<p><strong>Professor Michaela Rumlová, head of a research group of the National Institute of Virology and Bacteriology at UCT Prague (and Vice-Rector for doctoral studies), holder of a licensed patent &nbsp;for antiretroviral testing assays, and recipient of the Czech Science Foundation’s President’s Award for HIV research, has also become the principal investigator of the bilateral INTER-EXCELLENCE II project “</strong><i><strong>Cellular factors regulating the export and packaging of unspliced ​​retroviral RNA</strong></i><strong>”, starting this year. In this interview, she explains how retroviruses such as HIV “hack” the cell in order to export their own genome from the nucleus into the cytoplasm, and then to the site where it is specifically packaged into nascent viral particles, a key step in viral replication. Her team is collaborating with the NIH (National Institutes of Health) and Rockefeller University to elucidate mechanisms that may help reveal new vulnerable points in the retrovirus replication cycle while simultaneously clarifying related cellular processes.</strong></p><h5><strong>Could you describe your project and its main objectives?</strong></h5><p>Retroviruses, such as HIV, are viruses that carry genetic information in the form of RNA; however, after entering a cell, they are able to reverse transcribe it into DNA and integrate it into the host genome. In order to replicate, they must transport their genetic information—in the form of unspliced RNA—from the nucleus into the cytosol. There, it serves both as a template for the production of viral proteins and as the genome of new viral particles. This is highly unusual, as the cell very carefully controls which RNA molecules are allowed to leave the nucleus. Normal protein-coding cellular RNAs, known as messenger RNAs (mRNAs), must undergo splicing—a processing step in which certain segments are removed—before being exported. When this process fails, mRNA that retains these segments is held back in the nucleus as part of quality control and subsequently degraded. Retroviruses have managed to outwit this cellular control machinery, allowing their unspliced RNA to pass into the cytoplasm.</p><p>Our project focuses on identifying the cellular proteins that assist retroviruses in this step and on the mechanism by which protein complexes bound to viral RNA are remodelled during RNA transport. The goal is to understand how the virus uses cellular mechanisms to its advantage and how this pathway is linked to the subsequent fate of the RNA, i.e. to its translation and packaging into new viral particles.</p><h5><strong>Why is this export of unspliced ​​RNA so important for retroviral replication?</strong></h5><p>Because this RNA has a dual role for the virus. On the one hand, it serves as a template for synthesizing viral proteins in the cytoplasm; on the other, it represents the genetic material itself, which must be packaged into new viral particles. The virus must package its RNA genome in its unspliced form, as only this RNA contains essential sequences without which further viral replication would be impossible.</p><p><strong>Simply put: </strong>the virus needs to get a complete copy of its genetic instructions out of the nucleus. Once the viral RNA is in the cytoplasm, it can be used as a template for synthesizing viral proteins; and a portion of these RNA molecules is then selected and packaged into a new particle as a genome. We are interested in how the virus forces the cell to allow viral RNA that is, from the cell’s perspective, “unfinished,” that is unspliced, to leave the nucleus. We are also investigating how the cell and the virus together - albeit unintentionally from the cell’s perspective - determine which RNA molecule will be translated into proteins and which will be packaged as the genome. This is one of the key, yet still not fully understood, steps of retroviral replication.</p><h5><strong>What do you think it was about your project that impressed the Ministry of Education, Youth and Sports’ jury the most?</strong></h5><p>I believe the decisive factor was a combination of several elements. First, the reviewers were primarily interested in the scientific problem itself. We are investigating a mechanism that is essential for retroviruses, but still not sufficiently understood, which may also have broader implications for the regulation of cellular processes. In addition, the quality of the team, our strong collaboration with the<strong> NIH </strong>and <strong>Rockefeller University</strong>, and the use of modern methods that will allow us to track viral RNA in great detail also played a role. It was also important that the project has clear objectives and a realistic plan.</p><h5><strong>How will this research contribute to the development of new treatments for retroviral infections?</strong></h5><p>It is important to say that this is primarily basic research. Rather than developing a new drug directly, we are trying to understand, at the molecular level, the step that the virus cannot do without. Only when we fully understand the mechanism of these cellular processes and identify the essential factors for the export of unspliced ​​RNA and how they function, will we be able to better determine which interactions or mechanisms represent potentially vulnerable points. Similar mechanisms associated with the export of unspliced ​​mRNA also appear in some diseases, such as cancer, so I &nbsp;see the benefit of our research mainly in that it will refine our understanding of these pathological processes in the cell, and retroviruses help us do so. Therefore, our work on retroviruses not only shows us how the virus works, but also how the cell itself works.</p><h5><strong>What responsibilities does your UCT Prague team have? How does the collaboration with your American partners work?</strong></h5><p>At UCT Prague, we are conducting the project’s primary experimental work. Our research involves cell cultures, viral models, and various biochemical techniques ranging from immunochemistry to microscopy that allow us to track viral RNA and identify its cellular partners. In collaboration with colleagues from <strong>NIH, Frederick</strong>, we are developing single-particle tracking methods to observe viral RNA movement in living cells across both space and time. Furthermore, we are working with colleagues from <strong>Rockefeller University to employ iCLIP </strong>methods for the precise mapping of protein-binding sites on RNA. Both collaborations are valuable to us, because they are not just about data sharing, but also about the transfer of expertise and detailed know-how.</p><h5><strong>Who else is collaborating on the project?</strong></h5><p>At UCT Prague, we collaborate closely with the Department of Biotechnology and the Department of Biochemistry and Microbiology. Externally, we work with colleagues from the Institute of<strong> Organic Chemistry and Biochemistry of the CAS (IOCB Prague)</strong> and the <strong>Central European Institute of Technology in Brno (CEITEC)</strong>. Our research naturally necessitates the integration of diverse fields of expertise; it requires combining virology, cell biology, and biochemistry with advanced imaging and modern approaches to study RNA-protein interactions. This is exactly what makes our project capable of addressing complex questions that a single laboratory cannot tackle on its own.</p><h5><strong>Are there plans to continue the project? Have you brought on new collaborators, and are you looking to welcome new colleagues to the team?</strong></h5><p>Yes, definitely. In basic research, it often happens that one answered question opens up several more. High-quality, properly interpreted data provide a foundation you can build on — and that is what moves scientists forward. It is already clear that the topic of retroviral RNA export and packaging has broader implications and may reveal more general principles of how RNA functions within the cell. We also hope the project will be attractive to students, as it connects classical molecular biology, biochemistry, and virology with cutting-edge biotechnological methods. Students are already involved in the project as part of their bachelor’s and master’s theses.</p><h5><strong>What was the personal turning point that motivated you to pursue a long-term investigation into retroviral particles?</strong></h5><p>I first encountered retroviruses in the early 1990s during my diploma thesis, when I was working at IOCB Prague on HIV-1 protease inhibitors. At that time, AIDS could not yet be effectively treated, and retrovirus research had a completely different dimension. From the very beginning, I was fascinated by how such a relatively small and simple virus can outsmart evolutionarily highly optimized cellular processes and turn them to its own advantage. And that has never stopped fascinating me. From a scientific point of view, retroviruses are remarkable; just consider one of their enzymes, reverse transcriptase, without which no diagnostic or research laboratory in the world could function today. Without it, it would be impossible to diagnose RNA viruses like SARS-CoV-2, influenza, or HIV using RT-PCR tests, or, for example to produce recombinant human proteins in bacterial cells.</p>

Tracking unspliced RNA: Professor Rumlová studies how retroviruses “hack” cells

News from LabRulezLCMS Library - Week 20, 2026

<p><strong>🎤Lindsay Repka&nbsp;</strong></p><p><strong>Lindsay Repka is a chemistry professor at Middlebury College whose research explores photoredox chemistry, catalyst stability, and visible-light-driven transformations. Her lab emphasizes both mechanistic insight and hands-on student training in advanced analytical instrumentation.</strong></p><p>In this episode of Concentrating on Chromatography, we sit down with Lindsay Repka to discuss how LC-MS and GC-MS transformed her lab’s approach to photoredox chemistry.</p><p>What began as a project to develop a visible-light photocrosslinking handle unexpectedly led to a major discovery: the solvent (DMF) was reacting with the photocatalyst itself. Using high-resolution LC-MS, Lindsay’s team observed multiple solvent adducts forming — sometimes with complete catalyst consumption. That discovery reshaped their research direction.</p><p>This episode is a rare deep dive into both LC-MS and GC-MS within the same research project, showing how chromatography-driven insight can turn unexpected degradation into productive new reactivity.</p><p><strong>If you work in:</strong></p><ul><li>Photoredox chemistry</li><li>Reaction optimization</li><li>Mass spectrometry method development</li><li>Catalyst screening</li><li>Academic synthetic chemistry</li></ul><p>…this conversation will resonate.</p><h4><strong>Key Topics Covered</strong></h4><ul><li>Photocatalyst stability in DMF, DCE, and MeCN</li><li>Demethylation under mild visible-light conditions</li><li>High-resolution Q-TOF LC-MS quantitation</li><li>Internal standard methodology in GC-MS</li><li>Signal-to-noise improvement using extracted ion chromatograms</li><li>Reaction reproducibility and quality control strategy</li></ul><h3><strong>Video Transcription</strong></h3><p>In this interview, Lindsay Repka discusses the role of photoredox chemistry in method development, photocrosslinking, solvent activation, catalyst stability, and the use of LC-MS and GC-MS to understand reaction pathways.</p><h4><strong>What is photoredox chemistry?</strong></h4><p>Photoredox chemistry uses light to activate a photocatalyst, which can either donate or accept an electron from a substrate. This generates radical species that can undergo new types of reactivity.</p><p>Repka explains that one of the key advantages of photoredox chemistry is its ability to operate under mild conditions, often using visible light. This makes it attractive for sensitive molecules and biological systems, including potential applications in drug release, live-cell chemistry, and drug development.</p><h4><strong>Photocrosslinking and drug-target interactions</strong></h4><p>Repka’s group initially aimed to develop visible-light photocrosslinking methods to study drug interactions with biological targets. Photocrosslinking can covalently link a drug to its target, making it easier to identify the biological target of a compound.</p><p>Traditional photocrosslinking often uses UV light, but visible light offers milder and more biologically compatible conditions.</p><h4><strong>From photocrosslinking to solvent activation</strong></h4><p>The project shifted direction when the desired photocrosslinking reactions gave very low yields. Instead of reacting with amino acids as intended, the radical intermediate mainly reacted with itself.</p><p>The team explored different solvents to stabilize the radical, but found that some solvents, such as DMF, were not inert. Instead, the solvent reacted with the photocatalyst or radical species, revealing unexpected solvent activation chemistry.</p><h4><strong>Why LC-MS was critical</strong></h4><p>LC-MS became essential because the reaction mixtures were too complex and low-yielding for NMR or GC-MS. High-resolution LC-MS allowed the team to detect minor products, identify photocatalyst modifications, and observe solvent addition products with high confidence.</p><p>Repka emphasizes that LC-MS was both a qualitative and quantitative tool. It was used quantitatively to measure how much photocatalyst remained after light exposure, and qualitatively to identify degradation products and new reactivity pathways.</p><h4><strong>Photocatalyst stability workflow</strong></h4><p>The team developed a systematic workflow to evaluate photocatalyst stability under light exposure. Because catalyst degradation was time-sensitive, reproducibility required strict control of experimental conditions.</p><p>Key steps included using a glove box, commercial deoxygenated solvents, accurate weighing on a microbalance, controlled light exposure in a photoreactor, and temperature control with a chiller. These precautions helped reduce day-to-day variability and enabled reproducible measurements of catalyst remaining.</p><h4><strong>Quantifying catalyst remaining by LC-MS</strong></h4><p>Calibration curves were prepared using pure photocatalyst solutions and LC-MS ion areas. The team worked in the nanomolar range to maintain linearity.</p><p>Instead of relying only on total ion chromatograms, they used extracted ion chromatograms (EICs) to improve signal-to-noise for the photocatalyst signal. Baseline subtraction was also applied to reduce noise and improve quantitation at low concentrations.</p><p>Calibration curves and quality control samples were prepared and measured on the same day as the reaction samples, because LC-MS signal intensity can vary significantly from day to day.</p><h4><strong>Why GC-MS was used later</strong></h4><p>For demethylation studies, the group moved to GC-MS because the substrates and products were small and volatile enough for gas chromatography. GC-MS was better suited for rapidly screening many reaction conditions, including different photocatalysts, solvents, and oxidants.</p><p>Instead of generating full calibration curves for every experiment, the team used an internal standard to streamline analysis and determine percent conversion based on relative response factors.</p><h4><strong>Practical lessons from the project</strong></h4><p>Repka highlights the importance of careful sample preparation, especially when working with very small quantities of photocatalysts. A high-quality microbalance, stable granite platform, antistatic tools, and aluminum weigh boats improved weighing accuracy and reproducibility.</p><p>She also argues that mass spectrometry should be a standard part of photoredox method development because it can reveal minor products, support reaction optimization, and provide insights that may be difficult or impossible to obtain by NMR alone.</p><h4><strong>Final perspective</strong></h4><p>Beyond the chemistry, Repka emphasizes the importance of student training and mentoring. For her, research is not only about discovering new reactivity but also about giving students practical experience, helping them develop independence, and preparing them for future scientific careers.</p><blockquote><p><strong>This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.</strong></p></blockquote><blockquote><h4><strong>Concentrating on Chromatography Podcast</strong></h4><p>Dive into the frontiers of chromatography, mass spectrometry, and sample preparation with host David Oliva. Each episode features candid conversations with leading researchers, industry innovators, and passionate scientists who are shaping the future of analytical chemistry. From decoding PFAS detection challenges to exploring the latest in AI-assisted liquid chromatography, this show uncovers practical workflows, sustainability breakthroughs, and the real-world impact of separation science. Whether you’re a chromatographer, lab professional, or researcher you'll discover inspiring content!</p><p>You can find <strong>Concentrating on Chromatography Podcast </strong>in podcast apps:</p><ul><li><a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL"><strong>Spotify</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podcasts.apple.com/tz/podcast/concentrating-on-chromatography/id1788429185"><strong>Apple podcast</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podscan.fm/podcasts/concentrating-on-chromatography"><strong>podscan</strong></a></li></ul><p>and on <a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@ChromatographyTalk/videos"><strong>YouTube channel</strong></a></p></blockquote>

Using LC-MS & GC-MS to Decode Photoredox Catalyst Stability

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<p><strong>The technique of Gel Permeation Chromatography is conceptually simple: It is a separation based on size where no enthalpic interactions are observed.</strong></p>
<p>In practice, GPC is often much more involved yielding involved method development and data that can be difficult to interpret. Though GPC can seem mystifying at times 90% of the success is achieved by using the proper columns for your application.</p>
<p>During this e-seminar we will discuss what is new in GPC column technology, different types of stationary phases, and how to choose the best columns which yield:</p>
<ul>
<li><p><strong>Improved resolution</strong></p>
</li>
<li><p><strong>Improved reproducibility</strong></p>
</li>
<li><p><strong>Improved accuracy</strong></p>
</li>
<li><p><strong>Improved analysis time</strong></p>
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<p>The goal of the e-seminar is to illustrate how proper GPC column selection can make the difference between good and great chromatography.</p>
<blockquote>
<p><strong>Presenter:</strong> Michael Kruger (Application Engineer, Agilent Technologies, Inc.)</p>
</blockquote>


Agilent Technologies: Achieving Superior GPC Results Through the Correct Column Selection

Achieving Superior GPC Results Through the Correct Column Selection

What is new in GPC column technology, different types of stationary phases, and how to choose the best columns.

LCMS webinars focusing on Columns - page 33

Achieving Superior GPC Results Through the Correct Column Selection

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Using LC-MS & GC-MS to Decode Photoredox Catalyst Stability

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News from LabRulezLCMS Library - Week 20, 2026