Our Stakeholders
Dr.Bryan Brensinger
Background
Dr. Brensinger is a molecular biologist at JHU APL with expertise in projects involving development of a paper-based diagnostic for point-of-care use using RNA toeholds in a cell-free system. He is a primary performer for all droplet microfluidics projects, including DARPA SBIRs, CDC projects, and IRADs. He has demonstrated the ability to successfully infect cells with Influenza A virus in droplets. Bryan has also performed lab work across dozens of projects, including mammalian cell culture, virology, molecular biology, protein purification, microbiology, microfluidic chip fabrication, and others. He has trained new staff across multiple projects, as well as assisted interns for their projects while at APL.
Questions
1. How can we optimize the readout of multiple color outputs in a visually interpretative manner, given the limited resources available in point-of-care settings? 2. Are there any specific challenges associated with using urine samples for miRNA-based diagnostics, Or even Synthetic Urine? And how can we optimize the detection sensitivity in this context?
Takeaway
We learned after meeting with Dr. Brensinger the importance of fully modularizing every aspect of the assay, first showing the toehold trigger reaction is working, and having some output like GFP, RFP, that is detectable visually and through PCR. By confirming each step is independently working, then it is easier to figure out where the problem is. The question then became if RFP and GFP were going to be effective for us? If you produce enough of it it will be visible, and it needs to be fluorescent (only downside). Another approach for lateral flow devices, they use antibodies that are against something that is there, antibodies can be against anything so if we use GFP or RFP, the antibodies can be against the GFP and RFP. So you can directly detect RFP which is indirectly detecting the miRNAs which will tell you if they were there. Moving on we had questions about using urine samples for miRNA- based diagnostics and were curious about the best approach in order to optimize the detection sensitivity. Bryan recommended starting as simple as possible, using water at various dilutions, once it is working well he recommended trying it with urine. In the end it will depend heavily on the assay itself. He also told us to be wary about what will be in the urine, for example a urine sample that contains a lot of glucose, a result from someone who had diabetes, may affect the detection of miRNAs. Bryan also recommended us to in vitro transcription and to try to determine what we need our results to be and then look at how to get there.
Mr.Will Stone
Background
Mr. Will Stone is a molecular biologist at JHU APL with expertise in synthetic plant engineering, protein engineering, and biochemistry.
Questions
1.What are the key considerations when working with biomarkers in diagnostic assays, and how can we ensure the specificity and sensitivity of the detection method? 2.What are the critical factors to consider when designing a multiplexed detection system, and how can we ensure reliable and accurate results? 3.How can we optimize the readout of multiple color outputs in a visually interpretable manner, given the limited resources available in point-of-care settings? 4.What aspects of the miRNA-based colorimetric assay can be effectively modeled computationally, and how can these models aid in optimizing the tool's performance?
Takeaway
After meeting with Will Stone, we learned how to work with biomarkers while ensuring the specificity and sensitivity of the specific detection method. This, we found, could be done by buying synthesized/purified RNAs, which could be used to validate assays and toeholds. He also suggested we use existing literature to assist us with this. Furthermore, after testing, we could get iteratively closer to the desired goal. An alternative would be to put RNA into a stimulant, such as urine, to see if we could achieve the desired result. We then considered the design of a multiplexed detection system, and how we could ensure reliable and accurate results. We were advised to make sure the non-multiplexed system had no cross-talk before we tested each system on the target. We could then cross-reference the data from the multiplexed system with data from a non-multiplexed system. Next, we wanted to know how we could optimize the readout of multiple color outputs in an interpretable manner, given the limited resources available in our prospective point-of-care settings. We learned that we could use microfluid architecture to spatially separate distinct assays on the same kit. By putting the sample in one point and allowing it to flow through, the sample would reach different toeholds on different sides of the paper, allowing us to visualize results in an understandable manner. The next question we asked Dr. Stone was what aspects of the miRNA-based colorimetric assay could be effectively modeled with computational methods, and how we could use the models to aid in optimizing the performance of the actual tool. We were told that due to miRNA being specifically sequenced, computational modeling could be used to determine how big of a complement we would need for the RNA toehold to change structure. We could then do further biophysical modeling. In specific relation to our project, we could look at the miRNAs present in urine and test the RNA toeholds to see how much complementarity exists. Dr. Stone suggested that we include our source of the biomarkers to elaborate on the specificity, sensitivity, and coupling of miRNAs to enhance the communication of our methods. He also recommended that we start our in vitro protein synthesis tests with a commercially available test kit so that we can be sure that the final product can be replicable using market-available products. Finally, we should include a slide in the final product that discusses these methods and the tests we conducted at length in order to maximize our project’s delivery.
Dr. Jenna Mueller
Background
Dr. Jenna Mueller is a biomedical engineer who has experience with creating screening devices for cervical pre-cancers. The research that Dr. Muller primarily performs in her lab is the development of devices that can be used for managing cervical cancer.
Questions
1.What would be your best advice, as we will soon be presenting our research and its significance, for us to keep in mind while speaking on difficult subjects? 2.In your Ted Talk, you mentioned the importance of developing a device to diagnose patients in middle and low-income communities. Based on your knowledge of the logistics related to solving this problem in order to help people, do you believe our device could serve this purpose to realistically solve this problem? 3.From your background knowledge on getting biomedical devices to these countries, what do you believe the likelihood could be for us getting our device into the hands of the healthcare workers there? 4.Can you explain how routine screenings, such as Pap tests and HPV tests, play a role in the early detection of cervical cancer? 5.In low and middle income countries, not everyone has access to quality medical attention. How would women know to get tested for cervical cancer before it progresses? 6.What should we be doing with our project now to ensure that we can implement this into actual people?
Takeaways
To proceed with our project, Dr. Mueller suggested we generate a concept device that is capable of accurately detecting the difference between cancerous and benign cells. The device must also be able to differentiate cancer from pre-cancers. To begin testing, she suggested we find collaborators that can help us meet our objectives as the regulatory burdens surrounding the collection of urine prevents us from obtaining samples. To overcome this, she suggested we speak with nearby clinics to generate IRB protocols and get approval between UMD and the clinic. Once approved, we can then start urine sample collection and run it through our test. We will compare the results of the test with a standard from a clinically accepted test such as the pap smear. We will avoid using HPV as a standard because it is only capable of indicating infection. Approximately 80% of women who are screened for precancer and are asymptomatic either do not seek treatment or do not have access to treatment. Our project will reduce the issue of accessibility by creating a highly accessible and accurate screening device. This is especially important since the existing methods for identifying cervical cancer, like pap smears, are uncomfortable and in lower/middle income countries there are sometimes fewer female doctors or qualified technicians in general, presenting a barrier for women to access these services. Dr. Mueller noted that it is important to communicate effectively when talking about the project, for example by using the correct language. When presenting, it should be made clear whether cancer and precancer is being referred to, to avoid unnecessary confusion. Also, the project should be known as a screening tool, not a diagnostic. It is also key that we summarize how and why we decided to use miRNA.
Sharmista Mohapatra
Background
Sharmistha is the Cancer Screening Programs Unit Manager at the Center for Cancer Prevention and Control at the Maryland Department of Health and she was able to give us more insight on testing in a clinical setting and the process for cervical cancer screening and diagnosis.
Questions
1. Can you provide an overview of the cervical cancer screening programs currently offered in Maryland? 2. What are the recommended cervical cancer screening tests for women in Maryland, and at what ages should they start and how often should they be conducted? 3. What efforts are in place to raise awareness about the importance of cervical cancer screening among underserved communities in Maryland? 4. Can you explain the role of primary care physicians and gynecologists in cervical cancer screening, and how can they be encouraged to promote regular screenings? 5. What resources and support are available for healthcare providers to help them educate and counsel patients about cervical cancer screening? 6.Are there any specific challenges or barriers that healthcare providers face in promoting and delivering cervical cancer screening services, and what solutions are being explored to address these challenges? 7. What role could a device like the one proposed by UMaryland iGEM (us) play in addressing barriers to cervical cancer screening in Maryland, especially in underserved communities?
Takeaway
After meeting with Sharmistha Mohapatra, we gained a broader understanding of the recommended screening and diagnostic process for cervical cancer. Maryland follows the ASCCP guidelines which provides an algorithm for cervical cancer screening in terms of HPV screening and PAP smears. She described that age is a key factor in determining the use of a HPV test as younger patients tend to have a greater capacity to clear their HPV status. Specifically, those under 30 will typically only receive a cytology (PAP smear) test, those between 30 and 65 will typically receive both a cytology test and HPV test, and those over 65 will typically not receive routine screening due to likely comorbidities and relatively low mortality rate of cervical cancer. If a patient has abnormal cells in a PAP smear or if they report symptoms of cervical cancer, they will receive a biopsy to confirm a diagnosis. We also learned about the general process for receiving screening in America which can consist of many spaced out doctor’s visits. First, the patient will go to their primary care physician for screening and then be referred to a GYN for a PAP smear. If the primary care physician does family medicine, they can do the PAP smear themselves but it is important to note that the PAP does not always occur during initial consultation. Second visit is with the GYN to perform the PAP smear and/or the HPV test to screen the patient. Afterwards the specimen has to be taken to a lab and results can come back in a month, if there are abnormal results, you will likely be asked to come back for another visit or call back. During the third visit, you will have positive PAP or HPV, and ideally they will tell you to do the cervical biopsy. Then that specimen goes to the lab, results may take a week again. The results of the biopsy will be the diagnosis which determines the grade of cancer the patient has: abnormalities, CEN1, 2 or 3 which are the middle areas, or cancer. If it is cancer, you will be referred to a surgeon or oncologist, which requires a hospital visit. The fourth visit is called a leap where the surgeon excises the affected area of the cervix, which is followed by further surveillance. Additionally, she stressed the importance of the distinction between screening and diagnosis in that screening is a preventative measure that incurs no costs under the Affordable Care Act whereas diagnostic tests confirm a diagnosis when screening results are abnormal or when the patient experiences symptoms of cervical cancer in any consultation. She also discussed the barriers that health care providers face when trying to give cervical cancer screening such as appointment time constraints due to financial restrictions, strict charting measures that force doctors to spend more time on other routine measures such as STI and hypertension testing, patient discussions about affordability, and the need to refer patients out to specialists if the primary care physician can not perform the screening themselves. This also leaves physicians with little time to educate patients about cervical cancer screening so patients often have to educate themselves or find other medical staff to educate them. In order to combat this, the CDC funded Breast and Cervical Cancer Program in Maryland is covering screening costs for underinsured and uninsured patients so that they can access screening measures. Additionally, their outreach efforts in counties have been successful in primary care practices. Ultimately however, she stressed that primary care providers have the most responsibility and power to refer undocumented and uninsured patients to resources to receive care as they are the primary point of contact with patients. She explained that our project, once tested, could bypass issues to access a primary care visit as the test could be taken at home instead of potentially requiring multiple visits to the doctor. Additionally, this screening device could bypass social barriers in conservative countries where women may not feel comfortable going to male providers. However, she noted that by using this test, patients would have to commit to completing the whole screening and diagnostic sequence as they have to decide if they then go in for a diagnosis.