Team:IISER TVM/HP IHP

IGEM-IISERTVM

Human Practices


Integrated Human Practices

Understanding the Problem

Fungal diseases have been on a rise with over 1 billion cases of fungal infections every year. Invasive fungal infections (IFIs) result in approximately 1.7 billion deaths per year making it one of the most vicious diseases in the current scenario.[1] During the Covid-19 pandemic, there was a rapid increase in the IFI cases worldwide, especially in our country, India. IFIs also carry a 60-90% mortality rate among all ICU patients. Although fungal infections have been on the rise, it wasn't until the wake of Covid-19 that this issue was brought to the limelight. Even before the pandemic, fungal infections had have been on a rise due to increasing usage of medical devices such as catheters or cardiac valves (formation of biofilms), immunosuppressants and organ transplants.There are currently five classes of antifungal drugs such as Azoles, Polyenes, Echinocandins etc. Most of these antifungal drugs target similar pathways involving ergosterol biosynthesis. Tackling these infections has proven difficult due to a limited repertoire of antifungal drugs, their costs, drug interactions, the routes of administration, and side effects like nephrotoxicity. Furthermore, the rampant use of these limited drugs raises the alarming possibility of developing drug-resistant fungal strains.


Aware of this issue, our team at IISER-TVM set out to develop a solution to alleviate the suffering of millions of people across the world. We embarked on a journey to understand the underlying principles of fungal infections and tried to address the root cause of the issue. Almost immediately, the stark inadequacy of existing antifungal drugs was glaringly apparent. Existing therapeutics not only foreshadow the emergence of drug-resistant fungal strains, as proven by many studies but may also have significant cytotoxic and nephrotoxic effects 8


Our efforts culminated in the development of "Moldemort," a novel antifungal agent, which we believe holds the potential to reduce the global burden of invasive fungal infections significantly.

Designing the Solution

Once we understood our problem, we conducted in-depth literature studies to formulate recombinant chitinases. We laid out the experimental plans planned out through intensive brainstorming sessions. To better understand and assess the problem, the team met various experts from diverse fields and significant stakeholders.


The expert interactions we conducted greatly influenced our experimental plan. Our parallel interaction with stakeholders gave us insights into the potential broader impact of our project and methods to address the gaps between what society wanted and what we had. Furthermore, we shifted the project’s initial goal of tackling fungal infestation on walls to develop a novel antifungal therapeutic solution solely based on our stakeholders’ inputs. Additionally, we added BC2-our fourth recombinant chitinase combo, to our initial list of three. This gene was purely designed against Rhizopus spp., which is the causative agent of Mucormycosis.


Further studies on this matter led us to realize that India has a relatively higher load of invasive fungal infections among the affected Asian population. The need for affordable and effective antifungal drugs with minimum side effects dramatically increased the relevance of our project, particularly in the Indian scenario.


Our human practices can be broadly divided into two categories, namely expert interactions and stakeholder interactions.


Expert Interactions:


  1. Prof. M. R. N. Murthy and Prof. H. S. Savithri (Expert:Wetlab)

    Designation:

    Prof. M. R. N. Murthy, Distinguished Professor and AstraZeneca Chair, Indian Institute of Bioinformatics and Applied Biotechnology, Bengaluru

    Prof. H. S. Savithri,Faculty, Department of Biochemistry, Indian Institute of Science, Bengaluru


    “It is not only bacteria that produce chitinases, certain plants naturally produce chitinases too.”

    Prof. M.R.N. Murthy is a renowned crystallographer who retired as a professor of molecular biophysics at the Indian Institute of Science, Bangalore. He is currently the Distinguished Professor and AstraZeneca Chair at the Institute of Bioinformatics and Applied Biotechnology. His research interests include protein structure and functions, X-ray crystallography and virus structure and assembly. Prof H. S. Savithri is a member of the biochemistry faculty at the Indian Institute of Science, Bangalore. She works on plant virology, enzymology and protein chemistry. We had an opportunity to interact with them during the initial phase of our project.


    They were inevitable during the initial stages of the project. They advised us to work with recombinant plant chitinases along with the bacterial recombinant chitinase genes we had already designed. They also enlightened us about the advantage of using endochitinases over exochitinases as the former can lyse the substrate at several locations unlike the latter. They also stressed the importance of choosing an enzyme that would work at room temperature in a neutral pH range.



    Know more:


    Status of project before the discussion:

    1. We chose bacterial chitinase based on our literature review/studies.
    2. We were confused regarding the choice of exo and endo chitinases.
    3. Domains were not connected using linkers.
    4. The focus of the project was solely on black mould on walls.

    Suggestions:

    1. Domains could be connected using linkers to preserve the activity of individual domains.
    2. Check about plant chitinases also.[Many plants use their own chitinases to resist fungal infections.
    3. Consider temperature and pH-dependent activity of chitinases
    4. The cutoff weight of the protein constructs should be taken into account because larger proteins are difficult to fold and express.
    5. Choice of endo-chitinases over exo-chitinases would be better as the former attack the substrate at several locations and hence can provide better antifungal proteins
    6. The project could be expanded to prevent fungal contamination in laboratory settings.

    Status of the project after incorporation of suggestions:

    1. Domains were connected using linkers.
    2. Shifted the attention from bacterial chitinases to both bacterial and plant chitinases.
    3. The temperature and pH activity of enzymes were reviewed and chosen in such a way that they are active at room temperature and within a neutral pH range.
    4. The cutoff size was decided to be at 100kDa.
    5. Endo-chitinases was chosen over exo-chitinases.

  2. Prof. J. N. Moorthy(Expert: Project Design)

    Designation:Prof. J. N. Moorthy Director, Indian Institute of Science Education and Research, Thiruvananthapuram



    “How broad is ‘broad-spectrum’?”

    Initially, our project aimed to develop an antifungal in the form of a broad-spectrum chitinase. We hypothesized that our recombinant chitinases would have the ability to be effective against a broad range of fungal species, considering that chitin is an essential component of fungal cell walls. However, we did not have any strict definition of the species we would be targeting.


    Our interaction with Prof. Moorthy made us rethink our goals. We then decided to streamline our project to a more specific target. Therefore, we focused on obtaining a recombinant chitinase with a higher chitinolytic activity than the wildtypes.




    Know more:

    Status of project before the discussion:

    1. Wanted to make a master chitinase, effective against all fungi i.e, target species of our master chitinase was vague.

    Suggestions:

    1. What advantages does our chitinase have to offer over the currently available chemical antifungals/fungicides?
    2. How broad is “broad-spectrum”?

    Status of the project after incorporation of suggestions:


    1. Master chitinase to the broad spectrum
    2. Defined “broad” in broad-spectrum i.e., specified what all species our chitinase will target. Species: Penicillium, Aspergillus niger , Aspergillus flavus, Fusarium, Rhizopus, Cladosporium.
    3. Decided to analyze the side effects and mode of action of current antifungals available in the market.

  3. Dr. Binod Parameswaran(Expert: Wetlab)

    “Your project can have a greater impact on the community. Why not try a different track?”

    Dr Binod Parameswaran is a Principal Scientist at the National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala. His lab primarily focuses on various industrial applications of microbial enzymes such as biocatalysts, biofuels, production of biopolymers etc. He had also worked extensively on chitinases during his Master’s Degree. The team had numerous interactions with him regarding the project and its applications in the real world. He has also helped us in troubleshooting various experimental aspects of our chitinases.


    Initially, our project aimed to remove the black mould thriving on walls using our recombinant chitinases. However, Dr Binod briefed us about the challenges we could face during the external application of our recombinant chitinases, out of which the enzyme stability in an external environment seemed to be of great concern. He had also suggested looking at the therapeutic or agriculture applications for our project.


    His expertise in working with chitinases helped us in choosing E. coli as our chassis and was instrumental in designing the experimental setup for antifungal assays.


    Know more:



    1st Interaction:


    Status of project before the discussion:


    1. The team had designed 4 different recombinant chitinases that include domains from various plants and bacteria.
    2. The primary application of our chitinase was to remove moulds from walls. There were some ideas of using it as a therapeutic drug or as a biocontrol agent in Agriculture.
    3. If a therapeutic drug was the choice, we planned on using nanoparticles as a drug delivery vehicle.

    Suggestions:


    1. Using only enzymes on walls had several challenges like:
      • Enzyme optimum temperature and pH might not be maintained.
      • Enzyme loses stability quickly.
      • The cost of production would be very high.
      • The protein solution might induce the growth of some other microbes.
      • The enzyme solution might have non-specific activity (affect insects).
    2. Some types of stabilizers (polysaccharides) could be added like sorbitol or starch to increase the shelf life of our protein.
    3. Could look into further applications like
      • Therapeutic Drug with the proper delivery mechanism.
      • Biocontrol agent in Agriculture.
      • As an ointment or anti-dandruff product.
    4. If planning on a therapeutic drug, we need to account for the immune response, the localization of the product and the cost of developing it.
      • Using magnetic nanoparticles to allow localization of a drug to the target site.
      • Using a cocktail of enzymes to make it more effective.
    5. If the idea is still to be used on walls, then we can think about using the engineered organism to produce our enzyme upon induction.
      • The organism can be incorporated into bricks or paints.
    6. For expressing bacterial chitinases, E. coli is a good choice whereas Eukaryotic chitinases are best produced in Eukaryotic cell lines like Pichia pastoris.

    Status of the project after incorporation of suggestions:


    1. We decided to focus more on the interior walls of hospitals to protect the enzyme from the extreme conditions outside.
    2. Began looking further into the idea of using chitinase as a therapeutic drug and its delivery using nanoparticles.

    2nd Interaction:


    Status of project before the discussion:


    1. The team had made progress on successfully cloning one of the proposed recombinant proteins.
    2. A crude design of the upcoming experimental assay was made but a lot of doubts regarding the method to be used arose. Hence a second meeting was required with Dr. Binod to clarify the doubts.

    Suggestions:


    1. Suggested to use DNS (3,5-Dinitrosalicylic acid) assay which was a common and conventional method to measure chitinase activity.
    2. While conducting temperature and pH assay of the enzyme, it is important to note that,
      • The optimum pH of most bacterial and plant chitinases are within 5-9
      • The optimum temperature of most bacterial and plant chitinases are within 30°C-60 °C
    3. While performing the Zone of inhibition, temperature and pH assay, the crude enzyme could also be used instead of purified protein.
    4. Explained in detail how to perform a Zone of inhibition assay.

    Status of the project after incorporation of suggestions:


    1. The DNS method was chosen to measure the chitinase activity of our enzyme.
    2. A zone of inhibition assay was carried out with crude enzyme first.

    3rd Interaction:


    Status of project before the discussion:


    1. The team was facing difficulties carrying out the assays with the crude enzyme.
    2. Could not estimate activity using DNS assay.
    3. Colloidal chitin solution was precipitating.

    Suggestions:


    1. Dr. Binod reviewed our method of experiment and asked us to change the preparation of colloidal chitin.
    2. A water bath should be used instead of an incubator as the heating would be uniform.
    3. The moisture content of Colloidal chitin could be determined without drying the entire product.
    4. Zone of inhibition to be conducted with spore suspension instead of inoculum.
    5. Adding stabilizers (like sorbitol, starch) to increase the stability of our enzyme.
    6. Provided us with a formula to calculate Enzyme units from OD540 absorbance data.

    Status of the project after incorporation of suggestions:


    1. Colloidal chitin was prepared again using the modified method.
    2. Water baths were used instead of incubators to heat the solutions.
    3. A zone of inhibition was performed with spore suspensions as well.
    4. Calculated Enzyme Activity in units from the OD540 absorbance data.


  4. Dr. Robin Allshire(Expert:Project design)


    Designation:Dr. Robin Allshire, Wellcome Principal Research Fellow and Professor of Chromosome Biology, University of Edinburgh


    “Targeting multiple molecules in the fungal cell wall at the same time would be a more efficient way to solve the issue of antifungal resistance.”

    Robin Allshire is a Wellcome Principal Research Fellow and Professor of Chromosome Biology at the University of Edinburgh. His research focuses on epigenetic inheritance: establishment and transmission of specialized chromatin domains. He discovered an epigenetic mechanism that allows fungi to develop resistance to antifungal drugs without alterations to their DNA, which helps understand how pathogenic fungi become resistant to the limited number of available antifungals agents in clinical and agricultural areas.


    Dr. Allshire enlightened us with the emergence of antifungal resistance and the consequent need for new antifungal agents. As a result, we wanted to ensure that our recombinant chitinases would help resolve this issue. An exact solution cannot be designed without rigorous laboratory experiments; however, we could conduct studies on antifungal resistance in 96-well plates. The cell can evolve mechanisms that increase the production of chitin and hence if the rate of formation of chitin equals the rate of degradation of chitin by our chimeric chitinase, then the fungi can develop resistance against our enzyme. Moreover, targeting multiple molecules in the fungal cell wall (glucan and chitin) simultaneously can increase the success percentage of the antifungal activity of the drug.


    Know more:


    Status of the project before the discussion:



    1. We aimed to target only chitin in the fungal cell wall.

    2. We didn’t know how to check for antifungal resistance.


    Suggestions:


    1. Targeting multiple molecules in the fungal cell wall such as glucan and chitin at the same time can decrease the chances of antifungal resistance.
    2. We can check for antifungal resistance using 96-well plates.

    Status of the project after incorporation of suggestions:


    1. We planned to make a cocktail of glucanases along with chitinases in a later stage of our project.


  5. Dr. Hariprasad P. (Expert:Safety)



    Designation: Dr. Hariprasad P.,Associate Professor, Centre for Rural Development and Technology, Indian Institute of Technology, Delhi


    “Why prepare your fungal culture media in petri plates? Make the media slant by preparing them in test tubes.”

    Dr. Hariprasad P is an Associate Professor, at the Centre for Rural Development and Technology, Indian Institute of Technology, Delhi. He had worked on fungal samples as a part of his research career. We got his expert advice on how to handle fungus safely derived from environmental origin.


    Dr. Hariprasad’s experience in working with fungal samples proved to be of great help to us. It was after his suggestion that we started preparing fungal cultures in a slant medium of Potato Dextrose Agar. Additionally, he was able to provide us with authoritative resources, which was a great success to identify and classify fungal species under different risk groups.


    Know more:


    Status of project before the discussion:


    1. Status of project before the discussion:
    2. Confusion regarding Biosafety Cabinet to handle fungal samples.
    3. Confusion regarding the procedure for inducing the wheat chitinase secretion in plants, using fungi.

    Suggestions:


    1. Using slant Potato Dextrose Agar (PDA) prevents the spreading of spores even while working in a biosafety cabinet.
    2. The biosafety cabinet of class II type A2 equipped with a HEPA filter is good enough to work with fungi belonging to risk groups 1 and 2.
    3. Suggested some sources to look upon the risk group of fungi found on the wall.
    4. He suggested spraying the spore suspension to induce wheat Chitinase secretion and also explained the protocol for making spore suspension.

    Status of the project after incorporation of suggestions:


    1. Fungal cultures are made in slant PDA for identification and assays.
    2. The wheat sample induced with spore suspension showed high chitinase secretion.


  6. Dr. Rachit Agarwal( Expert:Proposed Implementation)


    Designation:,Dr. Rachit Agarwal, Assistant Professor, Indian Institute of Science, Bengaluru


    “Considering the difficulty in manufacturing capsules, why don’t you try nanoencapsulation?”

    Dr Rachit Agarwal is an assistant professor at the Centre for Biosystems Science and Engineering, IISc Bangalore. His lab focuses on using a wide range of tools and concepts such as nano and micro particles, hydrogels, scaffolds and 3D organoids to generate treatment for diseases like tuberculosis and osteoarthritis. His insights were highly valuable to design an efficient drug-delivery system for the therapeutic we were developing.


    Our project would be successful only if it happens to be of any use to the common man. Our initial idea was to engineer a hollow capsule that would carry our recombinant chitinase to the human body. Dr. Agarwal’s expertise in nano-particle drug-delivery systems proved to be of great help to us. He stated that nano-encapsulation of our recombinant chitinase would be an easier and efficient technique than a capsule. His advice of using Poly Lactic-co-Glycolic Acid (PLGA) as the nano-polymer was incorporated into our project as it would elicit a very minimal immune response. Furthermore, he also stressed the importance of using stabilizers like Trehalose for increased shelf-life of the drug.


    Moreover, if we would want to expand our project to the realm of dermatological fungal infections, hydrogels would be a better approach than ointments and have an added benefit of not requiring stabilizers.



    Know more:


    Status of project before the discussion:


    1. Initial idea was to engineer a hollow capsule for the delivery of our recombinant chitinase inside the human body.
    2. If our recombinant chitinase is effective we thought to formulate an ointment for superficial skin infections in the future.
    3. We planned on using NIR [near infrared] light triggered drug delivery.

    Suggestions:


    1. Hollow capsules are difficult to manufacture.
    2. It would be better to formulate hydrogels than ointments because they don’t require stabilisers.
    3. Unsterilised nano vectors can host pathogens that can lead to infections and other associated problems in our body upon its administration.
    4. If the drug is designed to target lung tissue then NIR will not be of much use as the depth of penetration of NIR will be less compared to the thickness of lung tissue.
    5. Even though most of the drugs do not take into account active targeting during their manufacture, it is still a better option to use it. Active targeting is able to significantly increase the quantity of drug delivered to the target site compared to free drug or passively targeted nanosystems. This is achieved through the decoration of the nanocarrier surfaces with receptors (in our case dectin-1) binding to the beta-glucans found on fungal cell walls.

    Status of the project after incorporation of suggestions:


    1. We decided to use PLGA as the potential polymer for our nano-particle based drug delivery due to its ability to elicit a minimal immune response.
    2. We read more about active targeting and decided to include Dectin-1, a mammalian innate immune receptor on the surface of our nanocarrier.
    3. We plan to use hydrogels instead of ointments once our project expands.
    4. We dropped the idea of using NIR light-triggered drug delivery.


  7. Dr. Ravindra Ghooi (Expert:Proposed Implementation)


    Designation:Dr. Ravindra Ghooi, Director, Scientia Clinical Services


    “You can independently commercialize the antifungal drug you are developing; need not approach a pharmaceutical company.”

    Dr. Ravindra Ghooi was the Director of Scientia Clinical Services for the past 14 years. He was a professor at the Symbiosis School of Medical Sciences teaching and guiding students in Drug Development and Clinical Research. He has also worked as the DEAN of Bilcare Research Academy, an academy dedicated to Clinical Research Education. He was also the Vice President of CliniRx Research Pvt. Ltd. Having worked as a Medical Advisor in different Pharmaceuticals, his extensive knowledge in Clinical Research Education helped the team gain insights on marketing our product.


    Our interaction with Dr. Ghooi was primarily for us to learn about the procedures to commercialize the antifungal drug we were developing. His expertise in this area of clinical trials, market policies, and the explicit presentation of facts turned out to be really helpful for us. His suggestions were instrumental in devising a plan for implementing our project.



    Know more:


    Status of project before the discussion:


    1. We lacked a comprehensive understanding of the drug discovery process beyond the first steps; having a hypothesis and generating a new drug molecule.
    2. We had only a preliminary understanding about the classification of drug molecules, the definition of a new drug, various regulatory committees and bodies involved, different stages of preclinical and clinical trials, the regulatory and ethical approval required at each of these stages, regulations on the price of new drugs, timeline and cost of the drug discovery process, tie-ups with pharmaceutical companies, etc.

    Suggestions:


    1. The interaction with Dr. Ghooi opened our eyes to the practical side of drug development beyond the laboratory. He broadened our understanding of various topics, such as the ones mentioned earlier.
    2. We appreciated that most drugs take 12-15 years to reach the market, and millions of dollars are spent on their development.
    3. Thus, it becomes imperative to “see ahead of the curve” so to speak, and assess whether the drug is likely to be valuable in the future, more so than in the present.
    4. Dr Ghooi outlined a rough strategy for us to follow: a) decide which specific fungal species we are targeting,b) Identify incidences of infection by that particular species from medical literature to determine its prevalence, c) Identify existing drugs in the market and determine their annual sales from the IMS database; pay special attention to the rate at which the sales are going up/declining, d) Make a realistic comparison of our compound to the existing drugs on the market based on factors such as efficacy, safety, and price.
    5. He also suggested that we pay close attention to the nature of clinical trials which we need to follow: they are likely to differ from conventional drugs since we are targeting invasive fungal infections in immunocompromised patients.
    6. Dr Ghooi also directed us to explore the possibility of patenting our drug, and register our clinical trials on the clinical trials registry of India whenever applicable.

    Status of the project after incorporation of suggestions:


    1. Our project has become much more focused after interacting with Dr Ghooi. Now aware of the regulations in place for the development of new drugs, we have started working towards ensuring they will be met.
    2. Based on the activity of our recombinant enzymes, we have chosen species of Rhizopus, Aspergillus, and Candida to target.
    3. The antifungal activity of our recombinant enzymes will be contrasted against that of a commercial antifungal, Nipagin.
    4. A detailed literature review on the procedures to be followed for preclinical and clinical trials has been conducted; these have subsequently been included in our long term plans

  8. Dr. Harilal Madhavan (Expert:Survey)


    Designation:Dr. Harilal Madhavan, Ad-hoc Faculty (Humanities), Indian Institute of Science Education and Research, Thiruvananthapuram


    “People prefer short surveys, with more open-ended questions.”

    Harilal Madhavan is a development economist specialized in economic and interdisciplinary research on Asian indigenous medicines, health and pharmaceutical policies and intellectual property rights. Dr. Harilal helped us with numerous aspects of the project, especially the Human Practices department.


    Being a faculty of the Humanities department, he was able to guide us in framing the questions for the survey. He also helped us form the template that we followed for making all the surveys we released. As the world is becoming busier by every passing day, not everyone would find time to take part in surveys. Hence, it is important to include more objective questions, where the respondent wouldn’t have to think much before answering the questions. He also stressed on the importance of obtaining consent from the respondents before submitting the survey. He also helped us with structuring the handbook we made on the legal scenario of biotechnology in India.



    Know more:


    Status of project before the discussion:


    1. Our surveys contained more subjective questions than objective ones.
    2. Handbook lacked a proper framework.

    Suggestions:


    1. Include more objective and less time-consuming questions in the survey.
    2. He gave us insights about restructuring the handbook.

    Status of the project after incorporation of suggestions:


    1. We included objective questions in the survey than the earlier draft.
    2. We restructured the handbook by incorporating his suggestions.

  9. Dr. Subha C. C. (Expert:Legal Handbook)


    Designation:Dr. Subha C. C., Assistant Professor, School of Law, Christ University, Delhi NCR Campus


    “ 'Act' is not synonymous with 'policy' and 'rule.' ”

    Dr. Subha guided us throughout the process of making the handbook on the Indian legal scenario in biotechnology. She provided us with valuable resources to refer to and helped us validate the loopholes we identified in the legal provisions. She was able to teach us the difference between laws, acts, policies, rules, and bills, while we believed that all of them are the same.




    Know more:


    Status of project before the discussion:


    1. We used the words 'act', 'rule', 'policy', 'bill', synonymously.

    Suggestions:


    1. There is a difference between 'act', 'rule', 'policy' ,and 'bill'.

    Status of the project after incorporation of suggestions:


    1. We understood the proper usage of those words and rectified them in our handbook.

  10. Ms. Parvathy Suresh (Expert:Legal Handbook)


    Designation:Ms. Parvathy Suresh, Graduate student, National Law School


    “Giving sections of actual legal provisions will not serve your purpose. Write about them in your own words. “

    Parvathy was the first person to review the draft of our handbook on Indian biotechnological legal scenario. Back then, the content was redundant and we had given relevant sections of laws also in the handbook, which made the book unwantedly long and complex, which did not fulfill the purpose of our handbook. She also offered to connect us with various legal experts who could help us in this regard.



    Know more:


    Status of project before the discussion:


    1. The handbook was unwantedly long
    2. The book contained sections from laws and policies.
    3. It did not have a special section for iGEM teams

    Suggestions:


    1. Stick to a word limit, and reduce the length of each section.
    2. Remove the actual sections from laws, and briefly summarize them. You can provide links to the particular law if required.
    3. Add a special section for iGEM teams, so that more people can find it relatable.

    Status of the project after incorporation of suggestions:


    1. We consistently reduced the length of each section.
    2. We removed redundant content, especially when a particular legal provision came about in two chapters.
    3. We briefly summarized each legal provision
    4. We also added a special section - "What should you take care of as an iGEM team?" at the end of each chapter.


STAKEHOLDER INTERACTIONS



  1. Interaction with medical doctors

    1. Dr. Rashmi Mary Philip

      Designation:Dermatologist, Amala Institute of Medical Sciences, Thrissur, Kerala


      Dr. Rashmi Mary Philip is a consultant dermatologist and a Senior Resident in dermatology at Amala Institute of Medical Sciences, Thrissur. Dr. Rashmi’s proficiency and experience in dermatology became really useful to us. She was able to give us common names of the fungal infections seen so that it would become easier for us to understand if we had to talk to affected patients in the future.


      She discussed in detail about dermatophytes, and how they cause superficial fungal infections. Dermatophytes have the ability to invade keratinized tissues, such as the skin, hair, and nails. Classified on the site of infection, dermatophyte infections include tinea corporis (ringworm), tinea unguium (nail infection), tinea capitis (scalp ringworm), and tinea pedis (athlete's foot), among others. She also briefed us about different other fungal species affecting humans.


      She was genuinely impressed with our project idea but also suggested expanding it as a therapeutic because pathogenic fungal spores would persist in the environment even after removing fungi thriving on walls.


      She was genuinely impressed with our project idea but also suggested expanding it as a therapeutic because pathogenic fungal spores would persist in the environment even after removing fungi thriving on walls.


    2. Dr. Kiran N.

      Children are an important group of individuals affected by fungal infections. As a paediatrician, Dr. Kiran provided us with an overview of common fungal diseases affecting children.


      His department comes across children with topical fungal infections regularly. They are caused by dermatophytes. Immunocompromised children, especially those who have undergone solid or liquid organ transplants, are prone to life-threatening fungal infections. They are commonly infected by Candida spp., Aspergillus spp., and Mucorales. He discussed in detail the different types of infections caused by them, covering the following pointers:



      • Source of infection
      • Site of infection
      • The defense mechanism of the immune system
      • Prevalence in children and high-risk groups
      • Current antifungal treatment regimen and their side-effects
      • Antifungal resistance to existing drugs

      He also highlighted the prevalence of Candida infections in neonates and the prophylactic treatment given for its prevention. Also, fungal contamination in hospital settings, especially on ICU walls, can lead to severe infections in critical-care patients. Our team had identified Penicillium spp., Aspergillus spp., Cladosporium spp., and Fusarium spp. from the walls of our institute. He mentioned that other than Aspergillus spp., the other genera are not clinically significant and do not cause severe infections in the Indian Population.


      Dr. Kiran appreciated our idea of removing fungal infestations from walls, thereby reducing the concentration of harmful fungal spores in the atmosphere. However, he also stressed that fungal spores from other habitats of fungi would persist in the environment.


      The narrow range of antifungals available for the treatment compared to a wide range of antibiotics often leave doctors like him left with very few options to treat patients; ergo, the successful completion of our project would be helpful in the treatment of fungal infections.





    3. Dr. Sabu Stephen


      Being a general physician, Dr. Sabu Stephen was able to provide us with a general overview of fungal infections in various age groups. He talked to us extensively about superficial and systemic fungal infections. Dr. Stephen often deals with superficial fungal infections affecting the hair, skin, mucous membranes, and oral cavity.


      Systemic fungal infections are rare because normal immunocompetent individuals have the ability to defend themselves against fungi entering human organ systems. Therefore, immunocompromised, long-term ill, and individuals under corticosteroid treatment often fall prey to systemic fungal infections caused by Candida spp., Aspergillus spp., and Mucorales. He discussed in detail the following about fungal infections:


      • Types of infection
      • Sites of infection
      • Defense mechanisms of the body
      • Existing treatment methods
      • Side-effects of the existing treatment regimen
      • Antifungal resistance to current drugs

      The unresponsiveness of the Tinea versicolor against the imidazole drug ‘Clotrimazole’ is a clinical example of antifungal drug resistance, which he mentioned.


      India was witnessing an enormous soar in Mucormycosis infections as a post-Covid complication. He attributed this increase to the usage of steroids in the treatment of Covid. Moreover, pre-existing medical conditions like diabetes are also a factor.


      Removing fungal stains from walls using an eco-friendly solution sounded good to him. However, he was of the opinion that developing a drug would be more impactful as opportunistic fungal pathogens might already be present in the body as commensal organisms.



    4. Dr. Shinu

      Being a pulmonologist, Dr. Shinu was able to rightly describe fungal infections affecting lungs. He spoke to us about the following on fungi and fungal diseases -

      • Classification of fungi on a clinical basis
      • Types of fungal infections
      • Risk groups of various fungi
      • Diagnostic tools
      • Treatment regimens with side effects

      He was able to capture our attention by giving case studies of Aspergillus spp. causes allergic reactions and in extreme cases leads to Aspergilloma and invasive Aspergillosis. A major reason why the patients’ conditions deteriorate quickly and their recovery become difficult is due to the absence of proper diagnostic tests in the early stages of the disease. Also, the treatment of IFIs using existing antifungals may span several weeks, and patients facing a financial crisis may not be able to afford the treatment at times.


      He suggested we expand our project to developing a diagnostic tool as it was a major problem to be addressed while dealing with fungal infections. Even though we did not do much in this, we looked in the literature about the possibility of using chitinases as markers for fungal infection diagnostic tests. Since the initial studies sounded quite promising, we would explore this idea further in the future phase of our project.


      What did we learn from them?

      We learned that removing molds from walls will not reduce the fungal contamination in the atmosphere. The remnant fungal spores in the atmosphere can still lead to health complications in immunocompromised individuals. Manufacturing a therapeutic to fungal infections is a better solution than removing black mold from walls to prevent infections caused by them.


  2. Interaction with farmer


    Pathogenic fungi cause havoc in several kingdoms; the plant kingdom is not an exception to this. Rubber is a major crop cultivated in our state Kerala and hence is a major source of income for many families here. The sight of the spraying of fungicides in rubber plantations made us curious about the fungal infections in rubber trees. To know more about the diseases caused by fungi in plants, we contacted a local rubber farmer Gigi Kurian. He cultivates rubber, nutmeg, coffee and various other crops. From the conversation and a booklet he shared with us, we learned a lot about the diseases found in rubber.



    FUNGAL INFESTATION IN RUBBER: A FARMER’S PERSPECTIVE

    “Infestation by Phytophthora spp. causes rotting of fruits and abnormal leaf fall during the early monsoon period. We generally spray the Bordeaux mixture prior to monsoon to prevent infestation. However, the practice is often not appropriately done considering the labor cost and profit. Lack of treatment often leads to heavy loss in production. These days, oil-based copper oxychloride is substituting Bordeaux mixture. In nursery plants, during heavy monsoon, Phytophthora causes shoot rot. From January to March, mist leads to an outbreak of powdery mildew. Oidium haevea is thought to be responsible for this. Tender leaf edges roll inwards to fall, and old leaves show necrotic shots. The overall production of rubber trees decreases. To prevent the outbreak of powdery mildew, sulfur dust is sprayed along with early morning mist. Canker of the bark occurs in tapping channels in moist weather, leading to unnecessary loss of latex. During heavy rains, black rot occurs in renewed barks of tapping channels. Phytophthora and Pythium cause both these diseases. Disinfection of tapping channel with Mancozeb and Dithane is done weekly to prevent infection.”


    We could have dived deep into the issue, but several constraints held us back. We worked on fungal samples that we collected from the walls of our institute. We observed that these cultures did not contain plant pathogenic fungi. Therefore, it would have been challenging to test the activity of our recombinant chitinases. Furthermore, the stability of enzymes in external settings was a serious concern then. However, we plan to expand our project to the agricultural sector also.


  3. Literature Survey

    Due to the COVID-19 pandemic and data protection and privacy issues, it was very difficult for us to interview patients in person who are the major stakeholders of our project. In order to overcome this difficulty, we did literature surveys on studies done to analyse fungal infections in different medical conditions namely COVID-19, Renal diseases, Organ transplants, AIDS, Diabetes and those undergoing chemotherapy and radiotherapy who are the major victims of fungal infections.


    The summarised analysis of the survey is given in the below table and graph .


    Medical condition Most affected age group Most affected gender(Incidence % on average) Major Infections/Infectious sources
    COVID-19 50-75 Males (79.2 %) Aspergillus species causes 3/4th of the infections followed by Mucor and Rhizopus. 59% of Mucormycosis incidence was seen in Covid-19 patients
    Diabetes 55-62 Almost Equal (Males : 44.59 %; Females : 44.29%) Candida , Aspergillus and Fusarium species
    Renal diseases 36-53 Males (62.14%) except in case of CAPD patients where females are more prone to fungal infections Candida, Aspergillus and Mucor species
    Chemotherapy and radiotherapy 40-60 Males (76.5% ) Candida followed by Aspergillus species
    AIDS 30-40 Males (66.82) Candidiasis, Aspergillosis, Cryptococcus pneumonia
    Organ transplant 35-55 Males (67%) Aspergillus and Candida followed by Mucor species

    *Rough estimate from the research articles.


  4. Dear Fungi



    India celebrates the 19th of June as Reading Day every year. This celebration is marked in the memory and honor of P. N. Panicker, father of Kerala's Library and Literacy movement. As part of the occasion, we conducted 'Dear Fungi,' a letter-writing competition for which children between the ages of 11 and 16 ( mainly high school students) were required to write a letter to any fungus/fungi of their interest.


    Children are often affected by fungal infections, especially dermatological fungal infections. This makes them a principal class of stakeholders, and thus, educating them about different kinds of fungi also becomes essential. Taking into account the fact that most Indian education syllabi do not discuss fungi in their high school curriculum, the objectives of 'Dear Fungi' included:


    1. Educate the students about various kinds of fungi:
      • Edible and non-edible
      • Pathogenic and non-pathogenic
    2. Make the students able to distinguish between fungi and other lower microscopic organisms.

    We received 36 entries for the contest. As mentioned in the guidelines, the letters were addressed to a fungus/fungi of their choice. While some wrote about the havoc caused by fungi, others wrote about its benefits. The judgment criteria were the following:


    • Depth of knowledge about fungi
    • Writing style

    The winner was awarded 'Women in Science: 50 Fearless Pioneers Who Changed the World', a book authored by Rachel Ignotofsky which highlights notable women's contributions to STEM.


    Here are a few letters we received for the contest:



  5. DSoF: Dark Side of Fungi Series



    Event brochure:



    “Dark Side of Fungi” (DSoF), as the name suggests, aimed at educating the general public, one of our critical stakeholders, about the plight caused by fungi. Fungal diseases/infections are generally not an issue of prime importance, which justifies the lack of awareness programmes held in this regard. Moreover, the results of the survey we conducted during the initial stages of our project validated our understanding.


    DSoF mainly focused on fulfilling the below-mentioned objectives:


    1. Increase public awareness about the causes and types of fungal diseases, and also about methods to prevent the diseases.
    2. Understand the gravity of the issue of antifungal resistance.
    3. Elaborate on the difference/change Moldemort can bring about in this scenario.
    4. Demonstrate the relevance of our project.

    It is only when we present our problem in an explicit and clear-cut manner can the solution be understood better. Accordingly, the talks on the first two days of the series focused on highlighting the issue of fungal infections and antifungal resistance. A confab followed this on the third and final day about our project and how it is a novel and effective solution to the problems raised in the first two talks.


    The series was conducted in an online mode through Google Meet. We received an enormous 160 registrations ranging from people of 16 years to 50 years. However, the registered participants majorly fell under the age group of 20-25 years, i.e., undergraduate students. The talks were around 45-60 minutes long and were followed by a question-answer session. The question-answer session was also lively and all doubts raised were cleared by the speaker.


    Know more:


    Schedule:


    Day 1: 6:30 PM IST, Jul 30, 2021 Topic: Invasive fungal infections Speaker: Dr. Shareek P. S. Designation: Infectious disease specialist. MBBS, MD (General Medicine), FID. Sree Uthradom Thirunal (SUT) Hospital, Pattom, Thiruvananthapuram, Kerala
    Day 2: 6:30 PM IST, Jul 31, 2021 Topic: Increased antifungal resilience in the older generation of pathogenic fungal cells Speaker: Dr. Somanon Bhattacharya Designation: Postdoctoral Associate, Antifungal Drug Resistance, Stony Brook University
    Day 3: 6:30 PM IST, Aug 1, 2021 Topic: Project Presentation Speaker(s): iGEM team from IISER Thiruvananthapuram

  6. Interaction with care-taker

    “Distributing medicines through ART centers will ensure that they reach the people in need of it.”

    AIDS patients form a significant section of immunocompromised individuals and thereby are our direct stakeholders. However, directly contacting them was an issue because most NGOs and similar organizations would not share the contact details of their patients due to data privacy issues. This is precisely why we contacted Fr. Jeejo, the Director at Mar Kundukulam Memorial Research and Rehabilitation Centre. As the name suggests, the organization was established for the rehabilitation and care of terminally ill AIDS patients in 1999. Fr. Jeejo spoke to us on behalf of the AIDS patients at the center.


    Antiretroviral Therapy (ART) is available free of cost to anyone availing it. The ART therapy for patients is done in phases depending on their physical conditions and age. They have witnessed infections (or adverse reactions) in patients while shifting from one drug to another. The advancements in ART and constant follow-ups of the patients’ conditions by healthcare workers have improved the conditions of AIDS patients in younger generations. Transmission from affected mothers to babies has also reduced drastically.


    Moreover, the ART center is equipped with skin specialists, psychiatrists, and other medical professionals for the well-being of the patients. AIDS patients in their organization are well taken care of. Fungal infections in AIDS patients are reported at times, but timely treatment has not led to severe complications. He suggested distributing our antifungal drug through ART centers would help make sure that these medicines reach the right individuals.



  7. Interaction with animal microbiologist

    “Sell your drug at a lower prices, because generally people do not spend too much for treating animals.”

    Dr. Chintu comes across fungal infections in dogs and cats, and most of them are skin infections caused by dermatophytes. Even though subcutaneous and deep mycoses infections are found in animals, they are not commonly detected.


    Apart from dermatological infections, Aspergillosis is another fungal infection that commonly affects birds, especially poultry. It affects chicken during the cold season, due to improper hatching of eggs and less care given. Birds infected with Aspergillosis may cough or can have other respiratory distress. Other deep-seated and invasive fungal infections are present in animals but are not detected often due to our inability to diagnose without them showing any visible symptoms. Hence, they are revealed only during the post-mortem of deceased animals.


    Azoles are the most common drug used for the treatment of animals. However, there have been cases of unresponsiveness to antifungals due to antifungal resistance. Also, dogs and cats are more susceptible to fungal infections than other domestic animals.


    He suggested developing an ointment targeting dermatological infections in animals. Furthermore, the market price should be reasonably low, as people generally do not spend much for treating animals.



  8. Interaction with GAFFI (an NGO)



    The Global Action Fund for Fungal Infections (GAFFI) is the only international NGO focussed on raising awareness of and collecting worldwide data on fungal disease. Their mission is to reach the poor and vulnerable through access to rapid tests and antifungal drugs.


    Dr. Denning pointed out that our project was essential in the global scenario, mainly because of the terrible outbreak of Mucormycosis India witnessed. According to him, the reasons were many:


    • The existing therapeutics are not good enough and are administered late.
    • The current antifungal drugs have acquired resistance. Example: Candida spp. are developing resistance to azoles.
    • The patient is immunocompromised.
    • Inability to diagnose IFIs at an early stage.

    He gave us insights into how chitinases, being large molecules, can be allergenic and lead to asthma in some instances. This might raise regulatory concerns. We should also have a solution to the consequences of our nanoparticles crossing the blood-brain barrier and leading to meningitis or other inflammations. Dr. Denning introduced us to TPP (Target Product Profile) and gave us tips to make one for a drug.


  9. Human Subject Research

    Surveys are an integral part of iGEM projects. They serve as a convenient medium for understanding common public opinion. Like most iGEM teams, our team also required surveys to understand public interest and preferences, which helped shape our project. According to iGEM’s human subject research policies, teams are required to obtain approval from the Institutional Review Board (IRB) or Research Ethics Committee (REC) before performing any sort of non-invasive experiments on humans/human subject research.


    Prior to the public release of our surveys, we consulted with Dr. Harilal Madhavan, a health economist and a faculty member of our institute. He helped us in redesigning and reframing specific questions in our surveys. He also stressed the importance of including objective questions rather than open-ended and subjective ones. This survey was then subject to thorough scrutiny by our PIs and other senior authorities of our institute, following whose approval we released it to the general public.


    The initial description of the survey mentioned that the data collected would be solely used for record-keeping purposes and gathered the respondents’ consent through the survey. Moreover, the participants were free to withdraw from the survey anytime in between. Parents or guardians could fill the survey on behalf of minors, patients, or anyone who couldn’t attend the survey only with the individual’s prior consent.


    1. Survey on the incidence of fungal infections among people residing in Kerala

      This survey aims to check the incidence of fungal infections and their awareness among the general public in Kerala. The survey was solely for the residents of Kerala, as we wanted to analyze the prevalence of fungal infections in our local community. Since the survey was directed only towards the residents of Kerala, it was translated into Malayalam. Mrs. Ajitha K., a lecturer in Malayalam at Victoria College, Palakkad, Kerala, verified our translation.


      From analyzing the survey, we realized that most respondents were not aware of fungi and fungal infections. Since the respondents were from varying age groups, we conducted the following specific awareness programmes for each of them.

      • The World of Fungi - A book on fungi for children.
      • Dear Fungi - A letter-writing competition for children of ages between 11 and 16 years.
      • The Dark Side of Fungi - A talk series on fungi and fungal infections for the general public.

    2. Survey on Drug preferences of the public

      Our project aimed to develop a therapeutic solution to Invasive Fungal Infections. Before being marketed as a therapeutic, it was necessary to understand public opinion about the same. Therefore, the purpose of this survey was to analyze the public’s preferences in the medicines they use. The survey was open to people all over the world.



      The image clearly shows that most people preferred a chemical/protein-based drug over a GMO. Moldemort is also therapeutic, which will be marketed as a protein-based drug.


      The detailed analysis of the survey can be found here: Survey Analysis


    3. World of Fungi

      Children are an important group of stakeholders of the antifungal therapeutic we are developing. Therefore, it is necessary to spread awareness among children about different fungi, specifically pathogenic and non-pathogenic fungi. ‘The World of Fungi’ is a fun and illustrious book curated by the team to educate children, especially children below 11 years, about the same. The outreach department of our team went through several children’s books. ‘Alice in Wonderland’ was a massive inspiration to us, and we thought of introducing several species of fungi in an imaginary fungal forest.



      Features of the book:


      1. Target audience: The book is mainly targeted at kids aged 7 to 10 years old. Dermatophyte infections are common in kids, and hence educating them regarding the same is essential to prevent such infections in them.
      2. Illustrations: Fungi species are introduced as attractive illustrations to gather the attention of kids. Visual representations of concepts often enable kids to understand them better.
      3. Nature of language: The book has been written in easy language to understand it better. We tried our very best to keep our content short and informative.
      4. Games: Books with just content alone might be boring to kids. We included games to increase their interest further.
      5. Q&A sections: This segment was included to improve the critical thinking skills of kids further. The questions were framed to enable the kid to think and answer them by understanding the concepts introduced in the book.
      6. Safety guidelines: This section explains methods to prevent and control common fungal infections. This will help kids to remain healthy and protect them from harmful fungi.

      Testimonials:


      “I liked the book very much. Fungi are pretty to see. I loved it”, ~Ameya T P, Fourth Grader, PGMMLP School, Kallai



      “ I read the informative booklet named “The World of Fungi” published by iGEM IISER TVM team. The book is written in a simple language so that children from class 2 to class 5 can easily comprehend the contents. The book is highly informative for children With attractive illustrations. I am appreciating the huge efforts taken by the team to bring out the book”-Jayalakshmi C J, Head Mistress, PGMMLP School, Kallai


      Here are the images of “The World Of Fungi” book interactive section filled out by the kids.

      Children’s feedback:


      Daniel:




      Mesheal :



Legal Landscape of Biotechnology in India


'Legal Landscape of Biotechnology in India' is a compendium of Indian biotechnology laws and International protocols of which India is a signatory, along with gaps in them aimed at novice scientists/researchers in India. Furthermore, it can serve as a reference/guide for first-time iGEM teams of the country. This handbook is a collaborative effort of the iGEM IISER_TVM and IISER-Tirupati_India.


As a first-time iGEM team and the first from our state, we faced our fair share of obstacles and hurdles to commence our work. During those times, the Human Practices handbook by iGEM Stockholm 2017 served as a guide and provided a foundation on where we could work. Thinking on similar lines, the idea of our handbook arose.


Being students of science, most of our knowledge was restricted to STEM alone. As a result, the handbook initially aimed at simply compiling the existing synthetic biology laws in the country. Our instructor, Ankit Kumar Pradhan, proposed to bring a different perspective by looking for loopholes/gaps in the laws we identify. Keeping this in mind, along with the iGEM team of IISER Tirupati, we identified 14 relevant biotechnology laws of the country and classified them into four categories, namely:


  • Animal Experimentation and Clinical Trials
  • Export-Import Policies
  • Environmental Safety
  • Biocontainment and Biowarfare

Each category corresponds to each chapter which contains relevant laws related to the topic and briefly summarizes each of them. It is followed by the gaps/loopholes we identified for the laws. Furthermore, each chapter concludes with a few directions for future Indian iGEM teams.


During the making of the book, we understood that most of the laws in 'Biocontainment and Biowarfare' overlapped with those in the other three sections. Hence, we decided to omit the last section, but its components would be appropriately available in the other three sections. Moreover, the augmentation of relevant international protocols and treaties of which India is a signatory has also helped us in gaining a broader idea of the legal system of the country.


This handbook does not have any section particularly related to Intellectual Property Rights, Patenting Rules, Biowarfare etc. We have not deliberately obliterated those sections. iGEM teams in the future can work on making a second volume to the book.


The present state of the handbook would not have been possible without the help of several experts. The inputs from individuals in numerous fields helped in shaping the handbook.


  • Dr. Harilal Madhavan, School of Humanities, IISER Thiruvananthapuram, helped us during the initial stages of the making.
  • Ms. Parvathy, a graduate of National Law School, helped us a great deal by restructuring and refining the framework of the handbook.
  • Dr. Subha C. C., Assistant Professor, School of Law, Christ University, Delhi NCR campus, guided us in validating the loopholes we identified for each law/rule.
  • Dr. Satish Khurana, Assistant Professor, School of Biology, IISER Thiruvananthapuram.
  • Dr. Poonam Thakur, Assistant Professor, School of Biology, IISER Thiruvananthapuram.
  • Dr. V. Stalin Raj, Associate Professor, School of Biology, IISER Thiruvananthapuram.
  • Dr. Ullasa Kodandaramaiah, Associate Professor, School of Biology, IISER Thiruvananthapuram.
  • Dr. Jishy Varghese, Assistant Professor, School of Biology, IISER Thiruvananthapuram.
  • Mr. Sudin Babu, Deputy Registrar, Purchase & Stores, IISER Thiruvananthapuram.


Human Practices: The Conclusion

Each advancement in the project was a result of in-depth literature studies and several interactions we had among ourselves, with experts and stakeholders. Human Practices are inevitable in any iGEM project because this is where the developers interact with society.


The various interactions we held helped us discover several sides to the problem we aimed to tackle. Human practices aided us in understanding the broader impact of our project and the potential adverse effects our project can have on society. This highlights the dual nature of the project,i.e., we significantly contribute to its development by possibly trying or making efforts to improve further and reduce the harm. Being from a scientific background, solely focussing on the subject matters was a usual routine for us. We were entirely new to the market regulations of our country. We understood the importance of data protection, privacy, and consent while conducting research.


We missed out on several stakeholders due to the time constraints and the Covid scenario in the country. Our project is still under development, and we hope to meet various experts and stakeholders we have not reached out to in the future phases.