. 2021 Jun;40(23):4033-4049.

doi: 10.1038/s41388-021-01824-3. Epub 2021 May 20.

Fragile X mental retardation protein in intrahepatic cholangiocarcinoma: regulating the cancer cell behavior plasticity at the leading edge

Simone Carotti^#^{1

2}, Maria Zingariello^#¹, Maria Francesconi¹, Laura D'Andrea¹, M Ujue Latasa³, Leticia Colyn³, Maite G Fernandez-Barrena^{3

4}, Rocco Simone Flammia¹, Mario Falchi⁵, Daniela Righi², Giorgia Pedini⁶, Francesco Pantano⁷, Claudia Bagni^{6

8}, Giuseppe Perrone^{2

9}, Rosa Alba Rana¹⁰, Matias A Avila^{3

4}, Sergio Morini¹¹, Francesca Zalfa^{12

13}

Affiliations

¹ Research Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, Campus Bio-Medico University, Rome, Italy.
² Predictive Molecular Diagnostic Unit, Department of Pathology, Campus Bio-Medico University Hospital, Rome, Italy.
³ Hepatology Program, Center for Applied Medical Research (CIMA), University of Navarra and IdiSNA, Pamplona, Spain.
⁴ Centro de Investigación Biomédica en Red, Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid, Spain.
⁵ National AIDS Center, Istituto Superiore di Sanità, Rome, Italy.
⁶ Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
⁷ Medical Oncology Department, Campus Bio-Medico University, Rome, Italy.
⁸ Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland.
⁹ Research Unit of Pathology, Campus Bio-Medico University, Rome, Italy.
¹⁰ Medicine and Aging Science Department, University G. D'Annunzio, Chieti-Pescara, Italy.
¹¹ Research Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, Campus Bio-Medico University, Rome, Italy. s.morini@unicampus.it.
¹² Research Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, Campus Bio-Medico University, Rome, Italy. f.zalfa@unicampus.it.
¹³ Predictive Molecular Diagnostic Unit, Department of Pathology, Campus Bio-Medico University Hospital, Rome, Italy. f.zalfa@unicampus.it.

^# Contributed equally.

PMID: 34017076
PMCID: PMC8195741
DOI: 10.1038/s41388-021-01824-3

Fragile X mental retardation protein in intrahepatic cholangiocarcinoma: regulating the cancer cell behavior plasticity at the leading edge

Simone Carotti et al. Oncogene. 2021 Jun.

. 2021 Jun;40(23):4033-4049.

doi: 10.1038/s41388-021-01824-3. Epub 2021 May 20.

Authors

Affiliations

¹ Research Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, Campus Bio-Medico University, Rome, Italy.
² Predictive Molecular Diagnostic Unit, Department of Pathology, Campus Bio-Medico University Hospital, Rome, Italy.
³ Hepatology Program, Center for Applied Medical Research (CIMA), University of Navarra and IdiSNA, Pamplona, Spain.
⁴ Centro de Investigación Biomédica en Red, Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid, Spain.
⁵ National AIDS Center, Istituto Superiore di Sanità, Rome, Italy.
⁶ Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
⁷ Medical Oncology Department, Campus Bio-Medico University, Rome, Italy.
⁸ Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland.
⁹ Research Unit of Pathology, Campus Bio-Medico University, Rome, Italy.
¹⁰ Medicine and Aging Science Department, University G. D'Annunzio, Chieti-Pescara, Italy.
¹¹ Research Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, Campus Bio-Medico University, Rome, Italy. s.morini@unicampus.it.
¹² Research Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, Campus Bio-Medico University, Rome, Italy. f.zalfa@unicampus.it.
¹³ Predictive Molecular Diagnostic Unit, Department of Pathology, Campus Bio-Medico University Hospital, Rome, Italy. f.zalfa@unicampus.it.

^# Contributed equally.

PMID: 34017076
PMCID: PMC8195741
DOI: 10.1038/s41388-021-01824-3

Abstract

Intrahepatic cholangiocarcinoma (iCCA) is a rare malignancy of the intrahepatic biliary tract with a very poor prognosis. Although some clinicopathological parameters can be prognostic factors for iCCA, the molecular prognostic markers and potential mechanisms of iCCA have not been well investigated. Here, we report that the Fragile X mental retardation protein (FMRP), a RNA binding protein functionally absent in patients with the Fragile X syndrome (FXS) and also involved in several types of cancers, is overexpressed in human iCCA and its expression is significantly increased in iCCA metastatic tissues. The silencing of FMRP in metastatic iCCA cell lines affects cell migration and invasion, suggesting a role of FMRP in iCCA progression. Moreover, we show evidence that FMRP is localized at the invasive front of human iCCA neoplastic nests and in pseudopodia and invadopodia protrusions of migrating and invading iCCA cancer cells. Here FMRP binds several mRNAs encoding key proteins involved in the formation and/or function of these protrusions. In particular, we find that FMRP binds to and regulates the expression of Cortactin, a critical regulator of invadopodia formation. Altogether, our findings suggest that FMRP could promote cell invasiveness modulating membrane plasticity and invadopodia formation at the leading edges of invading iCCA cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. FMRP is overexpressed in human iCCA.**
a FMRP immunohistochemical expression in non tumoral hepatic tissues (NT region) and in iCCA (T region). In left panel, FMRP expression is higher in tumoral tissue (T, dashed line) compared to non tumoral liver (NT). The box in left panel indicates the high power field in right panel. In the surrounding non tumoral liver, cholangiocytes of biliary ducts (asterisk) in portal tracts do not show a significant FMRP expression (D = biliary ducts, V = portal vein, A = hepatic artery). Calibration bars: 200 µm (left panel), 40 µm (right panel). b FMRP immunohistochemical expression in human iCCA samples was observed with low (left panel) or high (right panel) expression levels. Calibration bars: 20 µm. c FMRP expression was evaluated by IHC in 48 human iCCA samples and seven non-tumoral hepatic tissues (NT). Each sample was scored by using a semi-automated method and the IHC score of iCCA and NT tissues was reported in the left histogram. The columns represent median values and the bars, standard deviations (*p < 0.05; Mann–Witney test). In the right histogram the percentage of samples (iCCA or NT) with 0, 1, or 2 scores was plotted. d A representative IHC of an iCCA sample with low FMRP expression, showing the heterogeneous staining of FMRP in the neoplastic nests. The box in the left panel indicates the high-power field in the right panel. Arrowheads indicate FMRP immunopositivity at the boundaries of neoplastic nests. Calibration bars: 100 µm (left panel), 20 µm (right panel). e FMRP immunohistochemical expression in human iCCA cohort was compared with metastatic (Met) or primary (Prim) nature of tumor samples. The columns represent median values and the bars, standard errors (*p < 0.05; Mann–Witney test). f Representative western blotting for FMRP and β-Actin in intrahepatic normal human biliary epithelial cell line H69, in three eCCA cell lines (KMBC, TFK-1, and MzChA1) and in two iCCA cell lines (HuCCT and HuH28) protein extracts (10 μg). In the boxplot the densitometric quantification of FMRP bands (normalised for β-Actin bands) in three independent western blotting experiments. The boxes represent quartiles, the ×, the median and the whiskers, the variability beyond the upper and lower quartiles (**p < 0.01; Student’s t test). g *FMR1* mRNA expression levels were evaluated by RT-qPCR in the same cell lines of panel (f). In the boxplot, the boxes represent quartiles, the ×, the median and the whiskers, the variability beyond the upper and lower quartiles (*p < 0.05 and **p < 0.01; Student’s t test).

**Fig. 2. Immunohistochemical and ultrastructural localization of FMRP at the leading edge in iCCA cells.**
a Representative images of a IHC for FMRP in an iCCA sample, showing FMRP staining mainly localized beneath plasma membrane of iCCA cells, with a strong enhancement of signal towards the basal lamina (upper panels, black arrowhead). Cortactin staining exhibits a similar intracellular pattern (lower panels, white arrowhead). Calibration bars 20 µm. Original magnification 20× (left panels), high power fields 100× (right panels). b Ultrathin sections of HuCCT cellular pellets were subjected to immunogold electron microscopy with specific FMRP and Cortactin antibodies. The box in left panel indicates the high-power field in right panel. 25 nm gold particles (FMRP, white arrowheads) and 15 nm gold particles (Cortactin, black arrows) colocalized at the base of membrane protrusions of HuCCT cells. The white arrows indicate ribosomes densities. Nu Nucleus, *Cyt* Cytoplasm. Left panel: original magnification ×18,500. Scale bar: 1 μm. Right panel: original magnification ×37,000. Scale bar: 500 nm.

**Fig. 3. FMRP localizes in cytoplasmic granules inside Cortactin and MT1-MMP –enriched cell protrusions.**
a HuH28 or HuCCT cells were grown on fibronectin-coated chamber slides for 48 h and then IF was performed. Cells were stained with FMRP (red), Cortactin (green) and nuclei were stained with DAPI (blue). In the last right panels, enlarged images of white box in MERGE panels. Red arrowheads, FMRP granules; green arrowheads, Cortactin positive foci; yellow arrowheads, FMRP-Cortactin colocalization foci, inside cell protrusions. Bars, 20 μm in HuH28 panels; 10 μm in HuCCT panels; 4 μm in enlarged panels. b HuH28 or HuCCT cells were grown on fibronectin-coated chamber slides for 48 h and then IF was performed. Cells were stained with FMRP (red), MT1-MMP (green) and nuclei were stained with DAPI (blue). In the last right panels, enlarge images of MERGE panels. Red arrowheads, FMRP granules; green arrowheads, MT1-MMP positive foci; yellow arrowheads, FMRP-MT1-MMP colocalization foci, inside cell protrusions. Bars, 20 μm in HuH28 panels; 10 μm in HuCCT panels; 4 μm in enlarged panels. c HuCCT cells overexpressing FMRP-GFP were analysed with time-lapse fluorescence microscopy. FITC signal (green) corresponding to FMRP-GFP was acquired every 10 min for 12 h. A total of 72 acquisitions were assembled to produce a live imaging movie (see Supplementary Information). Frames 20–23 (series 1, upper panels) or 47–50 (series 2, lower panels) of Movie 1 are shown. White arrow points cellular leading edge, white arrowheads point filopodia at the leading edge and yellow arrow, the direction of cell migration. Original magnification 20×.

**Fig. 4. FMRP is localized inside invadopodia of HuH28 and HuCCT cells.**
a A total of 40% confluent HuH28 cells were sown in Cy3-gelatin coated chambers slides and incubated for 19 h for a GDA. Then an IF was performed, and the images were captured using a confocal microscope. Cy3-gelatin is red, FMRP staining green and DAPI staining blue. Representative images of a 60× magnification field (field 15 of 27 total fields). Scale bars: 10 μm. To the right and under the MERGE image, orthogonal views along Y (YZ) and X (XZ) axes are shown, respectively. White arrowheads indicate FMRP staining inside black areas of Cy3-gelatin (degradation areas). b 40% confluent HuCCT cells starved for 24 h in 2% FBS medium were sown in Cy3-gelatin coated chambers slides, treated for 16 h with the MMP inhibitor GM6001 and then incubated for 2 h without MMP inhibition for a GDA. IF and image acquisitions were performed as reported for HuH28. Representative images of a 60× magnification field (field 18 of 28 total fields). Scale bars: 10 μm. To the right and under the MERGE image, orthogonal views along Y (YZ) and X (XZ) axes are shown respectively. White arrowheads indicate FMRP staining inside black areas of Cy3-gelatin (degradation areas). c 3D reconstruction of Z stack (along Y axis) reported in panel (b).

**Fig. 5. Silencing of FMRP affects migration and invasion in HuH28 and HuCCT cells.**
a Confluent cell monolayers of HuH28 cells untransfected (CTR) or transfected with *FMR1* siRNAs (*FMR1* siRNA) or scrambled siRNA (scr siRNA) were wounded and after 0 (T0), 12 (T1), or 24 (T2) h the scratch spreads were evaluated. In the left panels, one defined area per group at initial (T0) and final (T2) times is shown. Scale bars: 300 μm. In 10 defined areas per group and in three independent experiments, the scratch sealing was quantified and shown (in %) in the boxplot (right panel). The boxes represent quartiles; the ×, the median; the dots, the individual data points and the whiskers, the variability beyond the upper and lower quartiles. *p < 0.05; ***p < 0.001 compared to CTR or scr siRNA (Student’s t test). b Confluent cell monolayers of HuCCT cells untransfected (CTR) or stable transfected with *FMR1* shRNAs (*FMR1* shRNA) or control shRNA (CTR shRNA) were wounded and after 0 (T0), 8 (T1), or 12 (T2) h the scratch spreads were evaluated. In the left panels, one defined area per group at initial (T0) and final (T2) times is shown. Scale bars: 300 μm. In 10 defined areas per group and in three independent experiments, the scratch sealing was quantified and shown (in %) in the boxplot (right panel). The boxes represent quartiles; the ×, the median; the dots, the individual data points and the whiskers, the variability beyond the upper and lower quartiles. *p < 0.05 compared to CTR or CTR shRNA (Student’s t test). c Previously cited HuH28 or HuCCT cells were sown into trans-well motility chambers (50,000 cells/well; upper panels) or Matrigel-coated trans-well invasion chambers (100,000 cells/well; lower panels) and after 24 h migrating or invading cells were counted. In left panels, a representative field (4× magnification) per group is shown. Scale bars: 300 μm. In right panels, cells present in ten magnification fields per group and per experiment were counted. Data derive from three independent experiments. The boxes represent quartiles; the ×, the median; the dots, the individual data points and the whiskers, the variability beyond the upper and lower quartiles. ***p < 0.001 compared to CTR or scr siRNA or CTR shRNA (Student’s t test).

**Fig. 6. Silencing of FMRP reduces the ability of HuH28 and HuCCT cells to degrade the Cy3-gelatin in GDA assays.**
a HuH28 cells were untransfected (CTR) or transfected with three specific *FMR1* siRNAs (*FMR1* siRNA) or with a scrambled nonspecific siRNA (scr siRNA), incubated for 72 h and then sown on Cy3-gelatin coated chamber slides (4000 cells for chamber) for a GDA assay. After 24 h, cells were fixed and stained with phalloidin (F-actin) and DAPI (nuclei). Representative images of 20× magnification fields of nuclei (blue), Cy3-gelatin (red), and F-actin (green). In the last right panels, merged fluorescence was shown. Scale bars: 60 μm. b HuCCT cells were untransfected (CTR) or stable transfected with a specific *FMR1* shRNA (*FMR1* shRNA) or with a control shRNA (CTR shRNA) and then starved for 24 h in 2% FBS medium. The cells were then sown on Cy3-gelatin coated chamber slides (4000 cells for chamber), treated for 16 h with MMP inhibitor GM6001 and then incubated without MMP inhibitor for a GDA assay. After 4 h, cells were fixed and stained with phalloidin (F-actin) and DAPI (nuclei). Representative images of 60× magnification fields of nuclei (blue), Cy3-gelatin (red), and F-actin (green). In the last right panels, merged fluorescence was shown. Scale bars: 20 μm. c Quantification of GDs (dark areas in Cy3-gelatin) present in 10 fields of both HuH28 (left panel) and HuCCT (right panel) cell lines. N = 3. The boxes represent quartiles; the ×, the median; the dots, the individual data points and the whiskers, the variability beyond the upper and lower quartiles. ***p < 0.001 compared to CTR or scr siRNA or CTR shRNA (Student’s t test).

**Fig. 7. FMRP binds cortactin mRNA and regulates its expression.**
a Cytoplasmic extracts of HuH28 cells were used for immunoprecipitation experiments using the specific anti-FMRP antibody ab17722 (IP FMRP) or nonspecific IgG antibodies (IP IgG). The co-immunoprecipitated RNAs were extracted from each IP and *ACTR3B, CTTN, FMR1, LRP4, MMP1, MMP10, MT1-MMP, MMP2, MMP9, MUSK, SH3PXD2A, SRC, TIMP-2, N-WASP, GAPDH, HPRT1,* and *TUBB* mRNAs were quantified by NanoString nCounter^® approach. The enrichment of these mRNAs (expressed as log₁₀ fold change) in FMRP IP versus IgG IP is reported in the boxplot, where only the positive values are shown. N = 3. Dashed line represents the confidence threshold chosen for log₁₀ fold change that correspond to a fold change = 2. b HuH28 cells were untransfected (CTR) or transfected with three specific *FMR1* siRNAs (*FMR1* siRNA) or with a scrambled nonspecific siRNA (scr siRNA) and incubated for 72 h. Representative images of a western blot probed with anti-FMRP, anti-Cortactin, anti-MMP9, anti-MMP1, anti Src, and anti-β-Actin antibodies from protein extracts of the three cellular conditions (left panel). In right panel, a quantitation of FMRP, Cortactin, MMP9, MMP1, and Src expressions, compared to β-Actin expression, is shown. Columns, mean; bars, SE. **p < 0.01 compared to CTR or scr siRNA (Student’s t test). c IHC data were obtained by The Human Protein Atlas library. Four iCCA samples (A1–A4) and three healthy livers (C1–C3) were evaluated for both Cortactin and FMRP expression and scored from +0 to +3. d Cortactin expression was evaluated by IHC in three human iCCAs with low FMRP expression and three human iCCAs with high FMRP expression. Representative images of FMRP and Cortactin immunostaining in a FMRP low expression sample (left panels) and a FMRP high expression sample (right panels) were shown. Scale bars: 150 μm. e Representative images of FMRP and Cortactin IHCs in xenograft tumors from *FMR1* shRNA (left panels) or CTR shRNA (right panels) transfected HuCCT cells. Original magnification 10×; scale bars: 250 μm. FMRP and Cortactin IHC expression in *FMR1* shRNA and CTR shRNA xenograft tumors are highly correlated (right panel); E, ρ = 0.34, *p < 0.05 with Spearman’s correlation Test. Double-IF labeling experiments show most cells co-expressing FMRP and Cortactin proteins (lower panels). FMRP was stained in red, Cortactin in green and nuclei in DAPI (blue). Original magnification 40×; scale bars: 60 μm.

**Fig. 8. Working model for FMRP action in invadopodia.**
FMRP in invading iCCA cells could regulate local metabolism (mRNA stability, transport, translation etc.) of specific mRNAs (such as *Cortactin* or *MMP* mRNAs) in invadopodia, favoring invadopodia formation and membrane plasticity at the leading edges of cancer cells during invasion processes (left panel with enlarged box). Silencing of FMRP reduced cell invasiveness, reducing their front-rear morphology and their ability to migrate and to degrade the ECM (right panel).

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Fragile X mental retardation protein in intrahepatic cholangiocarcinoma: regulating the cancer cell behavior plasticity at the leading edge

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Fragile X mental retardation protein in intrahepatic cholangiocarcinoma: regulating the cancer cell behavior plasticity at the leading edge

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