Connective tissue fibroblasts from highly regenerative mammals are refractory to ROS-induced cellular senescence

doi:10.1038/s41467-019-12398-w

. 2019 Sep 27;10(1):4400.

doi: 10.1038/s41467-019-12398-w.

Connective tissue fibroblasts from highly regenerative mammals are refractory to ROS-induced cellular senescence

Sandeep Saxena¹, Hemendra Vekaria^{2

3}, Patrick G Sullivan^{2

3

4}, Ashley W Seifert^{5

6}

Affiliations

¹ Department of Biology, University of Kentucky, Lexington, KY, 40506, USA.
² Department of Neuroscience, University of Kentucky, Lexington, KY, 40506, USA.
³ The Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY, 40506, USA.
⁴ Lexington VA Medical Center, Lexington, KY, 40506, USA.
⁵ Department of Biology, University of Kentucky, Lexington, KY, 40506, USA. awseifert@uky.edu.
⁶ The Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY, 40506, USA. awseifert@uky.edu.

PMID: 31562333
PMCID: PMC6764955
DOI: 10.1038/s41467-019-12398-w

Connective tissue fibroblasts from highly regenerative mammals are refractory to ROS-induced cellular senescence

Sandeep Saxena et al. Nat Commun. 2019.

. 2019 Sep 27;10(1):4400.

doi: 10.1038/s41467-019-12398-w.

Authors

Sandeep Saxena¹, Hemendra Vekaria^{2

3}, Patrick G Sullivan^{2

3

4}, Ashley W Seifert^{5

6}

Affiliations

¹ Department of Biology, University of Kentucky, Lexington, KY, 40506, USA.
² Department of Neuroscience, University of Kentucky, Lexington, KY, 40506, USA.
³ The Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY, 40506, USA.
⁴ Lexington VA Medical Center, Lexington, KY, 40506, USA.
⁵ Department of Biology, University of Kentucky, Lexington, KY, 40506, USA. awseifert@uky.edu.
⁶ The Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY, 40506, USA. awseifert@uky.edu.

PMID: 31562333
PMCID: PMC6764955
DOI: 10.1038/s41467-019-12398-w

Abstract

A surveillance system in mammals constantly monitors cell activity to protect against aberrant proliferation in response to damage, injury and oncogenic stress. Here we isolate and culture connective tissue fibroblasts from highly regenerative mammals (Acomys and Oryctolagus) to determine how these cells interpret signals that normally induce cellular senescence in non-regenerating mammals (Mus and Rattus). While H₂O₂ exposure substantially decreases cell proliferation and increases p53, p21, p16, and p19 in cells from mice and rats, cells from spiny mice and rabbits are highly resistant to H₂O₂. Quantifying oxygen consumption and mitochondrial stability, we demonstrate that increased intracellular H₂O₂ is rapidly detoxified in regenerating species, but overwhelms antioxidant scavenging in cells from non-regenerative mammals. However, pretreatment with N-acetylcysteine (NAC) protects mouse and rat cells from ROS-induced cellular senescence. Collectively, our results show that intrinsic cellular differences in stress-sensing mechanisms partially explain interspecific variation in regenerative ability.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1**
Fibroblasts from *Acomys*, *Rattus,* and *Oryctolagus* exhibit enhanced proliferative ability. a–d PDs for *Mus*, *Acomys*, *Rattus,*and *Oryctolagus* fibroblasts cultured under ambient (20%) and physiological (3%) O_2. a, b 3% O₂ enhances proliferative capacity of *Mus* and *Acomys* fibroblasts: *Mus* (n = 5) cell lines at 3% O₂: PDs = 20.8 ± 0.93, vs. 20% O₂: PDs = 4.4 ± 0.97 and *Acomys* (n = 5) cell lines at 3% O₂: PDs = 60 ± 3.4 vs. 20% O₂: PDs = 20.1 ± 0.34. c–d O₂ concentration did not affect mean proliferative ability of *Rattus* (n = 4) or *Oryctolagus* (n = 4) fibroblasts. e Cross-species comparison of proliferative ability at 3% O₂ shows that *Mus* cells still senesce while fibroblasts from *Acomys*, *Rattus,* and *Oryctolagus* proliferate for at least 140 days. f At 20% O₂, the proliferative population (EdU+) of P2 *Mus* cells (~25%) was significantly lower compared to *Acomys* (82%)*, Rattus* (95%) and *Oryctolagus* (95%) (ANOVA, F = 143.3982, P < 0.0001) (n = 4/species). Percent EdU + cells in *Acomys* were slightly lower compared to *Rattus* (Tukey-HSD, t = −4.38, P = 0.0043) and *Oryctolagus* (Tukey-HSD, t = −4.87, P = 0.0019) (n = 4/species). At 3% O_2, mean percent EdU + cells in P2 cultures are significantly lower in *Mus* (75%) compared to *Acomys* (88%)*, Rattus* (91%) and *Oryctolagus* (91%) (ANOVA, F = 17.3085, *Mus* vs. *Acomys*, P = 0.0003, *Mus* vs. *Rattus*, P = 0.0023 and *Mus* vs. *Oryctolagus*, P = 0.0002) (n = 4/species). g P2 cells co-labeled with EdU and the general fibroblast marker Vimentin demonstrate that > 95% of cell cultures from all four species are fibroblasts (n = 4/species). Scale bars = 50 µm. Graphics for *Acomys*, *Mus*, *and Rattus* were made by corresponding author and the *Oryctolagus* image is available free for comercial use. ***P < 0.0001, **P < 0.001 and *P < 0.05. Error bars = S.E.M. Source data are provided as a Source Data file

**Fig. 2**
In vitro resistance to senescence is not restricted to regenerating mammals. a, b In line with proliferative ability, cells cultured at 3% O₂ were more resistant to cellular senescence. Percent SA-βgal + cells were measured in *Mus*, *Acomys, Rattus,* and *Oryctolagus* (n = 4–5 species) at progressive passages. *Mus* cells senesced at P13 and > 90% of cultures were SA-βgal+. There were significantly more SA-βgal + cells compared to *Acomys* (~37%)*, Rattus* (~22%) and *Oryctolagus* (~4%) (P13 Tukey-HSD, *Mus* vs. *Acomys*, t = −23.81, P < 0.0001; *Mus* vs. *Rattus, t* = 29.29, P < 0.0001 *Mus* vs. *Oryctolagus, t* = 39.98, P < 0.0001). Red arrows indicate SA-βgal + cells (a). c *Acomys* and *Mus* fibroblasts (n = 3/species) from P2 co-labeled with γ-H2AX and EdU to differentiate senescent cells. Yellow arrows indicate nuclei positive for γ-H2AX and EdU and white arrows represent nuclei positive for EdU only. d–f P2 *Acomys* and *Mus* fibroblasts (n = 3) labeled with p16 (d), p53 (e), and p21 (f), and DAPI. Yellow arrows show marker positive cells while white arrows show negative cells. g Quantified cell counts for panels c–f. There were significantly more senescent cells in *Mus* cultures positive for: γ-H2AX+, p21+, p53+, and p16+ (Supplementary Table 4). Representative scale bars in panels a and c–f = 50 µm and 20 µm, respectively. ***P < 0.0001, **P < 0.001, *P < 0.05. Error bars = S.E.M. Source data are provided as a Source Data file

**Fig. 3**
H₂O₂ exposure does not induce senescence in *Acomys* fibroblasts. a–e P2 *Acomys* and *Mus* (n = 4/species) fibroblasts treated with sub-lethal doses of H₂O₂ (0 μM-control, 75 μM, 150 μM, and 300 μM) for 2 h and then cultured for 24 and 48 h. The horizontal line within the box represents the median sample value of the box plot. The ends of the boxes represent the 25th and 75th quantiles and the lines extending from each end of the boxes are whiskers representing the highest and lowest observations. a *Mus* fibroblasts showed no significant change in the percent EdU + cells compared to control after 24 h, but at 48 h experienced a significant decrease in response to 150 μM (Tukey-HSD, t = 4.71, P = 0.0019) and 300 μM H₂O₂ (Tukey-HSD, t = 7.94, P < 0.0001). No significant changes in *Acomys* EdU + cells at 24 h. A small, but significant decrease in percent EdU + cells after 48 h was detected in response to 300 μM H₂O₂ compared to control (Tukey-HSD_, t = 3.32, P = 0.0493). b H₂O₂ exposure had no effect on cellular senescence in *Mus* and *Acomys* fibroblasts after 24 h in culture. After 48 h in culture, *Mus* fibroblasts exhibited significant increases in SA-βgal + cells at all H₂O₂ concentrations, while *Acomys* fibroblasts registered a small, but significant increase at 300 μM only (Tukey-HSD, t = −3.75, P = 0.0189). c *Acomys* fibroblasts followed a similar trend for γ-H2AX + cells after H₂O₂ treatment while *Mus* fibroblasts significantly increased γ-H2AX + cells at 24 h in response to 300 μM H₂O₂ in addition to significant increases at 48 h in response to all concentrations. d Representative cultures for results in panels a–c stained with EdU and γ-H2AX. Scale bars = 20 µm. White arrows show double-positive cells and yellow arrows show γ-H2AX + senescent cells. e *Acomys* fibroblasts do not upregulate the senescence markers p16, p19, p53, and p21 in response to any concentration of H₂O₂. *Mus* fibroblasts show significant increases in these markers compared to *Acomys* in response to all H₂O₂ treatments after 48 h (Two-way ANOVA, p16, F = 783.3681, P < 0.0001; p19, F = 1094.501, P < 0.0001; p53, F = 399.1625, P < 0.0001; p21, F = 526.55, P < 0.0001) (Supplementary Tables 11-14). ***P < 0.0001, **P < 0.001, *P < 0.05. Error bars = S.E.M. Source data are provided as a Source Data file

**Fig. 4**
Fibroblasts from regenerating mammals are refractory to ROS-induced cellular senescence. a, b Fibroblasts from regenerating (*Acomys* and *Oryctolagus*) and non-regenerating mammals (*Mus* and *Rattus*) treated with increasing concentrations of H₂O₂ (0 μM-control, 75 μM, 150 μM and 300 μM) for 2 h and then cultured for 48 h in complete media (n = 4/species). a H₂O₂ induced a significant decrease in the percentage of proliferating cells (EdU+) in *Mus* at 150 μM (Tukey-HSD, t = 5.95, P < 0.0001) and 300 μM H₂O₂ (Tukey-HSD, t = 10.04, P < 0.0001), as well as in *Rattus* at 150 μM (Tukey-HSD, t = 8.25, P < 0.0001) and 300 μM H₂O₂ (Tukey-HSD, t = 12.88, P < 0.0001), a small but significant decrease in *Acomys* at 300 μM H₂O₂ (Tukey-HSD, t = 4.19, P = 0.0099) and no significant change at any H₂O₂ concentrations in *Oryctolagus* (Supplementary Table 15). The percentage of senescent cells (γ-H2AX+, SA-βgal+, p21+, and p53+) significantly increased in non-regenerating species and were unchanged in regenerating *Acomys* and *Oryctolagus* (Supplementary Tables 16–19). b Representative fibroblast cultures in panel a double-stained with p21 and p53 showing nuclear localization of these tumor suppressors in response to H₂O_2. Scale bars = 20 µm. ***P < 0.0001, **P < 0.001, *P < 0.05. Error bars = S.E.M. Source data are provided as a Source Data file

**Fig. 5**
Gamma irradiation induces DDR and cellular senescence in all species. a–d Cells from all four species were exposed to gamma radiation at 0 (control), 5, 15, and 30 Gy and fixed 6 h later (n = 4/species). The representative images show p21, p53, p16, and p19 positive cells in control (0 Gy) and after 30 Gy in *Mus* (a), *Rattus* (b), *Acomys* (c), and *Oryctolagus* (d). e Fibroblasts from regenerating species showed no significant increase in γ-H2AX + cells at 5 Gy, whereas all species demonstrated a significant increase in DNA damage at 15 and 30 Gy (Supplementary Table 22). Fibroblasts from all four species demonstrated a significant increase in p21+ and p53+ cells at all three radiation doses (Supplementary Tables 23–24). In contrast, fibroblasts from regenerating species showed minimal activation of p16 and p19 in response to gamma radiation, where fibroblasts from *Mus* and *Rattus* strongly activated p16 and p19 (Supplementary Tables 25–26). Scale bars in panels a–d = 20 µm. ***P < 0.0001, **P < 0.001, *P < 0.05. Error bars = S.E.M. Source data are provided as a Source Data file

**Fig. 6**
Mitochondria from regenerating species are resilient to H₂O₂. a Mitochondria stress testing measured as OCR (pmol/min/μg protein) for fibroblasts from all species after control (PBS) and 300 μM H₂O₂ treatment. Mitochondrial complex inhibitors were used to generate measures for basal respiration, maximal respiration, ATP production, and spare respiratory capacity. b Measured OCR was converted to percent normalized OCR and we found that all measured parameters were significantly decreased in fibroblasts from non-regenerating species: basal respiration (ANOVA, F = 15.8779, P < 0.0001), ATP production (ANOVA, F = 25.2168, P < 0.0001), maximal respiration (ANOVA, F = 20.2582, P < 0.0001), and spare respiratory capacity (ANOVA, F = 15.0920, P < 0.0001) (n = 5/species). c The mitochondria were isolated from all the species after 0 μM H₂O₂ (control) or 300 μM H₂O₂ treatment (n = 3/species). Mitochondria assays were performed on a XF96 analyzer using ADP + pyruvate/malate and mitochondrial inhibitors oligomycin, FCCP and Antimycin A. State III respiration was achieved through ADP stimulated respiration whereas State IV respiration was calculated after oligomycin inhibition. The Respiratory Control Rate (RCR) was calculated as the ratio of State III and State IV respiratory rate. The OCR for State III and RCR was significantly decreased after H₂O₂ treatment in purified mitochondria of non-regenerators while no significant effect was shown by regenerators: State III respiration (ANOVA, F = 19.1281, P < 0.0001) and RCR (ANOVA, F = 19.1558, P < 0.0001). Graphics for fibroblast and mitochondria were adapted from open source material. ***P < 0.0001, **P < 0.001, *P < 0.05. Error bars = S.E.M. Source data are provided as a Source Data file

**Fig. 7**
Regenerators rapidly detoxify exogenous hydrogen peroxide via increased GPx activity. a Representative images for a single live-imaged cell transfected with HyPer which captured every 30 min over 6 h. b Fluorescence intensity ratios (F/F0) were calculated for >20 cells/cell lines for all four species (n = 3 lines/species). A linear model was used to detect H₂O₂ detoxification rate and ANOVA was used to detect significant differences between species. F/F0 quantified 30 min after H₂O₂ treatment were not significantly different between species (ANOVA, F = 1.2153, P < 0.3651 and Supplementary Table 34). H₂O₂ was detoxified significantly faster in *Acomys* and *Oryctolagus* (yellow box) (Linear model, F = 3.9693, P < 0.0137 and Supplementary Tables 33, 34). After 30 min, F/F0 were significantly different between species until 4 h post treatment (Supplementary Table 34). In *Acomys* and *Oryctolagus*, F/F0 returned to baseline levels after ~2.5 h (Tukey-HSD, *Acomys*: t = −1.42, P = 0.1592 and *Oryctolagus*: t = −1.70, P = 0.0929), whereas it did not return to baseline in *Mus* and *Rattus* until ~5 h post-treatment (Tukey-HSD, *Mus*: t = −1.74, P = 0.0853 and *Rattus*: t = −1.91, P = 0.0588). Asterisk refers to significant differences between species from an ANOVA. c Catalase activity was significantly decreased in all species following H₂O₂ exposure (Supplementary Table 35). Comparing catalase activity between regenerating and non-regenerating species revealed a significantly greater decrease in non-regenerators (Two-way ANOVA, LS Means, F = 20.5504, P = 0.0003). d GPx activity was significantly increased in response to 300 μM H₂O₂ in all species (Supplementary Table 36). However, GPx activity increased to a greater extent in regenerators compared to non-regenerators (Two-way ANOVA, LS Means-, F = 172.5647, P < 0.0001). e Pre-treatment with NAC protected *Mus* and *Rattus* cells from mitochondrial destabilization upon H₂O₂ exposure. Percent normalized OCRs were not significantly different among non-treated and NAC + H₂O₂ (pre-treatment with 2 mM NAC followed by 2 h of 300 μM H₂O₂ treatment) treated samples in *Mus* and *Rattus* for all the measured components: basal respiration, maximal respiration, ATP production and spare respiratory capacity (Supplementary Tables 37–40). f–h Pre-treatment with NAC prevents ROS-induced senescence in *Mus* and *Rattus* cells as measured by percent p21 + (ANOVA, *Mus*, F = 1309.3470, P < 0.0071 and *Rattus*, F = 258.1558, P < 0.0924) and SA-βgal + cells (ANOVA, *Mus*, F = 669.5451, P < 0.1465 and *Rattus*, F = 125.3335, P < 0.0522) staining. Representative scale bars in panel a = 50 µm and in panel f = 20 µm. ***P < 0.0001, **P < 0.001, *P < 0.05. Error bars = S.E.M. Source data are provided as a Source Data file

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References

1. Hay ED, Fischman DA. Origin of the blastema in regenerating limbs of the newt Triturus viridescens. An autoradiographic study using tritiated thymidine to follow cell proliferation and migration. Dev. Biol. 1961;3:26–59. doi: 10.1016/0012-1606(61)90009-4. - DOI - PubMed
1. Tanaka EM, Reddien PW. The cellular basis for animal regeneration. Dev. Cell. 2011;21:172–185. doi: 10.1016/j.devcel.2011.06.016. - DOI - PMC - PubMed
1. Owlarn S, et al. Generic wound signals initiate regeneration in missing-tissue contexts. Nat. Commun. 2017;8:2282. doi: 10.1038/s41467-017-02338-x. - DOI - PMC - PubMed
1. Simkin J, Seifert AW. Concise review: translating regenerative biology into clinically relevant therapies: are we on the right path? Stem Cells Transl. Med. 2018;7:220–231. doi: 10.1002/sctm.17-0213. - DOI - PMC - PubMed
1. Gawriluk, T. R. et al. Comparative analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in mammals. Nat. Commun.7, 11164 (2016). - PMC - PubMed

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[1] Hay ED, Fischman DA. Origin of the blastema in regenerating limbs of the newt Triturus viridescens. An autoradiographic study using tritiated thymidine to follow cell proliferation and migration. Dev. Biol. 1961;3:26–59. doi: 10.1016/0012-1606(61)90009-4. - DOI - PubMed

[2] Hay ED, Fischman DA. Origin of the blastema in regenerating limbs of the newt Triturus viridescens. An autoradiographic study using tritiated thymidine to follow cell proliferation and migration. Dev. Biol. 1961;3:26–59. doi: 10.1016/0012-1606(61)90009-4. - DOI - PubMed

[3] Tanaka EM, Reddien PW. The cellular basis for animal regeneration. Dev. Cell. 2011;21:172–185. doi: 10.1016/j.devcel.2011.06.016. - DOI - PMC - PubMed

[4] Tanaka EM, Reddien PW. The cellular basis for animal regeneration. Dev. Cell. 2011;21:172–185. doi: 10.1016/j.devcel.2011.06.016. - DOI - PMC - PubMed

[5] Owlarn S, et al. Generic wound signals initiate regeneration in missing-tissue contexts. Nat. Commun. 2017;8:2282. doi: 10.1038/s41467-017-02338-x. - DOI - PMC - PubMed

[6] Owlarn S, et al. Generic wound signals initiate regeneration in missing-tissue contexts. Nat. Commun. 2017;8:2282. doi: 10.1038/s41467-017-02338-x. - DOI - PMC - PubMed

[7] Simkin J, Seifert AW. Concise review: translating regenerative biology into clinically relevant therapies: are we on the right path? Stem Cells Transl. Med. 2018;7:220–231. doi: 10.1002/sctm.17-0213. - DOI - PMC - PubMed

[8] Simkin J, Seifert AW. Concise review: translating regenerative biology into clinically relevant therapies: are we on the right path? Stem Cells Transl. Med. 2018;7:220–231. doi: 10.1002/sctm.17-0213. - DOI - PMC - PubMed

[9] Gawriluk, T. R. et al. Comparative analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in mammals. Nat. Commun.7, 11164 (2016). - PMC - PubMed

[10] Gawriluk, T. R. et al. Comparative analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in mammals. Nat. Commun.7, 11164 (2016). - PMC - PubMed

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Connective tissue fibroblasts from highly regenerative mammals are refractory to ROS-induced cellular senescence

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Connective tissue fibroblasts from highly regenerative mammals are refractory to ROS-induced cellular senescence

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