Overview
EXTRAgalin, iBowu-China 2022's project, was inspired to cure the severe neurodegenerative disease, Alzheimer's disease. After extensive research and interview with the doctors and experts, we learned the heartbreaking, unfortunate, and difficult experiences faced by these people. Around the globe, there are more than 44 million people experiencing the same distress. Understanding this, iBowu_China aimed to use our knowledge of synthetic biology and genetic engineering to synthesize Astragalin. In order to create the best product, we interviewed medicinal experts, government officials, pharmaceutical corporations, potential users, and doctors for feedback and suggestions on the product. After obtaining valuable information on comments for further improvements, we applied the suggestions to better improve and create our project and achieve our goal of healing the infamous Alzheimer's disease.
Interview with Prof. Guoqiang Chen
Prof. Chen’s main research areas are synthetic biology, microbial metabolic engineering, biomaterials, and industrial biotechnology. He is affiliated with School of Life Sciences, Tsinghua University, as a tenured professor. He is the associate editor and editorial board member of several authoritative journals.
He confirmed our proposal to use synthetic biology pathway to manufacture the target substance, astragalin. He commented that by synthesizing biological organisms, we can improve the expression efficiency of a substance and the efficiency of cells. Biosynthetic methods are also driving global advances in environmental protection. Therefore, synthetic biology methods are promising in replacing traditional chemical method of high temperature and high-pressure manufacturing with costly toxic exhausts.
Public Survey about Alzheimer’s Disease
Key results: low awareness of the disease, and lack of effective treatment
In the initial brainstorming of our project, we paid attention to Alzheimer's disease (AD), which is a typical neurodegenerative disease with memory decline as its early symptom, but aphasia, apraxia, agnosia, visual-spatial skills impairment, executive dysfunction, personality and behavior changes, and other comprehensive dementia manifestations were included in the later stage. We read a lot of scientific papers and reports related to the disease. The knowledge led us to think of the status-quo in the public, regarding the awareness of AD symptoms, and the availability of treatment.
We designed a few survey questions and disseminated the survey online through our friends and family. The survey received 403 responses, and according to our data check, all 403 responses are valid.
Figure: Survey results indicated that even though most people (99%) heard of Alzheimer’s disease, the majority had not a full knowledge of its early symptoms. The lack of such knowledge would lead incorrect decision of the public as of whether to see a doctor for proper diagnosis. Based on the result, we proposed a few public engagement projects and finally completed an important Role Play Game project. Please refer to our Education and Science Communication page for more details.
AD patients at later stages are at high risk of lost their way home. According to the survey response, around 90% of people will help an AD patient. This response result not only indicated that AD patients will get help, but also implies that our education product and our synthetic biology treatment proposals will likely to be actively evaluated by most people. We decided that the revenues from selling our art works and designs and educational items will go to a charity donation. Another survey results helped us choose the best items to donate: people think it is more likely to be helpful to use location devices and prepare contact card for AD patients.
This survey results is alarming to us. When asked about how to delay the progression of AD, the majority of people thought of AD as a natural aging degeneracy rather than a treatable disease. The lack of proper knowledge of AD and also the lack of widely received treatment strategies invited us to keep moving our project to synthesize astragalin for a potential drug, and at the same time working on educating people about AD.
Our response to the surveyWe realised that even if we gathered this input, it would be very difficult to improve the status quo of AD in China any time soon. Therefore, we decided to make a practical contribution to AD seniors.
At the same time, our public Alzheimer's questionnaire (Figure 2) showed that 92.56% of people chose "Make your own card with contact details" and 92.31%, 91.32% chose "Have someone with you when you go out" and "Carry a location device" respectively on the question of how to prevent people with dementia from getting lost. Based on these voting results, we have decided to use all the profits from this iGEM season to purchase "Lost and Found Forms" with contact information to help the seniors with AD from a social care perspective.
Including our educational programs and merch, we have collected a totla of around CNY 4,000 this year. Therefore, we started to research and select lost-preventitive bracelets in the market for the seniors.
After the search, we picked two products shown in the figure below. We will purchase the products and distribute them to the senior as soon as possible. At the moment, we are looking for a community charity who has the needs. We look forward to making a real contribution to the situation of AD in China!
Online interview with Dr. Zhuohao He
Our feedback: We should focus on the effect of medicine, other than the causing of AD.
In order to find a cure for AD, our team members agreed that we should start with the cause first. If there is an enzyme acting in the pathogenesis of AD, we can use synthetic biology to influence that enzyme, and if there are metabolites, we can also use synthetic biology to interfere with the production of those products. Therefore, in order to understand the etiology of AD, we first contacted Dr. Zhuohao He, a researcher at the Intersection Research Center of Biology and Chemistry, Chinese Academy of Sciences, who has been engaged in the research of neurological diseases for a long time and is very familiar with the pathogenesis of neurodegenerative diseases.
We conducted an online interview with Dr. He, who introduced the current pathogenic hypothesis of AD in detail. He proposed that there are two kinds of cognition in the mainstream scientific community.
1) Aβ toxicity hypothesis is the dominant theory in the pathogenesis of AD. Abnormal deposition of extracellular Aβ forms neuro-senescent plaques. Aβ fiber deposition can cause the increase of reactive oxygen species (ROS) in neurons, inducing cell apoptosis, and also triggering the phagocytosis behavior of microglia, leading to the abnormal release of inflammatory factors, and finally inflammatory reaction.
2)Tau protein is a normal component of the cytoskeleton, and the highly phosphorylated Tau protein in cells abnormally aggregates to form neuronal fiber knots, thus causing microtubules distortion and degeneration and failing to normally transport nutrients, leading to dystrophic atrophy of dendrites and axons at the end of neurons. However, Dr. He pointed out that although the above two hypotheses are dominant, the latest research may overturn the two hypotheses at any time. We are still not sure whether the expression of Aβ and tau in AD patients is pathogenic mechanisms or pathological manifestations. Therefore, whether the key pathologies (Aβ and tau) are fundamental drivers in AD onset are huge speedbumps in the field.
The communication with Dr. He denied our initial idea. Since the pathogenic mechanism of AD is still unclear, we cannot start from the etiology. Dr. He suggested that we look for the drugs currently on the market and pay attention to the effects of drugs so as to determine the direction of the project.
Face-to-face interview: Prof. Hu from Minzu University of China
Our feedback: Using synthetic biology to produce astragalin
We went back to our lab and read some papers, but we were still confused about how to find a proper substance to cure AD. We reached out to many experts looking for a suggestion. What we are looking for is something that can be produced via synthetic biology pathways, and it had better be a new candidate drug with special features not seen in the on-market drug. Among the experts we contacted, Prof. Hu from the Minzu University of China replied to us and invited us to his lab.
Prof. Hu had done neurobiology experiments for a long time and he gave us a key point that “the efficiency of a medicine to treat AD would depend on the nature of the compound, its stability, and especially the ability to cross the barrier between the blood and brain as well as epithelium.”
The new word “blood-brain barrier” broadened our horizon, Prof. Hu told us “many medicines worked pretty well in vitro, but show no effect in in vivo experiment.”, that is because these medicines are blocked outside the blood-brain barrier. So when we are thinking of an effective way to treat AD, Whether it crosses the blood-brain barrier is an important thing to verify.
Prof. Hu told us he's been looking for drugs that can cross the blood-brain barrier for a long time, and one of those drugs is astragalin which is a flavonoid naturally occurring in plants and has been shown to improve the cognitive abilities of mice, diminish Aβ plaque deposition, and have other neuroprotective properties. However, using the chemical synthesis methods to produce astragalin is very costly. Therefore, although it has a good effect, astragalin medicine is still difficult.
We were very excited to hear that because synthetic biology could exactly help. We could produce astragalin from microorganisms, and that could significantly reduce the cost.
Partnership with SHSBNU_China
Our feedback: Using E.coli to produce astragalin, other than yeast.
We had online meetup with our partner SHSBNU_China team during the project. Students from the SHSBNU_China team raised a lot of questions to us, some of which were new to us.
We would like to produce astragalin from yeast initially, but they proposed to preferentially use E.coli for protein expression. Because compared with yeast, E.coli is more efficient and convenient for protein expression. We took their advice and changed the expression system into E.coli.
Online interview with Dr. Xianbo Zhou
Our feedback: Using 2-3 plasmids is a better choice.
After reading papers, we found that plants can convert naringenin into astragalin through F3H, FLS and UGT enzymes. So we planned to construct linkers between three genes all in one plasmid to transform into E.coli, because we do think this is a way to give E. coli less pressure when expressing proteins.
However, although we have tried for many times, we still could not construct an intact plasmid. We were a little bit upset. So we went to ask Dr. Xianbo Zhou, who has founded Zhongze Pharmaceutical, an innovative drug discovery company. After hearing our question, Dr. Xianbo Zhou denied our proposal, he said “A pretty big plasmid could bring even larger express pressure. Microorganism are intend to eliminate foreign gene.” “The better choice is to divide them into 2 or 3 plasmids.”
This interview changed our experiment plans, and instead of obsessively constructing one complete plasmid, we constructed two or three plasmids separately. Subsequent results suggest that there is indeed a significant benefit and detailed information is displayed on our results page.
Online interview with Dr. Yong Zu
Key advice: Temperature gradient and time gradient experiments should be performed to optimize the expression level.
The second problem of our experiment come to be the expression level of our enzymes was low. We had tried to do codon optimization for E.coli but still did not improve.
We took our result to visit Dr. Yong Zu, who got his doctor’s degree in biochemistry and molecular biology. He recommended us to find specific quantitative data on how many astragalin has been produced under different condition, and how labs have improved the synthetic process of astragalin, for example, purity of astragalin or efficiency of producing. According to his advice, we underwent a time-course and a temperature-course experiment to find the best expression condition.
Detailed information is displayed on our results page.
Face-to-face interview: Prof. Hu from Minzu University of China
Our feedback: Conducting HPLC for product verifying.
At the last stage of our project, we encountered a new problem, that is, the expression product of astragalin did not have any color, so we had no means to figure out whether astragalin was successfully generated. We found Prof. Hu from Minzu University of China again. After Prof. Hu checked our experimental results, he said that there were two problems.
First, we were not sure whether the astragalin was successfully synthesized by bacteria, and second, we did not know whether there were any other intermediates in it.
He suggested that we conduct HPLC experiments on the expression products and compare the peaks of the products with those of the standards to find our answers. Since the HPLC apparatus from the University could not be operated by students, Prof. Hu offered to help us run the HPLC experiments. Fortunately, we got relatively pure astragalin, which was undoubtedly very exciting. Detailed experiment information is displayed on our results page.
Prof. Hu taught us the basic principles of HPLC in the lab.
Online interview with Timothy Huang
Our feedback: We would do the contrast test for further study in the future.
In order to promote our project, we took our result to Timothy Huang, who is a research assistant professor, Neuroscience Initiative, Neuroscience and Aging Research Center, Sanford Burnham Prebys Medical Discovery Institute. Prof. Huang is a well-known expert in this field.
In our communication, Timothy Huang told us it is promising to find therapeutic compounds from natural plant. There has been a long history of using drugs derived from plant extracts to target specific signaling pathways.
He also suggested a related component named Galantamine. Galantamine is extracted from Galanthus nivalis (snowdrop), which is an Ach inhibitor, has been shown to effectively delay the progression of AD. Similarly, our research showed that astraglin from Tabernaemontana pandacaqui flowers worked as a natural source that can inhibit Ache as well.
Prof. Huang reminded us there is a need to compare how stable, how potent and whether astraglin can produce superior effects compared to galantamine.
Because of the limited time, although we were willing to do this experiment, we didn’t have this chance to conduct further experiments. We planned to do the contrast test in the future.
Online interview with Dr. Ridong Wang
Dr. Ridong Wang is from Biogen Pharma, a company that has pushed a drug for AD on the market. We aim to seek technical support from drug development professionals to constructively evaluate the scientific, translational, and practical aspects of our project. Dr. Wang’s research and development did not involve astragalin or plant-based medines, but he raised our awareness of the quality of life of AD patients. The community doesn’t show enough sympathy for the patients, and there are many mistakes in people’s knowledge about AD. He suggested we use all types of communication activities to raise awareness of AD symptoms and treatments.
Online interview with Deputy Director Qingli Wang of the Department of Pharmacology and Toxicology of the Drug Review Center of the State Food and Drug Administration
Safety issues are always our biggest considerations. In order to learn what safety concern we should explore, we interviewed Director Wang from the state administration. Director Wang kindly introduced the whole procedure for drug safety trials and clinical trials, and praised our emphasis on the drug safety. We invited our partner SHSBNU_China to join this interview. They verified with Director Wang that their eye-protection products proposals observe the state safety regulations for similar products.
Online Interview with Pan Yu：Professor at Tsinghua University School of Clinical Medicine
Our feedback: inspired us to make the anti-loss bracelet at the end of the HP project
Pan Yu, Chief Physician of Rehabilitation Medicine, Tsinghua Changgeng Hospital, Beijing, and Professor at Tsinghua University School of Clinical Medicine, specialising in neurorehabilitation and brain-computer interface. In our preliminary interview, we asked the specialists about their treatments in a very general way, but not in a systematic way. Therefore, we approached Pan Yu with two questions 1. What is the treatment for AD in the middle and late stages from a Western medical point of view? 2. What methods can improve the quality of life of AD patients from the perspective of rehabilitation medicine? She gave us a very professional answer: In Western medicine, there is no specific medicine for patients with Alzheimer's disease in the middle and late stages, so there is no fixed treatment. In rehabilitation medicine, it is recommended to intervene as early as possible. The objectives of early rehabilitation interventions are as follows: 1) To maintain the patient's existing function as far as possible 2) To improve the patient's residual function as much as possible in order to provide quality of life, usually through exercise therapy, such as improving the patient's balance and walking ability. In addition, there is language and cognitive training. However, Pan also suggests that the disease itself is a continuous progression and that rehabilitation itself is very difficult to reverse. In rehabilitation medicine, the goal is to maintain and improve the patient's existing function and quality of life as much as possible, with this goal in mind. Pam's answer was very useful for us to learn about the treatment of AD and therefore inspired us to make the anti-loss bracelet at the end of the HP project
Interview with Mr. Sun: head of GreenValley Pharmaceutical.
GreenValley Pharmaceutical is a company that focuses on producing new-generation saccharide medicine for treating chronic diseases, and one of its most well-known product is the medicine 971, which is an anti-Alzheimer medicine that targets Aβ plaques. Realizing that the mechanism of Astragalin is similar to GreenValley’s medicine, we decided to interview Mr. Sun, head of GreenValley, about the detailed development history of medicine 971 to acquire some insights into the medicine-developing process of Alzheimer’s Disease. From the interview, we learned that medicine 971 can actually achieve a broad-spectrum anti-Alzheimer effect by not only targeting Aβ, but also the intestinal flora and alleviate inflammation. Such innovative broadens our horizons of Alzheimer medicines and would inspire us in further implementations of our product.
meeting with Prof. Shen: iGEM advisor of Beijing University of Chemical Technology
Our feedback: Provides ideas for future enhancements to the project
When we just finished our last experiments and start to finalize our Wiki contents, we hope to review from another professional perspective other than our advisors, so we turned to Prof. Shen at Beijing University of Chemical Technology. We thoroughly elaborated our background research, experimental designs and experiment results collected to Prof. Shen and received extremely professional and incisive opinions. For example, she corrected some of our misunderstandings of the nature of MCS (multiple cloning site) in our plasmids and pointed out some follow-up experiment designs that we can adopt for further implementations.
Online Interview with Deloitte：one of the world's top four accounting firms
Our project this year is dedicated to the efficient synthesis of the potential Alzheimer's drug viologenoside from E. coli. Therefore, we envisage a potential use of asiaticoside as a drug. To do this, we interviewed experts at the drug approval level to understand how the drug would be marketed and the current status of AD drugs from the perspective of both Chinese and Western medicine. However, in order to make our assumptions more accurate, we spoke to Deloitte, one of the world's top four accounting firms, to gain a deeper understanding of how a marketed drug would be priced on the market, a challenge we had been struggling with. Deloitte's answers to our questions The pricing of drugs in China is very much dependent on access to medical insurance, and the lead time to market also has a significant impact on the price of a drug. When pricing a drug, there are three dimensions to consider - price peer group / patient perception of price / local market pricing rules. In addition, we also look at dosage forms and specifications We learned that the packaging of a drug, whether it comes in 5 or 10 tablets and whether it is a drug or an injection can affect the price. In summary, the pricing of a drug on the market is complex from a number of perspectives, and it is important to cover the costs of promotion and total spend while ensuring safety, efficacy and other issues.
Online Interview With Lihua Gu: Expert in Utilizing Chinese medicine in Neuro-recovery
Doctor Gu is a specialist in combining the Chinese traditional medicine and healing with western methods in the field of nerve system recovery. In this interview, we were seeking for information of precedents of treating Alzheimer’s disease in traditional Chinese medicine. Though his field of research and application does not directly relate with the Alzheimer’s disease, he does provide his understanding of two ways in treating Alzheimer’s disease, medicine and acupuncture. The medicine is often customized for each patient with different herbs and shows some effect on Alzheimer’s Disease. They also use the medicine to improve the patient’s body condition to better treat AD. Acupuncture stimulates the acupuncture point and heals AD. He further talks about using computer simulated programs to help improve and counter the effects of AD, as well as training in physical abilities that often weaken with AD. He mentioned that Astragalin is a unknown way to his knowledge in treating Alzheimer’s disease and it could be a new and improved way in healing AD.