Overview
Human practice activities broaden our knowledge, expand our thought, and clarify the purpose and content of our project. All presented on this page is how we improved our iGEM project through human practice activities, and decided that the topic of our study is on early warning diagnosis of atherosclerosis (AS) by detecting TMAO and miRNAs in urine, ,and how we use our proiect to enable the elderly to undergo early warning test for AS, increasing the qulity of their lives and making the world more beautiful. Human practice activities have played a huge role in smoothing progress of our iGEM project.
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1. Survey online
After brainstorm, we are going to focus on the cardiovascular and cerebrovascular diseases (CCVD) caused by atherosclerosis (AS) in our project. In order to broadly understand this topic, we designed a survey questionnaire and put it online to interview the young and middle-aged people who could use smart phone about their understanding of CCVD and AS. Some mobile screenshots of the survey work online (the last update is on July 30th, 2023) are showed as follows (Fig.1).
Fig.1 Some mobile screenshots of survey work online about CCVD and AS.
There were 321 people who responded online to the survey questionnaire. We choose some questions’ responses to show here in the followings:
The first question is that “Do you know anything about AS before?” From the result we can see that only 5.92% of the respondents know about it clearly, and 66.67% of people know a little bit about it (Fig.2), indicating that most people are not very clear to AS, and more publicity of AS is required for the young and middle-aged population.
Fig.2 Responses to the question “Do you know anything about AS before?”
(a) Have no idea; (b) Know a little bit; (c) Yes, it’s clear.
The second question is that “What do you think are the harms of AS? (multiple choices question)" From the results we can see that the top two choices are myocardial infarction and coronary heart disease (Fig.3), indicating that most respondents understand the severity of AS.
Fig.3 Responses to the question “What do you think are the harms of AS? (multiple choices question)”
(a) Myocardial infarction; (b) Coronary heart disease; (c) Angina; (d) Intractable hypertension; (e) Stroke; (f) Brain atrophy.
The third question is that “Do you think it is convenient to go to the hospital to check for AS?" The result showed that about 73% of respondents believe that it is inconvenient or a bit inconvenient (Fig.4), indicating that they are not willing to go to the hospital for AS examination, especially if there is no symptom.
Fig.4 Responses to the question “Do you think it is convenient to go to the hospital to check for AS?”
(a) Convenient; (b) A bit inconvenient; (c) Inconvenient.
The fourth question is that “Do you want an available detector to make early warning for AS at home?" All respondents answered “YES”. We continued to ask the following question, “Do you think it is possible to use synthetic biology methods for the diagnosis or treatment of AS?” 48.3% of people believe it is possible although they don’t know synthetic biology very well (Fig.5), they hope it can solve some problems of AS.
Fig.5 Responses to the question “Do you think it is possible to use synthetic biology methods for the diagnosis or treatment of AS?”
(a) Possible although I don’t know it very well; (b) Not sure for it; (c) No idea.
To sum up for survey work online: (1) Most people know a little bit about AS, but they know it can cause CCVD; (2) Many people think it is possible to use synthetic biology for AS diagnosis or treatment.
These results of survey work online strengthened our determination to solve some problems of AS using synthetic biology methods.
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2. Interview with a doctor in hospital
To deeply understand the clinical situation including the diagnosis and development progress of AS, we interviewed Dr. Peng Jie in Jinan Cardiovascular and Cerebrovascular Disease Hospital (Fig.6). Dr. Peng is an expert who is very familiar with CCVD and AS.
Fig.6 Interview with Dr. Peng in Jinan Cardiovascular and Cerebrovascular Disease Hospital
Here are summarized some contents of the interview with Dr. Peng:
(1) Situation: The CCVD is a common disease that poses a serious threat to human health, especially for middle-aged and elderly people over 50 years old, with characteristics of high incidence, high disability rate, and high mortality rate. Even with advanced and comprehensive treatment methods, more than 50% survivors of cerebrovascular accidents cannot fully take care of themselves. The number of deaths of CCVD is as high as 15 million worldwide each year, ranking the first among various causes of death.
(2) Progress: AS is a slow progressive disease which is the most important cause of CCVD. As time goes on and age increases, the arterial wall becomes thickening, and the lesions of the affected arteries start from the intima, with local lipid accumulation and fibrous tissue proliferation, forming plaques, even clogging blood vessel (Fig.7).
Fig.7 Pattern map of AS and patient's arteriogram showing the clogged artery caused by AS (provided by Dr. Peng).
• Coronary AS can lead to angina pectoris, myocardial infarction, arrhythmia, or even sudden death if the coronary artery is more than 75% narrow;
• Cerebral AS can cause cerebral ischemia, brain atrophy, or rupture and hemorrhage of cerebral vessels;
• Renal AS often causes nocturia, intractable hypertension, and renal insufficiency in severe cases;
(3) Diagnosis: At present, the diagnosis of AS is mainly based on clinical symptoms, companied by the following examinations:
• X-ray examination: The aorta may be prolonged, dilated and distorted, and calcinosis may be seen. With arteriography, stenosis in the limbs, the kidneys, and the coronary arteries can be observed.
• Ultrasound examination: including carotid artery B ultrasound, lower extremity artery B ultrasound, etc. More obvious AS can be found.
• CTA angiography and magnetic resonance angiography: can see clear calcified plaque and assess the severity of AS.
Note: According to Dr. Peng’s introduction, the current diagnostic period for AS is usually when patients experience severe symptoms which is very danger. That’s because no symptom occurs if vessel is less than 75% narrow. If seeking medical diagnosis earlier, it is still possible to save lives, otherwise one may lose his life due to AS. Early diagnostic warning of AS is crucial.
Opportunity and challenge: From the interview with Dr. Peng, we figure out that it is extremely important to give early warning for AS and the degree of vascular blockage caused by AS for preventing the sudden onset of CCVD. So, it is particularly urgent to develop a sensitive and specific testing method to give the early warning, helping clinicians make early screening and early intervention for AS.
It is an opportunity and challenge, making us to make a decision that develop a testing method for AS early warning in our iGEM project.
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3. Survey work in community
Generally, only the young and middle-aged people could use the smart phones to respond our questionnaire online. In order to understand the elderly people's cognition to AS and facilitate our project design, we set a paper questionnaire and interview them face to face in community to explore the feasibility of our project (Fig.8A and Fig.8B).
Fig. 8A Interview with elderly face to face in community
Fig. 8B Interview with elderly face to face in community
Our survey questions include the cognition, harm, prevention, health desire, and early warning of AS at home etc., with the aim of which item would be focused on our project. We choose the responses to some questions in the survey as follows:
Responses to the question "Do you understand the hazards of AS?" showed that 74.5% understand, 21.3% heard but not understand, and 4.2% completely don't know (Fig.9), indicating that the majority of elderly people understand the hazards of AS, which is better than the young and middle-aged people. Maybe some of elderly experienced AS.
Fig.9 Responses to the question “Do you understand the hazards of AS?”
(a) Understand; (b) heard but not understand; (c) No idea.
The next question is that "Are you willing to do a test for AS early warning at home or to the hospital?" 95.2% of eldly like to be tested at home for early warning of AS due to the overcrowding hospital and complex procedures for testing in hospitals (Fig.10). It provided us the direction of design for our product.
Fig.10 Responses to the question “Are you willing to do a test for AS early warning at home or to the hospital?”
(a) At home; (b) To hospital.
For responding to the question “Would you like to inform your children about the testing result of AS early warning at home?", the result showed that 96.3% of eldly would like their children know the result (Fig.11). And for this reason, we should design a mobile APP that can forward the testing result to their children automatically.
Fig.11 Reponses to the question “Would you like to inform your children about the testing result of AS early warning at home?”
(a) Inform their children; (b) Not mind.
Conclusion: From the survey work online, the interview with a doctor, and interview with the elderly face to face, we know that:
(1) AS is the most risk factor to cause CCVD which ranks the first among various causes of death worldwide;
(2) Early warning of AS is very crucial for saving lives, especially in the asymptomatic period.
(3) Most people have understanding for harm of AS. They wish to do the early warning test of AS at home and inform the result to their children.
All these findings confirmed us to develop a testing method for AS early warning at home using synthetic biology. It should be simple, convenient and portable, in addition, an APP is expected to use in the detector to forward the testing result to their children.
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Integrated human practice
4. Interview with a Professor of Shandong University
After making our decision, we need to know some knowledge about AS. For example, what is the main pathogenesis and research hotspots of AS at present? What are the typical molecular targets of AS? What molecules are expected to be the molecular markers for clinical diagnosis of AS? We were confused and disoriented with many questions although we browsed a lot of literature. To broaden our knowledge, bringing some questions, we interviewed professor Wang Jianxun who works in Shandong University. Due to the working time arrangements, we held a virtual meeting with Dr. Wang (Fig.12).
Fig.12 Interview with pressor Wang in virtual meeting.
First, professor Wang highly praised our project. He thought it has special significance to develop a test method of AS early warning for the elderly, especially for “the empty nest elderly”.
According to our demand, professor Wang introduced that biomarkers could be serving as the diagnostic target molecules, including proteins, nucleic acids and some small molecules. Trimethylamine N-oxide (TMAO) is one of biomarker of AS, and it is related to the AS formation. It’s reported that TMAO can upregulate the expression of macrophage scavenger receptor CD36, SR-A, leading to the accumulation of cholesterol in macrophages and the formation of foam cells, even the formation of vascular plaque. In addition, TMAO is excreted in urine. Testing TMAO in urine is very convenient and suitable for the elderly at home. TMAO may be a good candidate of molecular diagnostic marker of AS.
Professor Wang also suggested that it would be better if we use lacZ marker gene instead of the chromoprotein to display the testing result, because lacZ gene encodes β-galactosidase which catalyzes the enzymatic reaction to amplify the signals. Many different substrates are available for this enzyme. We can use the substrate X-Gal to test β-galactosidase activity, indicating the level of TMAO in the samples. This is really helpful!
To solve the problem that people are not willing to go to the hospital for the complex detection process, professor Wang suggested that we may develop a filter paper biosensor using cell free system, so that people can detect the changes of TMAO in urine by himself at home.
Through interview with professor Wang, we determined to detect TMAO in urine sample using lacZ (β-galactosidase) as the marker protein which can catalyze the substrate X-gal to produce blue color product, in which the signals are amplified. Using the color change to display the testing result on the filter paper biosensor is very intuitive and acceptable for the elderly at home.
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5. Communication and education in the community
From the interview with the elderly face to face in community last time, we know that most people do not have the common sense of AS although they understand the harm of it. For publicity of the AS knowledge, we prepared a poster to communicate and educate them regarding the AS topic in community (Fig.13). For example, how to prevent AS? how to delay the progress of AS by changing the diet habit, exercises, or changing lifestyle? and how to deal with emergency of CCVD occurrence. This activity is very welcome to the elderly in community. We hope it works to decrease the occurrence rate of AS and CCVD in these elderly population!
Fig. 13 Communication and education with elderly people in community
In addition to the publicity, we also introduced the design of our project to them. Regarding the testing samples, we communicated with them, and most people prefer the urine sample to blood sample due to its accessibility and painless (data not shown)，confirming the acceptability of our product.
These communicaton and education activities in community made our project more meaningful. We helped them to know some common sense about AS and CCVD, increasing the qulity of their lives, and they gave us more suggestion and support to fufil and improve our project.
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6. Consulting professor Wang again to solve problems
In the process of conducting experiments and reading literature, it was found that patients with other diseases such as chronic kidney disease also have elevated level of TMAO in their urine, which would decrease the specificity of TMAO testing for AS early warning. How to increase the testing specificity for AS early warning? To solve this problem, we consulted professor Wang again with telephone.
Professor Wang introduced that non-coding microRNA (miRNA), one kind of nucleic acids, has attracted more and more attention in molecule diagnosis in recent years. miRNA is a class of endogenous single strand non-coding RNAs that widely exist in eukaryotes. miRNAs can block or inhibit the translation of target mRNAs by completely or partially complementary base pairing with the 3' non-coding region, so as to regulate the expression of target genes, involved in a wide range of physiological and pathological processes.
He also told us some specific miRNAs expression is upregulated or downregulated in AS patients, compared to normal people. These miRNAs are also present in urine which could be used as biomarker for testing. It is very helpful!
With the guidance of professor Wang, we searched for some references and found some differently expressed miRNAs in AS. We chose two of them miRNA-17-5p and miRNA-146a-5p combined with TAMO to serve as biomarkers, increasing AS detection specificity. We really appreciated professor Wang who gave us a great enlightenment and less detours.
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7. Consulting engineers of two companies
Since our product would be used by the middle-aged and elderly people at home, and the filter paper biosensor on which biomarkers are tested would be put into a detector which detects the color change to show biomarkers’ concentration, how to design it to be a sensitive, convenient, portable device is important. Based on this idea, we visited two companies to consult the design of our detector. They are Weili Intelligent Technology Co., Ltd and XIcheng Co., Ltd .
In Weili Intelligent Technology Co., Ltd (Fig.14), they first led us to visit the production line of their company's products, and introduced the disign and productive process. Then we introduced our project, telling them we want to design a hardware to detect the color change on a biosensor. In the communication with them, the engineer suggested that it would be better if we convert the color (divided into three primary colors) values on the biosensor into electrical signals, and then amplify and quantify the electrical signals to enhance the accuracy of the detection results. In addition, he introduced the principle and structure of the converter. This suggestion is in line with our wishes to a more accurate and more sensitive detector. It really improved the design of our hardware.
Based on their guidance, we designed and created a single chip processor (Fig.15), which provided great assistance for the design of our detector.
Fig.14 Visiting Weili Intelligent Technology Co., Ltd for hardware design.
Fig.15 The construction of single chip processor of detector in our project.
In the second company XIcheng Co., Ltd (Fig.16), in order to design a convenient and compact detector for AS early warning, with the demand from user, we communicated with two technicians. The technical engineers suggested that the power supply for various accessory elements in the Detector is very important. They introduced their company's power supply design theory and practice. Based on their help, we designed and manufactured a power supply (Fig.17), which played an important role in the preparation of the detector.
They also suggested that we’d better design a mobile APP used in our detector. It can calculate, store and forward the testing results to others, for example hospital, family doctor or relatives of users. This APP can perform cumulative statistics and parallel comparisons, facilitating to find the trends of test results and the occurrence of AS, get timely warning, and allow their children to see the results, which is conducive to providing necessary care and assistance.According to their suggestion, we designed the pattern graph of mobile APP (Fig.18).
Fig.16 Visiting Xicheng Co., Ltd for hardware design.
Fig.17 The construction of power supply of detector in our project.
Fig. 18 The pattern graph of APP designed to use in the detector.
From visiting companies, we got some inspiration to make a detector for detecting some biomarkers on the filter paper biosensor, which is convenient, concise and portable. They are very interested in our project and look forward to further cooperation in the future.
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8. Participation in 10th CCIC
In July 2023, the 10th Conference of China iGEMer Community was held in Hainan University. Some team members of HSASNU participated in it and presented our project including the design of hardware in the conference.
With the communication with other iGEMers, our project was modified and improved. In addition, we practiced our presentation for Jamboree.
Fig.18 The certificate of participation and some pictures taken in 10th CCIC.
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9. Publicity and education in middle school
To better promote synthetic biology development and publicize our project, we established a synthetic biology club in High School Attached to Shandong Normal University. In the recruitment activities, we introduced the history, development, application and prospect of synthetic biology, as well as iGEM competition. In addition, we introduced the purpose, principle, design and significance of our project.
Many new students are interested in these introductions, showing great enthusiasm in it. We hope the new students of our club will participate in iGEM compitition next year.
Fig.19 Some pictures taken in the recruitment activities.
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Conclusion:
(1) survey work online strengthened our determination to solve some problems of AS using synthetic biology methods.
(2) From interview with a doctor, we found an opportunity and challenge, making us to make a decision that develop a testing method for AS early warning in our iGEM project.
(3) Survey work in community provided us the direction of design for our product. Most people wish to do the test of AS early warning at home and forward the result to their children.
(4) After interview with professor Wang, we determined to detect TMAO in urine sample using lacZ (β-galactosidase) as the marker protein to catalyze the substrate X-gal, producing blue color product on the filter paper biosensor to show the testing result.
5) Communication and education activities in community made our project more meaningful. We helped them to know some common sense about AS and CCVD, increasing the quality of their lives, and they gave us more suggestion and support to fulfil and improve our project.
(6) With the guidance of professor Wang, we used miRNA-17-5p and miRNA-146a-5p combined with TAMO to serve as biomarkers, increasing AS detection specificity. Professor Wang gave us a great enlightenment and less detours.
(7) From visiting companies, we got some inspiration to make a detector, which is convenient, concise and portable. They are very interested in our project and look forward to further cooperation in the future.
(8) Communication with other iGEMers in 10th CCIC helped us to modify and improve our project. And we practiced our presentation for Jamboree.
(9) We established a synthetic biology club in High School Attached to Shandong Normal University. We publicized and educated synthetic biology, iGEM and our project to the new students in recruitment activities.