MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1

doi:10.26508/lsa.202301947

. 2023 Sep 19;6(11):e202301947.

doi: 10.26508/lsa.202301947. Print 2023 Nov.

MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1

Kensuke Sakaji¹, Sara Ebrahimiazar^{1

2}, Yasuhiro Harigae¹, Kenichi Ishibashi³, Takeya Sato^{1

4}, Takeo Yoshikawa^{5

6}, Gen-Ichi Atsumi³, Ching-Hwa Sung^{7

8}, Masaki Saito^{9

3}

Affiliations

¹ Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
² Department of Developmental Neuroscience, Tohoku University Graduate School of Medicine, Sendai, Japan.
³ Department of Molecular Physiology and Pathology, School of Pharma-Science, Teikyo University, Itabashi-ku, Tokyo, Japan.
⁴ Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine, Sendai, Japan.
⁵ Department of Neuropharmacology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
⁶ Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
⁷ Department of Ophthalmology, Margaret M. Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY, USA chsung@med.cornell.edu.
⁸ Department of Cell and Developmental Biology, Weill Cornell Medicine, New York, NY, USA.
⁹ Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan saitou.masaki.nb@teikyo-u.ac.jp.

PMID: 37726137
PMCID: PMC10509483
DOI: 10.26508/lsa.202301947

MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1

Kensuke Sakaji et al. Life Sci Alliance. 2023.

. 2023 Sep 19;6(11):e202301947.

doi: 10.26508/lsa.202301947. Print 2023 Nov.

Authors

Kensuke Sakaji¹, Sara Ebrahimiazar^{1

2}, Yasuhiro Harigae¹, Kenichi Ishibashi³, Takeya Sato^{1

4}, Takeo Yoshikawa^{5

6}, Gen-Ichi Atsumi³, Ching-Hwa Sung^{7

8}, Masaki Saito^{9

3}

Affiliations

¹ Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
² Department of Developmental Neuroscience, Tohoku University Graduate School of Medicine, Sendai, Japan.
³ Department of Molecular Physiology and Pathology, School of Pharma-Science, Teikyo University, Itabashi-ku, Tokyo, Japan.
⁴ Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine, Sendai, Japan.
⁵ Department of Neuropharmacology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
⁶ Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
⁷ Department of Ophthalmology, Margaret M. Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY, USA chsung@med.cornell.edu.
⁸ Department of Cell and Developmental Biology, Weill Cornell Medicine, New York, NY, USA.
⁹ Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan saitou.masaki.nb@teikyo-u.ac.jp.

PMID: 37726137
PMCID: PMC10509483
DOI: 10.26508/lsa.202301947

Abstract

The primary cilium undergoes cell cycle-dependent assembly and disassembly. Dysregulated ciliary dynamics are associated with several pathological conditions called ciliopathies. Previous studies showed that the localization of phosphorylated Tctex-1 at Thr94 (T94) at the ciliary base critically regulates ciliary resorption by accelerating actin remodeling and ciliary pocket membrane endocytosis. Here, we show that microtubule-associated serine/threonine kinase family member 4 (MAST4) is localized at the primary cilium. Suppressing MAST4 blocks serum-induced ciliary resorption, and overexpressing MAST4 accelerates ciliary resorption. Tctex-1 binds to the kinase domain of MAST4, in which the R503 and D504 residues are key to MAST4-mediated ciliary resorption. The ciliary resorption and the ciliary base localization of phospho-(T94)Tctex-1 are blocked by the knockdown of MAST4 or the expression of the catalytic-inactive site-directed MAST4 mutants. Moreover, MAST4 is required for Cdc42 activation and Rab5-mediated periciliary membrane endocytosis during ciliary resorption. These results support that MAST4 is a novel kinase that regulates ciliary resorption by modulating the ciliary base localization of phospho-(T94)Tctex-1. MAST4 is a potential new target for treating ciliopathies causally by ciliary resorption defects.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

**Figure 1.. MAST4 is localized in the cilium and required for ciliary resorption.**
**(A)** RPE-1 cells transfected with the GFP alone (control), MAST4-shRNA1-IRES-GFP (MAST4-sh1), or MAST4-shRNA2-IRES-GFP (MAST4-sh2) were starved and added serum back for the indicated time periods. Representative confocal images of the Ac-Tub–labeled cilium (red) and the γ-Tub–labeled basal body (cyan) in GFP⁺ cells (green) are shown. Cell nuclei were stained with DAPI (blue). n = 16–31 cells from three independent experiments. Scale bar: 5 μm. **(B, C)** Quantification of (A). Histogram shows the cilium length (measured by Ac-Tub) (B) and the fraction of the GFP⁺ cells expressing a cilium (C). Values are means ± S.E.M. *P < 0.05 and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. ^#P < 0.05 and ^##P < 0.01, two-way ANOVA followed by Bonferroni’s test. n = 16–31 cells (B) and n = 100 cells in each experiment, with three independent experiments (C). **(D)** RPE-1 cells transiently transfected with GFP (control) or GFP together with MAST4^WT were starved and followed by serum readdition. The quantification of the percentage of GFP⁺ cells, harvested at different time points post-serum stimulation, is shown. Values are means ± S.E.M. *P < 0.05 and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. n = 100 cells in each experiment, with three independent experiments. **(E)** Representative confocal images of MAST4-mCherry (mC) in quiescent transfected RPE-1 cells stained for mCherry (red), Ac-Tub (green), and γ-Tub (cyan). White dashed lines demarcate the cell border (DAPI: nuclear). The high-power view highlights the ciliary distribution of MAST4-mCherry. Scale bars: 10 μm (E, low-power magnification) and 1 μm (E, high-power magnification). **(E, F)** Segmented profiles of fluorescence intensities (FIs) along the white dashed arrow shown in (E). BB, basal body. The y-axis FI unit is defined by the acquired values divided by 10⁴ using Zen software (Zeiss). The x-axis describes the distance from the proximal part of the basal body (considered as 0). The representative line scan from the images of 16 cilia is shown.

**Figure S3.. Super-resolution images of MAST4^WT-mCherry in RPE-1 cells.**
A representative super-resolution Airyscan image of MAST4^WT-mCherry in serum-starved RPE-1 cells. MAST4^WT-mCherry signals were detected in the ciliary base (arrow) and the ciliary axoneme (arrowhead). The asterisk marks the MAST4^WT-mCherry signal juxtaposing to the ciliary axoneme, possibly representing the ciliary pocket. White dashed lines in the low-power view demarcate the cell border. Scale bars: 5 μm (low power) and 1 μm (high power).

**Figure 2.. Characterization of Tctex-1-MAST4 interaction and mapping Tctex-1–binding motif of MAST4.**
**(A)** Representative immunoblot of total HEK293 cell lysates containing transiently transfected MAST4^WT-mCherry and FLAG-Tctex-1^WT (input) and the immunoprecipitants (IP) pulled down by anti-mCherry or anti-GFP (control) antibodies probed with anti-RFP and anti-DYKDDDDK (FLAG) antibodies. **(B)** Diagram showing different domains and corresponding amino acids of MAST4. Different regions (Fr.1–Fr. 7) of MAST4 fused with GST for pull-down assays are also labeled. S/T_kinase, serine/threonine kinase domain. **(C)** MBP-Tctex-1–containing bacterial lysates were mixed with bacterial extracts containing GST or GST-MAST4 fragments (with indicated molecular weights) and then incubated with glutathione beads. The eluates from the glutathione beads were electrophoresed and immunoblotted (IB) with anti-MBP antibody, or GST or GST-MAST4 fragments in the elutes were stained with Coomassie brilliant blue (CBB). The expected molecular weights of GST (26 kD) and GST-MAST4 are shown. Note the roughly equivalent amounts of GST and GST-MAST4 fragments were loaded in the inputs.

**Figure 3.. MAST4 regulates Tctex-1–mediated ciliary resorption and the appearance of phospho-(T94)Tctex-1 at the ciliary base.**
**(A)** RPE-1 cells transfected with GFP alone (control), or MAST4-sh1-IRES-GFP (MAST4-sh1) together with FLAG-tagged Tctex-1^WT, Tctex-1^T94E, or Tctex-1^T94A were subjected to a ciliary resorption assay. The histogram shows the percentage of GFP⁺ cells expressing Ac-Tub–labeled cilium at the indicated time points post-serum restimulation. Values are means ± S.E.M. *P < 0.05 and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. ^##P < 0.01, two-way ANOVA followed by Bonferroni’s test. n = 100 cells in each experiment, with three independent experiments. **(B)** Ciliary resorption assay of RPE-1 cells transfected with FLAG-Tctex-1^T94A with or without co-transfected MAST4^WT. The histogram shows the percentage of ciliated GFP⁺ cells at 0 and 1 h post-serum readdition. Values are means ± S.E.M. ^###P < 0.001, two-way ANOVA followed by Bonferroni’s test. ^§§P < 0.01, t test versus 0 h. n = 100 cells in each experiment, with three independent experiments. **(C, D)** Representative confocal images of phospho-(T94)Tctex-1 (cyan) and Ac-Tub (red) in RPE-1 cells transfected with GFP (control) or MAST4-sh1 under the starving (0 h) and 2-h serum-stimulated conditions. **(C)** Phospho-(T94)Tctex-1 was detected by Alexa Fluor 647–conjugated secondary antibody and is pseudo-colored in cyan for presentation. **(D)** Fluorescence intensity of phospho-(T94)Tctex-1 associated with the proximal end of Ac-Tub–labeled cilium was quantified and is shown in (D). Scale bar: 1 μm (C). Values are means ± S.E.M. ^###P < 0.001, two-way ANOVA followed by Bonferroni’s test. ^§§§P < 0.001, t test. n = 25–39 cells from three independent experiments.

**Figure 4.. Mapping catalytic motif residues of MAST4 required for the ciliary base activation of phospho-(T94)Tctex-1.**
**(A)** Alignment of the kinase domain of MAST2 and PKA catalytic subunit α (PKACA) with the predicted catalytic domain of MAST4. Bold letters were used to highlight the amino acid residues that were changed to alanine by site-directed mutagenesis. The predicted catalytic motif (H502–N509), DFG motif (D522–G524), and autophosphorylation residues (T524 or T535) of MAST4 are marked by a bar. **(B)** Ciliary resorption assay of RPE-1 cells transfected with GFP alone (control), or GFP together with MAST4^WT or the indicated MAST variants. The histogram shows the percentage of ciliated GFP⁺ cells at different time points after serum readdition. Values are means ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. n = 100 cells in each experiment, with three independent experiments. **(C)** Representative confocal images of phospho-(T94)Tctex-1 (magenta) and Ac-Tub (green) in RPE-1 cells transfected with GFP together with WT or indicated mutant MAST4-mCherry under the starving (0 h) and 2-h serum-stimulated conditions. **(C)** Phospho-(T94)Tctex-1 was detected by Alexa Fluor 647–conjugated secondary antibody and is pseudo-colored in magenta for presentation. n = 12–16 cells from three independent experiments. **(D)** Fluorescence intensity of phospho-(T94)Tctex-1 associated with the proximal end of Ac-Tub–labeled cilium was quantified (D). Values are means ± S.E.M. ^##P < 0.01 and ^###P < 0.001, two-way ANOVA followed by Bonferroni’s test. ^§§§P < 0.001, t test. n = 12–16. Scale bar: 1 μm (C).

**Figure S4.. 3D structural prediction of the MAST4 kinase domain.**
The structure of the MAST4 kinase domain was predicted using the AlphaFold2 tool. **(A, C)** MAST4 kinase domain without (A) or with (C) side chain structure of amino acids is shown. **(B, D)** Boxed areas are highlighted in (B, D), respectively. The positions of R503, D504, and K506 are labeled.

**Figure S5.. Immunoblots of overexpressed MAST4 variants.**
Representative immunoblots of RPE-1 cells transfected with GFP alone (control), or GFP together with MAST4^WT, MAST4^R503A, MAST4^D504A, MAST4^K506A, MAST4^D522A, MAST4^T534A, or MAST4^T535A. The blots were probed with anti-MAST4 or anti-GAPDH antibody.

**Figure S6.. Ciliary localization of MAST4^R503A and MAST4^D504A.**
**(A, B, C, D)** Representative confocal images of serum-starved RPE-1 cells transfected with the indicated mutant MAST4-mCherry (mC) (red) and stained for Ac-Tub (green) and γ-Tub (cyan). **(A, C)** Low-power views show a single transfected cell. The high-power view highlights the ciliary distribution of MAST4-mCherry. **(B, D)** Profile blots of fluorescence intensities (FIs) are also shown. BB, basal body. White dashed lines in the low-power view demarcate the cell border (DAPI: nuclear). Scale bars: 10 μm (low-power magnification) and 1 μm (high-power magnification). The y-axis FI unit is defined by the acquired values divided by 10⁴ using Zen software (Zeiss). The x-axis describes the distance from the proximal part of the basal body (considered as 0). The representative line scans from the images of 11–16 cilia are shown.

**Figure 5.. MAST4 promotes ciliary resorption through Cdc42 activation.**
**(A)** Ciliary resorption assay of RPE-1 cells transfected with GFP alone (control), or MAST4^WT together without (control) or with HA-tagged Cdc42^T17N (Cdc42-DN). The histogram shows the percentage of GFP⁺ cells with Ac-Tub–labeled primary cilia at the indicated time points. Values are means ± S.E.M. ^##P < 0.01 and ^###P < 0.001, two-way ANOVA followed by Bonferroni’s test. ^§P < 0.05, t test. n = 100 cells in each experiment, with three independent experiments. **(B)** RPE-1 cells expressing GFP alone (control), MAST4^R503A, or MAST4^D504A (co-expressed with GFP), in the presence or absence (control) of HA-tagged Cdc42^G12V (Cdc42-CA). The cells were subjected to a ciliary resorption assay. The histogram shows the percentage of ciliated GFP⁺ cells at different time points post-serum readdition. Values are means ± S.E.M. *P < 0.05 and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. ^###P < 0.001, two-way ANOVA followed by Bonferroni’s test. n = 100 cells in each experiment, with three independent experiments. **(C, D, E, F)** Cdc42 activity assays. **(C, E)** Representative Cdc42 immunoblots containing total Cdc42 and active Cdc42 pulled down by PAK-PBD beads from RPE-1 cells transfected with pCAG (control) and pCAG-MAST4-sh1 (C) or MAST4 variants (E). **(D, F)**. Ratios of active Cdc42 to total Cdc42 in cells harvested at different time points post-serum readdition are shown in (D, F). Value at the 0-h time point was considered as 100. Values (D, F) are means ± S.E.M. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001, two-way ANOVA followed by Bonferroni’s test. n = 3 (D, F).

**Figure S7.. Cdc42 activity assay.**
The whole blots of Fig 5C–F. **(A, B)** Immunoblots show the active Cdc42 and total Cdc42 expressed in RPE-1 cells transfected with MAST4-shRNA1 (A), and MAST4^R503A or MAST4^D504A (B).

**Figure S8.. ARPC2 acts downstream of MAST4-mediated ciliary resorption.**
A ciliary resorption assay of RPE-1 cells transfected with GFP alone (control), or GFP together with MAST4^WT, in the presence or absence of ARPC2-shRNA-IRES-GFP (ARPC2-sh). The histogram shows the percentage of cilium-expressing GFP⁺ cells at the indicated time points. Values are means ± S.E.M. ^§P < 0.05, t test. n = 100 cells in each experiment, with three independent experiments. N.S., not significant.

**Figure 6.. MAST4 regulates the periciliary membrane endocytosis working model.**
**(A)** Representative SR-SIM images show Ac-Tub–labeled cilium (green) and the periciliary signal of internalized Alexa Fluor 594–conjugated transferrin (Tf-594) (red) in RPE-1 cells transfected with GFP alone (control), and GFP together with MAST4-sh1 1 h post–IGF-1 (10 nM) stimulation. Ac-Tub was detected by Alexa Fluor 405–conjugated secondary antibody and is pseudo-colored in green for presentation. Reprehensive images of 14 cilia (in control) and 8 cilia (in MAST-sh1–transfected cells) are shown. Scale bar: 1 μm. **(B)** Ciliary resorption assay of RPE-1 cells transfected with GFP alone (control), or MAST4-sh1-IRES-GFP (MAST4-sh1) together with FLAG-tagged Rab5^Q79L (Rab5-CA). The histogram shows the percentage of GFP⁺ cells with Ac-Tub–labeled primary cilia at the indicated time points. Values are the mean ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. ^#P < 0.05, two-way ANOVA followed by Bonferroni’s test. n = 100 cells in each experiment, with three independent experiments. **(C)** Schematic diagram shows the periciliary location and the processes by which MAST4 mediates the ciliary resorption, through the activation of phospho-(T94)Tctex-1, Cdc42/Arp2/3 activity (probably branched actin organization), and periciliary membrane endocytosis.

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References

1. An SWA, Choi ES, Hwang W, Son HG, Yang JS, Seo K, Nam HJ, Nguyen NTH, Kim EJE, Suh BK, et al. (2019) KIN-4/MAST kinase promotes PTEN-mediated longevity of Caenorhabditis elegans via binding through a PDZ domain. Aging Cell 18: e12906. 10.1111/acel.12906 - DOI - PMC - PubMed
1. Beretov J, Wasinger VC, Millar EK, Schwartz P, Graham PH, Li Y (2015) Proteomic analysis of urine to identify breast cancer biomarker candidates using a label-free LC-MS/MS approach. PLoS One 10: e0141876. 10.1371/journal.pone.0141876 - DOI - PMC - PubMed
1. Chen JL, Chang CH, Tsai JW (2019) Gli2 rescues delays in brain development induced by Kif3a dysfunction. Cereb Cortex 29: 751–764. 10.1093/cercor/bhx356 - DOI - PubMed
1. Chuang JZ, Yeh TY, Bollati F, Conde C, Canavosio F, Caceres A, Sung CH (2005) The dynein light chain Tctex-1 has a dynein-independent role in actin remodeling during neurite outgrowth. Dev Cell 9: 75–86. 10.1016/j.devcel.2005.04.003 - DOI - PMC - PubMed
1. Clement CA, Ajbro KD, Koefoed K, Vestergaard ML, Veland IR, Henriques de Jesus MP, Pedersen LB, Benmerah A, Andersen CY, Larsen LA, et al. (2013) TGF-β signaling is associated with endocytosis at the pocket region of the primary cilium. Cell Rep 3: 1806–1814. 10.1016/j.celrep.2013.05.020 - DOI - PubMed

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[1] An SWA, Choi ES, Hwang W, Son HG, Yang JS, Seo K, Nam HJ, Nguyen NTH, Kim EJE, Suh BK, et al. (2019) KIN-4/MAST kinase promotes PTEN-mediated longevity of Caenorhabditis elegans via binding through a PDZ domain. Aging Cell 18: e12906. 10.1111/acel.12906 - DOI - PMC - PubMed

[2] An SWA, Choi ES, Hwang W, Son HG, Yang JS, Seo K, Nam HJ, Nguyen NTH, Kim EJE, Suh BK, et al. (2019) KIN-4/MAST kinase promotes PTEN-mediated longevity of Caenorhabditis elegans via binding through a PDZ domain. Aging Cell 18: e12906. 10.1111/acel.12906 - DOI - PMC - PubMed

[3] Beretov J, Wasinger VC, Millar EK, Schwartz P, Graham PH, Li Y (2015) Proteomic analysis of urine to identify breast cancer biomarker candidates using a label-free LC-MS/MS approach. PLoS One 10: e0141876. 10.1371/journal.pone.0141876 - DOI - PMC - PubMed

[4] Beretov J, Wasinger VC, Millar EK, Schwartz P, Graham PH, Li Y (2015) Proteomic analysis of urine to identify breast cancer biomarker candidates using a label-free LC-MS/MS approach. PLoS One 10: e0141876. 10.1371/journal.pone.0141876 - DOI - PMC - PubMed

[5] Chen JL, Chang CH, Tsai JW (2019) Gli2 rescues delays in brain development induced by Kif3a dysfunction. Cereb Cortex 29: 751–764. 10.1093/cercor/bhx356 - DOI - PubMed

[6] Chen JL, Chang CH, Tsai JW (2019) Gli2 rescues delays in brain development induced by Kif3a dysfunction. Cereb Cortex 29: 751–764. 10.1093/cercor/bhx356 - DOI - PubMed

[7] Chuang JZ, Yeh TY, Bollati F, Conde C, Canavosio F, Caceres A, Sung CH (2005) The dynein light chain Tctex-1 has a dynein-independent role in actin remodeling during neurite outgrowth. Dev Cell 9: 75–86. 10.1016/j.devcel.2005.04.003 - DOI - PMC - PubMed

[8] Chuang JZ, Yeh TY, Bollati F, Conde C, Canavosio F, Caceres A, Sung CH (2005) The dynein light chain Tctex-1 has a dynein-independent role in actin remodeling during neurite outgrowth. Dev Cell 9: 75–86. 10.1016/j.devcel.2005.04.003 - DOI - PMC - PubMed

[9] Clement CA, Ajbro KD, Koefoed K, Vestergaard ML, Veland IR, Henriques de Jesus MP, Pedersen LB, Benmerah A, Andersen CY, Larsen LA, et al. (2013) TGF-β signaling is associated with endocytosis at the pocket region of the primary cilium. Cell Rep 3: 1806–1814. 10.1016/j.celrep.2013.05.020 - DOI - PubMed

[10] Clement CA, Ajbro KD, Koefoed K, Vestergaard ML, Veland IR, Henriques de Jesus MP, Pedersen LB, Benmerah A, Andersen CY, Larsen LA, et al. (2013) TGF-β signaling is associated with endocytosis at the pocket region of the primary cilium. Cell Rep 3: 1806–1814. 10.1016/j.celrep.2013.05.020 - DOI - PubMed

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MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1

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MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1

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