Human Practices | Shanghai_city

Our project is to develop antimicrobial peptide products that eliminate and inhibit fungi and bacteria to clean the fish tank, and cure those sick fishes who are bacterial infected[1]. In addition, people could even save water by using our products to maintain the water environment clean of farmed fish instead of changing the water frequently.
In order to fully understand the prospects of antimicrobial peptide products in the market,we did background research and confirmed that our stakeholders include aquariums, fish farms, pet fish lovers, etc.
For gathering more information from the public, especially stakeholders, we conducted surveys online and offline (Flower, Bird, and Fish Market). More than 30 questionnaires were received offline and 600 questionnaires were received online.
To have a deeper understanding of how the stakeholders think about our product, we paid a trip to Haichang Ocean Park and talked to the breeding technician, Mr. Ke.
Besides, we also sought professional insights on our project from two experts through online meetings. From Prof. Wang, who is an associate professor from Nanjing University of Traditional Chinese Medicine, we learned more about antimicrobial peptides in the synthetic biology view, including its application and many technical advice on the product implementation; From Doctor Lu, we acquired many information regarding the prospects and safety of the antimicrobial peptide products.

Below is the mind map of our Integrated Human Practice:

We mainly distributed the questionnaire through WeChat to invite people for completing our survey and finally we received 600 completed samples.

*More details regarding each question’s result are listed in -Appendix-Online Survey.

Through the analysis of the data, the following conclusions are drawn:
(1) The main group of the questionnaire respondents is young people
(2) Most people understand the effects and side effects of antibiotics
(3) Most people have never heard of antimicrobial peptides
(4) Most people expect antimicrobial peptide products

In conclusion, most young people have experience in fish farming and people who raise fish think that the biggest problem with raising fish is frequent water changes and fish disease. So our products - Pepsick, which can clean the water and heal fish are very marketable. Most people generally do not frequently use antibiotics, but a few might become dependent on them, which indicates that the bacteria’s or fungus’ drug resistance has increased. Nowadays, people have realized the side effects of antibiotics. In this case, our products that will not cause drug resistance could stand out and replace antibiotics someday and people might be willing to try this new product.

However, most people do not know about antimicrobial peptides at all. Therefore, we consider that it is meant to enhance the general public’s knowledge of antimicrobial peptides by launching a series of educational activities.

On July 2nd, our team carried out an offline survey at Shuangji Flower, Bird, and Fish Market, which radiated over 35 potential future customers. Before the trip, we designed a 12-question questionnaire concerning the public's view on existing similar products, price expectations, and so on. We divided our team members into three groups with different designated areas to invite the shop owners or customers to answer our questionnaires.
Since these respondents are the main stakeholders of our project, this offline survey activity could not only increases our survey coverage rate and lowers the co-selection rate, but also provided a great opportunity to communicate with the stakeholders who could present a certain view. Of course, we still met some difficulties, like getting refused. But we didn't give up and even concluded a good tip: younger people and people that were not so busy doing their work were more likely to accept our request.

*More details regarding each question’s result are listed in -Appendix-Offline Survey.

After we finished the offline survey, we returned to conduct data analysis of the questionnaires made by the respondents. Through data analysis, we found that most of the fish raisers are between 18 and 35 years old and they spend about half an hour cleaning the fish tank. Actually we found that people don't spend much time cleaning fish tanks.
They prefer the cleanning pills to clean the fish tank which will be very convenient, by just throwing it into the tank. These data has played a huge role for us, and we can optimize and develop our products from these conclusions.
We also summarized the difficulties encountered in asking passers-by to do the questionnaire. For example, many people will be disgusted with filling out the questionnaire, so they immediately refuse to fill in the questionnaire. And we have come up with countermeasures for these difficulties. For example, if people refuse to fill in, we can patiently explain our identity to him and explain that this is just an anonymous public welfare questionnaire, so that they will be less disgusted. In this survey, we gained a lot of experience in surveys which we also shared with other teams and greatly promoted our unity.

We decided to pay a visit to Haichang Ocean Park, the biggest ocean park in Shanghai. We also interviewed Mr. Ke, who is a technician in charge of breeding aquatic animals in Haichang Ocean Park. He has very rich experience in breeding aquatic animals and plants. He provided us with many useful suggestions on aquatic animal breeding and water cleaning, which further inspired the future development direction of our project.

After this conversation, we understand that it is very difficult to take care of the seawater environment, and improper medication will affect the survival of the fish. Therefore, for products that clean water, the most important indicative goal is: safety. We need to do experiments and have different discussions according to different water bodies. Our products work to make it easier for people to take care of their fish tanks.

In seawater fish tanks, there are many species of marine parasitic bacteria, including harmful bacteria and beneficial bacteria. And namely, the goal of our products is to eliminate and inhibit fungi and bacteria to protect beneficial bacteria^[2].
In large tanks, Mr. Ke mentioned that medicine is generally not used. Because putting in these drugs requires employees to dive down, which can cause physical harm to employees. Therefore, technicians use methods such as water change, cooling, ultraviolet and ozone disinfection, and 100-toxic filtration to resist harmful bacteria with beneficial bacteria.
The most important way to ensure water quality is to manually control the amount. The quantities that need to be paid attention to are: the proportion of the dose, seawater recoil (gravity pool, sand tank), ozone and ultraviolet sterilization.

Therefore, the application of our antimicrobial peptides in aquariums has a market.
1) Antimicrobial peptides can eliminate and inhibit fungi and bacteria.
2) If our product is in powder state, it is very convenient to only put it into the cylinder circulation system to solve the problem of medication. Teacher Ke reminded us: "Your products are small in size and expensive. Such small products cannot be used in large tanks, but they are suitable for small tanks."

Mr. Ke said that he respected the concept of environmental protection. If the product price conditions allow, he will accept environmentally friendly products. However, at present, the domestic market does not pay much attention to environmental protection. The concept of environmental protection is more attractive to young people. The concept of environmental protection needs to be improved from generation to generation.

1. The diseases are divided into three categories: viral diseases, bacterial parasitic diseases, and diseases affected by the environment.
1) Viral infectious diseases are currently untreatable, such as iridescent viruses.
2) Bacterial diseases are generally caused by individuals. For example: physical health, infection caused by appearance damage, fish fight injuries, etc. This type of treatment is only required for the individual. Parasitic diseases such as "white spots" are also related to excessive bacteria to a certain extent.
3) Environmental issues such as uneven water temperature, high ozone concentration, inappropriate water ph value, etc. After feeling the stimulus, they will feel irritable and leave by themselves.
2. Drug use: broad-spectrum and fine-spectrum drugs
1) Broad-spectrum drugs: diseases that are not very obvious in the short term, such as subcutaneous bleeding. The technician needs 24 hours to analyze. This is what we need for different diseases and medicines are broad-spectrum medicines.
2) Fine-spectrum drugs: When the disease of the body produces obvious characteristics, it proves to be a certain disease. At this time, we need to prescribe the right medicine and use the medicine corresponding to the disease.
In this regard, antimicrobial peptides can destroy bacteria and fungi as well as viruses and parasites. Therefore, antimicrobial peptides can also be used in the treatment of fish diseases.

1. Water temperature: Mr. Ke mentioned that the water temperature is generally controlled at about 24 degrees Celsius. Tropical shades need to be around 30 degrees.
Q: The effect of water temperature on corals also needs to be considered. Because many fish tanks have corals in them.
2. pH: The pH of the water body will affect the reproduction and survival of fish. Unsuitable pH can cause fish to suffocate and result in violent hear beast which can be fatal for ectothermic animals.
Suitable pH for seawater and freshwater:
1) The pH of seawater is stable at around 7.5.
2) The pH of fresh water will be acidic. For example, the living environment required by the food in the Amazon region is relatively acidic. In comparison, the water in the Changjiang River Basin is not so acidic. Therefore, when we cultivate freshwater plants, we put dead leaves in the pool to make the water thinner to increase the acidity of the water.
In summary, we found that fish survival temperature and pH are also important. The function of "maintaining pH balance" can also be considered in our product development. It is worth further thinking here.

1. Water quality has a great influence on the healthy survival of fish. There is a market for products to clean up the water quality.
2. Our products can consider adding more functions to our products, including: preventing diseases, extending the frequency of water changes, adding environmental protection concepts, maintaining the pH of water, etc.
3. There are harmful bacteria and beneficial bacteria in the water, so we can use a variety of antibacterial peptides combined to eliminate specialiy fungicidal and antibacter to protect beneficial bacteria .

On July.7, dry lab interviewed doctor Lu Cheng in order to understand the side effects of current clinical use of antibiotics, the future of antibiotics, and the possibility of using antimicrobial peptides in the clinic.

There are many antimicrobial therapies available in the clinic：
There are many antibacterial used clinically, such as penicillin, cephalosporin, saxin, erythromycin, and some synthetic ones. Antibiotics that have been eliminated on the market: chloramphenicol, because of its side effects on people, it is used locally for ophthalmic drugs and the treatment of tuberculosis.

In China, the clinical use of antibiotics in China is very common. Oral injection and external use in ENT are widely available. There is a regulation in China that the amount of antibiotics used in internal medicine is stipulated within 20%, and some pediatric departments may account for slightly more, which about 30%. At present, in China, large tertiary hospitals use antibiotics very cautiously. However, in second- and third-tier cities, antibiotics are abused, and antibiotics are used in hanging bottles in hospitals, whether it is bacterial infection or viral infection. Primary hospitals are not up to the conditions for antibiotic susceptibility testing. There are even patients who ask them to give themselves antibiotics without the doctor prescribing them, just for the sake of faster results. But they didn't know that antibiotics can lead to severe resistance. Therefore, it is necessary to increase public awareness of the harmful effects of antibiotics.

In terms of preservation, antibiotics should be stored in the dark and the temperature should not be higher than 24 degrees Celsius. The packaging should be protected from light, such as black paper packaging.

Side effects caused by antibiotics in the current clinical practice: With the widespread use of antibiotics, the drug resistance is getting stronger and stronger, and penicillin, etc. are not used. Because of the evolution of the current bacteria, the current antibiotics are useless. Penicillin, etc. have developed to carbapenems, carbapenems and other antibiotics in the medical insurance payment (multi-drug resistance: resistance to all three antibiotics, carbapenems can be used.)
At present, the methods that can alleviate the disadvantages of antibiotics include the use of traditional Chinese medicine, and the use of heat-clearing and detoxifying drugs such as isatis root to reduce the dependence and use time of antibiotics.
At present, there are different views on antibiotics at home and abroad. In foreign countries, hospitals often do not prescribe antibiotics to patients with a cold and fever. They believe that antibiotics are not necessary to treat minor illnesses and can be solved by drinking more hot water. In China, in the case of only a cold and fever, in order to quickly relieve symptoms, The hospital will use antibiotics to relieve symptoms

Regarding the development prospects of antibiotics, Director Lu believes that due to the emergence of drug-resistant bacteria, the bacteria of Nakiyanxinen, Krebsiella, will infect organisms. These bacteria are difficult to kill and easy to mutate. It is difficult for humans to completely suppress them.

The answer is positive. There are many ways to produce antimicrobial peptides, like microbial extraction and chemical synthesis,. But antimicrobial peptides (from bioengineering) are different from chemical classes.

There are three drugs: Dolin ointment, which has the ability of healing; Sulfate, which mainly for intestinal infections resistant to other drugs; intestinal infections, which are for systemic infection.
Since the drug resistance becomes serious, cephalosporins lose their efficiency. The use of antimicrobial peptides is strictly limited, for instance, polymyxins, general hospitals do not have the right to use polymyxins. Only those with advanced support have the right to use polymyxins .
Besides, Polymyxin (only for severe infections)used in plant animals, mold resistance, and veterinary drugs. Also, feed additives are given to them. The current concept is not only the concept of antibacterial drugs, but also can improve immunity and enhance antiviral ability, which has gone beyond the scope of ordinary antibiotics. Advantages included: spectrum antibacterial, antifungal, Antiviral. Polymyxin covers all functions, accelerates wound healing, is very good against viruses, anti-HBV, and inhibits the replication of HBV.
Director Lu attaches great importance to the application of antimicrobial peptides. If it can be widely used clinically, it will have a subversive effect on cancer. there is a Dharma phase effect, which means good cells and bad cells are killed. Antibacterial peptides only kill the harmful cells of the human body, and are good for the good cells of the human body, also they have no effect on normal cells, and kill cancer cells. Chemotherapy and radiation therapy for cancer, because current drugs can also harm normal and abnormal drugs. Take existing medicines to yourself and kill cancer cells.

(1) The extraction of antimicrobial peptides will not be used in large quantities due to the resource limit
(2) Lessons learned from the abuse of antibiotics, antimicrobial peptides are only the last trump card, and the existing antibiotics are ineffective as the last defense. It can only be used in anticancer drugs, pathogenic bacteria, sepsis, etc. It will be used when it is clinically important

In the clinical trial stage,antimicrobial peptides has no effect on normal cells. Compared with antibiotics, The advantage of antimicrobial peptides are natural active substances, mostly composed of 20-60 amino acids, including antibacterial, antifungal, and antibacterial viruses and other different roles are ideal alternatives to antibiotics

At the beginning of the project, we interviewed Professor Wang, who is currently working at the Shanghai Institute of Biological Sciences, about the current status and prospects of antimicrobial peptides. Before the interview, we were worried about the current development of antimicrobial peptides and wondered what our team could do to achieve the purpose of promoting market development.

At present, antimicrobial peptides are mainly used in animal breeding, even in the pharmaceutical industry, there are studies on antimicrobial peptides, but the current research level is not enough. He noted that antimicrobial peptides are rarely used in other industries, suggesting that their use in fish purification environments is rare. As for the development of antibacterial peptides in China, Professor Wang suggested starting from the basic principles of antibacterial peptides.

As for how synthetic biology can be applied to the study of antimicrobial peptides and how to produce them, Wang pointed out that to extract them from plants and animals, production efficiency needs to be considered and how to efficiently synthesize proteins with living organisms. When studying fish tanks, Wang suggests using combinations because these antimicrobial peptides can be used to kill bacteria in more situations.

For Pepsick's project, Professor Wang said, for the feasibility of our project temporarily did not think of the problem, currently feasible, in order to enhance our products, we need to choose a variety of antimicrobial peptides, can target a variety of classes of viruses bacteria, etc.

In more scenes of application, so as to have better results, and some constructive suggestions on our product transportation packaging, etc. [made into powder form easier to transport and preserve, protein temperature can not be too high (will deactivate, easy to degrade) recommended low temperature cold chain transport (-20 degrees), we have to control costs, keep room temperature transport, 4 degrees cold storage transport (recommended lyophilization, powder)], and positioning the product as a high-end commodity level.

[1] Chen Y , Wu J , Cheng H , et al. Anti-infective Effects of a Fish-Derived Antimicrobial Peptide Against Drug-Resistant Bacteria and Its Synergistic Effects With Antibiotic[J]. Frontiers in Microbiology, 2020, 11:602412.
[2] Shabir U , Ali S , Magray A R , et al. Fish antimicrobial peptides (AMP's) as essential and promising molecular therapeutic agents: A review[J]. Microbial Pathogenesis, 2018, 114:50-56.
[3] Antimicrobial peptides: recent insights on biotechnological interventions and future perspectives.