Browsing by Author "Rivera, Maira"

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    An Open One-Step RT-qPCR for SARS-CoV-2 detection
    (Public Library Science, 2024) Cerda Rojas, Ariel Patricio; Rivera, Maira; Armijo, Grace; Ibarra-Henríquez, Catalina; Reyes, Javiera; Blázquez Sánchez, Paula; Avilés, Javiera; Arce, Anibal; Seguel, Aldo; Brown, Alexander J.; Vásquez, Yesseny; Cortez-San Martín, Marcelo; Cubillos, Francisco A.; García, Patricia; Ferrés, Marcela; Ramírez Sarmiento, César Antonio; Federici, Fernan; Gutiérrez, Rodrigo A.
    The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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    An Open One-Step RT-qPCR for SARS-CoV-2 detection
    (2024) Cerda, Ariel; Rivera, Maira; Armijo, Grace; Ibarra-Henriquez, Catalina; Reyes, Javiera; Blazquez-Sanchez, Paula; Aviles, Javiera; Arce, Anibal; Seguel, Aldo; Brown, Alexander J.; Vasquez, Yesseny; Cortez-San Martin, Marcelo; Cubillos, Francisco A.; Garcia, Patricia; Ferres, Marcela; Ramirez-Sarmiento, Cesar A.; Federici, Fernan; Gutierrez, Rodrigo A.
    The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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    Developing and Implementing Cloud-Based Tutorials That Combine Bioinformatics Software, Interactive Coding, and Visualization Exercises for Distance Learning on Structural Bioinformatics
    (2021) Engelberger, Felipe; Galaz-Davison, Pablo; Bravo, Graciela; Rivera, Maira; Ramirez-Sarmiento, Cesar A.
    The COVID-19 pandemic has swiftly forced a change in learning strategies across educational institutions, from extensively relying on in-person activities toward online teaching. It is particularly difficult to adapt courses that depend on physical equipment to be now carried out remotely. This is the case for bioinformatics, which typically requires dedicated computer classrooms, as the logistics of granting remote access to a workstation or relying on the computational resources of each student is not trivial. A possible workaround is using cloud server-based computing resources, such as Google Colaboratory, a free web browser application that allows the writing and execution of Python programming through Jupyter notebooks, integrating text, images, and code cells. Following a cloud-based approach, we migrated the practical activities of a course on molecular modeling and simulation into the Google Colaboratory environment resulting in 12 tutorials that introduce students to topics such as phylogenetic analysis, molecular modeling, molecular docking, several flavors of molecular dynamics, and coevolutionary analysis. Each of these notebooks includes a brief introduction to the topic, software installation, execution of the required tools, and analysis of results, with each step properly described. Using a Likert scale questionnaire, a pool of students positively evaluated these tutorials in terms of the time required for their completion, their ability to understand the content and exercises developed in each session, and the practical significance and impact that these computational tools have on scientific research. All tutorials are freely available at https: //github.com/pb3lab/ibm3202.
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    Dimer dissociation is a key energetic event in the fold-switch pathway of KaiB
    (2022) Rivera, Maira; Galaz-Davison, Pablo; Retamal-Farfan, Ignacio; Komives, Elizabeth A.; Ramirez-Sarmiento, Cesar A.
    Cyanobacteria possesses the simplest circadian clock, composed of three proteins that act as a phosphorylation oscillator: KaiA, KaiB, and KaiC. The timing of this oscillator is determined by the fold-switch of KaiB, a structural rearrangement of its C-terminal half that is accompanied by a change in the oligomerization state. During the day, KaiB forms a stable tetramer (gsKaiB), whereas it adopts a monomeric thioredoxin-like fold during the night (fsKaiB). Although the structures and functions of both native states are well studied, little is known about the sequence and structure determinants that control their structural interconversion. Here, we used confinement molecular dynamics (CCR-MD) and folding simulations using structure-based models to show that the dissociation of the gsKaiB dimer is a key energetic event for the fold-switch. Hydrogen-deuterium ex-change mass spectrometry (HDXMS) recapitulates the local stability of protein regions reported by CCR-MD, with both ap-proaches consistently indicating that the energy and backbone flexibility changes are solely associated with the region that fold-switches between gsKaiB and fsKaiB and that the localized regions that differentially stabilize gsKaiB also involve regions outside the dimer interface. Moreover, two mutants (R23C and R75C) previously reported to be relevant for altering the rhyth-micity of the Kai clock were also studied by HDXMS. Particularly, R75C populates dimeric and monomeric states with a deute-rium incorporation profile comparable to the one observed for fsKaiB, emphasizing the importance of the oligomerization state of KaiB for the fold-switch. These findings suggest that the information necessary to control the rhythmicity of the cyanobacterial biological clock is, to a great extent, encoded within the KaiB sequence.
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    DNA facilitates heterodimerization between human transcription factors FoxP1 and FoxP2 by increasing their conformational flexibility
    (2023) Conuecar, Ricardo; Asela, Isabel; Rivera, Maira; Galaz-Davison, Pablo; Gonzalez-Higueras, Jorge; Hamilton, George L.; Engelberger, Felipe; Ramirez-Sarmiento, Cesar A.; Babul, Jorge; Sanabria, Hugo; Medina, Exequiel
    Transcription factors regulate gene expression by binding to DNA. They have disordered regions and specific DNA-binding domains. Binding to DNA causes structural changes, including folding and interactions with other molecules. The FoxP subfamily of transcription factors in humans is unique because they can form heterotypic interactions without DNA. However, it is unclear how they form heterodimers and how DNA binding affects their function. We used computational and experimental methods to study the structural changes in FoxP1's DNA-binding domain when it forms a heterodimer with FoxP2. We found that FoxP1 has complex and diverse conformational dynamics, transitioning between compact and extended states. Surprisingly, DNA binding increases the flexibility of FoxP1, contrary to the typical folding-upon-binding mechanism. In addition, we observed a 3-fold increase in the rate of heterodimerization after FoxP1 binds to DNA. These findings emphasize the importance of structural flexibility in promoting heterodimerization to form transcriptional complexes.
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    Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate
    (2023) Rivera, Maira; Burgos-Bravo, Francesca; Engelberger, Felipe; Asor, Roi; Lagos-Espinoza, Miguel I. A.; Figueroa, Maximiliano; Kukura, Philipp; Ramirez-Sarmiento, Cesar A.; Baez, Mauricio; Smith, Steven B.; Wilson, Christian A. M.
    BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis.
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    Temperature dependent mechanical unfolding and refolding of a protein studied by thermo-regulated optical tweezers
    (2023) Rivera, Maira; Mjaavatten, Are; Smith, Steven B.; Baez, Mauricio; Wilson, Christian A. M.
    Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic contributions are obtained by quantifying the conformational equilibrium based on melting experiments performed in bulk conditions. Such experiments are suitable only for those small single-domain proteins whose side reactions of irreversible aggregation are unlikely to occur. Here, we avoid aggregation by pulling single-protein molecules in a thermo-regulated optical tweezers. Thus, we are able to explore the temperature dependence of the thermodynamic and kinetic pa-rameters of MJ0366 from Methanocaldococcus jannaschii at the single-molecule level. By performing force-ramp experiments be-tween 2 degrees C and 40 degrees C, we found that MJ0366 has a nonlinear dependence of free energy with temperature and a specific heat change of 2.3 5 1.2 kcal/mol*K. These thermodynamic parameters are compatible with a two-state unfolding/refolding mechanism for MJ0366. However, the kinetics measured as a function of the temperature show a complex behavior, suggesting a three-state folding mechanism comprising a high-energy intermediate state. The combination of two perturbations, temperature and force, reveals a high-energy species in the folding mechanism of MJ0366 not detected in force-ramp experiments at constant temperature.