• Users Online: 307
  • Print this page
  • Email this page


 
 
Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 33  |  Issue : 3  |  Page : 155-159

A gene–gene interaction between the vascular endothelial growth factor a and brain-derived neurotrophic factor genes is associated with psychological distress in the Taiwanese population


1 Department of Biostatistics; Department of Electrical and Computer Engineering, University of Washington, Seattle, Washington, USA; Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan
2 Department of Public Health, Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, Taiwan
3 Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli County, Taiwan
4 Division of Interdisciplinary Medicine and Biotechnology, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, USA; Institute of Brain Science, National Yang-Ming University, Taipei, Taiwan
5 Division of Interdisciplinary Medicine and Biotechnology, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, USA; Department of Psychiatry, Taipei Veterans General Hospital; Division of Psychiatry, National Yang-Ming University, Taipei, Taiwan

Date of Submission24-Jun-2019
Date of Decision12-Jul-2019
Date of Acceptance12-Jul-2019
Date of Web Publication30-Sep-2019

Correspondence Address:
Shih- Jen Tsai
No. 201, Shih-Pai Road, Section 2, Taipei 1127

Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/TPSY.TPSY_30_19

Rights and Permissions
  Abstract 


Background: Vascular endothelial growth factor A (VEGFA) and brain-derived neurotrophic factor (BDNF), the most abundant and widely expressed neurotrophic factors in the brain, are believed to play an important rôle in depression and anxiety. In this study, we hypothesized that single-nucleotide polymorphisms (SNPs) within the VEGFA and BDNF genes would be linked with psychological distress through complex interactions in the general population. Methods: We analyzed 7,098 Taiwanese subjects from the Taiwan Biobank. Measures of anxiety and depression were evaluated using the Patient Health Questionnaire-4 (PHQ-4). Totally, 15 VEGFA and 43 BDNF polymorphisms were used in the genetic analysis. Results: In our analysis, an interaction was found between the VEGFA rs10434 and BDNF rs12418745 in significantly influencing depression state (p < 0.01). In addition, we found that influence of interaction existed between physical activity and VEGFA genetic variants including rs3025000, rs699947, rs833068, rs833069, rs3024998, and rs3025006 in depression state. But we found no association between 15 VEGFA genetic variants and depression state, after adjusting for age and gender. Furthermore, no VEGFA SNPs showed evidence of association with PHQ-4 scores. Conclusion: These results suggest that the VEGFA and BDNF genetic variants may contribute to psychological distress through gene-gene and gene-physical activity interactions in the general population.

Keywords: anxiety, depression, gene–gene interaction, exercise


How to cite this article:
Lin E, Kuo PH, Liu YL, Yang AC, Tsai SJ. A gene–gene interaction between the vascular endothelial growth factor a and brain-derived neurotrophic factor genes is associated with psychological distress in the Taiwanese population. Taiwan J Psychiatry 2019;33:155-9

How to cite this URL:
Lin E, Kuo PH, Liu YL, Yang AC, Tsai SJ. A gene–gene interaction between the vascular endothelial growth factor a and brain-derived neurotrophic factor genes is associated with psychological distress in the Taiwanese population. Taiwan J Psychiatry [serial online] 2019 [cited 2019 Nov 21];33:155-9. Available from: http://www.e-tjp.org/text.asp?2019/33/3/155/268318




  Introduction Top


Vascular endothelial growth factor (VEGF), initially known as vascular permeability factor, was originally discovered due to its involvement in the formation of new blood vessels [1]. Previous studies showed that VEGF is widely distributed in multiple cell types of the brain and is known to exert a trophic effect on neurons and glial cells [2],[3]. This effect has a rôle in the essential process for neuroprotection and neurogenesis. Actions of VEGF are mediated through binding to two high-affinity receptor tyrosine kinases, kinase insert domain receptor (or protein–tyrosine kinase receptor Flk-1) and FMS-related tyrosine kinase 1 [4]. Those kinases have been implicated to cause some degenerative diseases such as Parkinson's disease and Alzheimer's disease [4].

In 2007, VEGF was first reported to be induced by multiple classes of antidepressants at time points being consistent with the induction of cell proliferation and therapeutic action of these treatments [5]. VEGF's antidepressant-induced cell proliferation effect is through the Flk-1 receptor signaling [5]. A previous animal study further showed that the VEGF-induced antidepressant-like effects involve in modulating norepinephrine and serotonin systems [6]. A recent study showed that neuronal VEGF-Flk-1 signaling in the medial prefrontal cortex of mice plays a crucial rôle in the antidepressant actions of ketamine, to produce rapid antidepressant actions even in patients with treatment-resistant depression [7].

VEGF is also implicated in depression pathogenesis. In a study with chronic stress-induced depressive rats, the VEGF protein and mRNA expression levels in the hippocampus of the experimental group are lower than those in the control group [8]. In contrast to animal studies, most clinical studies have been reported to have high blood VEGF levels and mRNA expression in patients with major depressive disorder (MDD) compared to those in healthy controls [9].

VEGF is encoded by VEGFA gene, located on chromosome 6p12, which includes eight exons separated by seven introns [10]. Because VEGF signaling is required for antidepressant-induced behavioral response, we have done antidepressant pharmacogenetic study with 7 VEGFA polymorphisms in 351 MDD patients. We found that VEGFA genetic variants do not play a major rôle in response to selective serotonin reuptake inhibitors [11]. But a previous study in Finland has been found to have an association between VEGFA 2578 C/A polymorphism and treatment-resistant depression [12]. Recently, Xie et al. tested four VEGFA-related single-nucleotide polymorphisms (SNPs) identified through a genome-wide association study (rs10738760, rs6921438, rs6993770, and rs4416670) on depression [13]. The rs4416670 polymorphism has been found to be associated with increased risk for MDD, suggesting the existence of relationships between VEGFA genetic determinants and depression [13].

VEGF plays an important rôle in developing depression and its therapeutic mechanisms. To our knowledge, the VEGFA genetic effect in anxiety and depression is not studied in the general population. In this study, we hypothesized that VEGFA genetic polymorphisms would be linked with psychological distress. With a Taiwanese general population, we intended to assess the potential gene-gene and gene-physical activity effects of these associations on the mood states. Because brain-derived neurotrophic factor (BDNF), a homodimeric neurotrophic factor, is the most abundant and widely expressed neurotrophin in the brain, BDNF is thought to play an important rôle in stress response and mood disorder [14],[15],[16]. Thus, we also tested the gene-gene interaction between VEGFA and BDNF genetic variants in psychological distress.


  Methods Top


Study population

We analyzed data from 3,213 men and 3,885 women recruited in the Taiwan Biobank, a national biomedical research database that contains genetic information of the general population [17],[18]. Recruitment and sample collection procedures were approved by the institutional review board of the Taiwan Biobank before conducting the study, and the approved informed consent form was signed by each subject. IRB protocol number was 201506095RINC, and was approved on July 30, 2016.

Depression and anxiety assessment

Psychological distress assessment was done using the self-report Patient Health Questionnaire-4 (PHQ-4), which is an ultra-brief screening tool to detect emotional disorders in primary care [19]. PHQ-4 has four items (0 - 3 for each item; total score ranges from 0 to 12), to measure anxiety and depressive symptoms in the past two weeks and has been demonstrated to be a general marker of psychological distress [19].

Genotyping

DNA was isolated from blood samples using a QIAamp DNA blood kit following the manufacturer's instructions (Qiagen, Valencia, California, USA). The quality of the isolated genomic DNA was evaluated using agarose gel electrophoresis, and the quantity was determined by spectrophotometry. SNP genotyping was carried out using the custom TWB chips and run on the Axiom Genome-Wide Array Plate System (Affymetrix, Santa Clara, California, USA). To efficiently obtain maximal genetic information from Taiwanese Han Chinese samples, the custom Taiwan Biobank chips were designed using SNPs on the Axiom Genome-Wide CHB 1 Array (Affymetrix, Inc., Santa Clara, California, USA) with minor allele frequencies (MAFs) ≥ 5%, using SNPs in exons with MAFs > 10% on the HumanExome BeadChip (Illumina, Inc., San Diego, California, USA). In this study, quality criteria for BDNF SNP exclusion from further analyses were the following: due to the failure to achieve the Hardy–Weinberg equilibrium, a genotyping call rate < 90%, or to MAF < 1%. After the quality control procedure, a total of 15 VEGFA and 43 BDNF polymorphisms were used for further analysis.

Statistical analysis

To estimate the association of the investigated SNP with depression state, we did a logistic regression analysis to evaluate the odd ratios and their 95% confidence intervals, adjusting for covariates including age and gender. Furthermore, we estimated the associations of the investigated VEGFA SNPs with PHQ-4 scores using a general linear model using age and gender as covariates.

To investigate gene-gene and gene-physical activity interactions, we leveraged the generalized multifactor dimensionality reduction (GMDR) method [20]. We tested two-way interactions with 10-fold cross-validation. The GMDR software provided some output parameters, including testing accuracy and empirical p- values, to assess each selected interaction. Moreover, we provided age and gender as covariates for gene-gene and gene-physical activity interaction models in our interaction analyses. Permutation testing obtained empirical p- values of prediction accuracy as a benchmark based on 1,000 shuffles.

Multiple testing was adjusted using the Bonferroni correction. Data were presented as the mean ± standard deviation (SD). All the study data were computed using the Statistical Package for the Social Science version 22 for Windows (SPSS Inc., Chicago, Illinois, USA). The differences between groups were considered significant if p- values were smaller than 0.05.


  Results Top


This study included 7,098 subjects (male = 3,213, female = 3,885, age range = 30-70 years, and mean/SD = 49.9/11.0 years). The median PHQ-4 score was 0, and the interquartile range was 0-2.

A score of 3 in the 4th item of the PHQ-4 (feeling down, depressed, or hopeless) has been suggested as “current major depression and/or dysthymia” [21]. Among the studied subjects, 52 had score of 3 in this item [Table 1] were grouped as depression group, whereas the others were as normal state. Among the 15 VEGFA SNPs assessed in this study, we did not find group. As shown in [Table 2], no significant associations existed between 15 VEGFA SNPs and any significant association between 15 VEGFA SNPs and PHQ-4 scores [Table 3].
Table 1: Demographic and clinical characteristics of the study subjects

Click here to view
Table 2: Odds ratio analysis with odds ratios after adjustment for covariates between the Patient Health Questionnaire-4 (normal = 0, 1, and 2; depression = 3) and 15 single-nucleotide polymorphisms in the vascular endothelial growth factor A gene

Click here to view
Table 3: Linear regression models of associations between the Patient Health Questionnaire-4 (as a continuous outcome) and 15 single-nucleotide polymorphisms in the vascular endothelial growth factor A gene

Click here to view


Furthermore, the GMDR analysis with adjustment for age and gender was used to assess the impacts of SNP combinations between VEGFA and BDNF genes in depression state. A significant two-way model existed involving VEGFA rs10434 and BDNF rs12418745 (p < 0.01), indicating a potential gene-gene interaction between these two polymorphisms in influencing depression state.

Finally, physical activity and gene interaction models were evaluated using the GMDR method with adjustment for age and gender. As shown in [Table 4], significant two-way models existed involving physical activity and VEGFA SNPs including rs3025000 (p < 0.05), rs699947 (p < 0.05), rs833068 (p < 0.05), rs833069 (p < 0.05), rs3024998 (p < 0.05), and rs3025006 (p < 0.05), indicating potential physical activity and gene interaction in influencing depression state.
Table 4: Gene–physical activity interaction models identified by the generalized multifactor dimensionality reduction method with adjustment for age and gender

Click here to view



  Discussion Top


The risk of developing physical illnesses and the use of general medical services are increased through psychological distress, such as depression and anxiety [22],[23]. As shown in [Table 1], we included 52 participants for the depression group and 7,046 participants from controls in this study. We tested whether VEGFA genetic variants in the general population affects psychological stress. The major finding of this study [Table 4] showed significant interactions between physical activity and VEGFA genetic variants including rs3025000 (p < 0.05), rs699947 (p < 0.05), rs833068 (p < 0.05), rs833069 (p < 0.05), rs3024998 (p < 0.05), and rs3025006 (p < 0.05) in depression state. The positive influence of physical activity on mood has been well-studied [24]. The major causes of anxious and depressive moods, although they are known to have strong genetic determinants, are considered multifactorial consequences of rather complex interactions among environmental and genetic factors [25]. The GMDR analysis of gene-environment interactions in this study [Table 2] reflected the interplay among VEGFA and the physical activity in influencing depression state. Animal studies have focused mainly on the beneficial effects of exercise, including improved cognitive performance, memory, and mood, and on hippocampal function, with special emphasis on exercise-induced hippocampal neurogenesis [26],[27]. The physical response produced by exercise, which may directly or indirectly regulate the number of neuronal precursor cells in the hippocampus, has not been well-defined. A recent study using vegfa gene-ablated mice demonstrated that peripheral VEGF is necessary for exercise to stimulate hippocampal neurogenesis [28]. The mechanism by which peripheral VEGF induces hippocampal neurogenesis has yet to be clarified. Indeed, earlier animal studies also showed that peripheral inhibition of VEGF prevents exercise-induced hippocampal neurogenesis, suggesting that blood VEGF has the potential to induce direct effects on downstream pathways that affect neurogenesis [29]. Our finding is the first evidence from human study to suggest that exercise may interact with VEGFA gene to affect mood state.

Our second finding was that an interaction between the VEGFA rs10434 and BDNF rs12418745 was found in influencing depression state. The fact that most genetic studies are mainly focused on the associations between a single SNP with phenotype is a major problem [30]. This disregards the joint effect of multiple loci and the complex interaction network in between, underestimating the rôles of genetics in human diseases. Apart from the statistical significance, another concern was the potential biological mechanism under interaction models. These two factors, BDNF and VEGF, have been implicated in antidepressant action and depression pathogenesis [31],[32]. Because BDNF has been reported to stimulate VEGF expression and to be released in neuroblastoma cells [33], Deyama et al. tested if the VEGF signaling acts downstream of BDNF to produce the neurotrophic and antidepressant-like actions [34]. Investigators found that the antidepressant-like and neurotrophic effects of BDNF require VEGF signaling. In addition, BDNF stimulates the release of VEGF and that BDNF induction of dendrite complexity is blocked by a selective VEGF receptor antagonist. Our finding further suggests that BDNF may interact with VEGF in depression state.

In this study [Table 2], we found no significant associations between 15 VEGFA genetic variants and depression state after adjusting for age and gender. Furthermore, no VEGFA SNPs showed evidence of significant association with PHQ-4 scores [Table 3]. Thus, VEGFA alone may not affect mood state in the general population. Instead, it acts through interaction with BDNF and exercise to affect mood state.

Study limitations

This study presents four limitations which may cause over interpreting the results:

  • Our data showed no significant association between 15 VEGFA genetic variants and depression state after adjusting for age and gender. This can be due to small sample size in the study. Further studies with large sample size are warranted.
  • The self-report questionnaire PHQ-4 consists of a four-item inventory. Its reliability used by researchers is dependent on the honesty of the participants. But an issue which remains is the lack in introspective ability of the participant to give accurate response to a question. On the contrary, 84% of the total variance is explained by two discrete factors (depression and anxiety) confirmed by factor analysis. Functional impairment, health-care use, and disability days are strongly associated with increasing PHQ-4 scores [19].
  • The study data were from a genetic study bank. They may not effectively reflect the actual functional significance, which needs clinical studies with more vigorous design to support.
  • We cannot find the functional studies of the 15 VEGFA SNPs.


Summary

We have analyzed in detail the interactions as well as the genetic associations between VEGFA and BDNF genes with psychological stress in the Taiwanese general population samples. Based on the current study, the results demonstrated that the BDNF and VEGFA genetic variants may contribute to psychological stress through gene-physical activity and gene-gene interactions. Further insights into the rôle of the VEGFA and BDNF genes found in this study may be demonstrated in independent replication studies with larger sample sizes.


  Acknowledgment Top


The authors thank Emily Ting for English editing.


  Financial Support and Sponsorship Top


This work was supported by grant V108C-038 from the Taipei Veterans General Hospital.


  Conflicts of Interest Top


The authors declared that they have no conflicts of interest in writing this report.



 
  References Top

1.
Ferrara N, Henzel WJ: Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989; 161: 851-8.  Back to cited text no. 1
    
2.
Virgintino D, Errede M, Robertson D, et al.: VEGF expression is developmentally regulated during human brain angiogenesis. Histochem Cell Biol 2003; 119: 227-32.  Back to cited text no. 2
    
3.
Sun Y, Jin K, Xie L, et al.: VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest 2003; 111: 1843-51.  Back to cited text no. 3
    
4.
Storkebaum E, Lambrechts D, Carmeliet P: VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays 2004; 26: 943-54.  Back to cited text no. 4
    
5.
Warner-Schmidt JL, Duman RS: VEGF is an essential mediator of the neurogenic and behavioral actions of antidepressants. Proc Natl Acad Sci U S A 2007; 104: 4647-52.  Back to cited text no. 5
    
6.
Udo H, Hamasu K, Furuse M, et al.: VEGF-induced antidepressant effects involve modulation of norepinephrine and serotonin systems. Behav Brain Res 2014; 275: 107-13.  Back to cited text no. 6
    
7.
Deyama S, Bang E, Wohleb ES, et al.: Role of neuronal VEGF signaling in the prefrontal cortex in the rapid antidepressant effects of ketamine. Am J Psychiatry 2019; 176: 388-400.  Back to cited text no. 7
    
8.
Li B, Wang B, Chen M, et al.: Expression and interaction of TNF-α and VEGF in chronic stress-induced depressive rats. Exp Ther Med 2015; 10: 863-8.  Back to cited text no. 8
    
9.
Sharma AN, da Costa e Silva BF, Soares JC, et al.: Role of trophic factors GDNF, IGF-1 and VEGF in major depressive disorder: a comprehensive review of human studies. J Affect Disord 2016; 197: 9-20.  Back to cited text no. 9
    
10.
Tischer E, Mitchell R, Hartman T, et al.: The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 1991; 266: 11947-54.  Back to cited text no. 10
    
11.
Tsai SJ, Hong CJ, Liou YJ, et al.: Haplotype analysis of single nucleotide polymorphisms in the vascular endothelial growth factor (VEGFA) gene and antidepressant treatment response in major depressive disorder. Psychiatry Res 2009; 169: 113-7.  Back to cited text no. 11
    
12.
Viikki M, Anttila S, Kampman O, et al.: Vascular endothelial growth factor (VEGF) polymorphism is associated with treatment resistant depression. Neurosci Lett 2010; 477: 105-8.  Back to cited text no. 12
    
13.
Xie T, Stathopoulou MG, de Andrés F, et al.: VEGF-related polymorphisms identified by GWAS and risk for major depression. Transl Psychiatry 2017; 7: e1055.  Back to cited text no. 13
    
14.
Tsai SJ: Targeting brain-derived neurotrophic factor (BDNF) signaling pathway as a treatment strategy for depression. Taiwan J Psychiatry 2018; 32: 103-13.  Back to cited text no. 14
    
15.
Tsai SJ: Critical issues in BDNF val66Met genetic studies of neuropsychiatric disorders. Front Mol Neurosci 2018; 11: 156.  Back to cited text no. 15
    
16.
Hong CJ, Liou YJ, Tsai SJ: Effects of BDNF polymorphisms on brain function and behavior in health and disease. Brain Res Bull 2011; 86: 287-97.  Back to cited text no. 16
    
17.
Lin E, Yang AC, Tsai SJ: Association between metabolic syndrome and cognitive function in old adults in a Taiwanese population. Taiwan J Psychiatry 2017; 31: 232-40.  Back to cited text no. 17
    
18.
Lin E, Kuo P, Liu Y, et al.: A gene-based analysis of variants in the brain-derived neurotrophic factor gene with psychological distress in a Taiwanese population. Taiwan J Psychiatry 2019; 33: 33-8.  Back to cited text no. 18
    
19.
Kroenke K, Spitzer RL, Williams JB, et al.: An ultra-brief screening scale for anxiety and depression: the PHQ-4. Psychosomatics 2009; 50: 613-21.  Back to cited text no. 19
    
20.
Lou XY, Chen GB, Yan L, et al.: A generalized combinatorial approach for detecting gene-by-gene and gene-by-environment interactions with application to nicotine dependence. Am J Hum Genet 2007; 80: 1125-37.  Back to cited text no. 20
    
21.
Osório FL, Lima MP, Chagas MH: Screening tools for psychiatry disorders in cancer setting: caution when using. Psychiatry Res 2015; 229: 739-42.  Back to cited text no. 21
    
22.
McDonald WM, Salzman C, Schatzberg AF: Depression in the elderly. Psychopharmacol Bull 2002; 36 (Suppl 2): 112-22.  Back to cited text no. 22
    
23.
Rackley S, Bostwick JM: Depression in medically ill patients. Psychiatr Clin North Am 2012; 35: 231-47.  Back to cited text no. 23
    
24.
Basso JC, Suzuki WA: The effects of acute exercise on mood, cognition, neurophysiology, and neurochemical pathways: a review. Brain Plast 2017; 2: 127-52.  Back to cited text no. 24
    
25.
Tafet GE, Nemeroff CB: The links between stress and depression: psychoneuroendocrinological, genetic, and environmental interactions. J Neuropsychiatry Clin Neurosci 2016; 28: 77-88.  Back to cited text no. 25
    
26.
Vivar C, Potter MC, van Praag H: All about running: synaptic plasticity, growth factors and adult hippocampal neurogenesis. Curr Top Behav Neurosci 2013; 15: 189-210.  Back to cited text no. 26
    
27.
van Praag H: Neurogenesis and exercise: past and future directions. Neuromolecular Med 2008; 10: 128-40.  Back to cited text no. 27
    
28.
Rich B, Scadeng M, Yamaguchi M, et al.: Skeletal myofiber vascular endothelial growth factor is required for the exercise training-induced increase in dentate gyrus neuronal precursor cells. J Physiol 2017; 595: 5931-43.  Back to cited text no. 28
    
29.
Fabel K, Fabel K, Tam B, et al.: VEGF is necessary for exercise-induced adult hippocampal neurogenesis. Eur J Neurosci 2003; 18: 2803-12.  Back to cited text no. 29
    
30.
Yang CH, Chuang LY, Lin YD: Multiobjective multifactor dimensionality reduction to detect SNP-SNP interactions. Bioinformatics 2018; 34: 2228-36.  Back to cited text no. 30
    
31.
Newton SS, Duman RS: Regulation of neurogenesis and angiogenesis in depression. Curr Neurovasc Res 2004; 1: 261-7.  Back to cited text no. 31
    
32.
Sharma AN, da Costa e Silva BF, Soares JC, et al.: Role of trophic factors GDNF, IGF-1 and VEGF in major depressive disorder: a comprehensive review of human studies. J Affect Disord 2016; 197: 9-20.  Back to cited text no. 32
    
33.
Nakamura K, Martin KC, Jackson JK, et al.: Brain-derived neurotrophic factor activation of trkB induces vascular endothelial growth factor expression via hypoxia-inducible factor-1alpha in neuroblastoma cells. Cancer Res 2006; 66: 4249-55.  Back to cited text no. 33
    
34.
Deyama S, Bang E, Kato T, et al.: Neurotrophic and antidepressant actions of brain-derived neurotrophic factor require vascular endothelial growth factor. Biol Psychiatry 2019; 86: 143-52.  Back to cited text no. 34
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
   Abstract
  Introduction
  Methods
  Results
  Discussion
  Acknowledgment
   Financial Suppor...
   Conflicts of Int...
   References
   Article Tables

 Article Access Statistics
    Viewed130    
    Printed8    
    Emailed0    
    PDF Downloaded18    
    Comments [Add]    

Recommend this journal