<p><div><div><div><div data-show-faces="false" data-height="21" data-width="100" data-layout="button_count" data-send="true" data-href="https://www.r-bloggers.com/r-gene-regulatory-interaction-formulator-for-inquiring-networks/"></div></div><div></div></div><div></div></div>
<div>
(This article was first published on <strong><a href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/"> Fellgernon Bit - rstats</a></strong>, and kindly contributed to <a rel="nofollow" href="https://www.r-bloggers.com/">R-bloggers)</a> </div>
<p><em>This blog post was written by <a rel="nofollow" href="https://github.com/mar-esther23/" target="_blank">ME Martinez-Sanchez</a>, <a rel="nofollow" href="https://www.researchgate.net/profile/Stalin_Munoz" target="_blank">S Muñoz</a>, <a rel="nofollow" href="http://www.fciencias.unam.mx/directorio/33021" target="_blank">M Carrillo</a>, <a rel="nofollow" href="https://www.researchgate.net/profile/Eugenio_Azpeitia" target="_blank">E Azpeitia</a>, <a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/turing.iimas.unam.mx/~drosenbl/" target="_blank">D Rosenblueth</a> and originally posted at the <a rel="nofollow" href="http://www.comunidadbioinfo.org/r-gene-regulatory-interaction-formulator-for-inquiring-networks/" target="_blank">CDSB blog</a>.<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#fn1" target="_blank"><sup>1</sup></a></em></p>
<p>In this blog post we will describe the package <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> <a></a>(<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) that was one of the projects developed during the <a rel="nofollow" href="http://congresos.nnb.unam.mx/TIB2018/r-bioconductor-developers-workshop-2018/" target="_blank">TIB2018-BCDW</a>. We hope to continue developing <a rel="nofollow" href="http://turing.iimas.unam.mx/griffin/" target="_blank"><code>Griffin</code></a> and <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>). If you have ideas, suggestions or bugs, please contact us via <a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin GitHub repo</a>.</p>
<div>
<img src="https://i2.wp.com/lcolladotor.github.io/post/2018-09-13-r-gene-regulatory-interaction-formulator-for-inquiring-networks_files/header.jpg?w=450" data-recalc-dims="1">
</div>
<div>
<h3>The problem</h3>
<p>Boolean networks allow us to give a mechanistic explanation to how cell types emerge from regulatory networks. However, inferring the regulatory network and its functions is complex problem, as the available information is often incomplete. <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) uses available biological information (regulatory interactions, cell types, mutants) codified as a set of restrictions and returns the <a rel="nofollow" href="https://en.wikipedia.org/wiki/Boolean_network" target="_blank">Boolean Networks</a> that satisfy that restrictions. This Boolean networks can then be used to study the biological system.</p>
<p>The <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) package is an R connector to <a rel="nofollow" href="http://turing.iimas.unam.mx/griffin/" target="_blank"><code>Griffin</code></a> (Gene Regulatory Interaction Formulator For Inquiring Networks), a java library for inference and analysis of Boolean Network models. <a rel="nofollow" href="http://turing.iimas.unam.mx/griffin/" target="_blank"><code>Griffin</code></a> takes as inputs biologically meaningful constraints and turns them into a symbolic representation. Using a SAT engine, <a rel="nofollow" href="http://turing.iimas.unam.mx/griffin/" target="_blank"><code>Griffin</code></a> explores the Boolean Network search space, finding all satisfying assignments that are compatible with the specified constraints. The <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) package includes a number of functions to interact with the BoolNet package.</p>
</div>
<div>
<h3>A small example</h3>
<p>Let us suppose a cell, we know that this cell has three proteins called <strong>a</strong>, <strong>b</strong> and <strong>c</strong>. We know that <strong>a</strong> activates <strong>b</strong> and that <strong>b</strong> and <strong>c</strong> inhibit each other. We also suspect that <strong>b</strong> and <strong>c</strong> <strong>may</strong> have positive self-regulatory loops. We can add this interactions to the table as “OPU” (optional, positive, unambiguous). This dataframe is the topology of the network.</p>
<table>
<thead>
<tr>
<th>Source</th>
<th>Target</th>
<th>Interaction</th>
</tr>
</thead>
<tbody>
<tr>
<td>a</td>
<td>b</td>
<td>+</td>
</tr>
<tr>
<td>b</td>
<td>c</td>
<td>–</td>
</tr>
<tr>
<td>c</td>
<td>b</td>
<td>–</td>
</tr>
<tr>
<td>b</td>
<td>b</td>
<td>OPU</td>
</tr>
<tr>
<td>c</td>
<td>c</td>
<td>OPU</td>
</tr>
</tbody>
</table>
<p>Suppose we also have some information of what cell types have been observed. For example, there is a cell type that expresses <strong>b</strong>, but not <strong>a</strong> or <strong>c</strong> and an other cell type that expresses <strong>c</strong>, but not <strong>a</strong> or <strong>b</strong>. There might exist a third cell type that has not been fully characterized where we know that the cell expresses no <strong>a</strong> or <strong>c</strong> but we have NO information on <strong>b</strong>. This dataframe is the attractors of the network.</p>
<table>
<thead>
<tr>
<th>a</th>
<th>b</th>
<th>c</th>
</tr>
</thead>
<tbody>
<tr>
<td>0</td>
<td>1</td>
<td>0</td>
</tr>
<tr>
<td>0</td>
<td>0</td>
<td>1</td>
</tr>
<tr>
<td>0</td>
<td>*</td>
<td>0</td>
</tr>
</tbody>
</table>
<p>We can then use this information to create a query. <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) can include other types of information like transition between cell type, cycles, transitions between cell types or mutant cell types.</p>
<pre><code>## Install using:
# devtools::install_github('mar-esther23/rgriffin')
## Note that the package depends on rJava

library('rGriffin')</code></pre>
<pre><code>## Loading required package: rJava</code></pre>
<pre><code>genes = c('a','b','c')
inter = data.frame(source=c('a','b','c', 'b','c'), 
                  target=c('b', 'c', 'b', 'b', 'c'), 
                  type=c('+','-','-','OPU','OPU'),
                    stringsAsFactors = F )
q = create.gquery.graph(inter, genes)
attr = data.frame(a=c(0,'*',0), 
                 b=c(0,1,0), 
                 c=c(0,0,1),
                 stringsAsFactors = F )
q = add.gquery.attractors(q, attr)</code></pre>
<p>Then we can use <code>Griffin</code> to find the networks that behave according with our biological information.</p>
<pre><code>nets = run.gquery(q)
nets</code></pre>
<pre><code>## [1] &quot;targets,factors\na,false\nb,((((!a&amp;b)&amp;!c)|((a&amp;!b)&amp;!c))|((a&amp;b)&amp;!c))\nc,(!b&amp;c)\n&quot;                         
## [2] &quot;targets,factors\na,false\nb,(((((!a&amp;b)&amp;!c)|((a&amp;!b)&amp;!c))|((a&amp;b)&amp;!c))|((a&amp;b)&amp;c))\nc,(!b&amp;c)\n&quot;             
## [3] &quot;targets,factors\na,false\nb,((((((!a&amp;b)&amp;!c)|((!a&amp;b)&amp;c))|((a&amp;!b)&amp;!c))|((a&amp;b)&amp;!c))|((a&amp;b)&amp;c))\nc,(!b&amp;c)\n&quot;
## [4] &quot;targets,factors\na,false\nb,((((!a&amp;b)&amp;!c)|((a&amp;b)&amp;!c))|((a&amp;b)&amp;c))\nc,(!b&amp;c)\n&quot;                           
## [5] &quot;targets,factors\na,false\nb,((((((!a&amp;b)&amp;!c)|((a&amp;!b)&amp;!c))|((a&amp;!b)&amp;c))|((a&amp;b)&amp;!c))|((a&amp;b)&amp;c))\nc,(!b&amp;c)\n&quot;</code></pre>
<p>There are multiple options to integrate <code>BoolNet</code> and <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>). The function <code>get.net.topology()</code> can obtain the topology with interaction signs of a BoolNet network. The function <code>attractor2dataframe()</code> can be used to export a BoolNet attractor as a dataframe that <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) can use. The function <code>run.gquery()</code> includes the option <code>return=’BoolNet’</code>, that return the inferred networks as <code>BoolNet</code> networks.</p>
</div>
<div>
<h3>History</h3>
<p>The development of <a rel="nofollow" href="http://turing.iimas.unam.mx/griffin/" target="_blank"><code>Griffin</code></a> began in 2013 as a PAPIIT (Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica) project to solve the inference of Boolean Network models for the <strong>Arabidopsis thaliana</strong> root stem cell niche. It continued in 2015 with support of Conacyt grant 221341.</p>
<p>In January, 2017 we organized a course in <a rel="nofollow" href="https://www.c3.unam.mx/" target="_blank">C3-UNAM</a> to teach biologist how to use <a rel="nofollow" href="http://turing.iimas.unam.mx/griffin/" target="_blank"><code>Griffin</code></a>. We received two main comments: the input format was too complicated and it was uncomfortable to use the output with other packages. After some consideration we decided to create an <code>R</code> wrapper that could export and import <code>BoolNet</code> networks. We selected <code>BoolNet</code> as it has an good documentation and the package <code>BoolFilter</code> had been designed to work with it.</p>
<p>The development of <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) began during the <a rel="nofollow" href="http://fejer.ucol.mx/biomate/" target="_blank">EOBM 2017</a> in <a rel="nofollow" href="https://portal.ucol.mx/cuib/" target="_blank">CUIB</a>. For the following year we continued developing <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) as our schedules allowed. There were multiple challenges during the development: defining user-friendly inputs, using <code>Rjava</code>, and structuring the package. In August 2018, we attended the <a rel="nofollow" href="http://congresos.nnb.unam.mx/TIB2018/r-bioconductor-developers-workshop-2018/" target="_blank">TIB2018-BCDW</a> where we received valuable guidance from Martin Morgan and Benilton S Carvalho. It was during this workshop that the first version of <em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>) was finished.</p>
</div>
<div>
<h3>Acknowledgements</h3>
<p>Here you can find the tweet about the original blog post:</p>
<blockquote class="twitter-tweet">
<p lang="en" dir="ltr">Check our first contributed blog post: R Gene Regulatory Interaction Formulator For Inquiring Networks<a rel="nofollow" href="https://t.co/9azbElGZGH" target="_blank">https://t.co/9azbElGZGH</a></p>
<p>ME Martinez-Sanchez, S Muñoz, M Carrillo, E Azpeitia, D Rosenblueth</p>
<p>Thx <a rel="nofollow" href="https://twitter.com/mt_morgan?ref_src=twsrc%5Etfw" target="_blank">@mt_morgan</a> <a rel="nofollow" href="https://twitter.com/benilton?ref_src=twsrc%5Etfw" target="_blank">@benilton</a>!<a rel="nofollow" href="https://twitter.com/hashtag/CDSBMexico?src=hash&ref_src=twsrc%5Etfw" target="_blank">#CDSBMexico</a> <a rel="nofollow" href="https://twitter.com/hashtag/TIB2018?src=hash&ref_src=twsrc%5Etfw" target="_blank">#TIB2018</a> <a rel="nofollow" href="https://twitter.com/hashtag/LatAmBioc18?src=hash&ref_src=twsrc%5Etfw" target="_blank">#LatAmBioc18</a> <a rel="nofollow" href="https://twitter.com/hashtag/rstats?src=hash&ref_src=twsrc%5Etfw" target="_blank">#rstats</a> <a rel="nofollow" href="https://twitter.com/hashtag/TIB?src=hash&ref_src=twsrc%5Etfw" target="_blank">#TIB</a> <a rel="nofollow" href="https://twitter.com/hashtag/blog?src=hash&ref_src=twsrc%5Etfw" target="_blank">#blog</a> <a rel="nofollow" href="https://t.co/TbJv37fkWQ" target="_blank">pic.twitter.com/TbJv37fkWQ</a></p>
<p>— ComunidadBioInfo (@CDSBMexico) <a rel="nofollow" href="https://twitter.com/CDSBMexico/status/1031904001833414659?ref_src=twsrc%5Etfw" target="_blank">August 21, 2018</a></p></blockquote>
<p></p>
<p>This blog post was made possible thanks to:</p>
<ul>
<li><em><a rel="nofollow" href="https://cran.r-project.org/package=blogdown" target="_blank">blogdown</a></em> <a></a>(<a rel="nofollow" href="https://github.com/rstudio/blogdown" target="_blank">Xie, Hill, and Thomas, 2017</a>)</li>
<li><em><a rel="nofollow" href="https://cran.r-project.org/package=devtools" target="_blank">devtools</a></em> <a></a>(<a rel="nofollow" href="https://cran.r-project.org/package=devtools" target="_blank">Wickham, Hester, and Chang, 2018</a>)</li>
<li><em><a rel="nofollow" href="https://cran.r-project.org/package=knitcitations" target="_blank">knitcitations</a></em> <a></a>(<a rel="nofollow" href="https://cran.r-project.org/package=knitcitations" target="_blank">Boettiger, 2017</a>)</li>
<li><em><a rel="nofollow" href="https://github.com/mar-esther23/rGriffin" target="_blank">rGriffin</a></em> (<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#bib-Martinez-Sanchez_2018" target="_blank">Martinez-Sanchez, Muñoz, Carrillo, Azpeitia, et al., 2018</a>)</li>
</ul>
</div>
<div>
<h3>References</h3>
<p>
<a></a><a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#cite-Boettiger_2017" target="_blank">[1]</a><cite> C. Boettiger. <em>knitcitations: Citations for ‘Knitr’ Markdown Files</em>. R package version 1.0.8. 2017. URL: <a rel="nofollow" href="https://cran.r-project.org/package=knitcitations" target="_blank">https://CRAN.R-project.org/package=knitcitations</a>.</cite>
</p>
<p>
<a></a><a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#cite-Martinez-Sanchez_2018" target="_blank">[2]</a><cite> M. Martinez-Sanchez, S. Muñoz, M. Carrillo, E. Azpeitia, et al. <em>rGriffin: Gene Regulatory Interaction Formulator For Inquiring Networks</em>. R package version 0.1. 2018.</cite>
</p>
<p>
<a></a><a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#cite-Wickham_2018" target="_blank">[3]</a><cite> H. Wickham, J. Hester and W. Chang. <em>devtools: Tools to Make Developing R Packages Easier</em>. R package version 1.13.6. 2018. URL: <a rel="nofollow" href="https://cran.r-project.org/package=devtools" target="_blank">https://CRAN.R-project.org/package=devtools</a>.</cite>
</p>
<p>
<a></a><a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#cite-Xie_2017" target="_blank">[4]</a><cite> Y. Xie, A. P. Hill and A. Thomas. <em>blogdown: Creating Websites with R Markdown</em>. ISBN 978-0815363729. Boca Raton, Florida: Chapman and Hall/CRC, 2017. URL: <a rel="nofollow" href="https://github.com/rstudio/blogdown" target="_blank">https://github.com/rstudio/blogdown</a>.</cite>
</p>
</div>
<div>
<h3>Reproducibility</h3>
<pre><code>## Session info ----------------------------------------------------------------------------------------------------------</code></pre>
<pre><code>##  setting  value                       
##  version  R version 3.5.1 (2018-07-02)
##  system   x86_64, darwin15.6.0        
##  ui       X11                         
##  language (EN)                        
##  collate  en_US.UTF-8                 
##  tz       America/New_York            
##  date     2018-09-13</code></pre>
<pre><code>## Packages --------------------------------------------------------------------------------------------------------------</code></pre>
<pre><code>##  package       * version date       source                                
##  backports       1.1.2   2017-12-13 cran (@1.1.2)                         
##  base          * 3.5.1   2018-07-09 local                                 
##  bibtex          0.4.2   2017-06-30 CRAN (R 3.5.0)                        
##  BiocManager     1.30.2  2018-08-24 CRAN (R 3.5.0)                        
##  BiocStyle     * 2.9.6   2018-08-19 Bioconductor                          
##  blogdown        0.8     2018-07-15 CRAN (R 3.5.0)                        
##  bookdown        0.7     2018-02-18 CRAN (R 3.5.0)                        
##  colorout      * 1.2-0   2018-05-03 Github (jalvesaq/colorout@c42088d)    
##  compiler        3.5.1   2018-07-09 local                                 
##  datasets      * 3.5.1   2018-07-09 local                                 
##  devtools      * 1.13.6  2018-06-27 CRAN (R 3.5.0)                        
##  digest          0.6.16  2018-08-22 CRAN (R 3.5.0)                        
##  evaluate        0.11    2018-07-17 CRAN (R 3.5.0)                        
##  graphics      * 3.5.1   2018-07-09 local                                 
##  grDevices     * 3.5.1   2018-07-09 local                                 
##  htmltools       0.3.6   2017-04-28 cran (@0.3.6)                         
##  httr            1.3.1   2017-08-20 CRAN (R 3.5.0)                        
##  jsonlite        1.5     2017-06-01 CRAN (R 3.5.0)                        
##  knitcitations * 1.0.8   2017-07-04 CRAN (R 3.5.0)                        
##  knitr           1.20    2018-02-20 cran (@1.20)                          
##  lubridate       1.7.4   2018-04-11 CRAN (R 3.5.0)                        
##  magrittr        1.5     2014-11-22 cran (@1.5)                           
##  memoise         1.1.0   2017-04-21 CRAN (R 3.5.0)                        
##  methods       * 3.5.1   2018-07-09 local                                 
##  plyr            1.8.4   2016-06-08 cran (@1.8.4)                         
##  R6              2.2.2   2017-06-17 CRAN (R 3.5.0)                        
##  Rcpp            0.12.18 2018-07-23 CRAN (R 3.5.1)                        
##  RefManageR      1.2.0   2018-04-25 CRAN (R 3.5.0)                        
##  rGriffin      * 0.1     2018-09-13 Github (mar-esther23/rgriffin@04cdc39)
##  rJava         * 0.9-10  2018-05-29 CRAN (R 3.5.0)                        
##  rmarkdown       1.10    2018-06-11 CRAN (R 3.5.0)                        
##  rprojroot       1.3-2   2018-01-03 cran (@1.3-2)                         
##  stats         * 3.5.1   2018-07-09 local                                 
##  stringi         1.2.4   2018-07-20 CRAN (R 3.5.0)                        
##  stringr         1.3.1   2018-05-10 CRAN (R 3.5.0)                        
##  tools           3.5.1   2018-07-09 local                                 
##  utils         * 3.5.1   2018-07-09 local                                 
##  withr           2.1.2   2018-03-15 CRAN (R 3.5.0)                        
##  xfun            0.3     2018-07-06 CRAN (R 3.5.0)                        
##  xml2            1.2.0   2018-01-24 CRAN (R 3.5.0)                        
##  yaml            2.2.0   2018-07-25 CRAN (R 3.5.0)</code></pre>
</div>
<div>
<hr>
<ol>
<li>
<p>I’m re-posting their blog post here with their permission so that their post will be shared more widely via <a rel="nofollow" href="https://www.r-bloggers.com/" target="_blank">R-Bloggers</a> and <a rel="nofollow" href="https://rweekly.org/" target="_blank">R Weekly</a> among other sites since the CDSB blog is currently not linked to those aggregators. The original blog post has been edited so that it evaluates the R code and includes all the information needed for reproducibility.<a rel="nofollow" href="http://feedproxy.google.com/~r/FellgernonBit-rstats/~3/VHSZq_6H-Ys/#fnref1" target="_blank"><img alt="↩" src="https://s.w.org/images/core/emoji/2.4/72x72/21a9.png" class="wp-smiley"></a></p>
</li>
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