Team:Jilin China/Human Practices

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Human Practices
Preliminary Brainstorming
At the beginning of brainstorming, there were several research topics available, such as the treatment of vaginitis, the improvement of plant resistance and killing Beauveria bassiana. We expected to choose the most meaningful project that could be turned into a product.
Killing Beauveria Bassiana
Plant Resistance Improvement
Vaginitis Treatment
A lecture on Women's Health on March 8
Our team members attended a lecture on women's health on March 8. We found that there were many problems and troubles in the process of women's treatment. So we think it would be a quite meaningful project to study women's health.
Questionnaire
In order to understand the incidence of vaginitis and the prevalence of the various types of vaginitis, we conducted a nationwide questionnaire survey and received more than 1700 feedback questionnaires from all parts of the country.
Data show that more than 74% of those who have suffered from gynecological diseases have suffered from vaginitis. Of all vaginitis, vulvovaginal candidiasis (VVC) is the most prevalent.
Research Plan of VVC
After defining the research direction of vulvovaginal candidiasis, we decided to choose vulvovaginal candidiasis as our research subject. We intended to base our project on the current treatment methods and problems of vulvovaginal candidiasis. Therefore, we interviewed doctors and surveyed patients to get a full picture of this disease.
Current treatment situation
For the professional advice and actual situcation about this disease, we make the medical specialist and patient interviews to get the information we need.
Doctor interview
Zongxiang Leng, PhD, MD
Deputy Dean of Jilin Maternal and Child Health Hospital, Gynaecologist.
Process
Dr.leng have rich experience in treating VVC,we asked him about the disease.Dr. Leng told us that the major types of vaginitis have changed over the past few years.Trichomonas vaginitis used to be the leading variety of this disease. Nowadays, vulvovaginal candidiasis accounts for a relatively large population of vaginitis patients. He also mentioned that in the current treatment methods, patients are treated mainly with antifungal drugs. However, such treatment could easily cause recurrence. In addition, there were certain adverse reactions during the treatment.
Patient interview
We came to the outpatient department of the Second Hospital of Jilin University and interviewed some patients. They told us that there were side effects during the process of treatment. They also complained about its long treatment cycle and high likelihood of recurrence. They expected there would be new therapy methods.
Initial Design
With the shortcomings of the current treatment methods for VVC in mind, we conducted a mid-term brainstorming aiming at the problems of recurrence and adverse reactions, then we put forward initial design:
Sensing system
Therapeutic system
① -BDSF
② -Msp1
③ -β-1,3-Glucanase
④ - Nad1
Suicide switch
The polish of design
Exprimental promotion
Expert Consultant
Hongyu Xiang, PhD
Professor, School of Life Sciences, Jilin University, Microbiology, Microbial and Biochemical Pharmacy specialist.
Process
We introduced our project to Dr. Xiang. When Dr. Xiang knew that Nad1 antimicrobial peptide had not successfully expressed in engineered bacteria in the previous design, he suggested that we should continue to search for more literature on Nad1 expression or try to look for anthor antibacterial peptide which has been successfully expressed to replace Nad1. In addition, Dr. Xiang suggested that in the subsequent vector selection, we'd better choose a secretory expression vector to enable the enzymes in the therapeutic system to secrete extracellularly to function.
Improvement
1. We chose the antibacterial peptide LL-37, which has already expressed in the engineered bacteria to replace Nad1.

2. We selected the secretory expression vector pVE5523 as the destination vector.
Yinqiu Cui, PhD
Professor, Vice-Dean of College of Life Sciences, Jilin University, Molecular biology expert.
Process
In order to sense the presence of C. albicans, we designed the sensing system, which can detect the 4-HPA secreted by C. albicans, and then activate the PBC promoter to initiate transcription of various parts of the therapeutic system. However, while searching for the sequence of the PBC promoter, we found that there were much unintentional sequences between the start of the PBC promoter and the end of the previous gene. So we considered truncating the middle part of the sequence and asked Dr. Cui about this idea. Dr. Cui expressed appreciation for our design ideas but reminded us: "It is very likely that changing the sequence in front of the promoter may affect the normal start of the promoter." Therefore, she recommended that we keep all the sequences and do not delete them arbitrarily.
Improvement
1. We abandoned the idea of truncating the sequence in the front of the promoter.

2. We decided to change the promoter in the treatment system to a constitutive promoter so that we could easily conduct experiment to verify individual parts.
Comprehensive evaluation
Design determination
After interviewing two professors, our design has been greatly improved according to the proposal from Dr. Cui and Dr. Xiang. The design is as follows:
Sensing system
Therapeutic system
① -BDSF
② -Msp1
③ -β-1,3-Glucanase
④ - LL-37
Suicide switch
Therefore, we hope our project can be comprehensively evaluated from ethics, experimental safety and other aspects.
Overall evaluation
Feng Gao, PhD, MD
Professor, Director of National Engineering Laboratory for AIDS Vaccine, College of Life Sciences, Jilin University, Immunologist.
Process
Our project was basically completed and we hoped to find an expert to evaluate our project. Therefore, we asked Dr. Gao, an expert in the field, to give us some suggestions. We explained our project and presented the project book. Dr. Gao thought that our project was very innovative and close to people's lives. To enhance our project, he suggested that we'd better go to the local hospital and relevant places to collect the opinions of patient groups, which would make the project more practical and persuasive.
Improvement
1. We prepared to make the medicine into a vaginal capsule or vaginal gel by using a probioties preparation process. Engineered bacteria will exert their function in the vaginal environment and kill C. albicans for therapeutic purposes.

2. We have planned a series of interviews with pharmaceutical companies.
Xianghui Yu, PhD
Professor and Dean of School of Life Sciences, Jilin University, Biochemist.
Process
Because of the particularity of vulvovaginal candidiasis and C. albicans, after the establishment of the project system, we consulted Dr. Yu on project safety and ethics. In terms of safety, Dr. Yu reminded us to pay attention to the biological safety level of fungi. When cultivating C. albicans, we should always pay attention to the experimental operation specifications and the follow-up treatment of experimental materials. Both AIDS and vaginitis are closely related to sexual life. Dr. Yu mainly engaged in AIDS vaccine research. She reminded us to pay attention to the ethical aspect and suggested that we contact the experts of the Ethics Committee to get more professional advice.
Improvement
1. We prepared to make the medicine into a vaginal capsule or vaginal gel by using a Probioties preparation process. Engineered bacteria will exert their function in the vaginal environment and kill C. albicans for therapeutic purposes.

2. We have planned a series of interviews with pharmaceutical companies
Safety Operation Regulation
Since our experiment involves fungi, we need stricter laboratory safety regulations. With the assistance of laboratory safety experts, the laboratory safety specifications have been formulated and all the experimenters have been informed. Otherwise, the experiment will not be carried out. Dr. Jiang and Dr.Chen helped us with our regulations. Under the guidance of Dr. Jiang and Dr.Chen, we consulted the relevant laws of China. Organisms used in our project are commonly and widely used strains, so their safety are thus guaranteed. Click here for more information.
Ethics Consideration
Yanyan Li, PhD
Chief pharmacist, first Hospital of Jilin University, Ethics expert.
Process
Dr. Li first told us that biotherapy involves more and more complex ethical issues than traditional medication. Our project has transformed a natural bacteria. Dr. Li reminded us to clarify the safety issues of the project and submit relevant documents during the subsequent verification process. At the same time, in the process of treating vaginitis, it is necessary to inform the patients and their families of the novelty of this treatment and get their approval. For instance,pregnancy and sexual intercourse are not allowed throughout the treatment cycle.Finally, Dr. Li recommended that we go and learn about a type of cough capsule for treatment that shares similar mechanism as our project
Application perspective
We hope that our project will be applied in the future, so we have established interviews with pharmacies,Pharmaceutical Enterprises，and doctor to make our project more practical.
Pharmacy interview
Process
In order to understand the medicines for vaginitis in the current market, we interviewed dozens of pharmacies in Jilin, Yongsen and Yida pharmacies. We learned that there are two main types of medicines for vaginitis: oral and external use, such as fluconazole, terbinafine, Baofukang suppository and Ke. Myrazole and other drugs. However, oral drugs will cause some damage to the liver and the balance of vaginal flora. While using these drugs, there will be side effects like occasional local irritation, itching or burning sensation. In addition, Lactobacillus vaginal capsule is welcomed by patients and doctors. We introduced our project to pharmacists, and they said they are looking forward to our project.
Therepeutic evaluation
Tianmin Xu, PhD, MD
Deputy Director of Gynecology and Obstetrics, Second Hospital of Jilin University, Gynecologist.
Process
After we interviewed the pharmacy, we learned that there are many Lactobacillus vaginal capsules are better sold in the market for the treatment of VVC. So we contacted Dr Xu to evaluate our design by comparing with the common drugs. Dr. Xu told us that there were many mechanisms of fungal resistance to antifungal drugs at present, including drugs blocked at the cell wall level, reduced drug absorption or damage of drug and fungal targets. Current antifungal drugs usually have a single target for fungi, so Lactobacillus vaginal capsules are superior to antifungal drugs in terms of drug resistance. However, from the clinical effect, the treatment effect of Lactobacillus vaginal capsules is not ideal. The main reason is that the viability of probiotics in capsules is unknown in the vaginal microenvironment at 37 ℃, so the pharmacodynamics may not be obvious. For our project, Dr. Xu said that our method was a comprehensive approach to this disease, and would provide a new direction for the treatment of VVC.
Pharmaceutical Enterprises interview
Process
In order to make our project have more practical application value and make our verification more convincing, we contacted Minhang Factory of Shanghai Xinya Pharmaceutical Industry, which is a pharmaceutical factory specializing in the production of antifungal drugs. Zhijie Gu, our advisor, came to the pharmaceutical factory for an interview. Ding Shang, the general manager of the pharmaceutical factory, received us. In addition, we exchanged views with the technical personnel of the pharmaceutical factory. For the safety verification part of our experiment, technicians suggested that we used the bacteriostatic circle experiment, which provides us with a new idea. In order to verify whether the C. albicans was killed, we added a report-protein control experiment. In addition, in the process of communicating with the general manager of commerce, he suggested that we should compare our project with traditional health care products and medicines to reflect our advantages. At the same time, Ding Shang advised us to do experiments according to the relevant batches to meet the national standards.
Improvement
1. We added the experimental verification part of bacteriostasis circle.

2. We have adjusted our experimental proof scheme according to the relevant national batches.