The Advice

We posed several questions to Dr. Buspavanich, and here's what he shared:

Diagnosis Process for Bipolar: Dr. Buspavanich explained that diagnosing Bipolar can be challenging due to the complexity of the symptoms. It often starts as depression, followed by manic episodes. Different types of Bipolar exist, with Type 1 characterized by classic and severe fluctuations, making it potentially easier to diagnose. In contrast, Type 2 involves hypomanic phases and numerous depressive phases, which can be mistaken for depression, especially when the hypomanic phases give a false sense of improvement. He also highlighted the clinical difficulty in distinguishing between hypomania and normal behavior. Furthermore, he told us that patients are occasionally encouraged to maintain a journal to diligently monitor their mood fluctuations and external factors, aiding in the identification of potential triggers.

Commonly Prescribed Lithium Medications: He mentioned that Quilonum® is almost exclusively prescribed and readily available, while Hypnorex® availability varies.

Usage of Lithium Medication: Dr. Buspavanich outlined that Lithium can be used as a monotherapy for Bipolar, offering anti-manic, anti-suicidal, and anti-depressant effects. In such cases, no additional medication is typically required. However, for patients with treatment-resistant depression, Lithium in conjunction with antidepressants, is recommended. Using Lithium as a monotherapy in these cases may not yield satisfactory results.

Lithium Concentration Determination in Laboratories: Regarding the laboratory testing of Lithium concentration, Dr. Buspavanich described the use of flame photometry, a method involving a large device that requires trained personnel to operate. In rural areas, procuring such equipment can pose a challenge. Alternatively, samples may need to be sent to a distant laboratory, which can lead to delays in obtaining results. Using the device, however, allows for the rapid acquisition of results, typically within an hour.

As Dr. Buspavanich also treats patients who are diagnosed with Bipolar, he was the ideal person to ask about difficulties and prejudices patients may face in their daily lives. During a conference hosted by Bipolar UK, we discovered that in the United Kingdom, it can be considered more polite to refer to the condition simply as "Bipolar" rather than "Bipolar Disorder." This led us to wonder if a similar linguistic preference exists in the German terminology for the disease.

The response we received indicated that such a distinction is not particularly significant in Germany. In fact, having a name for the condition can be reassuring for patients, as it serves as an explanation for the feelings and behaviors they are experiencing.

Dr. Buspavanich's expertise shed light on these critical aspects of Bipolar diagnosis, Lithium medication, and laboratory practices.

How did we incorporate the feedback?

1. Language Choice: We decided to maintain the practice of referring to the condition as "Bipolar" when communicating in English. However, when speaking or writing in German, we continued to use the term "Bipolare Störung" (Bipolar Disorder). This choice allowed us to respect linguistic preferences while ensuring clarity and understanding in both languages.
2. Improving the Diagnosis Process: Inspired by the insights into the challenges of diagnosing Bipolar, we began brainstorming ways to contribute to improvement. Our brainstorming led to the concept of developing software that would empower patients to document their symptoms and automatically send them to their healthcare providers. This software would also enable patients to track recurring symptoms and receive trigger warnings, thereby enhancing their ability to manage their condition and communicate effectively with their medical professionals.

His Advice

During an interview, Prof. Schulze was able to provide important insights and groundbreaking advice:

• Influencing Factors

  He emphasised that factors such as heat, pregnancy, drinking behaviour and other medications can influence the concentration of lithium. Thus, conditions should be kept as equal as possible.

• Availability and Use of Lithium

  Prof. Schulze highlighted that lithium is an inexpensive yet highly effective treatment option for bipolar. Especially in warm developing countries where the infrastructure for storing refrigerated test systems is limited, the development of a reliable test system is of great importance.

• Correlation Between Saliva and Blood

  Prof. Schulze pointed out the significant correlation between the concentrations of lithium in saliva and blood, with saliva concentrations being 2-3 times higher. This could be important in monitoring and diagnosing lithium levels.

• Intake and Half-Life of Lithium

  We were told that lithium has a half-life of about 20 hours and is usually taken in the morning and evening or only in the morning to maintain a steady concentration in the body.

• Measuring Lithium

  When working out a test system, he advised not to measure only too high levels, but it would be better if we tested both too high and too low concentrations to get better readings and so even if the concentration is too low, we can show this as a result of the test. The test should be done about 12 hours after the last dose and the recommended dose is usually under 1000 mg. After the level has stabilised, the test at home can be very helpful in reducing the time-consuming measurements of the concentrations in the doctor's office, thus relieving both the laboratories and the patients. Prof. Schulze pleads for a calibration of the test, where the results have to be read individually. Test strips are not practical because of the necessary quantification, as the test system has to cover a certain range and measure the values accurately within this range.

These improvements adjustments were made with the aim of enhancing the precision and applicability of our project based on the input and expertise provided by the consortium from the Zentralinstitut für Seelische Gesundheit (Central Institute for Mental Health).

His Advice

Prof. Simmel gave valuable advice regarding riboswitches for our project.

• Stability of Riboswitches

  He pointed out that we have to consider nucleases in saliva as well as the improvement of stability by including secondary structures ("hairpins") downstream and upstream in our RNA construct. Furthermore, he recommended the use of in vitro transcription, as RNA is less stable than DNA and so we do not need to add unstable RNA molecules to the test. He also said that the loop with the ribosome binding site should be kept as short as possible to ensure stability, but still long enough to be able to switch to other conformations.

• Sensitivity

  In addition, he pointed out that sensitivity is not increased by tandem riboswitches, as it would be sufficient for lithium to bind to the riboswitch associated upstream of the reporter.

• Luciferase as Reporter

  He explained the advantages of luciferase enzymes over fluorescent reporter proteins such as GFP, as there is the possibility of an amplification response through a luciferase. This is because one enzyme can convert more substrates.

• Test System and Diversity of our Constructs

  He suggested using a blood glucose tester for the readout, as this is already established, and emphasised the need to screen several alternative structures. For this reason, we decided to screen 4 different variants of our riboswitch, each with 5 different reporters. He advised us to use kits with purified components and mentioned the importance of determining the detection limit by reducing the concentration as much as possible in our experiments until we no longer get a signal.

Her Advice

Prof. Süß gave valuable advice regarding riboswitches and the design of our test system.

• Which pH Value to Set

  One aspect we discussed with Prof. Süß is that the paper by White et al. (2022) that describes our riboswitch used a pH value of 9 for the tests, which is relatively high. She replied that we have to try out whether our experiments work at such a pH value because there might be problems, especially with translation in a cell-free system.

• Adjusting Sensitivity for our Test System

  Another thing we were wondering about is how to make our test system more sensitive. In the paper by White et al. (2022), from which we have the riboswitch, a concentration of lithium of 50mM was used for the tests. Prof. Süß explained that this concentration was unusually high and that normally the concentration is in the micro-mole or even nano-mole range.

  She suggested changing bases by mutations in the secondary structure to achieve higher sensitivity. In addition, she mentioned that possibly amplification could be done at detection - depending on the final test system - and that it could be quite helpful that the system is cell-free, as there are no disruptive or interfering cellular processes in this way.

• Identification of the Ribosome Binding Site (RBS)

  At the time of the interview, we had not yet identified the RBS. Prof. Süß then explained that in a translational riboswitch, the RBS should already be located in the sequence. She advised us to have another look at the paper by White et al. (2022). But in case this sequence would not be described there, one could look for the start codon in the sequence and thus localize the RBS. She noted that it must be taken into consideration from which organism the sequence originated and that, depending on this, the RBS must be adjusted so that the Shine Dalgarno sequence is not disturbed.

• What you Should Pay Attention to

  We asked Ms. Süß what is particularly important when designing DNA constructs, or when dealing with riboswitches. She told us that we should pay special attention to the flanking sequences because the context of structures in RNA is extremely important. She also recommended some structure prediction programs but mentioned that they are not fully reliable. She also suggested that we use spacers so that, for example, the transcription start is not located directly in the secondary structure.

• Other Things to Keep in Mind

  At the end of the interview, Prof. Süß gave us some more advice on things we should consider in our future procedure.

  1. Is the concentration of lithium in saliva sufficient for detection or is blood mandatory?
  2. Nanoluc could work as a reporter for our system, but normally only fluorescent reporters are used for cell-free systems.

His Advice

During an inspiring interview, he gave us valuable advice that significantly influenced our project.

• Optimising the Natural Riboswitch

  Prof. Mörl emphasised that the natural riboswitch is already optimised by evolution. This should be taken into account when we make modifications.

• Shine-Dalgarno Sequence After Lithium Binding

  It is crucial that the Shine-Dalgarno sequence is presented after lithium binding so that the desired signal can be expressed. This means that the riboswitch must change its conformation after binding of a ligand and make the Shine-Dalgarno sequence accessible to ribosomes.

• Use of Fast-Folding Fluorescent Proteins

  The choice of fast-folding fluorescent proteins was recommended to increase the efficiency of the system.

• Adaptation to the E. coli System

  Prof. Mörl pointed out that when working with E. coli, attention should be paid to codon usage, as a different open reading frame might be present in the E. coli strain. So, according to his advice, we should optimise codon usage.

• Challenges with Cell-Free Synthesis

  Using cell-free synthesis can be problematic as RNA is susceptible to degradation by RNases. These could get into the system simply by working with hands. Prof. Mörl recommended the use of a freeze-dried system containing only DNA and no mRNA.

• In vitro Transcription and Translation Coupled

  He explained that a coupled system for in vitro transcription and translation would be suitable for us, which would need to maintain active ribosomes, tRNAs, elongation factors and some other components and would be similar to what is found in a living cell. Freeze-dried RNA might be more stable here.

• In Silico Modelling

  Prof. Mörl mentioned that in silico modelling is challenging. It would be advantageous if one could predict the structure of an aptamer and thus a riboswitch on the basis of a ligand. Although he does not do it himself, he noted that Molecular Dynamic Simulation is a useful method for studying ligand interaction with RNA.

• Importance of Binding Pockets

  If the binding pockets responsible for ligand binding are already known, this facilitates randomised mutagenesis.

• Context Dependence of Riboswitches

  Riboswitches are context dependent, both in terms of sequence upstream and downstream of the riboswitch. Single base substitutions can impair the functionality of a riboswitch and even render it completely non-functional. He pointed out that in such a case, the flanking sequences could be the cause. To minimize risks, structural predictions by RNAFold or MFold should be used.

• Working with RNA

  Prof. Mörl emphasised the sensitivity of RNA and recommended always wearing gloves and preferably working in vivo. In vitro experiments should be carefully planned.

• Alternative Methods

  When working with translational riboswitches, he recommended transcription induction with T7 polymerase in E. coli DE3 strains that have the T7 gene integrated in the genome to obtain abundant riboswitch transcripts.

• pH and Stability of RNA

  The pH can be less critical inside the cell, but in vitro a pH of 9 is already problematic. RNA is very unstable in alkaline conditions, which is why we should not go above a pH value of 10.

• Sensitivity and Desensitisation

  It is much more difficult to make a riboswitch more sensitive than to desensitise it, Prof. Mörl emphasised.

• Reaction to Dissolved Ions

  He also pointed out that riboswitches only react to dissolved ions. This meant for us that we had to pay attention to this when choosing a lithium which is intended for our experiments.

Thanks to Prof. Mörl's experience, we were able to plan our experiments in a targeted way after answering our questions, and we incorporated many of his pieces of advice into our project.

The Advice

The initial contact with this consortium was established when they reached out to us after learning about our project. Their keen interest in our work led to comprehensive discussions. During our conversations, we presented our project idea, which received substantial positive feedback and support. They also posed thought-provoking questions that prompted us to consider making some improvements to our project:

One noteworthy question they raised was whether the riboswitch we were using for detection was sensitive to sodium ions, given that sodium is present in bodily fluids. This suggestion highlighted the potential revolutionary aspect of our project, as a riboswitch with lithium specificity could be a significant advantage over established ion testing methods.

The Advice

• Sustainable and Resource-Saving System

  On the one hand, he suggested that we use Freeze and Thaw as our test system for our experiments or afterwards in order to save resources.

• Dealing with Luciferases

  On the other hand, he was able to give us many tips and answer questions about working with luciferases as reporters. He mentioned that there is only a small time window in which measurements can be carried out. Accordingly, we would have to plan our experiments in such a way that the throughput remains high but not too much time passes before the luminescence is detected. Therefore, we should work quickly and keep the components cool. Since working with riboswitches is generally challenging, combining them with luciferases could make these enzymes susceptible to interference and lead to false positive results. Nevertheless, Dr. May emphasized that rapid tests are possible with luciferases.

• Threshold Quantification

  We also received the suggestion to use different spots with different riboswitch concentrations. The main aim is to measure a difference between a concentration that should be present in the therapeutic aspect and the concentration that is toxic for the organism, respectively the patient.

• Networking

  Besides these practical points, Dr. May also mentioned how important it is to get in touch with experts in case of problems and different concerns, as this is a much more efficient way to get fast and versatile information.

The Advice

• Target Group-Specific Communication

  One of the key insights from our conversation was the importance of targeting a specific audience or age group. To do this, it is crucial to conduct a thorough target group analysis and consider how we ourselves would best like to be reached. This allowed for tailor-made communication that had a lasting effect.

• Qualitative and Nationwide Reach

  Communication should not only be quantitative, but above all qualitative in order to stimulate people to think in a sustainable way. A regional approach would add to the quality of our education.

• Maintain Authenticity

  To be successful in science communication, authenticity is of utmost importance. It is crucial to remain authentic in our message and activities in order to build trust with our audience and develop a sustainable relationship. To do this, we should first and foremost choose formats that make us feel comfortable.

• Set Clear Goals

  Before we start our communication activities, we should be clear about what exactly we want to achieve. Do we want to get feedback, impart knowledge or motivate people and make them curious? Clearly defined goals help to plan and implement effective communication strategies. To get inspired, Gesa recommended the website "Wissenschaftskommunikation.de".

• Promote Interaction: House of Commons Debate and Co.

  Interactive formats are extremely effective. One idea that came up during the discussion was to hold a House of Commons debate with experts, in which the audience can also actively participate. Experts would give short talks in which the audience can, for example, declare themselves as opponents or supporters of a topic under discussion. This promotes dialogue and interest in actively engaging with the topic.

• Contacts and Lectures in Institutions

  The “Wissenschaft im Dialog” Team offered us their support in contacting suitable institutions to organize lectures and presentations and to collect feedback. They could also help in finding cooperation partners for joint events which would facilitate planning and implementation.

• Feedback in the University Botanical Garden

  Another piece of advice was to use feedback formats or methods to collect opinions during our activities in the Botanical Garden, where we talked to visitors about genetic engineering.

• Reaching Students in Class

  To reach students in class, it is advisable to analyze curricula and develop appropriate experiments or other active ways to spark interest about synthetic biology.

• Visual Representations and Playful Approaches

  Graphical overviews and pictograms are effective tools as many people learn visually. Similarly, playful approaches and interactive formats can increase participation and interest.

• Recommendations for Video Production

  When producing videos, it is advisable to focus on 1-2 selected platforms, as this is more resource-efficient. Quality is more important than quantity, said Gesa Hengerer.

Overall, the conversation with the “Wissenschaft im Dialog” deepened our understanding of effective science communication and gave us valuable tips for planning and implementing our activities.

• How does an authorisation procedure for medical devices work and how long do authorisation procedures take?

  The authorization procedure for medical devices in the European Union follows a comprehensive regulatory framework designed to ensure patient safety. In the case of in vitro diagnostic products, the process involves determining the risk class (ranging from A to D), which subsequently dictates the specific requirements and level of scrutiny.

  Notified Bodies, independent organizations like TÜV, are responsible for evaluating these medical devices. This evaluation process takes place within the private sector, assuring objectivity and impartiality.

  It's important to note that this regulatory framework for medical devices differs from that of pharmaceuticals, where separate approvals are required in each country, making the EU's approach more streamlined and harmonized.

• What costs are associated with an approval procedure?

  Costs in the 6-digit range upwards may be expected.

• What criteria would our test system have to fulfill in order to be approved?

  To obtain approval for our test system, several criteria need to be met. Firstly, the system must accurately detect the presence of lithium, ensuring reliable results.

  Additionally, it should adhere to the state-of-the-art technology standards. However, it's essential to acknowledge that in our case, we face a unique situation, as there are currently no comparable self-tests for lithium in existence.

  The precise criteria for approval should be discussed and determined in consultation with the Notified Body. They will provide the necessary guidance to ensure compliance with regulatory requirements, given the pioneering nature of our test system.

• Would there be specifications for the cell-free expression system for use on patients?

  The specifications for the cell-free expression system for use on patients are not explicitly defined. However, it's important to note that if intended for home use, higher requirements may apply, potentially leading to a higher risk class designation.

  Ensuring that laypersons can safely and effectively use the system is a key consideration. Therefore, the evaluation process is often done on a case-by-case basis.

  One critical aspect is that there should be no replication involved in the system's operation, and any chemicals used should ideally not come into direct contact with the user.

  In navigating the regulatory landscape, seeking guidance from a regulatory compliance consulting firm may be advisable to ensure compliance with applicable laws and regulations.

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