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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Description#background">Background</a></h5> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Achievements">Achievements</a></h5> |
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− | <h4><b>Cataracts</b> - the leading cause of blindness. Find out how we can | + | <h4><b>Cataracts</b> - the leading cause of blindness. Find out how we can noninvasively <b>treat</b> and <b>prevent</b> cataract formation.</b></h4> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Experiments#lensmodel">Lens Cataract Model</a></h5> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Experiments#construct">Prevention and Treatment Constructs </a></h5> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Experiments#prototype">Delivery Prototype</a></h5> |
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− | <h4>We | + | <h4>We don't just come up with great ideas. We show they work. Follow along our discovery of exciting science!</h4> |
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− | <a href="https://2016.igem.org/Team:TAS_Taipei/Modeling"><h4 class='dropdown-toggle disabled' data-toggle="dropdown"><b> | + | <a href="https://2016.igem.org/Team:TAS_Taipei/Modeling"><h4 class='dropdown-toggle disabled' data-toggle="dropdown"><b>MODEL</b></h4></a> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Model#crystallin">Cataract Damage</a></h5> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Model#gsr25hc">GSR/25HC Pathway</a></h5> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Model#nanoparticle">Nanoparticle Degradation</a></h5> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Model#eyedrop">Eyedrop Model</a></h5> |
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− | <h4><b>Computational Biology</b> provides us models that we cannot easily test. Click to | + | <h4><b>Computational Biology</b> provides us models that we cannot easily test. Click to find out the results of our modeling, and if you want, the math behind it!</h4> |
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− | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/ | + | <h5><a href="https://2016.igem.org/Team:TAS_Taipei/Team#members">Members</a></h5> |
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− | <h4>Behind every tough iGem project lies a | + | <h4>Behind every tough iGem project lies a hard-working yet cheerful group of students. <b>Meet the team!</b></h4> |
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<p>We answer two questions: How much GSR to maintain in the lens, and how to maintain that amount? We find the amount of GSR needed in the lens (Model 2) to limit crystallin damage so the resulting cataract is less than LOCS 2.5 (Model 1). Then, we find the optimal design of eyedrops (Model 4) and nanoparticles that will maintain this amount of GSR in the lens (Model 3). These models allow our team to understand the impact of adding GSR-loaded nanoparticles into the lens, and to design a full treatment plan on how to prevent and treat cataracts. </p> | <p>We answer two questions: How much GSR to maintain in the lens, and how to maintain that amount? We find the amount of GSR needed in the lens (Model 2) to limit crystallin damage so the resulting cataract is less than LOCS 2.5 (Model 1). Then, we find the optimal design of eyedrops (Model 4) and nanoparticles that will maintain this amount of GSR in the lens (Model 3). These models allow our team to understand the impact of adding GSR-loaded nanoparticles into the lens, and to design a full treatment plan on how to prevent and treat cataracts. </p> | ||
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+ | <li>Designed a simple calculator to find amount of GSR or 25HC eyedrops needed for a patient's LOCS score.</li> | ||
<li>Bridged the gap between the medical, biological, and chemical measurement of crystallin damage.</li> | <li>Bridged the gap between the medical, biological, and chemical measurement of crystallin damage.</li> | ||
<li>Predicted impact of adding GSR and 25HC on the amount of crystallin damage in the lens.</li> | <li>Predicted impact of adding GSR and 25HC on the amount of crystallin damage in the lens.</li> | ||
− | <li>Created Nanoparticle Customizer for | + | <li>Created Nanoparticle Customizer for doctors to find a full treatment plan.</li> |
− | <li>Generalized Customizer to allow other iGEM teams to | + | <li>Generalized our Customizer to allow other iGEM teams who wish to use nanoparticle drug delivery </li> |
− | <li>Analyzed sensitivity of prototype, and suggested insights into optimal manufacturing of prototype.</li> | + | <li>Analyzed sensitivity of prototype, and suggested insights into optimal manufacturing and clinical use of our prototype.</li> |
− | <li>Experimental data | + | <li>Used Experimental data to develop Models 1 and 3.</li> |
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<h2 id = 'crystallin'>Model 1: Crystallin Damage</h2> | <h2 id = 'crystallin'>Model 1: Crystallin Damage</h2> | ||
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There are four ways of measuring cataract severity, each used for a different purpose. | There are four ways of measuring cataract severity, each used for a different purpose. | ||
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<li><b>Opacity (%):</b> This is the physical, quantitative property of the LOC scale.</li> | <li><b>Opacity (%):</b> This is the physical, quantitative property of the LOC scale.</li> | ||
<li><b>Absorbance at 397.5 nm:</b> This is the experimental method, used by our team in the lab (c.d.).</li> | <li><b>Absorbance at 397.5 nm:</b> This is the experimental method, used by our team in the lab (c.d.).</li> | ||
+ | <li><b>Crystallin Damage: </b>This is a chemical definition. We quantify cataract severity as a function of how much oxidizing agents there are, as well as how long crystalline is exposed to oxidizing agents. We define 1 crystallin damage unit as the damage done to human crystallin when exposed to 1 M hydrogen peroxide, the main oxidizing agent, for 1 hour.</li> | ||
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<p>We use the unit of crystallin damage to connect cataract severity with the amount of GSR we add (in Model 2). We want to lower c.d. below so that the resulting cataract is of LOCS 2.5. For the rest of the model, our task is simple: relate each point of the LOCS scale to c.d., in order to connect to Model 2.</p> | <p>We use the unit of crystallin damage to connect cataract severity with the amount of GSR we add (in Model 2). We want to lower c.d. below so that the resulting cataract is of LOCS 2.5. For the rest of the model, our task is simple: relate each point of the LOCS scale to c.d., in order to connect to Model 2.</p> | ||
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<caption style='caption-side:top;'><b>Table 2: Results of Model 1 – Equivalent values for LOCS, Opacity, Absorbance, and Crystallin Damage.</caption> | <caption style='caption-side:top;'><b>Table 2: Results of Model 1 – Equivalent values for LOCS, Opacity, Absorbance, and Crystallin Damage.</caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
<th>LOCS</th> | <th>LOCS</th> | ||
− | <th>Opacity(%)</th> | + | <th>Opacity (%)</th> |
<th>Absorbance (@397.5 nm)</th> | <th>Absorbance (@397.5 nm)</th> | ||
<th>Crystallin Damage (M-h)</th> | <th>Crystallin Damage (M-h)</th> | ||
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− | <h2 id = 'gsr25hc'>Model 2: GSR/ | + | <h2 id = 'gsr25hc'>Model 2: GSR/25HC Chemical Pathway</h2> |
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− | + | <div class="row"> | |
− | + | <div class="col-sm-6" > | |
− | + | <h3> Abstract </h3> | |
− | <p> | + | <p> The key question: <b>How much GSR to add?</b> Now that we know how much we need to limit crystallin damage, we use systems of ordinary differential equation to model the GSR Pathway. We calculate the necessary GSR concentration to be maintained over 50 years so that the resulting cataract is below LOCS 2.5./p> |
+ | </p> | ||
+ | </div> | ||
+ | <div class="col-sm-6" > | ||
+ | <h3> Purpose </h3> | ||
+ | <p> How much GSR do we need to maintain in the lens so that the crystallin damage recorded over 50 years is below the threshold for LOCS 2.5? </p> | ||
+ | </div> | ||
+ | </div> | ||
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+ | <h3> Chemical Kinetics Model: Differential Equations </h3> | ||
+ | <div class="row"> | ||
+ | <div class="col-sm-12" > | ||
+ | <p> By the Law of Mass Action, Michaelis-Menten Enzyme kinetics, Ping-pong mechanism, and the Law of Passive Diffusion, we build a system of 10 differential equations based on 6 chemical reactions. All parameters, constants, and initial conditions are based off literature data. Estimates made are also shown with assumptions and reasoning. The details are shown in the collapsible for interested readers. </p> | ||
+ | </div> | ||
+ | </div> | ||
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+ | <div class="row"> | ||
+ | <div class="col-sm-6" > | ||
+ | <h3>Blackbox Approach: Testing GSR Impact </h3> | ||
+ | <p> Image</p> | ||
+ | </div> | ||
+ | <div class="col-sm-6" > | ||
+ | <h3> </h3> | ||
+ | <p> We will vary the input, Initial GSR concentration, from 0 to 100 uM, holding all other variables constant, and numerically solve for the amount of hydrogen peroxide over time. With this graph, we can find the amount of crystallin damage accumulated over 20 to 50 years if different levels of GSR is maintained. </p> | ||
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</div> <!-- End Menu --> | </div> <!-- End Menu --> | ||
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+ | <br><br><br><br><br> | ||
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+ | When determining the relationship between absorbance and crystallin, in Figure 1 the best fit line has a x – intercept that is nonzero. However, when converting each absorbance rating to equivalent crystallin damage in Table 2, we ignore the constant term. When doing the experiments, the fish lens may have contained GSH that is still active, so the fact that the crystallin is exposed to H2O2, the degradation reaction does not happen until all GSH is depleted, and crystallin damage begins to form. We subtract around 1 unit of crystallin damage from all values. | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
<h4> Conclusion</h4> | <h4> Conclusion</h4> | ||
<p> Conclusion</p> | <p> Conclusion</p> |
Latest revision as of 14:51, 6 October 2016
Modeling
Abstract
We answer two questions: How much GSR to maintain in the lens, and how to maintain that amount? We find the amount of GSR needed in the lens (Model 2) to limit crystallin damage so the resulting cataract is less than LOCS 2.5 (Model 1). Then, we find the optimal design of eyedrops (Model 4) and nanoparticles that will maintain this amount of GSR in the lens (Model 3). These models allow our team to understand the impact of adding GSR-loaded nanoparticles into the lens, and to design a full treatment plan on how to prevent and treat cataracts.
Achievements
- Designed a simple calculator to find amount of GSR or 25HC eyedrops needed for a patient's LOCS score.
- Bridged the gap between the medical, biological, and chemical measurement of crystallin damage.
- Predicted impact of adding GSR and 25HC on the amount of crystallin damage in the lens.
- Created Nanoparticle Customizer for doctors to find a full treatment plan.
- Generalized our Customizer to allow other iGEM teams who wish to use nanoparticle drug delivery
- Analyzed sensitivity of prototype, and suggested insights into optimal manufacturing and clinical use of our prototype.
- Used Experimental data to develop Models 1 and 3.
Outline
Introduction
Why Model?
In the lab, biologists are often unable to test everything experimentally. For example, in our cataracts project, cataract prevention occurs in the long-term, from 20-50 years. Obviously, although short experiments can provide us an idea of what prevention may look like, the power of computational biology allows us to model into the future. As a result, our modeling has been crucial in developing a prototype.
Focus
Most iGEM teams perform modeling on gene expression, which we accomplish in model 5. However, as our construct is not directly placed into the eyes, how our synthesized protein impacts the eyes after it is seperately transported is much more interesting. As a result, we spent the majority of our models on understanding the impacts on the eye.
Guiding Questions
- How much GSR do we want inside the lens?
- How do we use nanoparticles to control the amount of GSR in the lens?
- How do we synthesize GSR, package into NP, and send it into the eye?
Model 1: Crystallin Damage
Abstract
In our experiments, absorbance measurements are meaningless without understanding how severe a cataract that absorbance measurement means. We use literature research to relate LOCS, the physician's scale of cataract severity) to absorbance, which is how we quantified crystallin damage in experiments. We use experimental data to understand how crystallin damage can be quantified by measuring absorbance. With this model, we can calculate how much crystallin damage we have to limit to reduce LOCS to an acceptable level.
Purpose
How much do we need to limit crystallin damage so surgery is not needed?
Measurement of Cataract Severity
There are four ways of measuring cataract severity, each used for a different purpose.
- Lens Optical Cataract Scale (LOCS): Physicians use this scale, from 0 – 6, to grade the severity of cataracts.
- Opacity (%): This is the physical, quantitative property of the LOC scale.
- Absorbance at 397.5 nm: This is the experimental method, used by our team in the lab (c.d.).
- Crystallin Damage: This is a chemical definition. We quantify cataract severity as a function of how much oxidizing agents there are, as well as how long crystalline is exposed to oxidizing agents. We define 1 crystallin damage unit as the damage done to human crystallin when exposed to 1 M hydrogen peroxide, the main oxidizing agent, for 1 hour.
We use the unit of crystallin damage to connect cataract severity with the amount of GSR we add (in Model 2). We want to lower c.d. below so that the resulting cataract is of LOCS 2.5. For the rest of the model, our task is simple: relate each point of the LOCS scale to c.d., in order to connect to Model 2.
LOCS Equivalence to Absorbance: Literature Research
Past studies have done numerous studies on how absorbance measurements can be converted to the LOC scale that physicians end up using. With the results of ________ and ________, we construct the first three columns in Table 2.
Absorbance Equivalence to Crystallin Damage: Experimental Data
We use experimental data from our team’s Cataract Lens Model (link). In each trial, they added H2O2 to crystallin, and measured the resulting absorbance. The data used are shown in Table 1. We can calculate the theoretical c.d., and graph absorbance vs. crystallin damage in Figure 2.
With this relation in Figure 2, we calculate the equivalent crystallin damage to each LOCS rating and its equivalent absorbance.
Error Analysis
It may be surprising that only around 1 M-h is required to induce moderately severe cataracts. Remember that this is done in the absence of antioxidation systems (GSR) and at an extremely high oxidizing concentration of H2O2 (1M of H2O2). In the lens, H2O2 has a much lower concentration, so severe cataracts are induced over months to years.
There are some limitations of the model that arise from our assumptions. We assume that fish and human lens contain similar crystallin proteins that are degraded in the similar manner (Assumption 4). In addition, we made a rough adjustment of data based our diluting procedure. For better results to create a human cataract model, experiments will need to be done on human lens, even better if done in vitro, without any dilutions.
LOCS | Opacity (%) | Absorbance (@397.5 nm) | Crystallin Damage (M-h) |
---|---|---|---|
0.0 | 0.00 | 0.0000 | 0.0000 |
0.5 | 3.24 | 0.0143 | 0.1327 |
1.0 | 6.65 | 0.0299 | 0.2774 |
1.5 | 10.81 | 0.0497 | 0.4610 |
2.0 | 15.88 | 0.0751 | 0.6966 |
2.5 | 21.95 | 0.1076 | 0.9981 |
3.0 | 29.07 | 0.1492 | 1.3840 |
4.0 | 46.37 | 0.2706 | 2.5101 |
5.0 | 66.05 | 0.4691 | 4.3514 |
Conclusion
For surgery to not be needed, the LOCS value has to be below 2.5. This is equivalent to 21.95% in light opacity or 0.1076 abs units. Based on the results of our experiments, this is equivalent to 0.9981 units of crystallin damage, the damage done to crystallin if exposed to 0.9981 M of H2O2 for 1 hr. For future models, this value 0.9981 units of c.d. will be called the crystallin damage threshold for LOCS 2.5.
Model 2: GSR/25HC Chemical Pathway
Abstract
The key question: How much GSR to add? Now that we know how much we need to limit crystallin damage, we use systems of ordinary differential equation to model the GSR Pathway. We calculate the necessary GSR concentration to be maintained over 50 years so that the resulting cataract is below LOCS 2.5./p>
Purpose
How much GSR do we need to maintain in the lens so that the crystallin damage recorded over 50 years is below the threshold for LOCS 2.5?
Chemical Kinetics Model: Differential Equations
By the Law of Mass Action, Michaelis-Menten Enzyme kinetics, Ping-pong mechanism, and the Law of Passive Diffusion, we build a system of 10 differential equations based on 6 chemical reactions. All parameters, constants, and initial conditions are based off literature data. Estimates made are also shown with assumptions and reasoning. The details are shown in the collapsible for interested readers.
Blackbox Approach: Testing GSR Impact
Image
We will vary the input, Initial GSR concentration, from 0 to 100 uM, holding all other variables constant, and numerically solve for the amount of hydrogen peroxide over time. With this graph, we can find the amount of crystallin damage accumulated over 20 to 50 years if different levels of GSR is maintained.
When determining the relationship between absorbance and crystallin, in Figure 1 the best fit line has a x – intercept that is nonzero. However, when converting each absorbance rating to equivalent crystallin damage in Table 2, we ignore the constant term. When doing the experiments, the fish lens may have contained GSH that is still active, so the fact that the crystallin is exposed to H2O2, the degradation reaction does not happen until all GSH is depleted, and crystallin damage begins to form. We subtract around 1 unit of crystallin damage from all values.
Conclusion
Conclusion
Model 3: Nanoparticles
Abstract
Abstract
Purpose
Purpose
Conclusion
Conclusion
Model 4: Eyedrops
Abstract
Abstract
Purpose
Purpose
Conclusion
Conclusion
Conclusion
Yay
Citations
![](https://static.igem.org/mediawiki/2016/d/dd/T--TAS_Taipei--TaipeiBackground-reflected3.png)