figure S1. Absorbance spectra of TiO2 and N-TiO2 nanoparticles. Description: A shoulder was observed at the edge of the absorption spectra, which extended the absorption of N-TiO2 from380 nm to 550 nm.

http://www.biomedcentral.com/content/supplementary/1556-276X-8-96-S2.tiff

Web End =Additional file 2: Figure S2. The transmission spectrum of the 400 to 440 nm bandpass filter. Description: The filter could transmit some light with the wavelength below 400 nm, which could be absorbed by the pure TiO2 as shown in Additional file 1: Figure S1.

Competing interestsThe authors declare that they have no competing interests.

Authors contributionsZL carried out the experiments and drafted the manuscript. XP and TW participated in the confocal microscopy imaging. PW supervised the work, participated in the discussion of the results and in revising the manuscript. JC participated in the discussion of the results. LM designed the project and wrote the manuscript. All authors read and approved the final manuscript.

AcknowledgmentsThis work is supported by the National Natural Science Foundation of China (61008055, 11074053), the Ph.D. Programs Foundation of Ministry of Education of China (20100071120029), and the Key Subjects Innovative Talents Training Program of Fudan University.

Author details

1Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Department of Optical Science and Engineering, Shanghai Ultra-Precision Optical Manufacturing Engineering Center, Fudan University, 220 Handan Road, Shanghai 200433, China. 2State Key Laboratory of Surface Physics, Department of Physics, Fudan University, 220 Handan Road, Shanghai 200433, China.

Received: 14 December 2012 Accepted: 29 January 2013 Published: 22 February 2013

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doi:10.1186/1556-276X-8-96Cite this article as: Li et al.: Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation. Nanoscale Research Letters 2013 8:96.

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