HiLIFE Trainees

 

I’m Pinja Perkkiö, an aspiring microbiologist and a HiLIFE scholarship trainee in Reetta Satokari’s lab (University of Helsinki, Faculty of Medicine)! I’ve just finished my bachelor’s degree and I will start my studies in the master’s programme of Microbiology and Microbial biotechnology next autumn. This summer, I will be diving deep into the world of gut microbiota as I research the microbiota restoring potential of NGPs in intestinal inflammation and dysbiosis. 

 

How antibiotics disturb our gut microbiota 

The gut microbiota is defined as a community of microbes, e.g. bacteria, fungi, viruses and archaea residing within the gut. The microbiota is responsible for many important functions, such as breaking down complex carbohydrates and protecting the gut from infections. When a person takes antibiotics, it not only kills the bacteria causing the disease, but also the beneficial ones as antibiotics aren’t strain-specific. This allows potentially pathogenic bacteria to grow in larger numbers – the situation is called dysbiosis, meaning that the microbial community composition has been altered in a harmful way. 

Luckily, we have a tight layer of cells – the epithelium – that lines the walls of the gut and is covered by two layers of mucus. The mucus combined with the epithelium acts as a barrier between our intestines and the rest of our body, preventing intestinal pathogens from causing too much harm. Normally, it is very difficult for microbes to cross the mucus layer as it traps microbes and is loaded with antibacterial substances. In addition, the cells of the epithelium are tightly attached to each other, preventing any further movement of microbes. There are also lots of immune cells embedded in gut tissues that are ready for action, should a pathogen try to cause an infection. 

However, as dysbiosis leads to the growth of pathogenic bacteria, our immune cells (neutrophils, NK cells and macrophages, for example) activate inflammatory reactions. While these reactions are necessary for the elimination of pathogens, they also damage gut tissue. Pathogenic bacteria also activate the secretion of inflammatory molecules, cytokines, in a type of gut epithelial cell called enterocyte, which leads to the epithelium getting “leaky” – this means that microbes can cross the epithelium more easily and cause further inflammation in gut tissue. These bacteria can not only cause nasty infections, but they may also travel deeper in gut tissue or even to other sites in the body, increasing the risk of certain cancers (Bastos et al., 2023; Kouzu et al., 2022)! 

 

Next-generation probiotics against dysbiosis 

To fight antibiotic-induced dysbiosis and reduce the inflammation and leaky gut syndrome caused by it, researchers have turned their attention to potential new bacterial strains that could function as next-generation probiotics. They are defined as “live microorganisms identified on the basis of comparative microbiota analyses that, when administered in adequate amounts, confer a health benefit on the host” (Martín R, Langella P., 2019) – in simpler terms, microbes that can potentially help boost our health. Traditional probiotics, such as Bifidobacteria and Lactobacilli, have shown great potential in restoring a healthy microbial community in the gut after taking antibiotics, thus preventing dysbiosis and infections. They have also been shown to inhibit the production of inflammatory molecules in both immune cells and epithelium and boost the production of anti-inflammatory molecules.  

 

Probiotics exert anti-inflammatory effects on several cell types. Created with Biorender.com. 

 

There’s still lots of research to be done to bring next-generation probiotics into wide use. More information is needed on the different strains of potential probiotic bacteria and their specific effects on both the immune system and the gut epithelium. This is where my research project comes in! 

The project I’m collaborating on focuses on a mix of 14 different anaerobic bacterial strains that have the potential to restore the gut microbiota after antibiotic use and prevent or reduce inflammation. The next-generation probiotic mix was originally tested in an in vitro mucosal-simulator of the human gastrointestinal tract (M-SHIME(R)) model. A natural gut microbiota was obtained from the stool sample of a healthy human donor, after which it could grow. Then, the “gut” was exposed to antibiotics to induce dysbiosis. After the antibiotic treatment, one system was treated with the probiotic mixture and the other one with a negative control. Finally, samples from probiotic- and control-treated microbiotas were taken. Bacteria present on these will be studied to check the impact of the antibiotic intake in the microbiota and compare its possible recovery after the probiotic treatment. The production of short-chained and branched fatty acids, lactate and ammonia at different time points will also be measured since their levels indicate how much beneficial bacteria there are compared to harmful ones. 

To study how the anaerobic isolates might affect intestinal inflammation, I will culture two different intestinal epithelial cell lines: Caco-2 and HT-29. These cell lines were chosen because they are well-characterized and resemble enterocytes on the gut epithelium. Then, I will expose the HT-29 cell line to either the anaerobic bacterial blend or a placebo. After incubation, pro-inflammatory IL-8 production in the HT-29 cell line will be measured using ELISA assay. Later, the capability of a single freeze-dried strain in attenuating the IL-8 levels produced by HT-29 cell line will be assessed.  

Caco-2 cell line will be used to measure the effect of the microbiota samples collected from M-SHIME(R) on the barrier integrity of the Caco-2 (enterocyte) monolayer. The integrity will be assessed by measuring the TEER (transepithelial/transendothelial electrical resistance) of the cell monolayer, and the results will tell us if our strains can alleviate leakiness in the epithelial cell layer caused by dysbiosis. 

 

HT-29 cells under a microscope. 

 

Starting my research 

So far, I’ve read lots of literature related to the gut microbiota and treating inflammation with probiotics and next-generation probiotics. This has given me a good foundation on which to build my knowledge. The steps using M-SHIME(R) human gut simulator have been completed previously, so I’ve mainly been growing the intestinal cell lines required for our experiments. Luckily, I already have some experience with cell culture, so the start hasn’t been too difficult. My group has also been supportive and helpful, so I’m optimistic about the upcoming months! 

 

Seeding HT-29 cells for our experiments. 

 

The next step will be starting the IL-8 induction experiments, and once the initial results are ready, we can proceed to plan further. If you’re interested in our results, stay tuned for my next blog post! 

 

Sources: 

Bastos AR, Pereira-Marques J, Ferreira RM, Figueiredo C. Harnessing the Microbiome to Reduce Pancreatic Cancer Burden. Cancers (Basel). 2023 May 5;15(9):2629. doi: 10.3390/cancers15092629. PMID: 37174095; PMCID: PMC10177253. 

Kouzu K, Tsujimoto H, Kishi Y, Ueno H, Shinomiya N. Bacterial Translocation in Gastrointestinal Cancers and Cancer Treatment. Biomedicines. 2022 Feb 4;10(2):380. doi: 10.3390/biomedicines10020380. PMID: 35203589; PMCID: PMC8962358. 

Martín R, Langella P. Emerging Health Concepts in the Probiotics Field: Streamlining the Definitions. Front Microbiol. 2019 May 21;10:1047. doi: 10.3389/fmicb.2019.01047. PMID: 31164874; PMCID: PMC6536656. 

 

Some literature: 

Berta Bosch, Saliha Moutaharrik, Andrea Gazzaniga, Kaisa Hiippala, Hélder A. Santos, Alessandra Maroni, Reetta Satokari. Development of a time-dependent oral colon delivery system of anaerobic Odoribacter splanchnicus for bacteriotherapy. European Journal of Pharmaceutics and Biopharmaceutics. 2023; 190. 73-80. https://doi.org/10.1016/j.ejpb.2023.07.010. 

Cristofori F, Dargenio VN, Dargenio C, Miniello VL, Barone M, Francavilla R. Anti-Inflammatory and Immunomodulatory Effects of Probiotics in Gut Inflammation: A Door to the Body. Front Immunol. 2021; 26(12):578386. doi: 10.3389/fimmu.2021.578386.  

Marzorati M, Van den Abbeele P, Bubeck SS, Bayne T, Krishnan K, Young A, Mehta D, DeSouza A. Bacillus subtilis HU58 and Bacillus coagulans SC208 Probiotics Reduced the Effects of Antibiotic-Induced Gut Microbiome Dysbiosis in an M-SHIME® Model. Microorganisms. 2020; 8(7):1028. https://doi.org/10.3390/microorganisms8071028 

 

I’m Murat, a second year student in the Master’s programme in Life Science Informatics at the University of Helsinki, specializing in Biomathematics and Biostatistics. Last summer I embarked on a 4-month internship with the Environmental and Ecological Statistics group that was funded by HiLIFE, where I was able to apply my quantitative skills to a field I have been passionate about since I was a child: wildlife conservation. And what a journey it has been! Although my internship officially ended in September, I have kept working on the project until now.

My mission was to develop a Bayesian state-space model to help monitor Baltic ringed seals, a sub-species of ringed seals that was once on the verge of extinction due to unsustainable hunting practices and large-scale reproductive failure caused by pollution. Thankfully, the population has been recovering during the last few decades, at least in the northern region of the Baltic Sea¹. However, highly dynamic conditions brought on by changing sea ice patterns, decreasing pollution levels and the recent re-introduction of seal hunting have made reliable population monitoring increasingly difficult, and assessments of population status have not been possible for over a decade. The inability to estimate population size and growth rate has been a major obstacle in determining sustainable management practices.

My challenge was to create and parametrize a model that could accommodate all of these changing factors as well as the uncertainties associated with them, something conventional monitoring methods fell short of. Instead of analyzing different data sources separately, I integrated them into a single, unified model of ringed seal population dynamics – a method that is often called integrated population modeling³. By analyzing all available data holistically, integrated population models can exploit synergies between different data sources, making it possible to parametrize detailed and mechanistic population models.

The results we obtained with this approach were as exciting as they were important. For example, we discovered that the reproductive rates of seals might have fully recovered from the effects of past pollution, and the population may have increased from less than 5,000 to nearly 30,000 individuals! We also discovered that the recent re-introduction of seal hunting has had a notable impact on population growth, though not critical enough to ring any alarm bells for the seal population—yet. Another interesting finding was that seals may be behaving differently during years with low sea ice cover, hauling-out on ice in larger numbers during their annual molt in the spring. Since hauled-out ringed seals are counted each year during this time, population counts after warm winters may be significantly higher! This means that as sea ice patterns are altered due to climate change, reliably estimating the population size could become increasingly more difficult.

During the time I was working on this project, every day brought new and diverse challenges – a rich blend of mathematics, statistics, and ecology. Collaboration with researchers from other fields was not just helpful, but essential, making my days anything but repetitive. A year of hard work has finally culminated in a manuscript that is ready to be submitted for publication at a scientific journal! Reflecting back at the journey, it is amazing to see how much I have learned. I started my internship with many doubts – unsure whether I would enjoy doing research, and whether I had what it takes. I am now seriously considering a PhD, confident that a career in research is right for me. As I near the end of this project, I am filled with gratitude for the opportunity to have played a role in preserving these amazing animals, and I eagerly anticipate my next endeavor in the world of research.

References:

¹Sundqvist, L., Harkonen, T., Svensson, C. J., and Harding, K. C. (2012). Linking climate trends to population dynamics in the Baltic ringed seal: Impacts of historical and future winter temperatures. Ambio, 41:865–872.

²Kelly, B. P., Bengtson, J. L., Boveng, P. L., Cameron, M. F., Dahle, S. P., Jansen, J. K., Logerwell E.A., Overland J.E., Sabine C.L., Waring G.T., & Wilder, J. M. (2010). Status review of the ringed seal (Phoca hispida).

³Schaub, M. and Abadi, F. (2011). Integrated population models: a novel analysis framework for deeper insights into population dynamics. Journal of Ornithology, 152:227–237.

Transfer RNAs (tRNAs) are often described as humble, clover shaped molecular servants. They participate in protein synthesis by performing codon recognition on messenger RNAs (mRNAs) and by delivering amino acids necessary for translation. Despite technically being an accurate description, it underestimates the profound intricacies of the tRNAs. Humans have around 600 tRNA genes in the genome, and when expressed and processed, the beautiful clover leaf tRNA is reshaped to an L-shaped configuration and will acquire variable chemical modifications, increasing the diversity of tRNAs (Lant et al., 2019). Altogether, the modified L-shaped tRNAs comprise 15% of the total RNA found in the cell, whilst mRNA only comprises 1-5 % (Delaunay et al., 2024). Disruptions in tRNA expression, regulation and mutations have been linked with neurological and metabolic disorders and cancer (Lant et al., 2019). tRNAs are found in all forms of life (Delaunay et al., 2024), underscoring their fundamental role in biology. Understanding tRNAs is essential for understanding life: after all, would life even exist without the humble tRNA?

Modification of cellular macromolecules is crucial for accurate and efficient gene regulation. Many are familiar with DNA modifications, such as cytosine methylation, which may affect gene activity and chromatin structure (Liyanage et al., 2014). Cellular RNAs are also targets of post-transcriptional modifications (PTMs) with the N⁶-Methyladenosine (m⁶A) modification being one of the most widely studied (Lauman & Garcia, 2020). RNA modifications have essential regulatory implications: in mRNAs, certain modifications can affect transcript stability, localisation, splicing patterns, and translation (Delaunay et al., 2024). PTMs can be found in ribosomal RNA, long non-coding RNAs, and small non-coding RNAs (Delaunay et al., 2024). Unsurprisingly, PTMs are also seen in tRNAs. In fact, tRNAs are the most abundantly modified RNA species in the cell (Zhang et al., 2022). Our understanding of their effects is still limited but we know that the modifications can affect tRNA stability, tRNA-RNA interactions, tRNA-protein interactions, folding and mRNA decoding (Delaunay et al., 2024). Over 150 tRNA modifications have been identified (Delaunay et al., 2024) and as tRNAs are abundant in the cells, studying tRNA modifications becomes a very intriguing area of research.

During my time as a HiLIFE trainee I had the opportunity to delve into the science of tRNA modifications at the RNAcious laboratory, University of Helsinki. I participated in two projects, one where the aim was to produce hypomodified tRNAs and another where the aim was to determine the tRNA modification landscapes in different mouse tissues. You can find my first blog post here: https://blogs.helsinki.fi/hilife-trainees/2023/06/26/my-battle-against-rnases/

The making of plain cloverleaves: Hypomodified tRNAs

Modifications on tRNAs are so abundant that it would be difficult, or maybe even impossible, to extract hypomodified tRNAs from the cell: you see, in eukaryotes there are on average 13 modifications on each ~80nt long tRNA (Zhang et al., 2022). For methylations alone, there are around 40 proteins, known as modification writers (e.g. methyltransferases) and erasers (e.g. demethylases), which moderate tRNA modifications (Delaunay et al., 2024). One way to produce hypomodified tRNAs is through in vitro transcription (IVT), which essentially is a cell free transcription system. As IVT is cell free, it lacks the writer and eraser enzymes which modify the modification profile. My task was to develop a method to produce and isolate hypomodified tRNAs utilizing IVT, ribozyme- and MS2 based techniques.

Differentially decorated cloverleaves: tRNA modifications in different organs

We know that there are different tRNA modification landscapes in different organs (de Crécy-Lagard et al., 2019) but they have not yet been studied extensively. A tRNA modification landscape is the entire modification profile of the tRNAs of a specific organ. Uncovering the modification landscape would offer valuable insights into both the frequency and positional distribution of specific modifications within the tRNAs across various organs. Mass spectrometry is an effective tool to identify and study the location of modifications on single nucleotides (Lauman & Garcia, 2020), and certain reverse transcriptases can be used to study the location of modifications on the tRNA molecule (Zhang et al., 2022).

After my time as a HiLIFE trainee, I’ve truly gained a deep appreciation for the complexities of the humble tRNA. While I metaphorically refer to tRNA modifications as “decorations”, in reality, these modifications play essential roles in biological processes. Learning about them has been truly fascinating.

To end this journey, I would like to express my gratitude to Docent Peter Sarin and his group of bright researchers, especially my supervisor Jenni Pedor, who always supported me during my time in the research laboratory. The pioneering and motivational environment provided an invaluable experience and an inspiration for my career moving forward. I would encourage anyone interested in expanding their understanding of tRNA modifications and tRNA biology to explore the research conducted at RNAcious laboratory.

 

References

de Crécy-Lagard, V., Boccaletto, P., Mangleburg, C. G., Sharma, P., Lowe, T. M., Leidel, S. A., & Bujnicki, J. M. (2019). Matching tRNA modifications in humans to their known and predicted enzymes. Nucleic acids research, 47(5), 2143–2159. https://doi.org/10.1093/nar/gkz011

Delaunay, S., Helm, M., & Frye, M. (2024). RNA modifications in physiology and disease: towards clinical applications. Nature reviews. Genetics, 25(2), 104–122. https://doi.org/10.1038/s41576-023-00645-2

Lant, J. T., Berg, M. D., Heinemann, I. U., Brandl, C. J., & O’Donoghue, P. (2019). Pathways to disease from natural variations in human cytoplasmic tRNAs. The Journal of biological chemistry, 294(14), 5294–5308. https://doi.org/10.1074/jbc.REV118.002982

Lauman, R., & Garcia, B. A. (2020). Unraveling the RNA modification code with mass spectrometry. Molecular omics, 16(4), 305–315. https://doi.org/10.1039/c8mo00247a

Liyanage, V. R., Jarmasz, J. S., Murugeshan, N., Del Bigio, M. R., Rastegar, M., & Davie, J. R. (2014). DNA modifications: function and applications in normal and disease States. Biology, 3(4), 670–723. https://doi.org/10.3390/biology3040670

Zhang, W., Foo, M., Eren, A. M., & Pan, T. (2022). tRNA modification dynamics from individual organisms to metaepitranscriptomics of microbiomes. Molecular cell, 82(5), 891–906. https://doi.org/10.1016/j.molcel.2021.12.007

As a new year starts and new students are getting the opportunity to become HiLIFE trainees, I have been reflecting about what I did, learned and achieved during my own experience through this program.

I am Adrián Colino Barea, a (now) second year Master’s student of Ecology and Evolutionary Biology.  On March 2023, I was lucky enough to join the Integrative Evolutionary Biology (IntEvoBio) lab to answer what I first thought could be a trivial question that popped in my mind. Claudius Kratochwil, the PI of the group, helped me to plan a small project aiming to determine how light depletion determines sexual choice and preference in colorful cichlid fishes from the African Great Lakes. You can learn more about it reading my previous post, by clicking here.

In a nutshell, and if you are wondering, my short internship project didn’t come up with significant differences of mate preference under light and dark conditions. I did not find out how colorful fish fall in love in the dark. Instead, I found out how usual — and important — it is to not have results in science. It is crucial to get a glimpse on what questions are relevant to answer, and what research lines are worth following and investing on. There is just so much to find out, trial-and-error is necessary to keep going.

I learned new limits of the beauty of commitment. For my experiments, controlling fish schedules involved feeding them, and I decided to take care of it. I found myself showing up in the fish room every single day, sometimes in breaks between classes, sometimes during lazy Sundays, sometimes under absolutely crazy weather… And I loved it. Working March to June, I got to experience the Finnish weather every day at least for a bit. It was not too special at the moment, and anyone could do it if living in Helsinki. But now I am really glad I got to see how the seasons change. Very often, I stopped by the fish room only to go walking in the forest or birdwatching later, and experiencing nature changing with the seasons before my very eyes. Days became longer, snow melt, flowers sprung, everything sprung. And this experience is just so different to what I grew into, coming from the other side of the continent.

I also found myself becoming part of a community, formed by all the members of IntEvoBio lab. Over my working weeks, it was easy to be motivated to plan and act thanks to the great support and hospitality of all the members of the lab. I always felt encouraged to keep up and be up to date on meetings, presentations, and the deadlines I kept setting myself to meet ends — something vital in such a small project. Indeed, perfecting the art of managing time has been one of the most valuable assets I got out of the internship. But in this case, considering the inspiring time planning the whole lab follows, being organized myself was a piece of cake.

Another thing I learned and cherish is the value of kindness and humanity. During my internship, I always found a smile and willingness to help of the members of the lab I treated with, including people of all levels, from PI to Master’s students. Without this help, my project wouldn’t have developed after the very first stage. I received life-changing lessons on how to build my research career without falling into mistakes others fell into in the past. I learned how important it is to build and take care of relationships, as we got to welcome incoming researchers from distant countries and prepared the departure of some of IntEvoBio staff to other universities abroad. This is the spirit of the science of today: learning by sharing.

Although almost no one in the lab was a Finn during my stay, the spirit of properly balancing work and life was a rule throughout my internship, and I feel this is also part of the kindness and humanity I experienced. I felt welcome not only by having a cozy desk with plenty of lovely pictures and catchy fish jokes hung on the wall. I also loved lab outings. To wrap up the work of months, give farewell to a visiting researcher and say hello to summer, we went all together kayaking the Vanhankaupunginlahti in a hot day of June, right before the end of my internship, and then stayed for dinner. We really had a blast. And although few of this has to do with how fishes mate in the dark, it proved vital for the development of this project which tried to answer that question.

As the proverb says, ‘Give a man a fish and you’ll feed him for a day; teach a man to fish and you’ll feed him for a lifetime’. Thanks IntEvoBio and thanks HiLIFE for helping me so much on my journey to learn how to fish.