tag:blogger.com,1999:blog-49070776085737557832024-03-19T02:40:27.638-07:00QMVIEWSUnknownnoreply@blogger.comBlogger90125tag:blogger.com,1999:blog-4907077608573755783.post-73014664685486847332018-11-26T23:57:00.000-08:002018-11-26T23:57:04.266-08:00Shame on ScienceIn 2015 I wrote about Cas9 stuff:<br />
<br />
<a href="http://qmviews.blogspot.com/2015/09/literature-004-jinek-et-al-elife-2013.html">http://qmviews.blogspot.com/2015/09/literature-004-jinek-et-al-elife-2013.html</a><br />
<br />
In the last part, I mentioned very early experiements remotely related to humans. There were problems and most importantly, there was consensus among the scientific community that the technology should be handled with care and ethics in mind.<br />
<br />
None of that did prevent what happend. The guy from Standford did it. How come he wanted to do it? Why did the system fail to deliver ethical values to this guy? Why was the desire to be recognized, famous and prominent so much stronger than the respect for the ethics? Dont give me the medical reasons. There're just not credible here.<br />
In any case, we lost. The dam broke, the box is open. It feels hopeless. The only thing that's left is to feel sorry for these two kids. And for us.Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-52455358122721683512017-08-04T16:29:00.000-07:002017-08-04T16:29:29.375-07:00Machine LearningGreat fun<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiEHa2_JRRsh8INxD28YrJUkZR29U4608kKaF85x1bOSYkFW7jpJd2YhvO0PplY3rbCgCu6Rd635Ief-SwojFY1gQCpWH1NugesOb9Skjr10wTAe5PLm6RTfer0gJDx46Rl4AJ9-9PdspGh/s1600/Screen+Shot+2017-08-05+at+01.28.22.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="808" data-original-width="1052" height="488" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiEHa2_JRRsh8INxD28YrJUkZR29U4608kKaF85x1bOSYkFW7jpJd2YhvO0PplY3rbCgCu6Rd635Ief-SwojFY1gQCpWH1NugesOb9Skjr10wTAe5PLm6RTfer0gJDx46Rl4AJ9-9PdspGh/s640/Screen+Shot+2017-08-05+at+01.28.22.png" width="640" /></a></div>
<span id="goog_1768696607"></span><span id="goog_1768696608"></span><br />Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-40713376307748453962017-03-18T11:24:00.002-07:002017-03-18T11:24:15.065-07:00Open Traffic Data: Visualizing Cumulative Daily Delay of Tram Line 10 in ZurichFirst impression of visualizing the accumulated delay of tram line 10 in Zurich from late 2015 until end of January 2017 at every station of the line.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjhGZzMKlLqU72qC15i2mDOv2A6U4-P3IpHA00gOdbzq-LLofw0s3vcsIjNM8tEr-OACnUYLSUNPg9YVNMWjJD-0NE75C3KkAMUWO3tUEyhmERqweCWKzaq9IgIKBPGmHgcwCpxiVtgL7fE/s1600/img.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="480" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjhGZzMKlLqU72qC15i2mDOv2A6U4-P3IpHA00gOdbzq-LLofw0s3vcsIjNM8tEr-OACnUYLSUNPg9YVNMWjJD-0NE75C3KkAMUWO3tUEyhmERqweCWKzaq9IgIKBPGmHgcwCpxiVtgL7fE/s640/img.png" width="640" /></a></div>
The analysis is an early result of the <a href="http://zurich-r-user-group.github.io/hackathon.html" rel="nofollow" target="_blank">open data hackathon</a> earlier this month. To be continued...Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-73057303943376782532016-12-19T03:47:00.000-08:002016-12-19T03:47:36.224-08:00R: Clinical Trial SimulatorThe first version of the clinical trial simulator is online now.<br />
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It uses a dose-response model based on the Hill equation which basically is a saturation model. The assumption is that a cell is covered by a finite number of receptors. When the treatment is applied, a fraction of the receptors (depending on the dosage) is interacting with the treatment molecule and every interaction leads to a response. When every receptor is binding to the treatment, the response saturates.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjaaMhICt_6OFPdM6jSjKCVlI6Nwqz37YMdliN1NtCKTilc7jbNFga5KtuiD021ra2HljAPdOkld3jnftNDw5uk3dzAvScliHt6uhUcGnw9a-lEXCIXjetHhxvxp-6RSIjgXHFUZUoP7NDW/s1600/Screen+Shot+2016-12-19+at+12.45.03.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="633" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjaaMhICt_6OFPdM6jSjKCVlI6Nwqz37YMdliN1NtCKTilc7jbNFga5KtuiD021ra2HljAPdOkld3jnftNDw5uk3dzAvScliHt6uhUcGnw9a-lEXCIXjetHhxvxp-6RSIjgXHFUZUoP7NDW/s640/Screen+Shot+2016-12-19+at+12.45.03.png" width="640" /></a></div>
It's based on the MSToolkit package and is hosted online at shiny apps under<br />
<br />
<a href="https://mzhku.shinyapps.io/trialsim/" target="_blank">https://mzhku.shinyapps.io/trialsim/</a>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-23689468840917466772016-12-04T11:37:00.003-08:002016-12-04T11:47:53.357-08:00R: Sample Size CalculatorGiven a beta, how many patients need to be enrolled in a two-armed study of conventional against experimental therapy? Significance is set to 0.05, recruitment period and follow-up period can be defined together with median disease free survival times for the two study groups (implemented in R/Shiny):<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhechfZky-OOmJY8YmKXzJuX5Idu1ZS9LOa5P5rO8Vg910u3HbO8i2B1RDdzTvSZwHAxTL6sZYiC00dyfe0VK-dF_k9vwluDNo7cJEiGjWCPZgAXF6UsSv09-J00G9Sh7_2ij6RRvLWQrid/s1600/Screen+Shot+2016-12-04+at+Sonntag%252C+4.+Dezember+201620.33.56.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="347" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhechfZky-OOmJY8YmKXzJuX5Idu1ZS9LOa5P5rO8Vg910u3HbO8i2B1RDdzTvSZwHAxTL6sZYiC00dyfe0VK-dF_k9vwluDNo7cJEiGjWCPZgAXF6UsSv09-J00G9Sh7_2ij6RRvLWQrid/s640/Screen+Shot+2016-12-04+at+Sonntag%252C+4.+Dezember+201620.33.56.png" width="640" /></a></div>
<a href="https://github.com/mzhKU/size" rel="nofollow" target="_blank">Github repository</a><br />
<br />
Try it (also mobile)!<br />
<a href="https://mzhku.shinyapps.io/size/" rel="nofollow" target="_blank">https://mzhku.shinyapps.io/size/</a><br />
<br />Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-49664420503773221122016-12-04T08:24:00.001-08:002016-12-04T08:24:46.798-08:00R Shiny: Some reactivity by exampleStart R from the directory containing ui.r and server.r, import the shiny library and start the (development) server using runApp().<br />
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server.r<br />
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<div style="font-family: Monaco; font-size: 15px; line-height: normal; min-height: 20px;">
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<span style="color: #34bd26; font-variant-ligatures: no-common-ligatures;">function</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">input</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">,</span><span style="font-variant-ligatures: no-common-ligatures;"> output</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">)</span><span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">{</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> rv </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> reactiveValues</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">()</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal; min-height: 20px;">
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<div style="color: #5330e1; font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="color: black; font-variant-ligatures: no-common-ligatures;"> </span><span style="font-variant-ligatures: no-common-ligatures;"># Only modify and output reactive input</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> output$numOut1 </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> renderText</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">({</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> input$n+</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">10</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">})</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal; min-height: 20px;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span></div>
<div style="color: #5330e1; font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="color: black; font-variant-ligatures: no-common-ligatures;"> </span><span style="font-variant-ligatures: no-common-ligatures;"># Modify, reassign and output reactive input.</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> output$numOut2 </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> renderText</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">({</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> rv$a </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> input$n+</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">20</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">})</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal; min-height: 20px;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span></div>
<div style="color: #5330e1; font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="color: black; font-variant-ligatures: no-common-ligatures;"> </span><span style="font-variant-ligatures: no-common-ligatures;"># Use modified reactive input, modify and output.</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> output$num1 </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> renderText</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">({</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> rv$s </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> rv$a+runif</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">1</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">)</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">})</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal; min-height: 20px;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span></div>
<div style="color: #5330e1; font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="color: black; font-variant-ligatures: no-common-ligatures;"> </span><span style="font-variant-ligatures: no-common-ligatures;"># Assign reactive input locally, modify locally, print.</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> output$num2 </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> renderText</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">({</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> k </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> input$n</span></div>
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<span style="font-variant-ligatures: no-common-ligatures;"> paste</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">as.character</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">runif</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">1</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">)),</span><span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">"here"</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">,</span><span style="font-variant-ligatures: no-common-ligatures;"> k-</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">10</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">)</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">})</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal; min-height: 20px;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span></div>
<div style="color: #5330e1; font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="color: black; font-variant-ligatures: no-common-ligatures;"> </span><span style="font-variant-ligatures: no-common-ligatures;"># Use reactive value from another reactive function,</span></div>
<div style="color: #5330e1; font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="color: black; font-variant-ligatures: no-common-ligatures;"> </span><span style="font-variant-ligatures: no-common-ligatures;"># modify and print.</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> output$let </span><span style="color: #ce7924; font-variant-ligatures: no-common-ligatures;"><-</span><span style="font-variant-ligatures: no-common-ligatures;"> renderText</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">({</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> paste</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">as.character</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">rv$s</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">),</span><span style="font-variant-ligatures: no-common-ligatures;"> sample</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">letters</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">,</span><span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">1</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">),</span><span style="font-variant-ligatures: no-common-ligatures;"> sep=</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">""</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">)</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">})</span></div>
<br />
<div style="color: #d53bd3; font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;">}</span></div>
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<span style="font-variant-ligatures: no-common-ligatures;"><br /></span></div>
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<span style="font-variant-ligatures: no-common-ligatures;">ui.r</span></div>
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<span style="font-variant-ligatures: no-common-ligatures;"></span><br />
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<span style="font-variant-ligatures: no-common-ligatures;"><span style="font-variant-ligatures: no-common-ligatures;">fluidPage</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span></span></div>
<span style="font-variant-ligatures: no-common-ligatures;">
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> numericInput</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">inputId=</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">"n"</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">,</span><span style="font-variant-ligatures: no-common-ligatures;"> </span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">"Shiny Reactivity Example"</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">,</span><span style="font-variant-ligatures: no-common-ligatures;"> value=</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">25</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">),</span></div>
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<span style="font-variant-ligatures: no-common-ligatures;"></span><br /></div>
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<span style="font-variant-ligatures: no-common-ligatures;"> textOutput</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">outputId=</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">"numOut1"</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">),</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal; min-height: 20px;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> textOutput</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">outputId=</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">"numOut2"</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">),</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal; min-height: 20px;">
<span style="font-variant-ligatures: no-common-ligatures;"> </span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> textOutput</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">outputId=</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">"num1"</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">),</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="font-variant-ligatures: no-common-ligatures;"> textOutput</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">outputId=</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">"num2"</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">),</span></div>
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<span style="font-variant-ligatures: no-common-ligatures;"> textOutput</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">(</span><span style="font-variant-ligatures: no-common-ligatures;">outputId=</span><span style="color: #c33720; font-variant-ligatures: no-common-ligatures;">"let"</span><span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">)</span></div>
<div style="font-family: Monaco; font-size: 15px; line-height: normal;">
<span style="color: #d53bd3; font-variant-ligatures: no-common-ligatures;">)</span><span style="font-variant-ligatures: no-common-ligatures;"> </span></div>
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<span style="font-variant-ligatures: no-common-ligatures;"><br /></span></div>
</span></div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-75440516080709859082016-08-18T12:56:00.000-07:002016-08-18T12:57:18.349-07:00R: Plotting multiple categorical dataFrequently, values are plotted against a categorical argument. The categorical argument can be thought of something like a weekday label or a step in a specific technical procedure ("Step 1", "Step 2", ...). Of course, these arguments could be converted to a numeric value used for plotting and after plotting, one would overwrite the x-axis labels to get the categorical data on the axis. But if Excel can do it, then it should be possible to do it in R as well. Here's how.<br />
<div class="a-spacing-none" id="title">
The data in the example is from the book "<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><a href="https://www.amazon.com/gp/product/0470887656/ref=pd_cp_0_2?ie=UTF8&psc=1&refRID=DXYHCW28QG5J9SWPC6MC" target="_blank">Design and Analysis of Clinical Trials: Concepts and Methodologies</a>" by Chow (<a href="http://pastebin.com/AEUN0BRV" target="_blank">full data set</a>).</span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><br /></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle">Here, the categorical data (x-axis) are five repeated measurements of diastolic blood pressure, denoted "DBP1", ..., "DBP5", where DBP1 is the baseline value. 40 subjects have been treated with either a hypertensive agent (A) or placebo (B). After averaging over treatment groups we end up with two groups we want to inspect.</span></span></div>
<div class="a-spacing-none" id="title">
<br /></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><span style="font-family: "courier new" , "courier" , monospace;">> d <- aggregate(dat[, 3:7], by=list(TRT=dat$TRT), FUN=mean)</span></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><span style="font-family: "courier new" , "courier" , monospace;">> d<br /> TRT DBP1 DBP2 DBP3 DBP4 DBP5<br />1 A 116.55 113.5 110.70 106.25 101.35<br />2 B 116.75 115.2 114.05 112.45 111.95</span></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><br /></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle">How can now <span style="font-family: "courier new" , "courier" , monospace;">d</span> be plotted to immediately make the difference between the treatments visible? Lattice can plot multiple data records (the A- and B-rows) against categorical data, but for that, the data has to be in <i>long</i> format. This can be generated using the <span style="font-family: "courier new" , "courier" , monospace;">melt</span> function from the <span style="font-family: "courier new" , "courier" , monospace;">reshape2</span> package.</span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><br /></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><span style="font-family: "courier new" , "courier" , monospace;">> m <- melt(d, id="TRT", measure.vars=colnames(d[, 2:ncol(d)]))</span></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><span style="font-family: "courier new" , "courier" , monospace;">> m<br /> TRT variable value<br />1 A DBP1 116.55<br />2 B DBP1 116.75<br />3 A DBP2 113.50<br />4 B DBP2 115.20<br />5 A DBP3 110.70<br />6 B DBP3 114.05<br />7 A DBP4 106.25<br />8 B DBP4 112.45<br />9 A DBP5 101.35<br />10 B DBP5 111.95</span></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><br /></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle">The melted data.frame <span style="font-family: "courier new" , "courier" , monospace;">m</span> can now be plotted.</span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"><br /></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-family: "courier new" , "courier" , monospace;"><span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle">> xyplot(m$value~m$variable, type="o", group=m$TRT, auto.key=list(T))</span></span></span></div>
<div class="a-spacing-none" id="title">
<span style="font-weight: normal;"><span class="a-size-extra-large" id="productTitle"></span><span class="a-size-extra-large" id="productTitle"><br />The resulting figure looks like this:</span></span></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipV28Y0s-J5eYvoa09ylxkMuo0iuabbxYozJDoaTZU4o9-uI1-CPb7SqfeT-DjM1N9Yp5LADQzA2Inw6Abc3sYoDI5k6dG-20R1GSRh9asVoWZsBVXvNVj325HzkVJjtWEQFc6PjyX8_-Z/s1600/Screen+shot+2016-08-18+at+Donnerstag%252C+18.+August+201621.54.14.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="464" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipV28Y0s-J5eYvoa09ylxkMuo0iuabbxYozJDoaTZU4o9-uI1-CPb7SqfeT-DjM1N9Yp5LADQzA2Inw6Abc3sYoDI5k6dG-20R1GSRh9asVoWZsBVXvNVj325HzkVJjtWEQFc6PjyX8_-Z/s640/Screen+shot+2016-08-18+at+Donnerstag%252C+18.+August+201621.54.14.png" width="640" /></a></div>
<div class="separator" style="clear: both; text-align: center;">
</div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-88181584022747228112016-07-16T23:10:00.000-07:002016-07-16T23:10:01.657-07:00Book: Understanding Enzymes published!Finally the book is out! Happy to have in hands.<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgIu4vs9UP99TcorCOjTxnuQ5qRhmMSnss-b-wJfLOXhuqoZU54yTm0LgZ_bsxbO3mIE3ZifeqlbhcS0xAwciJi5Yiq-A56Dsp47_CeTzmvhcWwA8IFKiXzk4xnl4QDpnaxFAEHyUf3WT1C/s1600/Screen+shot+2016-07-17+at+Sonntag%252C+17.+Juli+201608.09.02.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="444" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgIu4vs9UP99TcorCOjTxnuQ5qRhmMSnss-b-wJfLOXhuqoZU54yTm0LgZ_bsxbO3mIE3ZifeqlbhcS0xAwciJi5Yiq-A56Dsp47_CeTzmvhcWwA8IFKiXzk4xnl4QDpnaxFAEHyUf3WT1C/s640/Screen+shot+2016-07-17+at+Sonntag%252C+17.+Juli+201608.09.02.png" width="640" /></a></div>
<br />Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-34128137605283464392016-07-07T12:14:00.004-07:002016-07-16T23:05:12.660-07:00Python: Concate code to a single stringUsing the code below, the code from a source file can be concatenated to a single string. This string however can still be printed and formates nicely on a JavaScript console.
It returns a string variable `s' and requires two command line arguments.
<br />
<pre># Usage:
# > python ~/path/to/script/multiline.py <name_of_ouput_variable> <source .js=""></source> <source_identifier>
</source_identifier></pre>
<pre class="brush:python"> filename = sys.argv[1]
f = open(filename, "r")
d = f.readlines()
f.close()
s = "var %s = " % sys.argv[2]
s = s.strip()
# s += '"' + "".join(d).replace("\n", "") + '";'
cnt = 0
n = len(d)
# '\\n' is explicitly written to the 'source'-file such that
# the JavaScript console can print the source code including line
# breaks.
for line in d:
# First line.
if cnt == 0:
s += "\n" + ' "' + d[cnt].replace("\n", "") + '\\n"\n'
cnt += 1
# Inbetween lines.
elif cnt > 0 and cnt < (n-1):
s += '+ "' + d[cnt].replace("\n", "") + '\\n"\n'
cnt += 1
# Last line.
else:
s += '+ "' + d[cnt].replace("\n", "") + '"'
</pre>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-23977695218796618812016-05-01T05:41:00.000-07:002016-05-17T11:03:29.466-07:00R: Replacement functionsIf you use R, you've been using so called <i>replacement functions</i> such as '<span style="font-family: "courier new" , "courier" , monospace;"><-</span>' to assign a value to a variable or '<span style="font-family: "courier new" , "courier" , monospace;">colnames</span>' to define names of columns in a data frame.<br />
Remember, in R everything operation is a function call (therefore also the assignment operations).<br />
In the following, the behaviour of the replacement functions are illustrated (and compared to a -failing- ordinary approach) and code is shown to list all replacement functions in the <span style="font-family: "courier new" , "courier" , monospace;">base</span> R package.<br />
<br />
Replacement functions act as if they modify their arguments in place such as in<br />
<br />
<pre class="brush:python">colnames(d) <- c("Input", "Output")</pre>
<br />
They have the identifier '<span style="font-family: "courier new" , "courier" , monospace;"><-</span>' at the end of their name and return a modified <i>copy</i> of the argument object (non-primitive replacement functions) or the <i>same</i> object (primitive replacement functions).<br />
<br />
At the R prompt, the following will not work:<br />
<br />
<span style="font-family: "courier new" , "courier" , monospace;">> `second` <- function(x, value) {<br />+ x[2] <- value<br />+ x<br />+ }<br />> x <- 1:10<br />> x<br /> [1] 1 2 3 4 5 6 7 8 9 10<br />> second(x) <- 9<br />Fehler in second(x) <- 9 : konnte Funktion "second<-" nicht finden</span><br />
<br />
As one can see, behind the scenes, R is looking for a function called '<span style="font-family: "courier new" , "courier" , monospace;">second<-</span>'. So lets do the same thing but using this name:<br />
<br />
<span style="font-family: "courier new" , "courier" , monospace;">> `second<-` <- function(x, value) {<br />+ x[2] <- value<br />+ x<br />+ }</span><br />
<br />
Now, the assignment at the second position of the vector works:<br />
<br />
<span style="font-family: "courier new" , "courier" , monospace;">> second(x) <- 9<br />> x<br /> [1] 1 9 3 4 5 6 7 8 9 10</span><br />
<br />
The following code allows to list all replacement functions in are and check if they are primitive:<br />
<br />
<br />
<pre class="brush:python">
# Get all objects from the base package, then
# functions from among those objects.
objs <- mget(ls("package:base"), inherits=TRUE)
funs <- Filter(is.function, objs)
# Replacement functions have '<-' at the end of their name.
get_rep_fun <- function(fun_i) {
if(substr(fun_i, nchar(fun_i) - 1, nchar(fun_i)) == "<-") {
fun_i
}
}
# The list returned by 'lapply' contains many 'NULL'
# entries. Only non-'NULL' function name strings remain
# after 'unlist'.
rep_funs <- lapply(names(funs), get_rep_fun)
rep_funs <- unlist(rep_funs)
# Identify primitive replacement functions.
get_prim_rep_fun <- function(rep_fun) {
if(is.primitive(rep_fun)) {
rep_fun
}
}
# Get the actual function object by 'sapply'-ing 'get' to
# each string identifier from the 'rep_funs' vector.
prim_rep_funs <- lapply(sapply(rep_funs, get), is.primitive)
# Prepare data frame for convenience.
k <- data.frame(fun_name = rep_funs, prim=unlist(prim_rep_funs))
# Rows are named after functions -> reduce redundancy.
rownames(k) <- NULL
</pre>
<br />
<br />
Unknownnoreply@blogger.com1tag:blogger.com,1999:blog-4907077608573755783.post-24043134855790096932016-04-03T12:23:00.000-07:002016-04-24T04:22:16.434-07:00Clustering data with k-means and plotting for exploratory analysisI clustered data from my paper <a href="https://peerj.com/articles/111/" target="_blank"><i>A computational method for the systematic screening of reaction barriers in enzymes: searching for Bacillus circulans xylanase mutants with greater activity towards a synthetic substrate</i></a> using k-means clustering and plotted the results using the `Lattice` library in R.<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgK0RGTDewgtdbwDNhbpmw481Mv4Fgkf81DVXbxNuScBEW5dnmVXc3jv9I90cl8UCka-3MG89jZWD9DrpMK2xRmpzl5SgB3u1l2GmNG6FkSDOkSDMqDdp-DSi5xsLcDQVua8iSbMDOdtgiy/s1600/xyplot_16.png" imageanchor="1"><img border="0" height="640" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgK0RGTDewgtdbwDNhbpmw481Mv4Fgkf81DVXbxNuScBEW5dnmVXc3jv9I90cl8UCka-3MG89jZWD9DrpMK2xRmpzl5SgB3u1l2GmNG6FkSDOkSDMqDdp-DSi5xsLcDQVua8iSbMDOdtgiy/s640/xyplot_16.png" width="640" /></a></div>
This analysis could be used to further automate selection of reaction barriers for including or excluding from analysis if the barrier profile is not physically meaningfull.<br />
<br />
Raw data:<br />
<a href="http://pastebin.com/41TrNNvH">http://pastebin.com/41TrNNvH</a><br />
<br />
A detailed report on the analyis is available here:<br />
<a href="http://rpubs.com/mzh/167769">http://rpubs.com/mzh/167769</a><br />
<br />
Github repository:<br />
<a href="https://github.com/mzhKU/Enzyme-Barrier-Clustering" target="_blank">https://github.com/mzhKU/Enzyme-Barrier-Clustering</a><br />
<br />
The critical part of the code is where the two plots (the k-means clusters, red lines, and the raw data of each cluster, blue lines) are overlayed using the lattice panel plot construct (in <a href="https://github.com/mzhKU/Enzyme-Barrier-Clustering/blob/master/scripts/003_execute.r" rel="nofollow" target="_blank">003_execute.r</a>):<br />
<br />
<pre class="brush:python"># 'group' required to prevent drawing of a continuous line connecting the last
# data point of a mutant with the first data point of the next mutant barrier.
xyplot(Barrier_Cl ~ x|Cluster_fac, data=kmdf, iedf_i=iedf,
ylim=c(-20, 50), ylab="Reaction Energy [kcal/mol]",
xlab="Reaction Coordinate", xlim=c(1, 12), strip=T,
# Note:
# 'x' and 'y' are the data from the cluster data frame 'kmdf'.
# 'iedf_i' is the 'iedf'-data frame provided to the 'i'-nner panel function.
panel = function(x, y, iedf_i)
{
panel.xyplot(x=iedf_i[iedf_i$Cluster==panel.number(), ]$x_axis,
y=iedf_i[iedf_i$Cluster==panel.number(), ]$Barrier_i,
group=iedf_i$id, subscripts=TRUE, type="o", col="blue")
panel.xyplot(x, y, type="l", col="red", lwd=2)
}
)
</pre>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-67702283233793950302015-09-20T06:17:00.000-07:002016-05-01T04:08:02.058-07:00Literature 004: Jinek et al., eLife 2013; 2:e00417<style>
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<br />
<h3 style="text-align: justify; text-justify: inter-ideograph;">
Introduction</h3>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
CRISPRs
have attracted enormous attention since the recent publication by <a href="http://elifesciences.org/content/2/e00471" rel="nofollow" target="_blank">Jinek <i>et al</i></a>. The discovery has been subject to numerous
reviews in both the <a href="http://www.sciencemag.org/content/346/6213/1258096" rel="nofollow" target="_blank">scientific literature</a> as well as popular and mass
media publications:</div>
<ul>
<li><a href="http://www.independent.co.uk/news/science/crispr-gene-therapy-scientists-call-for-more-public-debate-around-breakthrough-technique-8927606.html" rel="nofollow" target="_blank">The Independent</a> (fancy picture) </li>
</ul>
<ul>
<li><a href="http://www.economist.com/news/leaders/21661651-new-technique-manipulating-genes-holds-great-promisebut-rules-are-needed-govern-its" rel="nofollow" target="_blank">The Economist</a> ("[...]<i>where doctors put normal genes into the cells of people who suffer from genetic diseases such as Tay Sachs or cystic fibrosis.</i>"- note how simply doctors these days just "put" normal genes into the cells of people, of course only the right cells... you've had yours today already, haven't you?)</li>
</ul>
<ul>
<li><a href="http://www.nytimes.com/2014/03/04/health/a-powerful-new-way-to-edit-dna.html?_r=0" rel="nofollow" target="_blank">NY Times</a> (pragmatic report)</li>
</ul>
<ul>
<li><a href="http://www.srf.ch/wissen/sommerserie-pioniere/eine-dna-schere-revolutioniert-die-gentechnik" rel="nofollow" target="_blank">Swiss national television</a> (fancy picture)</li>
</ul>
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<span style="font-family: "cambria"; font-size: 12.0pt;">While providing information to the public about
this discovery is of tremendous importance, many reports however seem to emphasize promising future applications without giving any
understanding of how this development actually looked like when it was made in
the laboratory. I.e. the real data obtained by the scientists in those
breakthrough moments is rarely if ever shown.</span><br />
<span style="font-family: "cambria"; font-size: 12.0pt;">The eLife publication is open access so it can't be a mather of accessibility. Much rather I believe journalists prefer to present
fancy 3d-renderings of DNA and proteins with fluorescent numbers and
pseudo-code on a black background, like everything was in Matrix or so - for whatever reason. But, the real science is actually just as appealing and therefore in this post, the real data is shown and explained.</span><br />
<span style="font-family: "cambria"; font-size: 12.0pt;"><br /></span>
<br />
<h3>
<span style="font-family: "cambria"; font-size: 12.0pt;">Background</span></h3>
<span style="font-family: "cambria"; font-size: 12.0pt;">Subject of interest is a protein-RNA complex which can cut dsDNA at virtually any position and which is much cheaper and easier to use than any of the sofar existing methods. This complex is called the <i>CRISPR/Cas-9</i> system and it was discovered 1987[<a href="http://jb.asm.org/content/169/12/5429" rel="nofollow" target="_blank">Ishino <i>et al.</i>]</a> when its involvement in cutting dsDNA was yet completely unclear.</span><br />
<span style="font-family: "cambria"; font-size: 12.0pt;">The discovery back then looked like this:</span><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEicO5XfJkcUO2ws-lQzTFUj7JNRdKt1J8YLMl9EETJEuFvwyw6OGEmZWH99MHb5kmWr46Nebu1eG0uIUYD-phe4NmmbKGf777nTqmnGVJ5fe_wvyGgAwmxCnC1NC7ADaPVvpXVKptT5Oo_i/s1600/ishino+fig-5.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="160" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEicO5XfJkcUO2ws-lQzTFUj7JNRdKt1J8YLMl9EETJEuFvwyw6OGEmZWH99MHb5kmWr46Nebu1eG0uIUYD-phe4NmmbKGf777nTqmnGVJ5fe_wvyGgAwmxCnC1NC7ADaPVvpXVKptT5Oo_i/s640/ishino+fig-5.png" width="640" /></a></div>
<span style="font-family: "cambria"; font-size: 12.0pt;">A pattern is immediatly visible (the point being science is not always that difficult).</span><br />
<span style="font-family: "cambria"; font-size: 12.0pt;">The underlined repeats are of dyadic symmetry and regularly separated by (non-uniform) short spacer sequences. The authors wrote:</span><br />
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<i><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj4dFsW7tARPnG85v-BQ2nbn7IXEbaMorX45r0BXC9tqNeqac_WR9CoLullcGPU1sYocUaDMcmjxtt3R1N-wiKlxBFPKIAkIVsAsVeWHrxp8evISmrT_xHLvDuWB1YMDtnnnBLuNEybRdIl/s1600/ishino+fig-5+text.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="245" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj4dFsW7tARPnG85v-BQ2nbn7IXEbaMorX45r0BXC9tqNeqac_WR9CoLullcGPU1sYocUaDMcmjxtt3R1N-wiKlxBFPKIAkIVsAsVeWHrxp8evISmrT_xHLvDuWB1YMDtnnnBLuNEybRdIl/s400/ishino+fig-5+text.png" width="400" /></a></i></div>
<span style="font-family: "cambria"; font-size: 12.0pt;">This structure was "unusual" to them. I can only imagine how much time they spent wondering what this was about before they wrote this paragraph.</span><br />
<br />
<span style="font-family: "cambria"; font-size: 12.0pt;">Only 2004/2005 was it recognized that the non-uniform sequences were from foreign DNA.[<a href="http://mic.sgmjournals.org/content/journal/micro/10.1099/mic.0.27437-0" rel="nofollow" target="_blank">Pourcel </a><i><a href="http://mic.sgmjournals.org/content/journal/micro/10.1099/mic.0.27437-0" rel="nofollow" target="_blank">el al.</a></i>, <a href="http://link.springer.com/article/10.1007%2Fs00239-004-0046-3" rel="nofollow" target="_blank">Mojica </a><i><a href="http://link.springer.com/article/10.1007%2Fs00239-004-0046-3" rel="nofollow" target="_blank">et al.</a></i>, <a href="http://mic.sgmjournals.org/content/journal/micro/10.1099/mic.0.28048-0" rel="nofollow" target="_blank">Bolotin </a><i><a href="http://mic.sgmjournals.org/content/journal/micro/10.1099/mic.0.28048-0" rel="nofollow" target="_blank">et al.</a></i>] </span><span class="meta-value"><span style="font-family: "cambria"; font-size: 12.0pt;">T</span></span><span style="font-family: "cambria"; font-size: 12.0pt;">he random looking sequences to the right of the dyadic elements in the above figure are the foreign DNA fragments of previous
viral attacks on the cell. The dyadic elements are also referred to as <i>palindromic</i> because they can be folded and basepaired onto themselves.</span><br />
<span style="font-family: "cambria"; font-size: 12.0pt;">Gradually, attention was rising and in 2010 the first Science and Nature reviews on the topic appeared.[<a href="http://www.ncbi.nlm.nih.gov/pubmed/20056882" rel="nofollow" target="_blank">Horvath <i>et al.</i></a>, <a href="http://www.ncbi.nlm.nih.gov/pubmed/20125085" rel="nofollow" target="_blank">Marraffini <i>et al.</i></a>] Application to dsDNA was alluded to at that point, but nothing was certain.</span><br />
<blockquote class="tr_bq">
<i><span style="font-family: "cambria"; font-size: 12.0pt;">"</span></i><span style="font-family: "cambria"; font-size: 12.0pt;">[...]</span><i><span style="font-family: "cambria"; font-size: 12.0pt;">Other potential applications of CRISPRs await fur ther development to determine their plausibility. For example, a crRNP complex in P. furiosus50 can cleave a target RNA at a specific site dictated by the sequence of the crRNA guide. This activity could in principle have applications in molecular biology to specifically cleave RNA molecules in vitro, and <b>could be extended to DNA molecules if other crRNP complexes are proven to have DNA endonuclease activity.</b></span></i><span style="font-family: "cambria"; font-size: 12.0pt;">[...]"</span></blockquote>
Remarkably, neither of these reviews cites any work by either Jinek, Doudna or Charpentier.<br />
Then the two breakthrough papers were published.[<a href="http://www.ncbi.nlm.nih.gov/pubmed/22745249" rel="nofollow" target="_blank">Jinek<i> et al. 2012</i></a>, <a href="http://elifesciences.org/content/2/e00471" rel="nofollow" target="_blank">Jinek <i>et al.</i></a><a href="http://elifesciences.org/content/2/e00471" rel="nofollow" target="_blank"> 2013</a>] In the following, the results of the 2013 paper are summarized.<br />
<br />
<b>The Jinek Publication</b><br />
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In
the following, all images are taken from the eLife publication to show how the
results lead to conclusions. Assume as a scientist, you don’t know in detail how the CRISPR/Cas9
system works and what you can use it for. All you know is it has to be transfered to the nucleus and is
programmed by RNA. How can you prove your claim?</div>
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In the publication, the target gene is a clathrin gene, a protein participating in vesicle formation at the cell surface. Clathrin was subject to an <a href="http://qmviews.blogspot.ch/2014/08/literature-003-navaroli-et-al-pnas-2012.html" rel="nofollow" target="_blank">earlier post</a> on this blog. </div>
<br />
First, its good to show that the protein of interest can indeed be expressed by the target (human) cells.<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhV6NLOno1288c_JaMX6Qt9326IsAap1bzAHsDZUM2jgb8pLNRRsXRAT1IdEKorleK3p8OSpMaUsY-0rPLJSu4fRIETkWSjIXu-Wv2QFmqZDZj1Ilgws8IFlN4cD8Y2HCeraE-54RvDNOY6/s1600/fig-1a+Cas9-HA-NLS-GFP+Expression+construct.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="312" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhV6NLOno1288c_JaMX6Qt9326IsAap1bzAHsDZUM2jgb8pLNRRsXRAT1IdEKorleK3p8OSpMaUsY-0rPLJSu4fRIETkWSjIXu-Wv2QFmqZDZj1Ilgws8IFlN4cD8Y2HCeraE-54RvDNOY6/s400/fig-1a+Cas9-HA-NLS-GFP+Expression+construct.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
The black line at 170 kDa indicates a protein of roughly the weight one would expect for Cas9 being modified by a <a href="https://en.wikipedia.org/wiki/Cytomegalovirus#Genetic_engineering" rel="nofollow" target="_blank">CMV promoter</a>, an <a href="https://en.wikipedia.org/wiki/HA-tag" rel="nofollow" target="_blank">HA epitope</a> (facilitating detection/purification), a <a href="https://en.wikipedia.org/wiki/Nuclear_localization_sequence#Mechanism_of_nuclear_import" rel="nofollow" target="_blank">nuclear localization signal</a> (NLS) and a fluorescent signal (GFP). This experiment proves that transfection of human embryonic kidney cells with the Cas9 construct works and that cells express the desired protein. Of course, the cell could be expressing other proteins with similar weight, but this experiment makes it highly plausible that the results of the subsequent experiments are indeed due to the Cas9 activity.<br />
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<span style="mso-bidi-font-weight: normal;">Then, since the protein is believed to be active in the
nucleus where the DNA is, can it be shown that the protein gets to the nucleus
after human embryonic kidney cells ("HEK293T cells") have been transfected with a DNA-fragment („vector“) encoding the
above construct?</span></div>
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<span style="mso-bidi-font-weight: normal;">
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The
below images show separately the GFPs, the cells and an overlay. It is not easy
to see but it appears as if the GFPs shine from within the cell nucleus, and
therefore most likely the Cas9 is also in the nucleus.</div>
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiWZDFBXPOFh36L61TxnFSxzOWmTmrAdsYCAMkT6BmRAq5erVuwpEOxtj0EecmOlXLT86VGEDdj8hcIQCPe_sors-WOvRPMPTgjMiD-nKeZam4xZIzQD-zxwZW2P6sNgbDcS5pmTDsQP3V9/s1600/fig-1b+gfp_merge_overlay.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="171" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiWZDFBXPOFh36L61TxnFSxzOWmTmrAdsYCAMkT6BmRAq5erVuwpEOxtj0EecmOlXLT86VGEDdj8hcIQCPe_sors-WOvRPMPTgjMiD-nKeZam4xZIzQD-zxwZW2P6sNgbDcS5pmTDsQP3V9/s640/fig-1b+gfp_merge_overlay.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
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<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
Especially
the GFP image of four cells in the left bottom corner nicely overlaps with
their nucleus position.</div>
<br />
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
Now, <span style="mso-bidi-font-weight: normal;">since RNA is required to program the
nuclease, can the target HEK cell express the guiding RNA while at the same time be transfected with engineered Cas9?</span><br />
<span style="mso-bidi-font-weight: normal;">
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<span style="mso-bidi-font-weight: normal;">A Northern blot
shows that indeed, third column from the left, guiding RNA („CLTA1 sgRNA“) of 62
nucleotide length (= 20 nt of guiding RNA + 42 nt of RNA required to bind the
Cas9) is expressed by the cells.</span></div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiepqh-HR2yIMlg20RxaXDDXM9Md-9Q5gxCPmvsoBVr7UJ5H2S9eBsvYvSc8BKmU4Nk-IW5G720f8p0Ih2pGw8d_dPtxXkdRgFu8_lvYcRX6BSa256-8Cr7fyEQ15On3hDXFsu45kDHT-um/s1600/fig-1c+Expression+of+sgRNA.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="572" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiepqh-HR2yIMlg20RxaXDDXM9Md-9Q5gxCPmvsoBVr7UJ5H2S9eBsvYvSc8BKmU4Nk-IW5G720f8p0Ih2pGw8d_dPtxXkdRgFu8_lvYcRX6BSa256-8Cr7fyEQ15On3hDXFsu45kDHT-um/s640/fig-1c+Expression+of+sgRNA.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
<span style="mso-bidi-font-weight: normal;">In the fourth column from the left ("CLTA1 sgRNA + Cas9"), the signal is even a bit stronger, suggesting the stabilization of the sgRNA by Cas9. Possibly binding of the sgRNA by Cas9 protects it from degradation.</span></div>
<span style="mso-bidi-font-weight: normal;">
</span></div>
<br />
So, the pieces are in place. The cells can be transfected and they're shown to transcribe sgRNA and translate foreign Cas9 simultaneously. It remains to show that Cas9 is operative.<br />
<br />
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<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
<b style="mso-bidi-font-weight: normal;">What happens to the DNA if the sgRNA and Cas9 together
are expressed in a cell?</b></div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
Transfecting
HEK293T cells with a Cas9 vector and a vector for Clathrin directing sgRNA followed by isolation of cell
products resulted in the following cell-lysate image.</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh1Vwnkaf7KYFMroTqa-qI86yiPTvFoca6IjJwru6b7ZbrWSPjiOq6Hdc70pQ6RXozQU8M-kq26aB96mom9nVK-rDEIRz447aatgcJU2j0CnuwdHHILUyirNoOn5QZCcEcCb5CxXrvpRGUL/s1600/fig-1e+results+surveyor+assay.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="459" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh1Vwnkaf7KYFMroTqa-qI86yiPTvFoca6IjJwru6b7ZbrWSPjiOq6Hdc70pQ6RXozQU8M-kq26aB96mom9nVK-rDEIRz447aatgcJU2j0CnuwdHHILUyirNoOn5QZCcEcCb5CxXrvpRGUL/s640/fig-1e+results+surveyor+assay.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
For the moment, only consider columns under the "Cas9-mCherry" label. In all of these columns Cas9 is present (mCherry is another fluorescent labeling function).</div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
To demonstrate the workings of Cas9, the authors use the so called <i>Surveyor Assay</i> method. This method allows to identify breaks in double stranded DNA, albeit only indirectly. When an agent like Cas9 breaks dsDNA the cell immediately fixes the DNA using its own fixing mechanisms (like <i>non-homolguous end joining</i>, NHEJ). These mechanisms are however error prone and can introduce mismatches, just like a genomic scar.</div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
In the Surveyor assay then, the transfected cells are lysated and the DNA is extracted, amplified with PCR and then incubated <i>in vitro</i> with the nuclease Cel-1. This protein identifies mismatches resulting from NHEJ and again breaks the dsDNA at these positions. The products of this subsequent nuclease activity are what is analysed finally and shown in the above figure.</div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
In the figure above then, the authors show step-by-step what effect only Cas9, sgRNA + Cas9, sgRNA + Cas9 + Cel-1 have, respectively.</div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
If there is only Cas9 present (column 2, -/-), DNA of roughly 400 bp is found. Only Cas9 + Cel-1 again yields DNA of 400 bp. sgRNA + Cas9 also 400 bp (important: Cas9 was active here but in the absence of Cel-1 mismatches are not recognized). But then, in column 5 (+/+), a dim line at little less than 200 bp is visible. This dim line is what the whole excitement is about. The column with only sgRNA and Cas9 can be viewed as the control for the column with sgRNA, Cas9 and Cel-1 since it could be that Cel-1 itself has some sort of nuclease activity and cleaves DNA. But no, the third column (-/+) is negative.</div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
A positive confirmation is provided by comparison to the ZFN results. ZFN is a highly engineered, very expensive system that cuts at almost the same position in the DNA.</div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
<br /></div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
From the image, the next question immediately arises: How can the dim line be made stronger?</div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
<br /></div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
Is there enough Cas9? Is there enough sgRNA? Does the sgRNA bind sufficiently strongly to Cas9? Should the guiding sequence be longer?</div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
Checking for sgRNA availability, the authors found this:</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi_WGIPcBvkvEx_zY0vH7QgZrumUzQ-BkcWc3WgWqa0uKF0Lo68n2AZafBVMr5IB4-wK6bWct87vyiRgN79PoYL7PL_NHLU_YVoriOpj9x5Vq2atfqZF6PwI6M7kszGVl78lCz071327Z7k/s1600/fig-2a+in+vitro+RNA.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="350" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi_WGIPcBvkvEx_zY0vH7QgZrumUzQ-BkcWc3WgWqa0uKF0Lo68n2AZafBVMr5IB4-wK6bWct87vyiRgN79PoYL7PL_NHLU_YVoriOpj9x5Vq2atfqZF6PwI6M7kszGVl78lCz071327Z7k/s640/fig-2a+in+vitro+RNA.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
When adding additional sgRNA, in column 5 (Cas9-HA-NLS-GFP plasmid + in vitro transcribed CLTA1 sgRNA added to lysate), the lines are indeed stronger. Even stronger than the ZFN signal. Here, the authors first transfected cells with Cas9 and sgRNA plasmids (just like before). The cells were then lysated (so the Cas9 protein was available <i>in vitro</i>) and then mixed with additional sgRNA. The signal gets stronger. So more sgRNA is better, however this doesn't explain why, is it because then overall there is more active Cas9? Or is it just because expression of plasmid sgRNA is not efficient enough?</div>
The authors just added even more sgRNA:<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi3CtLFr40z90W9IBQZQXTVN00N6jXjZKtU_GiEiFjj0onrFxOW7cvVvm-MUkITGIP1PlT5VsNfx61D1ZE17CfP1kaWiJPLR-ByJWxMu_njtJYGz7TXZdmiQRIRbPzy5bDGSoDkMkJD8aha/s1600/fig-2b+in+vitro+RNA.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="240" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi3CtLFr40z90W9IBQZQXTVN00N6jXjZKtU_GiEiFjj0onrFxOW7cvVvm-MUkITGIP1PlT5VsNfx61D1ZE17CfP1kaWiJPLR-ByJWxMu_njtJYGz7TXZdmiQRIRbPzy5bDGSoDkMkJD8aha/s640/fig-2b+in+vitro+RNA.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
The signal is strongest when both the sgRNA transcribed from plasmids as well as <i>in vitro</i> transcribed sgRNA are added to the system (right-most column).<br />
It is concluded that either sgRNA expression or its loading into Cas9 is the limiting factor of Cas9 nuclease performance.<br />
<br />
At the time of doing these experiments, probably the simplest second thing to do was to extend the region of sgRNA which is involved in binding to Cas9. So they did.<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiBvuJ3mDKAIufCSrA3oHusbCme09Ocr6jlYKVaB9yxcGw0KUtGK_1biULPIHZtm8YS7txGbnF9ZEnzE9LJg6TQ5T0Ozw8Alcu5nKpDdDpZ9LS9MJYr8sDAHUXkDuPRlvKe9BM_aVAkMXoj/s1600/fig-3a+extended+sgRNA.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="428" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiBvuJ3mDKAIufCSrA3oHusbCme09Ocr6jlYKVaB9yxcGw0KUtGK_1biULPIHZtm8YS7txGbnF9ZEnzE9LJg6TQ5T0Ozw8Alcu5nKpDdDpZ9LS9MJYr8sDAHUXkDuPRlvKe9BM_aVAkMXoj/s640/fig-3a+extended+sgRNA.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
V1.0 represents the originally used system. In v2.1, the presumed Cas9 binding region is extended by 4 basepairs (red basepairs to the right of GAA) and the 3'-end was extended by 5 nucleotides. In v2.2, the Cas9 binding region was extended by 10 basepairs and the 3'-region by 5 nucleotides (compared to v1.0).<br />
Again, a Surveyor Assay was carried out.<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgmUtGYWzYTqEeG6f72WSY4-gbCW8RT7gzNeF9vIWV6iD72rvjLT8Q2NRMSiGBwEpW68IxTlRLRba6gblM5X7tpqK7UAqqJMi7KfAWJXwIGwRkvBUseBuNxtdnznjPLNnj0eIdjLFIAWq_p/s1600/fig-3b+results+extended+sgRNA.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="380" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgmUtGYWzYTqEeG6f72WSY4-gbCW8RT7gzNeF9vIWV6iD72rvjLT8Q2NRMSiGBwEpW68IxTlRLRba6gblM5X7tpqK7UAqqJMi7KfAWJXwIGwRkvBUseBuNxtdnznjPLNnj0eIdjLFIAWq_p/s640/fig-3b+results+extended+sgRNA.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
The results are not as clear as in the above case. Overall, v2.1 and v2.2 appear to have similar performance over v1.1 (7 - 8 % cleavage to 4 % cleavage in v1.1). This means that increasing the Cas9 binding region is more important than increasing the 3'-region of the sgRNA. Extension of the guiding sequence length was not examined. Furthermore, RNA can be stabilized in vivo by modifications at the 5'- or 3'-ends, both of which have not been further examined in these experiments.<br />
The authors suggest more research in this direction to be necessary.<br />
<br />
Finally, the above results are all in agreement with a visual model like this:<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi51y9sp_A0ajT5gzqpQvlAFlthjICVZ5IdORKnJmO-8nELZSBfXGJWSicmh0yjQbXG_kl9ulY8y9_j6Di8Px3WO9qUvpCJaSCEOnNmR-xxRDkdmESBJm7xahgiYTTQv4kXbehCuSYM9jOj/s1600/fig-1c+Cas9+operation.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="468" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi51y9sp_A0ajT5gzqpQvlAFlthjICVZ5IdORKnJmO-8nELZSBfXGJWSicmh0yjQbXG_kl9ulY8y9_j6Di8Px3WO9qUvpCJaSCEOnNmR-xxRDkdmESBJm7xahgiYTTQv4kXbehCuSYM9jOj/s640/fig-1c+Cas9+operation.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://elifesciences.org/content/2/e00471</td></tr>
</tbody></table>
<br />
<b>Discussion</b><br />
Hopefully, the above could provide some understanding of the research and make the paper more accessible. In any case it will be interesting to see where this leads.<span style="font-family: "cambria"; font-size: 12.0pt;"></span><br />
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
<span style="font-family: "cambria"; font-size: 12.0pt;"></span></div>
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
<span style="font-family: "cambria"; font-size: 12.0pt;"></span></div>
However, and this is very important to consider: what are the ethical implications of this technology. Given superb selectivity and sensitivity, the system could hypothetically be used to engineer the genome of living humans at will (and these changes could be inherited by later generations).<br />
<div class="MsoNormal" style="text-align: justify; text-justify: inter-ideograph;">
It is not yet conceivable, when the first genome engineering applications will be studied in clinical trials. To put it differently, who would want something injected that has the sole purpose of cutting the host DNA? Let
it be clear that to date the only „test“ in a (disfunctional) single-cell human
embryo was not perfectly successful[<a href="http://link.springer.com/article/10.1007%2Fs13238-015-0153-5" rel="nofollow" target="_blank">Liang <i>et al.</i></a>]:</div>
<blockquote class="tr_bq">
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--> </style><i style="mso-bidi-font-style: normal;"><span style="font-family: "cambria"; font-size: 12.0pt;">"</span></i><span style="mso-bidi-font-style: normal;"><span style="font-family: "cambria"; font-size: 12.0pt;">[...]</span></span><i style="mso-bidi-font-style: normal;"><span style="font-family: "cambria"; font-size: 12.0pt;">Off-target cleavage was also apparent in these 3PN
zygotes as revealed by the T7E1 assay and whole-exome sequencing</span></i>.[...]".</div>
</blockquote>
Most recently, in April 2015, at a conference in California a number of leading geneticists met to discuss the implications of Cas9-based technology. In a <a href="https://www.uam.es/personal_pdi/ciencias/jmsierra/documents/Baltimore2015Sci.pdf" rel="nofollow" target="_blank">perspectives publication</a>, the authors end by strongly recommending against "...<i>germline genome modifications for clinical applications in humans as long as societal, environmental and ethical implications of such activity</i>..." are not conclusively discussed.<br />
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It is left to hope that these recommendations are being taken into account by researchers when starting new Cas9 based research.<br />
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<br />
Edit (01. May, 2016):</div>
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A version of this post has been published on the blog of the Swiss based Think-Tank REATCH (Research and Technology in Switzerland).</div>
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<a href="https://reatch.ch/?q=de/content/detailed-analysis-jinek-et-als-seminal-crispr-publication" target="_blank">https://reatch.ch/?q=de/content/detailed-analysis-jinek-et-als-seminal-crispr-publication</a></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-57190174843492267432015-09-01T11:01:00.003-07:002015-10-11T05:24:36.411-07:00Coursera CertificateData Science Specialization Certificate.<br />
<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjduQSof9np35u8RdAuW90UsKDRGk8-O49aucfpB5PtIkTMHiX7tVWtKPuuoIFf4z2gIDHlHyvmp8OxQH3BYiLq0JUfDHTSaCxB58-oMG2eh4v1OI1eQr3lEOlzd4a_jI-TmARsYPOey4Mu/s1600/Coursera+Data+Science+2015.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="492" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjduQSof9np35u8RdAuW90UsKDRGk8-O49aucfpB5PtIkTMHiX7tVWtKPuuoIFf4z2gIDHlHyvmp8OxQH3BYiLq0JUfDHTSaCxB58-oMG2eh4v1OI1eQr3lEOlzd4a_jI-TmARsYPOey4Mu/s640/Coursera+Data+Science+2015.png" width="640" /></a></div>
<span id="goog_2018349161"></span><span id="goog_2018349162"></span><br />
<span id="goog_2018349161">Various Links to repositories from some of the courses:</span><br />
<br />
<span id="goog_2018349161"><a href="https://mzhku.shinyapps.io/04_SwiftXeyApp" rel="nofollow" target="_blank">Capstone Project App</a> / <a href="http://rpubs.com/mzh/103942" rel="nofollow" target="_blank">Slide Show</a> / <a href="http://rpubs.com/mzh/96515" rel="nofollow" target="_blank">Milestone Report</a></span><br />
<br />
<span id="goog_2018349161"><a href="https://github.com/mzhKU/loess_fit_app">Developing Data Products Repository</a> / <a href="https://mzhku.shinyapps.io/course_project/" rel="nofollow" target="_blank">App</a> / <a href="http://mzhku.github.io/#1" rel="nofollow" target="_blank">Slide Show</a></span><br />
<br />
<span id="goog_2018349161"><a href="https://github.com/mzhKU/ML_Project" target="_blank">Practical Machine Learning Repository</a> </span><span id="goog_2018349161"> </span>/ <a href="https://github.com/mzhKU/ML_Project/blob/master/ml-report.md" target="_blank">Report</a><br />
<a href="https://github.com/mzhKU/RepData_PeerAssessment1/blob/master/PA1_template.md" target="_blank"><br /></a>
<a href="https://github.com/mzhKU/RepData_PeerAssessment1/blob/master/PA1_template.md" target="_blank">Reproducible Research Report 1</a><br />
<a href="http://rpubs.com/mzh/25793" target="_blank">Reproducible Research Report 2 </a><br />
<br />
<a href="https://github.com/mzhKU/getdata_peer_assignment" rel="nofollow" target="_blank">Getting and Cleaning Data Repository </a><br />
<br />
<a href="http://rpubs.com/mzh/26062" target="_blank">Regression Models Report</a>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-76339708939635857862015-01-03T10:14:00.000-08:002015-01-04T01:01:23.112-08:00Excel like PivotTable and more with R<br />
A table contains values as below (download from link under figure):<br />
<br />
<style>table { }td { padding-top: 1px; padding-right: 1px; padding-left: 1px; color: black; font-size: 12pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri,sans-serif; vertical-align: bottom; border: medium none; white-space: nowrap; }.xl64 { }.xl65 { }.xl66 { border-width: medium medium 0.5pt; border-style: none none solid; border-color: -moz-use-text-color -moz-use-text-color windowtext; }.xl67 { }</style>
<br />
<table border="0" cellpadding="0" cellspacing="0" style="border-collapse: collapse; width: 441px;">
<colgroup><col style="mso-width-alt: 5205; mso-width-source: userset; width: 122pt;" width="122"></col>
<col style="mso-width-alt: 3157; mso-width-source: userset; width: 74pt;" width="74"></col>
<col style="mso-width-alt: 2048; mso-width-source: userset; width: 48pt;" width="48"></col>
<col style="mso-width-alt: 2176; mso-width-source: userset; width: 51pt;" width="51"></col>
<col style="mso-width-alt: 3584; mso-width-source: userset; width: 84pt;" width="84"></col>
<col style="mso-width-alt: 2645; mso-width-source: userset; width: 62pt;" width="62"></col>
</colgroup><tbody>
<tr height="15" style="height: 15.0pt;">
<td class="xl66" height="15" style="height: 15.0pt; width: 122pt;" width="122">Item</td>
<td class="xl66" style="width: 74pt;" width="74">Category</td>
<td class="xl66" style="width: 48pt;" width="48">Price</td>
<td class="xl66" style="width: 51pt;" width="51">Profit</td>
<td class="xl66" style="width: 84pt;" width="84">Actual Profit</td>
<td class="xl66" style="width: 62pt;" width="62">Calories</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Beer</td>
<td>Beverages</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$4.00 </td>
<td align="right" class="xl64">50%</td>
<td align="right" class="xl67" style="background: #BBE2C7; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right">200</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Soda</td>
<td>Beverages</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$2.50 </td>
<td align="right" class="xl64">80%</td>
<td align="right" class="xl67" style="background: #BBE2C7; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right">120</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Chocolate Bar</td>
<td>Candy</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right" class="xl64">75%</td>
<td align="right" class="xl67" style="background: #D1EBDA; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$1.50 </td>
<td align="right">255</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Ice Cream Sandwich</td>
<td>Frozen Treats</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$3.00 </td>
<td align="right" class="xl64">67%</td>
<td align="right" class="xl67" style="background: #BBE2C7; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right">240</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Bottled Water</td>
<td>Beverages</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$3.00 </td>
<td align="right" class="xl64">83%</td>
<td align="right" class="xl67" style="background: #A5D9B4; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$2.50 </td>
<td align="right">0</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Gummy Bears</td>
<td>Candy</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right" class="xl64">50%</td>
<td align="right" class="xl67" style="background: #E7F4ED; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$1.00 </td>
<td align="right">300</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Soda</td>
<td>Beverages</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$2.50 </td>
<td align="right" class="xl64">80%</td>
<td align="right" class="xl67" style="background: #BBE2C7; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right">120</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Hamburger</td>
<td>Hot Food</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$3.00 </td>
<td align="right" class="xl64">67%</td>
<td align="right" class="xl67" style="background: #BBE2C7; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right">320</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Popcorn</td>
<td>Hot Food</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$5.00 </td>
<td align="right" class="xl64">80%</td>
<td align="right" class="xl67" style="background: #63BE7B; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$4.00 </td>
<td align="right">500</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Licorice Rope</td>
<td>Candy</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right" class="xl64">50%</td>
<td align="right" class="xl67" style="background: #E7F4ED; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$1.00 </td>
<td align="right">280</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Hot Dog</td>
<td>Hot Food</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$1.50 </td>
<td align="right" class="xl64">67%</td>
<td align="right" class="xl67" style="background: #E7F4ED; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$1.00 </td>
<td align="right">265</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Licorice Rope</td>
<td>Candy</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$2.00 </td>
<td align="right" class="xl64">50%</td>
<td align="right" class="xl67" style="background: #E7F4ED; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$1.00 </td>
<td align="right">280</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Popcorn</td>
<td>Hot Food</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$5.00 </td>
<td align="right" class="xl64">80%</td>
<td align="right" class="xl67" style="background: #63BE7B; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$4.00 </td>
<td align="right">500</td>
</tr>
<tr height="15" style="height: 15.0pt;">
<td height="15" style="height: 15.0pt;">Popcorn</td>
<td>Hot Food</td>
<td align="right" class="xl65"><span style="mso-spacerun: yes;"> </span>$5.00 </td>
<td align="right" class="xl64">80%</td>
<td align="right" class="xl67" style="background: #63BE7B; mso-pattern: black none;"><span style="mso-spacerun: yes;"> </span>$4.00 </td>
<td align="right">500</td>
</tr>
</tbody></table>
<br />
It is very easy to summarize the data using the Excel PivotTable generator (even for multiple variables like "Profit" and "Actual Profit" simultaneously):<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgqEK9sTp-JKmsYllEj2EXLJOA0nVnXHo1YT0qulb-e6F_5GhV7w60Rr4ileD9BFhgdYQfwolqx_GKV-zUDIj6mGVlx6IxHie5Ho6bSZNjsprYvwtUntJY-cXa3roxR-_wNHedY-5QIWC4O/s1600/Screen+shot+2015-01-03+at+Samstag,+3.+Januar+201517.52.27.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgqEK9sTp-JKmsYllEj2EXLJOA0nVnXHo1YT0qulb-e6F_5GhV7w60Rr4ileD9BFhgdYQfwolqx_GKV-zUDIj6mGVlx6IxHie5Ho6bSZNjsprYvwtUntJY-cXa3roxR-_wNHedY-5QIWC4O/s1600/Screen+shot+2015-01-03+at+Samstag,+3.+Januar+201517.52.27.png" height="640" width="452" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Example taken from the <span id="goog_1001211631"></span><a href="http://eu.wiley.com/WileyCDA/WileyTitle/productCd-111866146X.html" rel="nofollow">Data Smart book<span id="goog_1001211632"></span></a>.</td></tr>
</tbody></table>
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It probably doesn't make a lot of sense to summarize the data by percentage, but this is just for illustration purposes.<br />
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A similar operation is the <span style="font-family: "Courier New",Courier,monospace;">aggregate</span> function in R (the Concessions.xlsx data frame is loaded as <span style="font-family: "Courier New",Courier,monospace;">con</span>):<br />
<span style="font-family: "Courier New", Courier, monospace;">> aggregate(cbind(Profit, Actual.Profit) ~ Category, data=con, FUN=sum)<br /> Category Profit Actual.Profit<br />1 Beverages 31.23333 98.5<br />2 Candy 23.25000 46.5<br />3 Frozen Treats 23.00000 69.0<br />4 Hot Food 45.21667 142.0</span><br />
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The same is possible using the <span style="font-family: "Courier New",Courier,monospace;">xtabs</span> function:<span style="font-family: "Courier New",Courier,monospace;"><br /></span>
<span style="font-family: "Courier New",Courier,monospace;">> xtabs(cbind(Profit, Actual.Profit) ~ Category, data=con)<br /> <br />Category Profit Actual.Profit<br /> Beverages 31.23333 98.50000<br /> Candy 23.25000 46.50000<br /> Frozen Treats 23.00000 69.00000<br /> Hot Food 45.21667 142.00000</span><br />
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Note, no special operation <span style="font-family: "Courier New",Courier,monospace;">FUN</span> is specified in xtabs.<br />
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Occurence of items can be done when specifing "Count" in the PivotTable builder:<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjBbjlrOX1ae0bZiI8zs2e6Wm0NdmSzDZ23GQOTywTZPJnkcruLdSP69m3B_g0v8jfwrQrUp_kjnqosfpnsYkaIF5xFqHm-rE0qfICnFH_14WWy62imtM1AI803R99KVA2fkf9t-EsxkmXn/s1600/Screen+shot+2015-01-03+at+Samstag,+3.+Januar+201518.23.22.png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjBbjlrOX1ae0bZiI8zs2e6Wm0NdmSzDZ23GQOTywTZPJnkcruLdSP69m3B_g0v8jfwrQrUp_kjnqosfpnsYkaIF5xFqHm-rE0qfICnFH_14WWy62imtM1AI803R99KVA2fkf9t-EsxkmXn/s1600/Screen+shot+2015-01-03+at+Samstag,+3.+Januar+201518.23.22.png" height="640" width="634" /></a></div>
<div class="separator" style="clear: both; text-align: center;">
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R can do this with the <span style="font-family: "Courier New",Courier,monospace;">plyr</span> package and its <span style="font-family: "Courier New",Courier,monospace;">count</span> function.<br />
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<span style="font-family: "Courier New",Courier,monospace;">> count(con$Item)<br /> x freq<br />1 Beer 20<br />2 Bottled Water 13<br />3 Chocolate Bar 13<br />4 Chocolate Dipped Cone 11<br />5 Gummy Bears 14<br />6 Hamburger 16<br />7 Hot Dog 15<br />8 Ice Cream Sandwich 10<br />9 Licorice Rope 13<br />10 Nachos 15<br />11 Pizza 17<br />12 Popcorn 16<br />13 Popsicle 13<br />14 Soda 13</span><br />
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This can also be done using the extremely powerful built-in functions of the data.table type:<br />
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<span style="font-family: "Courier New",Courier,monospace;">> con[, .N, by=Item]<br /> Item N<br /> 1: Beer 20<br /> 2: Bottled Water 13<br /> 3: Soda 13<br /> 4: Chocolate Bar 13<br /> 5: Gummy Bears 14<br /> 6: Licorice Rope 13<br /> 7: Popsicle 13<br /> 8: Ice Cream Sandwich 10<br /> 9: Chocolate Dipped Cone 11<br />10: Popcorn 16<br />11: Hamburger 16<br />12: Nachos 15<br />13: Pizza 17<br />14: Hot Dog 15</span><br />
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Here, <span style="font-family: "Courier New",Courier,monospace;">.N</span> is a built-in count function applied to the data.table object.<br />
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Or with a call to table where one can break down the items by profit:<br />
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<span style="font-family: "Courier New",Courier,monospace;">> table(con$Item, con$Profit)<br /> <br /> 0.25 0.5 0.67 0.75 0.8 0.83<br /> Beer 0 20 0 0 0 0<br /> Bottled Water 0 0 0 0 0 13<br /> Chocolate Bar 0 0 0 13 0 0<br /> Chocolate Dipped Cone 0 11 0 0 0 0<br /> Gummy Bears 0 14 0 0 0 0<br /> Hamburger 0 0 16 0 0 0<br /> Hot Dog 0 0 15 0 0 0<br /> Ice Cream Sandwich 0 0 10 0 0 0<br /> Licorice Rope 0 13 0 0 0 0<br /> Nachos 0 15 0 0 0 0<br /> Pizza 17 0 0 0 0 0<br /> Popcorn 0 0 0 0 16 0<br /> Popsicle 0 0 0 0 0 13<br /> Soda 0 0 0 0 13 0</span><br />
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I.e. 13 items of "Bottled Water" giving a profit of 83% each have been sold.<br />
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Excel can then apply eg. summation when breaking down items by category:<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEglGSsM8bjjrb7p1iKv3Dm3lAwpK4tbvU03_vQWWbAzqeDw7RPzRL8_XGBxgLIhHhoR4ppi6aW_pOYb4VN3lZlFTwFunN9v9PMlPF4Ugbx9C4yn7pEawAFPr5PvO83Kny7tSLe1ZAejRW68/s1600/Screen+shot+2015-01-03+at+Samstag,+3.+Januar+201518.37.54.png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEglGSsM8bjjrb7p1iKv3Dm3lAwpK4tbvU03_vQWWbAzqeDw7RPzRL8_XGBxgLIhHhoR4ppi6aW_pOYb4VN3lZlFTwFunN9v9PMlPF4Ugbx9C4yn7pEawAFPr5PvO83Kny7tSLe1ZAejRW68/s1600/Screen+shot+2015-01-03+at+Samstag,+3.+Januar+201518.37.54.png" height="334" width="640" /></a></div>
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The operation to carry out (sum, count, ...) is defined when clicking on the small "<i>i</i>" symbol in the Values field. So in total, beer earned 80 $.<br />
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Another <span style="font-family: "Courier New",Courier,monospace;">plyr</span> built-in function of the <span style="font-family: "Courier New",Courier,monospace;">data.table</span> object allows to quickly obtain the summarized prices of the category sales, broken down by category.<br />
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<span style="font-family: "Courier New",Courier,monospace;">> con[, sum(Price), by=list(Category, Item)]<br /> Category Item V1<br /> 1: Beverages Beer 80.0<br /> 2: Beverages Bottled Water 39.0<br /> 3: Beverages Soda 32.5<br /> 4: Candy Chocolate Bar 26.0<br /> 5: Candy Gummy Bears 28.0<br /> 6: Candy Licorice Rope 26.0<br /> 7: Frozen Treats Popsicle 39.0<br /> 8: Frozen Treats Ice Cream Sandwich 30.0<br /> 9: Frozen Treats Chocolate Dipped Cone 33.0<br />10: Hot Food Popcorn 80.0<br />11: Hot Food Hamburger 48.0<br />12: Hot Food Nachos 45.0<br />13: Hot Food Pizza 34.0<br />14: Hot Food Hot Dog 22.5</span><br />
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<br />Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-5138984105175073002014-08-03T14:01:00.002-07:002016-02-11T12:00:36.774-08:00Literature 003: Navaroli et al., PNAS, 2012, 109, 8, E471-480<div style="text-align: justify;">
<a href="http://qmviews.blogspot.ch/2014/04/literature-002-niewoehner-et-al.html" rel="nofollow">Niewoehner <i>et al.</i></a> state that "<i>The precise mechanism by which the sFab construct escapes lysosomal sorting and is released on the abluminal side is not known.</i>"</div>
<div style="text-align: justify;">
This is interesting because it essentially forms part of the frontier of the human understanding of how the cell works.</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
A paper by <a href="http://www.pnas.org/content/109/8/E471.abstract">Navaroli et al.</a> from 2012 discusses a specific internalization and sorting mechanism, the uptake of the Transferrin receptor (TfR).<br />
<br />
As one might guess, internalization
of cargo across the cell membrane is a highly complex process. It is
highly regulated because the cell wants to avoid internalizing
substances which are toxic or which are already present in the cell at
sufficient concentrations while at the same time it wants to promote
uptake of needed substances.<br />
TfR allows the cell to internalize Transferrin
(Tf) which again binds Fe. In the figure below, "receptor" would be TfR
(AP-2 is the so called adapter protein type 2 and PM is the plasma
membrane). Clathrin is a factor involved in vesicle formation. Conceptually, AP-2 can be understood to have a certain "directing" effect on the vesicle in a similar way as it is attempted to be established for Rabenosyn-5 in this article.<br />
<br />
(<i>In the following, the figure caption labels correspond to the figure captions in the article.</i>) <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhioHw2h5P2uzh_cR5xA6wObGOby7MdZExNOFNSJre0BvqHJ2E1j67shc3iiqmT7WbAofifSVV8o-srfB9Ga897tM6ZzpD_IBdBSuAgHoXeAVF4lB7_aCxhw1usKdybH12m1OB2Gw-LWVhp/s1600/Itrafig2.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="476" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhioHw2h5P2uzh_cR5xA6wObGOby7MdZExNOFNSJre0BvqHJ2E1j67shc3iiqmT7WbAofifSVV8o-srfB9Ga897tM6ZzpD_IBdBSuAgHoXeAVF4lB7_aCxhw1usKdybH12m1OB2Gw-LWVhp/s1600/Itrafig2.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://en.wikipedia.org/wiki/Receptor-mediated_endocytosis#mediaviewer/File:Itrafig2.jpg</td></tr>
</tbody></table>
</div>
<div style="text-align: justify;">
<br />
Rabenosyn-5 is another endosomal component implicated in early endosome fusion and, as reported in this paper, is found in the vicinity of clathrin coated cell surface regions (Fig. 2 in article).<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJdIFeQjw3RzHLGO1Bb1gbA3nfJAjeqaVceOlHNlUZrGxVO2P6hhO92wUchJWQvSPKqaWW4mlNk4vNllh7SN5zCQYuP6aQwFaIpCY-OGgZC_UEh7B7czDdqZn_LG7xo8p0Nl47Vlb8gcGG/s1600/fig2tesm.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="374" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJdIFeQjw3RzHLGO1Bb1gbA3nfJAjeqaVceOlHNlUZrGxVO2P6hhO92wUchJWQvSPKqaWW4mlNk4vNllh7SN5zCQYuP6aQwFaIpCY-OGgZC_UEh7B7czDdqZn_LG7xo8p0Nl47Vlb8gcGG/s1600/fig2tesm.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig. 2</b>: http://www.pnas.org/content/109/8/E471/F2.expansion.html<br />
Top panels: Clathrin<br />
Lower panels: Rabenosyn-5</td><td class="tr-caption" style="text-align: center;"></td></tr>
</tbody></table>
TESM refers to <i><b>T</b>IRF <b>ES</b>L <b>M</b>icroscopy</i>, where TIRF and ESL stand for <i>Total Internal Reflection Fluorescence</i> and <i>Epifluorescence Structured Light</i>, respectively. The top two panels show clathrin while the lower two show Rabenosyn-5 distribution. The scale, <i>z-position</i>, indicates how deep within the cell interior a pixel is measured.<br />
TIRF allows to observe fluorescent objects in the cell but can not provide information about the location relative to the cell surface because, as reported, the brightness of an object can result from either its distance to the cell surface or its size. ESL corrects for this and the overlay, TESM, shows the z-axis dependent distribution of clathrin and Rabenosyn-5 at the cell surface: most pixels are blue which means that the majority of the proteins reside at the surface and the surface is populated by both clathrin and Rabenosyn-5.<br />
<br />
Since the question is to figure out what Rabenosyn-5 does, the next question to ask is<b> what happens when Rabenosyn-5 is not present in the cell </b>(Fig. 4D in article)<b>?</b></div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEinI3nY9RmKyVhcrpeXo5kfX-QS7YNNJFc7x-8qU7GNPQvM4uERXEF6cF2GN0iCt3bFsfhnMymOKPubY4Gye25ButNpREBf8Li8ErlGT1y-MEPL8GLgsigVbJc18YBaJfqcVznW6qHvDqcj/s1600/fig4d.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="390" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEinI3nY9RmKyVhcrpeXo5kfX-QS7YNNJFc7x-8qU7GNPQvM4uERXEF6cF2GN0iCt3bFsfhnMymOKPubY4Gye25ButNpREBf8Li8ErlGT1y-MEPL8GLgsigVbJc18YBaJfqcVznW6qHvDqcj/s1600/fig4d.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig. 4D</b>: http://www.pnas.org/content/109/8/E471/F4.expansion.html<br />
Sc: Rabenosyn-5 present<br />
Si: Rabenosyn-5 not present</td></tr>
</tbody></table>
<div style="text-align: justify;">
Using siRNA (<i>small interfering</i> RNA), transcription of Rabenosyn-5 can be prevented. The white pixels are TfR. "Sc" is a control for the siRNA function (<i>sc</i>rambled siRNA).</div>
<div style="text-align: justify;">
Considering the TIRF images (where Sc means that Rabenosyn-5 is active because the surpressing siRNA is scrambled), it is seen that the cell has a higher TfR surface concentration compared to the case where the siRNA is active (right TIRF image). The difference is reported to be a decrease of 75% in the TIRF image. The decrease measured by EPI is less pronounced. Put differently, when Rabenosyn-5 is not produced, the TfR surface amount is reduced which also means that Tf uptake is reduced in TfR depleted cells.</div>
<div style="text-align: justify;">
<br />
This observation leads to the question, how Rabenosyn-5 controls the cell surface TfR level. <b>Does Rabenosyn-5 regulate TfR gene transcription, TfR gene translation or some other kind of post-translational mechanism?</b> This question is answered by panel A of Fig. 6A in the article.<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjyxWiUu9qOoONUsR4mViLTp3QhaMjTmR68d2tpqaH0ZiHk04heGZZGJ7z_GRVL2xcp5pEGMdpJ1pcRNvub3HnB0IYzmaijcl-lmOSKc-sCYzkgUd9qZLzHTJqqsx9W7Qahp3cBakKr3kAA/s1600/fig6a.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="302" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjyxWiUu9qOoONUsR4mViLTp3QhaMjTmR68d2tpqaH0ZiHk04heGZZGJ7z_GRVL2xcp5pEGMdpJ1pcRNvub3HnB0IYzmaijcl-lmOSKc-sCYzkgUd9qZLzHTJqqsx9W7Qahp3cBakKr3kAA/s1600/fig6a.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig. 6A</b>: http://www.pnas.org/content/109/8/E471/F6.large.jpg</td></tr>
</tbody></table>
</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
</tbody></table>
<div style="text-align: justify;">
GADPH is used to provide a scale for the measurement.<br />
<b> </b><br />
<br />
<b>Possibility of translational regulation.</b> It is known that translation of TfR can be regulated by mRNA parts before and behind the sequence coding for the protein (so called untranslated regions, UTRs).<br />
To test for translational effects of Rabenosyn-5, the host was transfected by TfR-GFP (green fluorescent protein) lacking these UTRs (that means translation of this TfR-GFP can not be regulated). As can be seen, when Rabenosyn-5 is active (Rbsn Sc column), both TfR and TfR-GFP are produced (right most panel, Sc column). However, when Rabenosyn-5 is surpressed (right most panel, Si column) both TfR-GFP (lacking the regulatory sequences) as well as native TfR are present at lower levels.</div>
<div style="text-align: justify;">
This means Rabenosyn-5 can not be having an effect on the translation of TfR, because then TfR-GFP levels would not have been reduced (since its translation can not have been regulated)- but the TfR-GFP levels <i>are</i> reduced. Put differently, regulation of TfR by Rabenosyn-5 must be post-translational. So how come the TfR-GFP levels be reduced when this can not be caused by translational regulation?</div>
<div style="text-align: justify;">
<b>Possibility of transcriptional regulation.</b> Although there is no experiement discussing this specifically, the question, if TfR levels are regulated at the transcriptional level, is answered by considering that the gene for TfR-GFP does not contain promoter sequences. Therefore it is not possible that Rabenosyn-5 has a transcriptional effect.<br />
<br />
Now, if not at the transcriptional nor translational level, <b>how else can Rabenosyn-5 control TfR levels?</b><br />
The hypothesis is that Rabenosyn-5 acts as a sort of label for how the cell should handle internalized TfR. Once TfR is internalized, it can be directed to the lyosome, i.e. degraded or it can be recycled, that means the TfR is returned to the cell surface and reused. The explanation for the lower levels of TfR in the absence of Rabenosyn-5 is that when Rabenosyn-5 is <i>not</i> present, a larger proportion of TfR gets directed towards the lysosome and subsequently degraded. Evidence for this mechanism is found in that recycling rates are significantly lower in Rabenosyn-5 deactivated cells (Fig. 5D in article).<br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjpO9S87y3U8i1JQOU8SFp2agTTcZEvhjX0rsE-OA3TPqNb7NBdelyQuROF32kZBIc0MMF2lR4FNWMjjvZO7rbaSnkDlWNz6SR7jeFuQ2DcF3wgG3mIKvxgnAGB62liU9llqYhsMRgpHKOC/s1600/fig5d.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="284" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjpO9S87y3U8i1JQOU8SFp2agTTcZEvhjX0rsE-OA3TPqNb7NBdelyQuROF32kZBIc0MMF2lR4FNWMjjvZO7rbaSnkDlWNz6SR7jeFuQ2DcF3wgG3mIKvxgnAGB62liU9llqYhsMRgpHKOC/s1600/fig5d.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Release rates (arbitrary units) of previously internalized Tf depending on time.<br />
http://www.pnas.org/content/109/8/E471/F5.expansion.html</td></tr>
</tbody></table>
Put differently, a lower proportion gets recycled to the cell surface resulting in apparent lower TfR cell surface amount. These mechanistic alternatives are illustrated by the red arrows in the figure below (Fig. 6C in article):<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgjx7H-OkkQRJOQ39-SUpxY-mAKItjIwgdxoGn3wmZQbDkvNSmAzl7bphWsTljq1z3cL7iBfaIE8HwUJ9rdIlXRMwYtPeFIoMRijMuSuyjV5VwlCxPcc8i2OzCxbjcRrMvYOkvnJ6YkhDE8/s1600/fig6c.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="352" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgjx7H-OkkQRJOQ39-SUpxY-mAKItjIwgdxoGn3wmZQbDkvNSmAzl7bphWsTljq1z3cL7iBfaIE8HwUJ9rdIlXRMwYtPeFIoMRijMuSuyjV5VwlCxPcc8i2OzCxbjcRrMvYOkvnJ6YkhDE8/s1600/fig6c.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig. 6C</b>: Alternative mechanisms of TfR sorting in presence and absence of Rabenosyn-5.<br />
Top panel: Rabenosyn-5 labels clathrin coated TfR vesicles for recycling (and reuse of TfR) to cell surface.<br />
Lower panel: Absence of Rabenosyn-5 results in TfR vesicles being directed to lysosome for degrading.<br />
http://www.pnas.org/content/109/8/E471/F6.large.jpg</td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
</tbody></table>
This is verified by comparing TfR levels between cells with activated and deactivated lysosomes (Fig. 6B in article).<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhOtyMG5XgX3Q4VcHZWBLGF7PCqzdXHoo48Qzg9y2T66q-5RReRFR9Thrx5wMIptqIvA30JWB-nTICwih9BD8MhomQ_RSr2TQe926oOKOB7aks_n7dxWFUZXGsAwE6VZSKxY8aeo1Pqjm0I/s1600/fig6b.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhOtyMG5XgX3Q4VcHZWBLGF7PCqzdXHoo48Qzg9y2T66q-5RReRFR9Thrx5wMIptqIvA30JWB-nTICwih9BD8MhomQ_RSr2TQe926oOKOB7aks_n7dxWFUZXGsAwE6VZSKxY8aeo1Pqjm0I/s1600/fig6b.png" width="322" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig. 6B</b>: Baf: Bafilomycin A1. <br />
Sc/Si refer to scrambled and siRNA of Rabenosyn-5.<br />
"+": Baf present, "-": Baf absent<br />
http://www.pnas.org/content/109/8/E471/F6.large.jpg</td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
</tbody></table>
Under the influence of Bafilomycin A1 ("Baf") an inhibitor of lysosome operation, the TfR level is higher than when the lysosome is operational: the TfR level in the column Baf+/Sc is most intensive because it reports the TfR levels on the cell surface as well as those in the disfunctional lysosomes.</div>
<div style="text-align: justify;">
<br />
An additional finding is that the absence of Rabenosyn-5 has an effect on clathrin dynamics. In the absence of Rabenosyn-5, the number of clathrin regions and the size of the clathrin regions are increased. In the absence of Rabenosyn-5, increased clathrin signals are reported in the region of the cell nucleus (Fig. 7C in article).<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgxdW_B7Z6POah1P1HDJ-nhYk6DD6Bsqyp153jtlA8PiBgMpU4sK1zlaSXMRu1OLo4L-ERvBP9AU1RkOoDbh8LzI1N7K0ctO0wzbHsOC947kFpmmj-HG-G0clcReEhrpTFR03VqQ7Scfbur/s1600/fig7c.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="259" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgxdW_B7Z6POah1P1HDJ-nhYk6DD6Bsqyp153jtlA8PiBgMpU4sK1zlaSXMRu1OLo4L-ERvBP9AU1RkOoDbh8LzI1N7K0ctO0wzbHsOC947kFpmmj-HG-G0clcReEhrpTFR03VqQ7Scfbur/s1600/fig7c.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig. 7C</b>: left panels: Rabenosyn-5 present<br />
right panels: Rabenosyn-5 deactivated<br />
http://www.pnas.org/content/109/8/E471/F7.expansion.html</td></tr>
</tbody></table>
However, as shown in Fig. 6C, the total cellular clathrin amount is not affected by the deactivation of Rabenosyn-5. It appears therefore as if the depletion of Rabenosyn-5 results in a redistribution of clathrin, which normally is also present as unassembled clathrin in the cytoplasma, to the cell surface and to the nuclear region. It is therefore reasonable to expect that such a redistribution of clathrin at the nuclear region is only observed because a specific, directing, not yet completely characterized interaction between Rabenosyn-5 and clathrin is <i>missing</i> in the Rabenosyn-5 deactivated cells. The authors contemplate that when Rabenosyn-5 is missing, this <i>results in delayed movement of cathrin coated vesicles away from the plasma membrane or a delay in their uncoating and fusion with endosomes.</i></div>
<div style="text-align: justify;">
<b>Summary.</b> The paper presents evidence for the hypothesis that Rabenosyn-5 <i>prevents</i> TfR from getting directed to the lysosome for degradation. The mechanism of this labeling is however yet to be characterized in detail.<br />
<br />
<b>Editorial.</b> I have contacted the senior author of the paper (Prof. Corvera) to ask if she agrees to calling Rabenosyn-5 a labeling agent as stated above. Prof. Corvera added that in Rabenosyn-5 deficient mice severe perinatal phenotypes are observed. Furthermore, she added that based on this work she was approached by geneticists <i>who have found Rabensyn-5 mutations in humans with severe neurological and developmental delays</i>.</div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-75749158694273804192014-05-13T04:34:00.000-07:002015-10-11T05:31:00.695-07:00Illustrating protein structuresPyMOL can produce really nice protein structures.<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiIgfVRTv_uSqHLnAlI6MxIIiFSeaPFq8wBQRAJxvW23TTTNnENhGSYMBfQqaILjg9ZrrelFRiaV89LbQ6s9SiPwuG2Nt3g-laezOJJu0aKug4fVrGf1j0WKLftmzTG75m7cFk4tLpV6whj/s1600/bcx-constraint-layers-ray-occlusion-2.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="574" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiIgfVRTv_uSqHLnAlI6MxIIiFSeaPFq8wBQRAJxvW23TTTNnENhGSYMBfQqaILjg9ZrrelFRiaV89LbQ6s9SiPwuG2Nt3g-laezOJJu0aKug4fVrGf1j0WKLftmzTG75m7cFk4tLpV6whj/s1600/bcx-constraint-layers-ray-occlusion-2.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Bacillus circulans xylanase with bound substrate (center).</td></tr>
</tbody></table>
This rendering uses ambient occlusion. The imporant settings are<br />
<br />
<span style="font-family: "Courier New",Courier,monospace;">set light_count, 10</span><br />
<span style="font-family: "Courier New",Courier,monospace;">set spec_count, 1</span><br />
<span style="font-family: "Courier New",Courier,monospace;">set shininess, 10</span><br />
<span style="font-family: "Courier New",Courier,monospace;">set sphecular, 0.25</span><br />
<span style="font-family: "Courier New",Courier,monospace;">set ambient, 0</span><br />
<span style="font-family: "Courier New",Courier,monospace;">set direct, 0</span><br />
<span style="font-family: "Courier New",Courier,monospace;">set reflect, 1.5</span><br />
<span style="font-family: "Courier New",Courier,monospace;">set ray_shadow_decay_factor, 0.1</span><br />
<span style="font-family: "Courier New",Courier,monospace;">set ray_shadow_decay_range, 2</span><br />
<span style="font-family: "Courier New",Courier,monospace;">unset depth_cue</span><br />
<br />
What seems a bit tricky to get are the black outlines and depends on the background color setting.<br />
The following command sequence seems to work, assuming the background is white when you start PyMOL.<br />
<br />
1) <span style="font-family: "Courier New",Courier,monospace;">set ray_trace_mode, 1</span> (color + outlines)<br />
2) <span style="font-family: "Courier New",Courier,monospace;">ray</span> (outlines will be white)<br />
3) <span style="font-family: "Courier New",Courier,monospace;">bg black</span><br />
4) <span style="font-family: "Courier New",Courier,monospace;">ray</span> (outlines will be white)<br />
5) <span style="font-family: "Courier New",Courier,monospace;">bg white</span><br />
6) <span style="font-family: "Courier New",Courier,monospace;">ray</span> (outlines should be black)<br />
<br />
This swapping of background is most likely not needed if you want to plot to a black background. I'm using PyMOL 1.3 and it could also be different in other versions.<br />
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If you use these instructions in your work, please be kind enough to credit this blog post. <br />
<br />Unknownnoreply@blogger.com2tag:blogger.com,1999:blog-4907077608573755783.post-86982008429390223642014-04-30T04:13:00.002-07:002016-02-11T12:00:57.077-08:00Literature 002: Niewoehner et al., Neuron, 2014, 81, 1, p49–60<div style="text-align: justify;">
A number of <a href="http://en.wikipedia.org/wiki/Central_nervous_system_disease" target="_blank">disorders</a> affect the central nervous system (CNS) such as inflammations, viral infections, Alzheimer's disease, cancers, Parkinson's disease and many, *many* more. In case of Alzheimer, which currently is not curable, people who suffer from the disease are found to have what is called Amyloid-beta plaques in the brain, aggregates of some kind of protein. These aggregates negatively affect the brain function but can be removed if targeted with special antibodies such as <a href="http://en.wikipedia.org/wiki/Aducanumab" target="_blank">Aducanumab</a>, <a href="http://en.wikipedia.org/wiki/Bapineuzumab" target="_blank">Bapineuzumab</a> or <a href="http://en.wikipedia.org/wiki/Gantenerumab" target="_blank">Gantenerumab</a>, possibly leading to a reversal of the disease.</div>
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The treatment of diseases of the CNS is difficult, because the body protects the CNS from the outside using a special kind of barrier. In the brain, this barrier is called blood-brain-barrier. The barrier in principle consists of special cells (brain-endothelial-cells, BECs/Endothelium in the figure below) which form the blood vessels and only permit certain substances to transpass from the vessel to the brain. For example, antibodies as those mentioned above are not as such allowed to pass the barrier.</div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: 0px; margin-right: 0px; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="http://clincancerres.aacrjournals.org/content/13/6/1663/F3.large.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://clincancerres.aacrjournals.org/content/13/6/1663/F3.large.jpg" height="555" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://clincancerres.aacrjournals.org/content/13/6/1663/F3.large.jpg</td></tr>
</tbody></table>
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In the following, out of the shown possible passways, the focus is on (<b>C</b>), transferrin receptor mediated transcytosis. In this pathway, a substance first binds to the transferrin receptor (Tfr) and is then transported across the cell. This is where the research of <a href="http://www.cell.com/neuron/fulltext/S0896-6273%2813%2901035-0" target="_blank">Niewoehner <i>et al</i>.</a> comes in. In short, they want to find a way of transporting an active substance such as one of the above mentioned antibodies to the brain.</div>
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In "<i>Increased Brain Penetration and Potency ofa Therapeutic Antibody Using a Monovalent Molecular Shuttle</i>", the researchers are able to do this by using a construct which consists of the therapeutically active antibody, a linker peptide and a Tfr active antibody fragment (Tabf). They test two versions of this construct, one with only one Tfr-active fragment, one with two such fragments. The control experiments are done using just the antibody without the linker unit or the Tfr active fragment.</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhj7npbvnFG_s0jQxMx3h7_CbGbxwBPfc2emR00hErwMjKfy1Wc2LBitKFGMfaXKOjGwUlpaC91zVp8poffmIRhmtNoP5o3-A9Eiyp7CHaD6A6r413SBzDXwuu-jWnqio1XX8Wqg9xbaoqM/s1600/shuttles.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhj7npbvnFG_s0jQxMx3h7_CbGbxwBPfc2emR00hErwMjKfy1Wc2LBitKFGMfaXKOjGwUlpaC91zVp8poffmIRhmtNoP5o3-A9Eiyp7CHaD6A6r413SBzDXwuu-jWnqio1XX8Wqg9xbaoqM/s1600/shuttles.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</td></tr>
</tbody></table>
<div class="separator" style="clear: both; text-align: center;">
</div>
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Here, dFab stands for double-Fab and sFab for single-Fab. mAb31 is the monoclonal antibody 31.</div>
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In the following, the series of experiments is addressed by the questions they answer in order to establish a causal chain of reasoning.</div>
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<b>1 Do the active parts of the construct interfere with their respective function?</b></div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjeDX6Em9cHwj5QS0mtU2EZ-FrW9ZNe10tud-AMB1YIR3J4txs5FdvZsU2JeXI-zhsbAyA6Zaur0GH50iJAnXBG7EsdHyxHaTBepCy2BYSWtyeoOwrTS57liGsPzEn7gAYmRxgeRPt6HJj6/s1600/binding.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="294" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjeDX6Em9cHwj5QS0mtU2EZ-FrW9ZNe10tud-AMB1YIR3J4txs5FdvZsU2JeXI-zhsbAyA6Zaur0GH50iJAnXBG7EsdHyxHaTBepCy2BYSWtyeoOwrTS57liGsPzEn7gAYmRxgeRPt6HJj6/s1600/binding.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</td></tr>
</tbody></table>
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In B, the solid and striped bars refer to two different coating densities used in the affinity experiment. As seen, the construct still can bind both ways (Abeta referes to Amyloid beta, the target protein aggregate). Notably, sFab and dFab have very similar binding affinity.</div>
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<b>2 Does the construct enter the BEC?</b></div>
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Using a laser to highlight cells where the construct entered, the number of cells at a given fluorescence intensity are counted.</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhUBHaUfQ1jHcxzSj07mtNZISY6t5IntFtg9WyCECDv5WCulkP9stKu-MV-toZYV8SxJaS07DCLXUxH5zeiNDsuvuFCYdgKFRRRkyOwLEB3YexU8oF1OPaDuYWY2DAlHVbNUsyXTSHju1ei/s1600/counts.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="277" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhUBHaUfQ1jHcxzSj07mtNZISY6t5IntFtg9WyCECDv5WCulkP9stKu-MV-toZYV8SxJaS07DCLXUxH5zeiNDsuvuFCYdgKFRRRkyOwLEB3YexU8oF1OPaDuYWY2DAlHVbNUsyXTSHju1ei/s1600/counts.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/cms/attachment/2012543313/2034747624/gr2.jpg</td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td></tr>
</tbody></table>
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In red the cells with two Tabf's are counted, in green with only one. The black count corresponds to no Tabf. Apparently two Tabf's lead to a slightly better internalization of the construct.</div>
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<b>3 What happens to the constructs (one or two Tabfs) once inside the cell?</b></div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjt3dGW9dlywA5qKHxii9SwJTtZHPtP-ujTTPpe5A-4OqkLMK_wV9nKtAOpAg3ZI5lnPysNRyOYD1bsFktnmE61Vpf9oRDsfVLiAjEN-D4_x_okx7GqQW7GH_YOajs5lxp1UAdCwHMMR915/s1600/internalizd.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjt3dGW9dlywA5qKHxii9SwJTtZHPtP-ujTTPpe5A-4OqkLMK_wV9nKtAOpAg3ZI5lnPysNRyOYD1bsFktnmE61Vpf9oRDsfVLiAjEN-D4_x_okx7GqQW7GH_YOajs5lxp1UAdCwHMMR915/s1600/internalizd.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</td></tr>
</tbody></table>
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One hour after exposure to the construct, it is measured how much of the construct is colocalized with the Lysosome associated membrane protein 2 (Lamp2). This gives an indication of where the construct is found in the cell.</div>
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The BECs have a mechanism to identify what has entered them. It is unclear how the cell identifies the dFab and moves it to the lysosome for digestion.</div>
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<b>4 How can one be sure that the cell does not digest the sFab construct?</b></div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgTlRF1EKGtOW_EPAQE8Ba2Lkf6GVcXj4FxtUV5VBm3tNvLFtgVf4Z4nZ4gDSpFebiGSOuLm5b02AWQYYo2woWQIv83P-HW8F15hq-q0GNSTnUZGJsLv6yuG5E8s5LbAoZ0XWZZBpb3ap6y/s1600/tfrexpression.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgTlRF1EKGtOW_EPAQE8Ba2Lkf6GVcXj4FxtUV5VBm3tNvLFtgVf4Z4nZ4gDSpFebiGSOuLm5b02AWQYYo2woWQIv83P-HW8F15hq-q0GNSTnUZGJsLv6yuG5E8s5LbAoZ0XWZZBpb3ap6y/s1600/tfrexpression.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</td></tr>
</tbody></table>
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Red and pink are two different concentrations of dFab.</div>
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After having entered the cell, the construct has three possible ways to continue: 1) to cross the cell, 2) be digested by the cell and 3) exit the cell where it has entered (see also the first figure).</div>
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In the first case, this would mean that the overall number of receptors on the surface should remain constant and this is observed (green bars). The surface concentration of receptors associated with dFab however decreases over time, meaning they do not reappear at the surface once internalized. After 72 hours, apparently new Tfr's are expressed by the cell.</div>
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<b><br /></b></div>
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<b>5 Do the constructs cross the cell?</b></div>
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An <i>in vitro</i> experiment is designed where a layer of human BECs separates two sections of liquid (the cells in the above experiments were from mice).</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi7DgDiZ8EfquXqe_CT6PjJW6rVvYi2cdUuWUP5Y3crfNoKhBP6tTcH7dszQBIjCRrF3kwHLb9GkRm5nEYv3pOPa4CFMCBszRl5HUipHKq5VrtAYcKR00sSHx4z7g25F7AMQT-2vUdTxWEn/s1600/trans.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi7DgDiZ8EfquXqe_CT6PjJW6rVvYi2cdUuWUP5Y3crfNoKhBP6tTcH7dszQBIjCRrF3kwHLb9GkRm5nEYv3pOPa4CFMCBszRl5HUipHKq5VrtAYcKR00sSHx4z7g25F7AMQT-2vUdTxWEn/s1600/trans.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><u>http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</u></td></tr>
</tbody></table>
<div style="text-align: justify;">
The construct is added to the Lumen compartment (model for the blood vessel inside) and allowed to enter the cells for 1 hour. Then both layers are washed, which controls for constructs entering the abluminal layer by channels inbetween the cells, and amount of construct over time in the two compartments is measured. sFab is observed to appear in both the abluminal and luminal part, while dFab is only found reappearing in the luminal part.</div>
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This is where I get confused.</div>
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The authors note that</div>
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EC50(sFab) = 3.8 nM</div>
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EC50(dFab) = 6.4 nM</div>
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but say "<i>This suggests that the monovalent binding mode to the Tfr, and not just the <b>reduced</b> affinity, is the key factor for efficient transcytosis.</i>" (p. 51) If they are referring to the measured affinities for the construct towards mouse Tfr (see question 1 where dFab has 5.3-8.9 times higher affinity than sFab), then it makes sense. Otherwise I would have said that sFab has higher affinity than dFab based on these numbers.</div>
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One more aspect I find strange:</div>
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</div>
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In my opinion, in (J), the yellow bars should also appear because the way it looks, it seems to indicate that the construct only returns to the lumen, i.e. it exits the cell only in one direction. Why should there be a preference for exiting the cell in only one direction?<br />
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Edit:<br />
After discussing this issue with fellow collegues, the following explanation arose. The cell layer is incubated with sFab/dFab for one hour. Then both the luminal and abluminal compartments are flushed. Any sFab or dFab that appears in either compartment after that has to be coming from within the cells. Since the cargo enters the cell on one side, it has to travel a distance through the cell, but because this is a diffusion process, it has no preference as to what direction it takes. But because the cell has a sorting mechanism, the dFab cargo gets filtered out while it is crossing the cell. That's why no yellow bars appear for dFab. dFab however does also appear in the luminal side, that's because this distance is too short for the cell to sort it out before dFab can exit the cell again on the side it entered. That's also the reason why the luminal bars for sFab are slightly higher than for the abluminal side. The spatial distance within the cell simply requires more time to cross.</div>
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An <i>in vivo</i> image of the vessels after 15 min and 8 h of exposure to the construct shows what happens.</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiE6DCVcqiZpcCgkzrW5Bcy3PHLim5hGK0x-IboU7Lc3LFZebAWtX6McQ9odkq1d6RIazGwJ0nOG2Th6XwqeYlTY-fL8piT_OHeD_r9QhnWknbzLE1RA-NGce8vtj4HuYRlYJvMxmAP9Rc9/s1600/exposure.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiE6DCVcqiZpcCgkzrW5Bcy3PHLim5hGK0x-IboU7Lc3LFZebAWtX6McQ9odkq1d6RIazGwJ0nOG2Th6XwqeYlTY-fL8piT_OHeD_r9QhnWknbzLE1RA-NGce8vtj4HuYRlYJvMxmAP9Rc9/s1600/exposure.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</td></tr>
</tbody></table>
<div style="text-align: justify;">
In the beginning, both constructs occupy the vessel cells (A, B). After 8 hours though, only sFab has crossed the vessel cells, i.e. they are not illuminating any longer (C), dFab has remained in the cells (D). In (C), the arrows point to accumulations of the construct on Amyloid-beta plaque aggregates.</div>
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<div style="text-align: justify;">
<b>6 Does the observed difference in BEC crossing between sFab and dFab depend on the rate at which they enter the BECs?</b></div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhE8rlCQehG2e8mESRNvCkNv5BstlTlbfBrpXLXf1F-idRfpEGvKzN7VV5QfLhVpOHRKec6KDWegbaKeHafZeoMtDKXpGB5CD_cWWiLf5jiQmF72Dew-TDHMh1JZHJwAYo733NmFyByqHcF/s1600/rates.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="359" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhE8rlCQehG2e8mESRNvCkNv5BstlTlbfBrpXLXf1F-idRfpEGvKzN7VV5QfLhVpOHRKec6KDWegbaKeHafZeoMtDKXpGB5CD_cWWiLf5jiQmF72Dew-TDHMh1JZHJwAYo733NmFyByqHcF/s1600/rates.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</td></tr>
</tbody></table>
<div style="text-align: justify;">
No.</div>
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<div style="text-align: justify;">
<b>7 Which construct binds more to Amyloid-beta plaque?</b></div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEieljl-KPt4IPNDRE3-PGobSVTASPVZqZgap9jV0zsuSKQXVhf9RgaGRfc5Kbra8y6teQ8c_EAAZ5dYdRUnnQ43qLykTgXPACR92Sevv0tVSizY6VvOJSm75PiEB1w2bmGIFknVHKwSzhnD/s1600/plaquebinding.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEieljl-KPt4IPNDRE3-PGobSVTASPVZqZgap9jV0zsuSKQXVhf9RgaGRfc5Kbra8y6teQ8c_EAAZ5dYdRUnnQ43qLykTgXPACR92Sevv0tVSizY6VvOJSm75PiEB1w2bmGIFknVHKwSzhnD/s1600/plaquebinding.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</td></tr>
</tbody></table>
<div style="text-align: justify;">
The measured increased plaque binding by sFab is therefore causal attributed to the fact that sFab binds to Tfr only by one Tabf.</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
<b>8 Does sFab cover plaque faster than only mAb31 even at lower concentration?</b></div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh_F3NNwJQppwyEtUcOHHTnqHgtOYQpLqNkyDOaFI5ROBYHEuCPdPp4m-CJUtGttpwk4d9GXpjnQ_wodAx-HEO9tykyr9Dasy4BHjlNIsN_2RD1LDFz26ydHlVnJVbtxG3BY4JiMBSnsnAQ/s1600/exposuretime.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh_F3NNwJQppwyEtUcOHHTnqHgtOYQpLqNkyDOaFI5ROBYHEuCPdPp4m-CJUtGttpwk4d9GXpjnQ_wodAx-HEO9tykyr9Dasy4BHjlNIsN_2RD1LDFz26ydHlVnJVbtxG3BY4JiMBSnsnAQ/s1600/exposuretime.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0</td></tr>
</tbody></table>
<div style="text-align: justify;">
</div>
<div style="text-align: justify;">
Yes. The concentration of the antibody alone (light blue is 5 times higher than green) when not having a Tabf attached does not provide any activity. Duration of exposure does not change this, in other words the blood-brain-barrier remains closed no matter how high the concentration.</div>
<div style="text-align: justify;">
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<div style="text-align: justify;">
<b>9 Is there a therapeutic effect of the construct?</b></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjletBMEWlIrJ4oeV6dEleej1o8FJotl8OyaJUc5MkNHL5CmBFxtRaxoj_Cf60boSyh_NxmTs_ZGxRMFmpwaJLPxIWvO931VzS0jRLyByOp88nhlSGSFhs8qAf1P3vQn4S6f3lF41B-pUvE/s1600/ther.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjletBMEWlIrJ4oeV6dEleej1o8FJotl8OyaJUc5MkNHL5CmBFxtRaxoj_Cf60boSyh_NxmTs_ZGxRMFmpwaJLPxIWvO931VzS0jRLyByOp88nhlSGSFhs8qAf1P3vQn4S6f3lF41B-pUvE/s1600/ther.png" /></a></div>
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Yes. The baseline shows the plaque number when the mice are 4.5 months old. Then, during 14 weeks, only the linker and the Tabf (vehicle), only the therapeutic antibody (mAb31) and the sFab are injected.</div>
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The plaque concentration increases in all mice, but when treated with sFab it increases at a lower rate.</div>
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<b>10 Remarks</b></div>
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I would like to better understand the experiment from question 5. I<b> </b> would expect that the next thing to do is to study dependence of therapeutic effect on amount of construct administered. What are the elimination rates of the construct? Interferences with other mechanisms? How stable is the construct? What is the exact nature of the cell sorting mechanism?<br />
Also, in case you're interested, you can find the patent for this vehicle is WO 2014/033074. </div>
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<br />
A number of questions were raised during the discussion:<br />
- If the cell sorting mechanism could be explained, maybe also dFab could be used as a carrier. It has some benefitial characteristics over sFab. Is this possible?<br />
- If I was to manage this project, what would I do if it turns out that the shuttle is not linked strongly enough to the cargo, meaning it lets it's cargo go and picks up something, possibly toxic, else and carries it to the brain. I would say this shuttle is too important and therefore the engineering efforts have to be directed towards making the shuttle more strongly attached to the cargo.<br />
- One short-coming of the study, in order to prove that it is not dependent on the type of cargo, is that no other cargo was used. This would have demonstrated independence of cargo nature. Of course, this would require a major additional effort. </div>
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Some out of context notes.</div>
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I was inspired to do this summary because I wanted to see how well I can understand and communicate a study from a totally different field. I believe it's important to be open to other fields and especially to be able to understand experimental approaches. Initially, the biology specific terms were difficult to understand but after a while I think I could identify the more important aspects.</div>
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Also, it seems sometimes that because of all the pharma-bashing, one doesn't notice that these guys actually do really amazing work.</div>
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Disclaimer: I have nothing to do with this research</div>
Unknownnoreply@blogger.com2tag:blogger.com,1999:blog-4907077608573755783.post-7877703278754474632014-04-28T06:41:00.000-07:002014-05-13T06:44:44.499-07:00Coursera Certificate<div class="separator" style="clear: both; text-align: center;">
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My first coursera certificate. Lot's of work, but totally worth the effort.<img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiugkH044Gj5eC7e6QrCaBZ4mHsEDeAsqrSqxP1_wx-yb8JGMWmxQO8_mmmR4F81w9_TvAEnW8hhJzK4xmcyGYBPcTzR72YCBBKWklJQI4R2C7iHJqo0bT_e3AtRpVtb8idfdWndfD9NEsS/s1600/certificate_stat_mol_thermo.png" height="494" width="640" /></div>
<br />Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-8759690985300782122014-03-18T04:09:00.002-07:002016-02-11T12:02:00.906-08:00Papers and ThesisCalB Paper 1 <i>PLOS ONE</i>:<br />
<a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0049849" target="_blank">http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0049849</a><br />
<br />
CalB Paper 2 <i>PEERJ</i>:<br />
<a href="https://peerj.com/articles/145/" target="_blank">https://peerj.com/articles/145/</a><br />
<br />
BCX Paper <i>PEERJ</i>:<br />
<a href="https://peerj.com/articles/111/" target="_blank">https://peerj.com/articles/111/</a><br />
<br />
BIOFET-SIM Online Paper <i>PLOS ONE</i>:<br />
<a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0045379" target="_blank">http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0045379</a><br />
<br />
BIOFET-SIM Online Chapter <i>Nanoscale Sensors</i>:<br />
<a href="http://link.springer.com/chapter/10.1007/978-3-319-02772-2_3" target="_blank">http://link.springer.com/chapter/10.1007/978-3-319-02772-2_3</a><br />
<br />
PhD Thesis:<br />
<a href="http://www.scribd.com/doc/213075474/MH-Thesis" target="_blank">http://www.scribd.com/doc/213075474/MH-Thesis</a><br />
<br />
PhD Defence Slides:<br />
<a href="http://www.scribd.com/doc/213105271/Presentation-Defence" target="_blank">http://www.scribd.com/doc/213105271/Presentation-Defence</a>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-6787339881362693802013-12-13T15:59:00.000-08:002013-12-16T02:55:08.049-08:00Chem 001: Nernst Equation<div style="text-align: justify;">
Consider a galvanic cell composed of two \(\text{H}^{+}\left|\right.\text{H}_2\)-half cells where \( \left[\text{H}^+\right] \) are \(0.025 \text{M} \) and \( 5 \text{M} \) in the first and second half-cell, respectively. Calculate the electromotive force (EMF).</div>
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It's good practice to first visualize such a problem before attempting to solve it in order to gain a clear understanding of the system. The galvanic cell could look like this:</div>
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<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiHksoDedPEWlrfhuMk5Dkvx55SxWb7MWWGfJR5IbJKQI5Njq3ENq84psq_9jaLQYF-aZlJr4GzX5_OEfs81cZiSQv2gps3LpQ2vV8mrCT2kNqMd6B2YEAeL15key1bUhYpjKRzQB7q_0HJ/s1600/hydrogen-cell.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="272" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiHksoDedPEWlrfhuMk5Dkvx55SxWb7MWWGfJR5IbJKQI5Njq3ENq84psq_9jaLQYF-aZlJr4GzX5_OEfs81cZiSQv2gps3LpQ2vV8mrCT2kNqMd6B2YEAeL15key1bUhYpjKRzQB7q_0HJ/s320/hydrogen-cell.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Concentration chain of two hydrogen electrodes. The blue arc indicates the salt-bridge.<br />The hydrogen pressures do not need to be the same (see text).</td></tr>
</tbody></table>
</div>
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A galvanic cell with two times the same system at different electrolyte concentration is refered to as <i>concentration chain</i>. At the platinum electrode, the hydrogen gas is oxidized and \(\text{H}^+\) ions enter into solution. A thermodynamic equilibrium is reached when \( \left[\text{H}^+\right] \) is equal in both cells.</div>
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We can recognize that in electrode 1, \( \left[\text{H}^+\right] \) is lower and so it is in electrode 1 where hydrogen is oxidized. By convention, the electrode where oxidation occurs is referred to as <i>anode</i>, the one where reduction occurs as <i>kathode</i>. The electrons thus exit the cell at electrode 1, run through the wire to electrode 2 where they enter the cell again and reduce hydrogen ions. Electrode 1 is therefore referred to as the <i>minus pole</i> of the cell ("where the electrons come from"). At the anode (electrode 1), the half-reaction is</div>
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\(\text{H}_2\text{(g)} \rightarrow 2\text{H}^+\text{(aq)} + 2\text{e}^-\)</div>
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In order to calculate the EMF, it is required to calculate the potential difference between both electrodes 1 and 2 compared to the standard hydrogen electrode. The EMF for the cell is then calculated as the sum of <i>reduction</i> potentials</div>
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\( \Delta E = E(\text{Kath.}) + E(\text{An.}) \)</div>
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and this is where we have to be careful to choose the sign correspondingly to the reaction we are formulating.</div>
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For the reaction at the kathode (where reduction of \(\text{H}^+\text{(aq)}\) occurs), the potential (compared to the normal hydrogen electrode) is given by the Nernst equation</div>
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\( E\text{(Kath.)} = E^0 - \frac{0.06}{2}\log{Q} \)</div>
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The \(2\) in the fraction is the number of transported electrons per reduction equivalent. The \( (-) \)-sign in front of the log-expression is used when the reaction quotient \( Q \) corresponds to a reaction formulated in <i>direction of reduction:</i></div>
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\( 2\text{H}^+ \text{(aq)} + 2\text{e}^- \rightarrow \text{H}_2 \text{(g)} \)</div>
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For the general reaction</div>
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\( k\cdot K + l\cdot L \rightarrow x\cdot X + y\cdot Y \)</div>
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the reaction quotient is always defined as</div>
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\( Q = \frac{a^x(X) \cdot a^y(Y)}{a^k(K) \cdot a^l(L)} \),</div>
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where the \(a^i\)'s are activities of the species. Here, we understand the hydrogen gas has unit activity, \(a(\text{H}_2\text{(g)}) = 1\), and so </div>
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\( E\text{(Kath.)} = E^0 - \frac{0.06}{2}\log \frac{1}{ \left[ \text{H}^+ \right] ^2 } \)</div>
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where \( \left[\text{H}^+\right] = 5 \text{M} \) and the exponent \(2\) is the stoichiometric coefficient. We emphasize again the minus sign in front of the \( \log \)-term used when the reaction is formulated in direction of reduction. \(E^0\) is the electrode potential for the normal-hydrogen electrode which is zero by definition.</div>
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A similar equation is formulated for the anode half-reaction:</div>
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\( E\text{(An.)} = E^0 + \frac{0.06}{2}\log \frac{\left[ \text{H}^+ \right]^2}{1} \)</div>
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however here a \((+)\)-sign is used in front of the \(\log\)-term because the reaction is formulated in direction of oxidation (so the oxidized species \(\left[H^+\right]\) appears in the numerator) and \( \left[\text{H}^+\right] = 0.025 \text{M} \).</div>
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The numeric values are \(E(\text{Kath.}) = +0.041 V\) and \(E(\text{An.}) = -0.096 V\). \(E(\text{An.})\) however corresponds to an <i>oxidation</i> potential and so in order to calculate the EMF, we need to take its negative (such that we are adding reduction potentials). This then provides us with</div>
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\( EMF = \Delta E(\text{Kath.}) + {}^-\Delta E(\text{An.}) = 0.041 + {}^-(-0.096) = 0.137 [V] \)</div>
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The question can now be extended by asking what the EMF will be, when the hydrogen gas pressures are different from atmospheric pressure (and so the activity \(a(\text{H}_2)\) is no longer unity):</div>
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Assume \(p(\text{H}_2) = 202.6 \text{kPa}\) at the anode and \(p(\text{H}_2) = 10.13\text{kPa}\) at the kathode.</div>
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Given these pressures, the activities are \(a(\text{H}_2, \text{Kath.}) = p(\text{H}_2)/101.3 \text{kPa} = 0.1 \) and \(a(\text{H}_2, \text{An.}) = p(\text{H}_2)/101.3 \text{kPa} = 2 \). By substituting these values in the Nernst equations for the respective half-cells (using the same concentrations for \(\left[H^+\right]\)), we arrive at</div>
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\( E\text{(Kath.)} = E^0 - \frac{0.06}{2}\log \frac{0.1}{ \left[ \text{H}^+ \right] ^2 } = 0.072 \left[V\right]\)</div>
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and</div>
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\( E\text{(An.)} = E^0 + \frac{0.06}{2}\log \frac{\left[ \text{H}^+ \right]^2}{2} = -0.105 \left[V\right] .\)</div>
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The EMF in this cell is therefore (after again switching the sign of the anode potential to obtain a <i>reduction</i> potential)</div>
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\( EMF = \Delta E(\text{Kath.}) + {}^-\Delta E(\text{An.}) = 0.071 + {}^-(-0.105) = 0.177 [V] .\)</div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-14552376626992504422013-12-04T03:22:00.000-08:002016-02-11T12:01:26.484-08:00Literature 001: Alexandrova et al., JACS, 2008, 130, 15907-15915In "<a href="http://pubs.acs.org/doi/abs/10.1021/ja804040s">Catalytic Mechanism and Performance of Computationally Designed Enzymes for Kemp Elimination</a>", Alexandrova et al. report calculated activities of enzymes that were previously prepared by the Baker group. The experimentally expressed enzymes are called KE07, KE10 (which contains the V131N mutation) and KE15.<br />
To recall, the Kemp elimination is this reaction:<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjZXNrfutNjqahkPEN0pMdaY6Vjh3VQFcrZzsAWfTichhc0zJ7eMLgjw5amtw_hWX7NC1zhER8rRG4ShBsUXqg-JOpWiIYxh1epOr0azSDJgr9hVCYK2rCvcQS79GNIXgWB7Q8EP0ztV1Ij/s1600/Screen+shot+2013-12-04+at+Mittwoch,+4.+Dezember+201311.07.34.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="160" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjZXNrfutNjqahkPEN0pMdaY6Vjh3VQFcrZzsAWfTichhc0zJ7eMLgjw5amtw_hWX7NC1zhER8rRG4ShBsUXqg-JOpWiIYxh1epOr0azSDJgr9hVCYK2rCvcQS79GNIXgWB7Q8EP0ztV1Ij/s400/Screen+shot+2013-12-04+at+Mittwoch,+4.+Dezember+201311.07.34.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">http://pubs.acs.org/doi/abs/10.1021/ja804040s</td></tr>
</tbody></table>
From semiempirical QM/MM calculations and transition state theory, the authors obtain \(k_{cat}\) or \(\Delta G^\ddagger\) which they compare to experiment.<br />
For example, for KE07 the authors report a calculated \(\Delta G^\ddagger\) of 8.1 kcal/mol while the measured activation free energy is 17.1 kcal/mol. That appears as quite a discrepancy between predicted and measured activation energy and one might wonder how come this made it into JACS (see below). Also for KE15, the calculated activation energy doesn't really accurately predict the measured activation energy (calc. / exp. [kcal/mol] \(\Delta G^\ddagger\): 12.3 / 17.0). For KE10, \(k_{cat}\) wasn't measured.<br />
<br />
However, what the authors claim is that they are not that much interested in exactly matching the measured activation energy (most likely they would use a more elaborate QM method), but much more if they can predict if the enzyme actually contributes to catalysis. I.e. they're interested in the ratio \(k_{cat}/k_{uncat}\) where \(k_{uncat}\) is the rate constant for the reaction in solvent using \(\text{OH}^-\) as base. And apparently they are able to correctly predict if this ratio is larger or smaller than 1 for all enzymes:<br />
<br />
\(k_{cat}^{KE07}/k_{uncat} = 1.6 \cdot 10^4\)<br />
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\(k_{cat}^{KE10}/k_{uncat}\) < 3.3 (assuming \(K_M\) to be comparable or smaller than for KE07)<br />
<br />
\(k_{cat}^{KE15}/k_{uncat} = 1.9 \cdot 10^4\)<br />
<br />
\(k_{cat}^{KE16}/k_{uncat} = 5.2 \cdot 10^3\) (by 2-step mechanism and requiring to impose the protonation state of a catalytic D48 residue - why do they not estimate the \(pK_a\) value of the residue?)<br />
<br />
For all enzymes, catalytic activity is observed, so qualitatively they are able to tell if an enzyme will be active or not (they calculate the uncatalyzed reaction to have an activation energy of 19.8 kcal/mol and the activation energies of all enzymes is calculated to be lower than that).<br />
Possible explanation for discrepancies provided: no backbone sampling, accuracy of SE QM (PDDG/PM3) - even if they claim this method to be suited for elimination reactions, no polarization in MM field.<br />
<br />
Another question can be raised: how can they be sure about their prediction if they don't consider that \(K_M\) might be different for all enzymes. So even if an enzyme might be able to catalyze the reaction, it could in principle be that it hardly binds the substrate. The way to answer this question these days is to say "<i>The catalytic efficiency, \(k_{cat}/K_M\), has not been computed owing to the technical challenges for \(K_M\), which requires computation of the absolute free energy of binding for the substrate.</i>" The substrate does not dissociate from the active site in the equilibration and so the working assumption is to believe that all enzymes can bind the substrate and do so comparably well.<br />
<br />
My impression: The paper provides elaborate discussions on observed mechanisms and explains them in minute (structural) detail. The weak points, which are very explicitly disclosed, are compensated by pointing out possible reasons and by the careful mechanistic analysis. A strong point is that the results provide suggestions for how to further improve the catalytic activity (i.e. increasing basicity of the catalytic base, optimization of substrate orientation and positioning of hydrogen bond donors should help increase activity). Furthermore, in the mindset that agreement with experimental activation energy is the key property to consider, the paper would not be considered good. But, the point is really to provide a method that allows to qualitatively tell if the enzyme is active or not, and it does so very well I think.Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-4907077608573755783.post-47318095111668695712013-03-10T12:53:00.002-07:002013-03-10T13:10:59.407-07:00QM 006: INDO One-electron IntegralsIn Frank, Computational Chemistry, Eq. 3.86 is<br />
$$ \langle \mu_A \left| \bf{h} \right| \mu_A \rangle = \left< \mu_A \left| -\frac{1}{2}\nabla^2 - \bf{V}_a \right| \mu_A \right> - \sum_{a \neq A}^{Nuclei} \langle \mu_A \left| \bf{V}_a \right| \mu_A \rangle $$.<br />
<br />
Is there a reason for not writing it as<br />
$$ \langle \mu_A \left| \bf{h} \right| \mu_A \rangle = \left< \mu_A \left| -\frac{1}{2}\nabla^2 \right| \mu_A \right> - \sum_{a = A}^{Nuclei} \langle \mu_A \left| \bf{V}_a \right| \mu_A \rangle $$?Unknownnoreply@blogger.com1tag:blogger.com,1999:blog-4907077608573755783.post-71428185452929515822013-02-26T02:05:00.002-08:002013-02-28T02:27:00.162-08:00QM 005: Comparing semi-empirical methodsIn a recent <a href="http://www.pnas.org/content/107/52/22523.full?sid=70c5afdd-b6d0-414a-8d3b-89bcd7c76cd0" target="_blank">paper</a> Liao <i>et al.</i> calculated the reaction profile for a newly suggested mechanism of acetylene hydratase. Conveniently, the atomic coordinates of all species along the reaction coordinate have been deposited in the supporting material.<br />
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Carrying out some SPE calculations of all species using MOPAC2012, we find the following profiles.</div>
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEguGPDSvzgwI003I4zT9Du69MtmK3A3u9gw9ZgYhIhlfNGH0yx7_W593JAVOJdhVBHbRYRtAa1YPCwvlJwAy30bKRtWJdIMe6Hve3OK-_FcT71UgcwcyyCNU-s5fF3niKoYFnc1iBMfUCX1/s1600/data.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="448" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEguGPDSvzgwI003I4zT9Du69MtmK3A3u9gw9ZgYhIhlfNGH0yx7_W593JAVOJdhVBHbRYRtAa1YPCwvlJwAy30bKRtWJdIMe6Hve3OK-_FcT71UgcwcyyCNU-s5fF3niKoYFnc1iBMfUCX1/s640/data.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Fig. 1: Calculated Reaction profiles.</td></tr>
</tbody></table>
<div>
In the SPE calculations, a dielectric constant of \(\epsilon=4\) was applied. In the reference ("B3LYP" in Fig. 1), the optimization was done using the LANL2TZ(f) pseudo-potential on W, 6-311+G(d) for S and 6-31G(d,p) for the other elements. The reference SPE calculations were carried out using 6-311+G(2d,2p) on all elements but W.</div>
<div>
Apparently, the semi-empirical methods greatly overestimate the interaction energy between W and the acetylene unit (going from 0 to 1) and underestimate the activation energy for the proton transfer in step 4.<br />
However, without reoptimizing the transition state, PM6 is does see a very clear transition state for the hydroxylation at step 2.<br />
<br />
Orbital diagram from supplementary material.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhX43gQWvAnS9mHUeSHNmUshvaDYu7rrBGdvZacNjJ6Rmnn3baDBMdAZGYQ23_ZloKAIDuJXWPmN-yJfxwhE5VVKxMeRYuoHVPDxSPb6hR1TBYCIiVa5uXXyDLtvj4mtMY_QtjDTrafbS2Z/s1600/orbitals.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="396" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhX43gQWvAnS9mHUeSHNmUshvaDYu7rrBGdvZacNjJ6Rmnn3baDBMdAZGYQ23_ZloKAIDuJXWPmN-yJfxwhE5VVKxMeRYuoHVPDxSPb6hR1TBYCIiVa5uXXyDLtvj4mtMY_QtjDTrafbS2Z/s400/orbitals.png" width="400" /></a></div>
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Unknownnoreply@blogger.com2tag:blogger.com,1999:blog-4907077608573755783.post-70145950628670456752013-01-16T01:06:00.000-08:002013-01-16T01:07:08.806-08:00PyMOL 007: New Feature<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">I recently wrote a mail to the PyMOL user mailing-list asking if PyMOL can write vector graphics based files of a session (similar to Molscript). Vector graphics are scalable, require less disc space (good for journal uploads) and look beautiful, allowing to focus attention on details that matter.</span><br />
<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">Here's the mail I wrote:</span><br />
<br />
<pre wrap=""></pre>
<pre wrap="">Hi PyMOL users
Can PyMOL write a vector based picture of a session? Does not require to
be very "fancy", but vector based would be cool. Something like in the
old days with molscript.
Best regards
Martin</pre>
<pre wrap=""></pre>
<pre wrap=""><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">In the following, a number of people responded suggesting SVG or EPS/PDF as file format. Within three days, Jason Vertrees of Schrödinger started a poll to check which format was most preferred by the users. Find it here:</span></pre>
<pre wrap=""><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">
</span></pre>
<pre wrap=""><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;"><a href="http://pymol.org/vector_poll" target="_blank">http://pymol.org/vector_poll</a></span></pre>
<pre wrap=""><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">
</span></pre>
<pre wrap=""><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">It looks as if this really is a feature that could be appreciated and it would be great if it was implemented.</span></pre>
<pre wrap=""><span class="Apple-style-span" style="font-family: Arial, Helvetica, sans-serif;">I guess there will be some management involved in the decision making, but in any case its another example of the great support by the PyMOL developers at Schrödinger. And sometimes this deserves mentioning.</span></pre>
Unknownnoreply@blogger.com1tag:blogger.com,1999:blog-4907077608573755783.post-21613042897095534152012-12-08T11:39:00.000-08:002012-12-08T11:45:26.399-08:00QM 004: Two-electron integral manipulationIn the independent-particle model, the electronic energy of an \(N\)-electron system is frequently written as<br />
\begin{equation}E = \sum_{i}^{N} h_i + 1/2 \sum_{i, j} J_{ij} - K_{ij}.\label{eq:energy}\end{equation}<br />
When inserting the expressions for the Coulomb and exchange operators, \(J\) and \(K\), and calculating the variation in energy, \(\delta E \), given by (in physicists notation)<br />
\begin{equation}\delta E = \sum_i \langle \delta \phi_i | h_i | \phi_i \rangle + \langle \phi_i | h_i | \delta \phi_i \rangle + 1/2 \sum_{i, j} \left[ \langle \delta \phi_i \phi_j | \phi_i \phi_j \rangle + \langle \phi_i \delta \phi_j | \phi_i \phi_j \rangle + \phi_i \phi_j | \delta \phi_i \phi_j \rangle + \langle \phi_i \phi_j | \phi_i \delta \phi_j \rangle \\<br />
- \langle\delta\phi_i\phi_j | \phi_j\phi_i\rangle - \langle\phi_i\delta\phi_j | \phi_j\phi_i\rangle - \langle\phi_i\phi_j | \delta\phi_j\phi_i\rangle - \langle\phi_i\phi_j | \phi_j\delta\phi_i\rangle\right], \label{eq:variation}\end{equation}<br />
it can be found that four pairs of terms in the second sum-expression are equal and thus the factor of \(1/2\) can be cancelled.<br />
For the Coulomb integrals this can be done by considering the definition of the bracket notation<br />
$$ \langle \delta\phi_i(1)\phi_j(2) | \phi_i(1) \phi_j(2)\rangle = \int d{\bf x}_1 d{\bf x}_2 \delta\chi_i^*(1)\chi_j^*(2){\bf r}_{12}^{-1} \chi_i(1)\chi_j(2)$$<br />
and noting that when the orbitals are understood to be real, i.e. \(\phi^* = \phi\), and the ordering of electron labels is considered, the orbitals of an electron \(m \in \{1, 2 \}\) can be swapped such that<br />
$$ \int d{\bf x}_1 d{\bf x}_2 \delta\chi_i(1)\chi_j(2){\bf r}_{12}^{-1} \chi_i(1)\chi_j(2) = \int d{\bf x}_1 d{\bf x}_2 \chi_i(1)\chi_j(2){\bf r}_{12}^{-1} \delta\chi_i(1)\chi_j(2) $$<br />
and thus, in bracket notation,<br />
\begin{equation} \langle \delta\phi_i\phi_j | \phi_i \phi_j \rangle = \langle\phi_i\phi_j | \delta\phi_i \phi_j \rangle \label{eq:manipulation_coulomb}\end{equation}<br />
which we recognize as the the first and third term in the second sum of Eq. \ref{eq:variation}.<br />
For the exchange integrals, a similar expression exists (Szabo, Ostlund, Eq. 2.94)<br />
$$\langle ij | kl \rangle = \langle ji | lk \rangle .$$<br />
To show this, we write the integral explicitly and after first exchanging the dummy variables \(1\), \(2\) and then reordering the orbitals in order to restore the conventional \(1, 2\) order of electrons (keeping "the orbital on the electron"), we obtain<br />
\begin{equation} \int d{\bf x}_1 d{\bf x}_2 \chi_i^*(1)\chi_j^*(2){\bf r}_{12}^{-1} \chi_k(1)\chi_l(2) = \int d{\bf x}_1 d{\bf x}_2 \chi_j^*(1)\chi_i^*(2){\bf r}_{12}^{-1} \chi_l(1)\chi_k(2). \label{eq:manipulation_exchange}\end{equation}<br />
Using this approach<br />
\begin{equation} \langle \delta\phi_i(1)\phi_j(2) | \phi_j(1)\phi_i(2) \rangle = \langle \phi_j(1)\delta\phi_i(2)|\phi_i(1)\phi_j(2) \rangle = \langle \phi_i(1)\phi_j(2)|\phi_j(1)\delta\phi_i(2) \rangle \label{eq:exchange}\end{equation}<br />
where Eq. \ref{eq:manipulation_exchange} was used to obtain the first equality and Eq. \ref{eq:manipulation_coulomb} was used to swap \(\phi_j(1)\) with \(\phi_i(1)\) and \(\delta\phi_i(2)\) with \(\phi_j(2)\) to obtain the second equality.<br />
The first and last integral are recognized as the first and fourth exchange integral of Eq. \ref{eq:variation}.<br />
Applying these operations on the remaining integrals allows to factor out a factor of \(2\) which then cancels with the \(1/2\).Unknownnoreply@blogger.com0