Team:IISER Berhampur/Human Practices
Human Practices
Inspiration
A major part of fighting a disease is having adequate knowledge and awareness about it. It is inevitably clear from widespread false news and unscientific myths surrounding the COVID-19 Pandemic, that not only a cure but also correct information and awareness about symptoms and procedures goes a long way to help out in saving lives. Similarly, for a disease like Dengue, it is equally essential to reach the masses with correct and appropriate information along with new techniques and innovative therapeutic approaches. So, for us, the human practices and outreach component of iGEM was as important as the project itself. And this could have been achieved substantially through science communication and outreach activities including awareness programs. This was something we had decided even before the pandemic forced us to rethink our project component. But after the pandemic, this aspect became even more essential as we could see all around us how misinformation can cost lives and spread unwarranted fear and panic. During this unprecedented year, it has been challenging for almost all the teams to physically reach out to the public and speak of the problem they are trying to mitigate. Thus, most of our activities were conducted online. The world scenario for Dengue is one of the sole reasons for choosing our project. Read on further to know how we have overcome this challenge of social awareness in the era of social distancing.
Even after days of brainstorming and research, our project was lacking a major constituent, namely, the input of experts and stakeholders. So we reached out to a number of scientists working in the field of Dengue and other infectious diseases, drug development and tried to understand each and every aspect of our strategy and review changes required. We also made a sincere effort to integrate them into our project accordingly. The integrated human practices are highlighted. We followed the rule of Triple I’s.
Meeting with Prof. Sudhir Krishna and Team
Introduction :
Prof. Sudhir Krishna and his team members Dr. Meenakshi Iyer and Dr. Arun Sankaradoss from National Center For Biological Sciences, Bengaluru are working on the strains of Dengue that are endemic to India, in search of an effective vaccine. We reached out to them to understand the reason why we do not have a successful vaccine against Dengue.
They have worked before on NS1 and NS2 proteins, and were looking to extend their work on the other NS Proteins as well. They have sequenced genomes from four regions of India: Mumbai, Delhi, Jodhpur and Bangalore to get a wider understanding of the genome and its mutations in order to develop a viable vaccine. We discussed our project with them and we received several suggestions from them.
Insights :
1- After appreciating our approach towards looking for peptide inhibitors, they suggested that small molecule inhibitors could also be looked at.
2- They told us to look into the replication complex, as a potential target, with NS4 being the centre of it.
3- As for designing the peptides, they encouraged us to perform the MD simulations and even offered the prospect of a possible future collaboration in the MDS aspect.
4- In case we did not end up getting a good score from protein docking, they suggested we look into possible modifications like adding certain molecules and groups to increase the success rate and make the interactions more stable, by increasing the binding energy. Overall, it was a productive meeting that enabled us to understand the improvements that we could make to various aspects of our project.
Integration :
1- Our Dry lab members looked at the possibility of incorporating small molecule inhibitors and after reviewing we came to the conclusion that small molecules will have certain off-target effects. Hence, it gave more clarity and drive to our project.
2- We incorporated MD simulations into our modelling component.
Meeting with Dr. Vinay Bulusu
Introduction :
Dr. Vinay Bulusu is an Assistant Professor at IISER Berhampur. His research interests mainly focus on protein engineering and cancer biology. We approached him as he has experience working with FRET, which is a core part of our proposed reporter system.
We began the meeting with a thorough yet concise description of our project. Several relevant and thought-provoking questions were asked. The answers to these questions helped us iron out some small discrepancies in our project idea.
Insights :
Dr. Bulusu then went on to explain how to choose a suitable FRET pair for our project. To optimize our reporter system, we must choose the best possible placement of the fluorophore and CID tags. Regarding this, there ensued a lengthy discussion.
Integration :
After viewing our docked structure of the DENV NS5 and hSTAT2 dimer, Dr. Bulusu helped us decide the appropriate termini at which to attach our tags. As a result of the discussion, we were also able to devise appropriate control experiments which would help normalize the FRET results and give us a reasonably accurate result concerning the strength of the protein-protein interaction and the efficiency of the peptide inhibitor.
Ultimately our meeting with Dr. Bulusu was very constructive. It helped us understand the FRET system better and refine our project idea. The insights were useful in designing the plasmids by tagging YFP to DENV NS-5 protein and CFP to the hSTAT2 in different orders with respect to FRB and FKBP, and creating controls for the same.
Meeting with Dr. Sunita Patel
Introduction :
Our project revolves around the study of various protein-protein interactions. Given the adverse situation the pandemic has brought about, we had to resort to verifying our proposed system by employing computer simulations. On this account, we approached Dr. Sunita Patel, an expert on Molecular Dynamics Simulations for protein folding and interactions.
Insights :
A thorough description of our project idea induced a didactic discussion regarding the use of Molecular Dynamic Simulations to validate and improve our reporter system. Dr. Sunita brought to our notice the hardware and software required for running these resource-intensive simulations. We received advice to implement our simulations using the open-source molecular dynamics package: GROMACS. Being novices in the field, Dr. Sunita suggested several tutorials for us to acquaint with the GROMACS commands; and understand the principles governing MDS.
Integration :
The team was assigned tasks with the motive of learning and preparing the prerequisites for the MD simulations. After we were done with all the tasks given by her, we went ahead with presenting to her what we learnt, which helped Dr. Sunita know how much we have understood the science behind molecular dynamics and are we now capable enough to go ahead with the actual MDS using GROMACS or not. Finally, after the presentation, she found our knowledge satisfactory to start with the MDS. We carried out MDS step by step and Dr. Sunita helped us troubleshoot all the errors we encountered while running the code. After carrying out all the steps successfully under the guidance of Dr. Sunita, finally got the desired results and our next step was to analyse them. In the analysis also Dr. Sunita was always with us; supporting and guiding in interpreting all the results that we had obtained. She not only advised us about possible plots for analysis but also gave us an insight into what the plots essentially portray. She also did not step back to help us in reviewing our final documentation and correct our errors in the document.
Meeting with Dr. Biswa Ranjan Meher
Introduction :
Dr. Meher has expertise in Computational Biology and Bioinformatics and his research area focus on molecular modelling and simulations of biomolecules of therapeutic importance, structure based drug and vaccine design, pharmacologically bioactive natural products, and application of computational tools to solve biological problems.
Insights :
The meeting began with a presentation of our project. Interaction of FKBP and FRB was discussed elaborately with him. He suggested to have cases for interaction with Rapamycin and without Rapamycin, and simulate by Molecular Dynamics Simulations (MDS). We discussed the effect of iPEP on DENV NS-5 and hSTAT2 by MDS. Taking many components together in a process may give erroneous results so it was decided to proceed with taking individual components separately. We then explained our previous modelling plan to him. Being new to the field of MDS, he acquainted us with the same by giving a brief introduction about it and software to be used. He appreciated the plan to be executed.
Interview with Dr. Gulam Hussain Syed
Introduction :
Dr. Gulam is a molecular virologist and works on the characterisation of virus-host interactions on the interface of mitochondrial processes. His primary research work also covers the understanding of viral life cycle and to identify viral and host protein interactions and the processes involved in virus propagation.
Insights :
The team approached him to get a better understanding of the life cycle of the dengue virus and how the proposed PPI is vital in the life cycle of the virus. We began the meeting with a brief introduction to our project and the reporter system that we plan to design. Dr. Gulam Syed gave us a very detailed insight on how the pathway that we intend to target works, which supported our point and gave us a boost as to why targeting the hSTAT-NS5 pathway for its PPI can be a promising intervention strategy. Several questions were also mentioned about the FKBP-FRB system which is a very crucial part of the reporter system, and he also suggested some necessary modifications that can help us in improvising the reporter system so that the native protein structures do not get compromised. Though the wet lab part of the project couldn't be executed this year, he also suggested some methodologies (like in vitro studies along with transfection in HEK cell lines) by which they can be carried forward further.
Apart from that, he also gave us an excellent understanding of why there haven't been any vaccines yet against dengue virus, even though it costs thousands of lives every year. For that concept, he explained to us how dengue virus being an RNA virus develops mutants and as a result, any vaccine designed against it wouldn't be effective for long. He also explained to us as to why choosing conserved domains/motifs, or conserved pathways are important for any kind of drug development initiative.
The interview ended with us asking him about how the COVID-19 pandemic has changed the way scientists are now tackling various virus-based pathogens. For that, Dr. Gulam helped us understand how the process of understanding any new virus works: by understanding its mechanism that contributes to the disease pathogenesis and how these processes should be understood at the cellular biology level, using animal models and also analyses of the clinical data.
The meeting gave us an idea about the pathogenesis pathway and how our reporter system can be of aid to researchers in accelerating the whole process of drug discovery and synthesis.
Meeting with Dr Puja Singh
Introduction :
We met with Dr. Puja Singh to discuss the challenges we might face while implementing our wet lab ideas, especially with our Chemically Induced Dimerization module, since she has had experience working with CID systems in subcellular targeting.
Insights :
Her suggestions included the incorporation of controls to check the localisation and expression patterns of our proteins of interest in mammalian cells. If one of the proteins has more affinity to certain cellular regions, the probability of them coming together to interact might get affected, and this was, therefore, one of the factors we should consider.
She also proposed using transduction as an alternative to introducing the constructs into our chassis. With CID systems, transfection is not always very reliable and can generate a lot of background, affecting the results. Virus based systems like lentiviruses or adenoviruses for transduction may offer better confidence in generating stable cell lines and helping in a controlled expression of both the proteins. Transfection may generate heterogeneity as not all cells will take up the plasmids uniformly. Because we are doing a co-transfection of two different plasmid constructs, the expression level of the two proteins is an important aspect that we will need to consider, otherwise, there might be biases.
Rather than using two different plasmids, if it was possible to make them in the same one with a linker, was another possibility we explored. The two constructs, in this way, would get cleaved in the cell after being expressed together, conferring more control over the expression level.
Finally, Dr. Puja conveyed that since we are not studying the native interactions, and enforcing the proteins to come together, the quantification of iPEP efficiency might not be reliable as it will have to overcome not only our PPI but also the powerful FKBP-FRB interaction to give a measurable readout. For screening peptide drugs, doing some co-localisation of co-immunoprecipitation based studies using only proteins of interest was an alternative. However, these are time-consuming and FRET can give an immediate readout instead.
Integration :
Based on these inputs, we revisited our design and revised our strategy as follows:
1. Clone constructs into E.coli.
2. Transfection to mammalian cells.
3. Electroporation for increased transfection efficiency.
4. Lentiviral system for constructs that show reliable results from above strategies.
Interview with Dr. Rindu Raveendran, Assistant Professor at DM WIMS medical college, Wayanad
Our first interview was with Dr. Rindu Raveendran, who works at the grass-root level of communities where Dengue infection is quite rampant. This interview helped us in understanding the disease from a medical professional's perspective, the challenges they face and their strategy.
Insights on the spread of Dengue
He stated that Wayanad, being a rural area is dominated by vector-borne diseases like Dengue and Leptospirosis in the monsoon. But this year, because of the pandemic, the incidence has taken a back seat. Usually, the state of Kerala gets two peaks for dengue, one in August-September and the other in January- February.
Since their local medical intervention strategies are quite good, there is effective control over the spread and early detection for Dengue is possible as diagnostic tests are available at every level. Local people use papaya leaves and stem bark as first aid for the infection, which is actually not scientifically proven, and as an ill effect, people have suffered from kidney failures.
Diagnosis and Treatment
Characteristics of Dengue that distinguish it from other viral diseases are high-grade fever and severe body ache. It is diagnosed by mainly two types of tests: Rapid Dengue Test, which is an antibody test, and qRT-PCR, which is a confirmational test. Sometimes the symptoms are very mild, so the patients are supplied with only hydrates. Only about 5 to 10 % of the total patients go to severe complications which includes Dengue Haemorrhagic Fever, marked by loss of body fluids from capillaries of lungs or abdomen. Most of the cases are cured, only a small percent of the Dengue Haemorrhagic Fever can be life-threatening. Second-time infection is observed rarely but is generally very severe.
Survey on Awareness and Spread of Dengue Fever in India
After the Interview with Dr. Rindu, we realised that not everyone would know about these different stages of Dengue, and hence we decided to start a survey to know where the common people stood in terms of their awareness of the potential health hazard. So we conducted a survey among people in India to check their awareness about Dengue fever and the Government initiatives to prevent its spread. We circulated the survey through our social media platforms. We obtained 730 responses of which about 92.5% people admitted to being aware of the Dengue fever and around 65% of the respondents asserted that they were taking preventive measures against the infection. The distribution of the responses for our survey was biased towards certain states. However, it was able to capture a modest glimpse of the situation in the country.
It was interesting to notice from the survey that around 36% of the respondents were indeed not aware of the Government initiatives taken for the prevention of Dengue. 33% of the respondents were from the state Odisha and hence to know about the various existing Government programs in the state, we got in touch with the District Malaria Officer (DMO) of Ganjam.
Dr. R Jagadeesh Pattnaik, DMO, Ganjam, Odisha
Being the District Malaria Officer, Dr. Jagadeesh is in charge of all the activities conducted for the prevention of infectious diseases in the district. We were really fortunate that he was able to give us some of his time amidst the hectic pandemic.
Insights on dealing with Dengue
He informed us that the post-monsoon time of the year is the peak time for the infection. This year due to COVID-19, the cases of dengue have drastically dropped in the district of Ganjam. He also pointed out that the patients first get tested for COVID-19 whenever they are brought in for any check-up, resulting in fewer dengue tests performed too (30-35 in number), whereas last year it averaged around 1500 tests out of which 70-80 turned out to be ‘+’ ve. He also clarified that Aedes mosquitoes are not easily found in the rural or hilly areas but rather in urban and suburban areas.
He also stated that Dengue spreads very fast if the breeding sites are not destroyed. The use of outdoor open containers to store water, which is generally done due to scarcity of water in parts of India (Odisha), plays a significant part in setting up the breeding sites. Marriage functions and parties where the dumped garbage is not taken care of also acts as a breeding site. However, this was avoided this year due to the COVID-19 restrictions.
He informed us that stagnant water from ponds that are not frequently cleaned and found around the city do not act as breeding sites The Berhampur Municipal Corporation looks after the cleanliness of ponds in general but those are not considered as breeding sites because either the pond is disturbed daily by locals or is far too dirty for the larvae to grow since the oxygen levels are low and due to other factors.
Activities organised by the Dengue control board for prevention
16th of May is celebrated as National Dengue Prevention Day and July being National anti-Dengue month, they start a series of activities led by Aanganwadi workers, health workers and other medical staff to search and destroy breeding sites of mosquito by doing door to door survey in every village, during which the people are made aware about essential information about the viral disease. The board also boasts about MDD- Malaria Dengue and Diarrhoea Campaign, which supervises these activities every year to be carried out enthusiastically. During the same period, students and teachers are encouraged to conduct activities such as literary competitions and rallies to make the society aware of the disease. This year’s campaign was merged with COVID-19 prevention activities.
Personal steps that are taken to prevent the disease
Knowing the behaviour of the vector is most important, it bites during dusk and dawn which can be avoided by closing doors and windows at the same time, wearing full-sleeved clothes, and the use of mosquito repellents and nets. Water storage practices should be closely monitored, since if the water container is not cleaned and the walls not scrubbed, it can lead to a peculiar case i.e., if the eggs were already infected by the virus and they were not scrubbed off while cleaning the containers, whenever the eggs come in contact with water (after few months) it will hatch and would already be carrying the virus and hence just changing the water proves to be useless. Going from a prone/ vulnerable area to a non-vulnerable area, one must take care of hygiene and make sure that he/she is not being a passive carrier of the disease. It is suggested for the benefit of all that the patient stays inside the nets all the time.
Understanding the different facets of health programs conducted by the government, we chalked out different levels at which we can interact with the public, make them aware, and also educate them about the problem.
Since the district authorities have been focusing on the COVID-19 control measures, we offered to assist with Dengue Awareness in prone areas.
We started our first level of awareness programs in the form of flyers, posters, and pamphlets on the prevention of mosquito-borne diseases and the various symptoms of dengue to look out for. These were spread out through our social media platforms at different time points of our project.
Survey conducted by CCU
From the results shared by CCU iGEM, we gained a little more insight as to how much the public knows about Dengue fever, in a country outside our own.
A total of 248 responses were recorded by iGEM CCU Taiwan team out of which nearly 98% of the respondents were from Taiwan itself and the remaining 2% represented people from other nationalities. About 5.6% of the respondents or their families had been infected by dengue fever which gave us an insight about the frequency of occurrence of dengue in Taiwan as compared to India. As for knowledge about its severity in Taiwan, about 32.4% of the respondents asserted that it is ‘quite serious.’ A majority of the population, around 54.3% thought that Dengue had ‘average’ severity.
Most of the respondents were well aware of the mode of transmission of dengue (around 97.6%). And with this knowledge, 27.5% of the people claimed they were in a setting, exposed to dengue fever.
Even though the majority of the people were aware of Dengue not having any vaccines till now, around 38.2% of the respondents were unaware about any current or past vaccines and 15.2% of the respondents believed that there was a vaccine available for it.
Keeping these statistics in mind, we felt that there was a dire need to educate the public about drug development and the situation of vaccines in case of infectious diseases like dengue besides just the disease itself. With this aim in mind, in collaboration with CCU, we now set about to create a GLOBAL survey form, that along with giving us insights about public knowledge, would also in a way help to educate them about Dengue and the nuances and steps involved in actually preparing antiviral drugs for these diseases.
Global Survey
Our global survey received close to 300 responses from around twenty countries all over the world. The maximum respondents were from India- 71%, but we also received significant responses from countries like Peru, Mexico, Greece, Taiwan and Puerto Rico. When asked to rate the severity of dengue out of 10, most people rated it from 7 to 10, which implies they were well aware of the danger and risks that dengue can pose. Yet, when it came to knowledge about treatments, only around 80(~26%) people were aware of all treatment strategies available, that is, IV fluids, fever medication and blood transfusion. Around 60 of them had no idea about any of these. The rest only had heard about some of them. 43.9% did not know that dengue did not have an antiviral drug yet and almost 80% underestimated the time required to actually produce a viable drug, which is more than nine years. So clearly, the current public knowledge about the various facets of antiviral drug development and the causes for such long delays in producing viable therapeutics is quite limited. So, our aim to educate the public about drug development strategies was further strengthened by these responses themselves.
After these background questions, the next section of the form focused on trying to raise awareness about the challenges faced by researchers working in drug development. We provided a list of details about this and then went on to explain how our project FRaPPe would help in improving and accelerating the current scenario. Thus, the survey form not only allowed us to glean how much the general population knows about antiviral drug production but also enabled us to convey some essential information about this field and how our project would come of use, in the long run. When asked whether they thought our iGEM project would be helpful, 97.4% responded "Yes".
There was also a section where iGEM CCU from Taiwan, with whom we collaborated on this survey, posed a few questions about Dengue detection and what the common man would be looking for in a detection kit since they are working on building one.
Both us and the CCU Taiwan, through this survey, have gained some truly valuable insights into our respective projects. Finally, we asked whether they too learnt anything new through the survey, 92% said "Yes". So, this survey and our collaboration has gone a long way in educating the public about Dengue, its detection and the avenues in antiviral drug research.
Crowd Funding
The interaction with Dr. R Jagadeesh (D.M.O, Ganjam) also helped us to know of certain sections of the society which are extremely vulnerable to mosquito-borne diseases due to their living conditions.
There are several communities living in mosquito prone and low-resource areas, which are frequently affected by vector-borne diseases. A lack of awareness, hygiene and healthcare amenities aggravates the situation.
To extend a helping hand to the less privileged sections of the society in and around our institute, we initiated a crowdfunding campaign to raise funds for providing mosquito nets to those people.
Fortunately, we were able to raise around INR 50,000 (680 USD) in 45 days for our cause. We distributed mosquito nets, face masks and awareness pamphlets in local language to around 70 families in and around Berhampur. The purchase of all the mosquito nets were from local craftsmen who earn their living by making the nets and with this association we believe the fund provided to us by the well wishers of our campaign was put to good use.
The activity was conducted in strict adherence to the COVID-19 protocols. We are really grateful to people who supported our campaign. It is because of them that we were able to spread smiles among needy people.
©iGEM IISER Berhampur
