. 2019 Feb;4(2):269-279.

doi: 10.1038/s41564-018-0300-x. Epub 2018 Dec 3.

Clostridium difficile toxins induce VEGF-A and vascular permeability to promote disease pathogenesis

Jun Huang^{1

2

3}, Ciarán P Kelly¹, Kyriaki Bakirtzi⁴, Javier A Villafuerte Gálvez¹, Dena Lyras⁵, Steven J Mileto⁵, Sarah Larcombe⁵, Hua Xu¹, Xiaotong Yang^{1

6}, Kelsey S Shields¹, Weishu Zhu⁷, Yi Zhang^{1

8}, Jeffrey D Goldsmith⁹, Ishan J Patel^{1

10}, Joshua Hansen¹, Meijin Huang², Seppo Yla-Herttuala¹¹, Alan C Moss¹, Daniel Paredes-Sabja¹², Charalabos Pothoulakis⁵, Yatrik M Shah¹³, Jianping Wang^{14

15}, Xinhua Chen¹⁶

Affiliations

¹ Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
² Department of Colorectal Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
³ Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
⁴ Division of Digestive Diseases, Inflammatory Bowel Disease Center, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, CA, USA.
⁵ Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia.
⁶ Institute of Microbiology and Immunology, Life and Environment Science College, Shanghai Normal University, Shanghai, China.
⁷ Mucosal Immunology and Biology Research Center, Massachusetts General Hospital for Children and Harvard Medical School, Charlestown, MA, USA.
⁸ Department of Gastrointestinal Surgery, The Third Xiangya Hospital, Central South University, Changsha, China.
⁹ Department of Pathology, Children's Hospital Boston and Harvard Medical School, Boston, MA, USA.
¹⁰ Icahn School of Medicine at Mount Sinai (Elmhurst) Program, New York, NY, USA.
¹¹ Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, University of Eastern Finland and Science Service Center and Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland.
¹² Microbiota-Host Interactions and Clostridia Research Group, Departamento de Ciencias Biológicas, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile.
¹³ Departments of Molecular and Integrative Physiology, Division of Gastroenterology, University of Michigan Medical School, Ann Arbor, MI, USA.
¹⁴ Department of Colorectal Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. wangjpgz@126.com.
¹⁵ Guangdong Institute of Gastroenterology, Guangzhou, China. wangjpgz@126.com.
¹⁶ Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. xchen1@bidmc.harvard.edu.

PMID: 30510170
PMCID: PMC6559218
DOI: 10.1038/s41564-018-0300-x

Clostridium difficile toxins induce VEGF-A and vascular permeability to promote disease pathogenesis

Jun Huang et al. Nat Microbiol. 2019 Feb.

. 2019 Feb;4(2):269-279.

doi: 10.1038/s41564-018-0300-x. Epub 2018 Dec 3.

Authors

Affiliations

¹ Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
² Department of Colorectal Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
³ Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
⁴ Division of Digestive Diseases, Inflammatory Bowel Disease Center, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, CA, USA.
⁵ Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia.
⁶ Institute of Microbiology and Immunology, Life and Environment Science College, Shanghai Normal University, Shanghai, China.
⁷ Mucosal Immunology and Biology Research Center, Massachusetts General Hospital for Children and Harvard Medical School, Charlestown, MA, USA.
⁸ Department of Gastrointestinal Surgery, The Third Xiangya Hospital, Central South University, Changsha, China.
⁹ Department of Pathology, Children's Hospital Boston and Harvard Medical School, Boston, MA, USA.
¹⁰ Icahn School of Medicine at Mount Sinai (Elmhurst) Program, New York, NY, USA.
¹¹ Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, University of Eastern Finland and Science Service Center and Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland.
¹² Microbiota-Host Interactions and Clostridia Research Group, Departamento de Ciencias Biológicas, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile.
¹³ Departments of Molecular and Integrative Physiology, Division of Gastroenterology, University of Michigan Medical School, Ann Arbor, MI, USA.
¹⁴ Department of Colorectal Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. wangjpgz@126.com.
¹⁵ Guangdong Institute of Gastroenterology, Guangzhou, China. wangjpgz@126.com.
¹⁶ Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. xchen1@bidmc.harvard.edu.

PMID: 30510170
PMCID: PMC6559218
DOI: 10.1038/s41564-018-0300-x

Abstract

Clostridium difficile infection (CDI) is mediated by two major exotoxins, toxin A (TcdA) and toxin B (TcdB), that damage the colonic epithelial barrier and induce inflammatory responses. The function of the colonic vascular barrier during CDI has been relatively understudied. Here we report increased colonic vascular permeability in CDI mice and elevated vascular endothelial growth factor A (VEGF-A), which was induced in vivo by infection with TcdA- and/or TcdB-producing C. difficile strains but not with a TcdA^-TcdB^- isogenic mutant. TcdA or TcdB also induced the expression of VEGF-A in human colonic mucosal biopsies. Hypoxia-inducible factor signalling appeared to mediate toxin-induced VEGF production in colonocytes, which can further stimulate human intestinal microvascular endothelial cells. Both neutralization of VEGF-A and inhibition of its signalling pathway attenuated CDI in vivo. Compared to healthy controls, CDI patients had significantly higher serum VEGF-A that subsequently decreased after treatment. Our findings indicate critical roles for toxin-induced VEGF-A and colonic vascular permeability in CDI pathogenesis and may also point to the pathophysiological significance of the gut vascular barrier in response to virulence factors of enteric pathogens. As an alternative to pathogen-targeted therapy, this study may enable new host-directed therapeutic approaches for severe, refractory CDI.

PubMed Disclaimer

Conflict of interest statement

Competing Interests

The authors declare no competing interests.

Figures

**Figure 1.. *C. difficile* infection led to increased colonic vascular permeability in mice.**
Antibiotic-exposed C57BL6 mice were infected with 0.5×10⁵ cfu *C. difficile* VPI10463. Colon and cecum tissue was harvested at 24 and 48 hours post-infection. (a) Colorectal tissue freshly obtained from control and CDI mice demonstrated more visible colonic vasculature. This experiment was repeated at least 10 times independently with similar results. (**b & c**) *C. difficile* infection led to increased colonic vascular permeability in mice. Vascular permeability was quantified by Evans Blue extravasation method. Data are presented as Mean ± SEM, ****P<0.0001. N=10 biological independent animals in each group. (**d & e**) Significant increase of colonic vascular staining in CDI mice. Representative images of vWF staining. Scale: 100 μm N=5 (control), N=6 (CDI) biologically independent animals. **P=0.0048. Data are presented as Mean ± SEM. Two-sided t-test was used for all statistical analysis above.

**Figure 2.. *C. difficile* toxins induced VEGF-A production in human colonocytes; this induction was mediated by HIFα, p38-MAPK and MEK1/2 signaling pathways.**
(a) Angiogenesis cytokines array demonstrated that VEGF-A and IL-8 were induced significantly in NCM460 cells treated with TcdA (1.0 μg/ml) for 6, 12 and 24 hours. IL-1α (P=0.184), IL-1β (P=0.096), IL-6 (P=0.094), IL-17 (P=0.074), VEGF-C (P=0.250), VEGF-D (P=0.427), TNFα (P=0.060), TNFβ (P=0.184), FGF-2 (P=0.250),VEGF-A (P=0.0008), IL-8 (P=0.0019). Three independent biological samples were used in each group. Data are presented as Mean ± SEM, two-sided t-test was used for all statistical analysis. (**b & c**) NCM460 cells were treated with TcdA or TcdB at various concentrations (0.01, 0.1, 1.0 and 10 μg/ml) for 12 or 24 hours. VEGF-A concentration in the media was quantified by ELISA. TcdA induces dose-dependent VEGF-A production in NCM460 colonocytes, P=0.070 (ctrl vs. TcdA0.01μg/ml), P=0.012 (ctrl vs. TcdA0.1μg/ml), P=0.019 (ctrl vs. TcdA1.0μg/ml), P=0.003 (ctrl vs. TcdA10μg/ml). TcdB at concentrations of 0.01, 0.1 and 1.0 μg/ml induced VEGF-A production significantly, although not at 10 μg/ml due to cell cytotoxicity. P=0.022 (ctrl vs. TcdB0.01μg/ml), P=0.007 (ctrl vs. TcdB0.1μg/ml), P=0.016 (ctrl vs. TcdB1.0μg/ml), P=0.217 (ctrl vs. TcdB10μg/ml) (**d & e**) HIFα, p38-MAPK and MEK1/2 signaling pathways are all involved in TcdA or TcdB-induced VEGF-A production. TcdA- or TcdB-induced VEGF-A production in NCM460 cells was inhibited by each of these inhibitors: p38-MAPK (SB203580, 10 μM), HIFa (FM19G11, 20 μM) and MEK1/2 (U0126, 10 μM or PD98059, 20 μM). P=0.001 (ctrl vs. TcdA), P=0.005 (TcdA vs. TcdA+U0126), P=0.0002 (TcdA vs. TcdA+SB230580), P=0.024 (TcdA vs. TcdA+PD98059), P=0.003 (TcdA vs. TcdA+ FM19G11), P=0.016 (ctrl vs. TcdB), P=0.004 (TcdB vs. TcdB+U0126), P=0.011 (TcdB vs. TcdB+SB230580), P=0.034 (TcdB vs. TcdB+PD98059), P=0.001 (TcdB vs. TcdB+FM19G11). Data are presented as Mean ± SEM, two-sided t-test was used for all statistical analysis above. *P<0.05, **P<0.01, ***P<0.001. N=3 independent biological samples. These experiments were individually repeated three times.

**Figure 3.. Toxins induced VEGF-A production in CDI mice *in vivo* and in human colonic mucosa *ex vivo*.**
(a) Antibiotic-exposed mice were infected with 0.5×10⁵ cfu *C. difficile* VPI10463. Tissue VEGF-A was quantified by ELISA. VEGF-A protein is significantly increased in both the colon and cecum of CDI versus control mice at 48 hours post-infection (P=0.003; P<0.0001, N=6 & 7 biologically independent animals). P=0.898 (ctrl vs. 24h, colon), P=0.001 (ctrl vs. 48h, colon), P=0.387 (ctrl vs. 24h, cecum), P<0.0001 (ctrl vs. 48h, cecum). Values are expressed as means ± SE, two-sided t-test was used for statistical analysis. (b) TcdA and TcdB induced VEGF-A production during infection. Mice were infected with wildtype and isogenic toxin mutants of *C. difficile* strain M7404. Colonic VEGF-A at 48 hours post infection quantified by ELISA was significantly elevated in mice infected by the wildtype (TcdA⁺B⁺), TcdA-B⁺ and TcdA⁺B⁻ mutant strains, but not in mice infected with the double negative TcdA⁻B⁻ mutant strain. Data were represented as mean ± SEM, two-sided t-test was used for statistical analysis, *P<0.05; **P<0.01, N=5 biological independent animals in each group except TcdA-B+ group (N=4). P=0.02 (uninfected vs. wildtype); P=0.02 (uninfected vs. TcdA-B+); P=0.0079 (uninfected vs. TcdA+B-); P=0.02 (TcdA-B+ vs. TcdA-B-); P=0.02 (TcdA-B+ vs. TcdA+B-) (**c, d**) VEGF-A was induced in human colonic mucosa exposed to TcdA or TcdB. Freshly obtained human colonic mucosal biopsies were placed in ex vivo culture and exposed to TcdA (0.1, 1.0 μg/ml) or TcdB (1.0, 10 μg/ml) for 24 hours. VEGF-A released into the conditioned media was measured by Milliplex Human Cytokine Magnetic Bead assays. Data were represented as mean ± SEM, two-sided t-test was used for statistical analysis, *P<0.05; **P<0.01, ****P<0.0001, NS: not significant, N=13 in TcdA & N=5 in TcdB. P=0.302 (ctrl vs. TcdA0.1μg/ml), P=0.006 (ctrl vs. TcdA1.0μg/ml), P=0.013 (ctrl vs. TcdB1.0μg/ml), P=0.012 (ctrl vs. TcdB10μg/ml). (e) Immunohistochemical analysis of eNOS in colonic tissue of normal or CDI mice. Yellow arrows indicate sub-mucosal vascular vessels (Scale bars: 100 μm). Bar chart shows increased expression score of vascular endothelial eNOS in CDI mice compared to control (P=0.005). N=8 biological independent animals in each group. (f) RT-PCR relative amounts of eNOS expression in control animals and those infected with CDI. Values are expressed as means ± SE, two-sided t-test was used for statistical analysis. *P =0.0228 compared with control, N=5 biological independent animals in each group.

**Figure 4.. Human colonocytes pre-exposed to TcdA or TcdB stimulated HIMEC proliferation, which was mediated by HIF signaling.**
(a) Human colonocytes pre-exposed to TcdA or TcdB stimulated HIMEC proliferation. NCM460 cells were pre-treated with Toxin A or Toxin B at 1.0 μg/ml for 24 hours. The supernatant were ultra-filtered to remove toxins before applying to HIMEC cells. After another 24 hours, tube formation of HIMEC was quantified. Compared to naïve cell, NCM460 cells exposed to either toxin A or B significantly enhanced HIMEC tube formation (P<0.0001 each). Data are presented as Mean ± SEM. Two-sided t-test was used for statistical analysis. (b) HIF-1a signaling in human colonocyes mediates toxin-induced HIMEC proliferation through VEGF. Human colonocytes HCT116 with wildtype HIF-1α (WT), HIF-1α knock down (KD), or HIF-1α over expression were stimulated by mixture of TcdA and TcdB at 1.0 μg/ml each. Using the same approach above in A, relative HIMEC tube formation were compared among all experimental groups. All cell lines responded to toxins in the subsequent HIMEC stimulation. Compared to wildtype cells (WT), HIF-1α knock down (KD) cells had significantly less stimulation of HIMEC proliferation (P=0.0065) whereas HIF-1α overexpression significantly enhanced the effect on HIMEC (P=0.04), which was attenuated by VEGF-A neutralizing antibody (P=0.03). Data are presented as Mean ± SEM. Two-sided t-test was used for statistical analysis. (c) Stabilization of HIF-1α expression exacerbated CDI severity; Inhibition of eNOS protected against CDI. CDI mice were treated with DMOG (400mg/kg ip, day 1 and day 2), L-NAME (20mg/kg ip, day 1, 2 and 3) or vehicle (saline, day 1, 2 and 3) respectively. DMOG treatment significantly exacerbated the death rate in CDI mice (P<0.0001). In contrast, inhibition of eNOS by in vivo administration of L-NAME significantly protected CDI mice (P=0.04). N=10 biological independent animals in each group. Log-rank (Mantel-Cox) test was used for survival analysis. (d) Knocking out HIF-1α specifically in intestinal epithelial cells reduced CDI symptom in mice. Both HIF1α-KO mice and wildtype littermate control were challenged with *C difficile* using the same protocol described above. Litters from both groups are more resistant against CDI compared to our other mice experiments. There is no significant difference in the mortality rate (P=0.456, upper panel, Log-rank test). The onset of diarrhea are significantly delayed and the percentage of diarrhea was reduced in HIF1α-KO mice (middle panel, P =0.03, Log-rank test). There is significantly less weight loss in HIF1α-KO group compared to wildltype (lower panel, on day 5, P=0.008, two-sided t-test). N=9 in wildtype littermate control. N=5 in HIF1α-KO group. n.s. nonsignificant; ** P<0.01.

**Figure 5.. Both VEGFR-2 kinase inhibition and Anti-VEGF-A treatment attenuated vascular permeability and protected mice from *C. difficile* infection.**
(a) VEGFR-2 kinase inhibitor SU1498 significantly attenuated vascular permeability in mice infected with *C. difficile*. Colon: P=0.0002 (Ctrl vs. CDI), P=0.006 (CDI vs. CDI+SU1498); cecum: P<0.0001 (Ctrl vs. CDI), P<0.0001 (CDI vs. CDI+SU1498); N=8 biological independent animals in each group. CDI mice were treated with SU1498 (a selective inhibitor of VEGFR-2 kinase, 30 mg/kg *i.p*. daily for 3 days). Colonic and cecal vascular permeability was quantified as described above. Data are presented as Mean ± SEM, two-sided t-test was used for statistical analysis. (b) SU1498 treatment significantly enhanced survival of CDI versus control mice (63% vs. 19%, P=0.001, N=22&21 biological independent animals, Log-rank test) (upper panel). Relative body weights were monitored daily and were compared using the Mann-Whitney U test. SU1498 treatment reduced the weight loss associated with CDI as compared to vehicle control. P=0.044 (day6), 0.012 (day7), 0.012 (day8). Data are presented as Mean ± SEM, two-sided t-test was used for statistical analysis, *P<0.05, **P<0.01, ***P<0.001. N=22 & 21 biological independent animals (lower panel). (c) Anti-VEGF-A treatment significantly attenuated vascular permeability in mice infected with *C. difficile* compared to isotype control (colon: P=0.0007; cecum: P <0.0001; N=6 biological independent animals in each group). CDI mice were either untreated or treated with anti-mouse VEGF-A antibody or isotype control by i.p. injection at 12h and 36h after *C. difficile* challenge. Colonic and cecal vascular permeability was quantified as described above. Data are presented as Mean ± SEM, two-sided t-test was used for statistical analysis. P=0.154 (untreated vs. isotype ctrl, colon), P=0.0005 (isotype ctrl vs. anti-mouse VEGF-A, colon), P=0.197 (untreated vs. isotype ctrl, cecum), P<0.0001 (isotype ctrl vs. anti-mouse VEGF-A, cecum). (d) Anti-VEGF-A antibody treatment protected mice from weight loss (P<0.0001, data are presented as Mean ± SEM, two-sided t-test) and increased the overall survival significantly relative to isotype control (62.5% vs. 10.0%, P=0.03, Log-rank test). N=8 in anti-VEGF-A group, N=8 in isotype group, & N=10 in untreated group.

**Figure 6.. Enhanced serum levels of VEGF-A in CDI patients and reduced VEGF-A level in follow-up sera after standard treatment.**
Serum VEGF-A were measured and compared in healthy control (N=35), active CDI patients (N=50) and 23 of these CDI patients’ follow-up sera (N=23). (a) Serum levels of VEGF-A are significantly higher in CDI patients than in healthy controls (P=0.001, two-sided Mann-Whitney test). Data are presented as Mean ± SEM. (b) VEGF-A is significantly lower in follow-up sera of CDI patients than their paired sera in active CDI (P=0.015, Wilcoxon matched pairs test). Data were presented as paired (pre- and post-treatment) VEGF-A concentrations in each patient’s sera.

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Clostridium difficile toxins induce VEGF-A and vascular permeability to promote disease pathogenesis

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Clostridium difficile toxins induce VEGF-A and vascular permeability to promote disease pathogenesis

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