Elena Hidalgo

To survive and thrive under stressful conditions, living organisms must adapt. Challenges such as nutrient deprivation disrupt normal cellular functions, requiring appropriate responses to restore ...

Since their discovery in the late 1980s as peroxidases that eliminate hydrogen peroxide (H ₂ O ₂ ), peroxiredoxins (Prxs) have been recognized not only for detoxification but also for roles in redox signaling. In fission yeast, the main Prx, Tpx1, maintains steady-state H ₂ O ₂ below toxic levels through its scavenging activity. Under excess peroxides, Tpx1 switches to a signaling role, transmitting oxidative signals to the transcription factor Pap1. Dissecting the relative contributions of Tpx1’s peroxidase and sensor functions is challenging. To address this, we used a synthetic biology strategy to separate these dual roles. Tpx1 or catalase (Ctt1) was expressed in distinct subcellular compartments, and their capacity to remove H ₂ O ₂ and activate Pap1 was assessed. Both scavengers can complement the growth defects of cells lacking Tpx1 in high oxygen. Combined with H ₂ O ₂ biosensors, our results show that Tpx1 efficiently controls H ₂ O ₂ levels from any compartment, whereas Ctt1 does not. However, cells expressing mitochondrial- or nuclear-targeted Tpx1 display reduced oxidative stress tolerance because Pap1 activation requires cytosolic Tpx1. Overall, Tpx1 is a highly effective peroxidase throughout the cell, but its subcellular localization is essential for proper redox signaling regulation.

Summary: Schizosaccharomyces pombe offers a powerful model for mitochondrial studies In vivo and in vitro methods to study mitochondrial function and homeostasis Assays to study mitochondrial metabolism, bioenergetics, and redox balance Description of biosensors for in vivo measurement of ATP, H ₂ O ₂ , NAD(P)H Tools to analyze morphology and respiratory supercomplexes.

The analysis of ribosome profiling (Ribo-Seq) data has provided evidence that many eukaryotic mRNAs contain translated upstream or downstream ORFs (uORFs/dORFs), but the biological significance of this translation activity remains, for the most part, unknown. One of the principal limitations has been the lack of Ribo-Seq data from several closely related species, precluding the identification of cases in which translation is phylogenetically conserved. Here, by combining Ribo-Seq data from 100 different experiments, we identify 2,332 translated uORFs and 1,008 translated dORFs in S. cerevisiae , which result in microproteins that tend to be highly hydrophobic or positively charged. To study their phylogenetic conservation, we have generated Nanopore direct RNA sequencing data, together with Ribo-Seq data, from six additional Saccharomyces species, spanning an evolutionary period of around 16 million years. We have identified 195 translated S. cerevisiae uORFs that are also translated in other Saccharomyces species; these uORFs are translated at levels comparable to the main coding sequence and display signatures of purifying selection at the level of the encoded microproteins. In contrast, dORFs are translated at very low levels and they are rarely conserved, suggesting much more limited microprotein functionalization. We have also discovered that uORF translation is associated with the formation of alternative transcript isoforms encompassing the region containing the uORFs but not the main protein coding sequence, implying that some microproteins can be produced independently of the main protein product. This work significantly advances our understanding of how initially pervasive uORF translation can result in new microproteins, providing many new candidates for further functional studies.

Background: The histone code, comprised of diverse histone post-translational modifications, intricately regulates nucleosome organization and gene expression. Histone marks also serve as binding sites for a diverse array of protein complexes and enzymes, orchestrating downstream cellular functions. Some modifications are crucial for enabling the complete activation of alternative gene expression programs in response to environmental changes. Results: Our study delves into the consequences of depleting histone mark erasers, the H2B deubiquitinases Ubp8 and Ubp16, in transcription efficiency in fission yeast. Cells lacking these enzymes exhibit resistance to oxidative stress, attributed to enhanced transcription of stress genes both under basal and induced conditions. Ubp8 is recruited to stress promoters as part of the activating SAGA complex, which also acetylates histone H3, while Ubp16 promotes H2B deubiquitination in coding regions. Cells lacking Ubp8 or Ubp16 display enhanced levels of both H2Bub and H3K4me, and chromatin structure at stress genes is already affected under basal conditions and is not timely restored in the wake of elongating Pol II. Depletion of the COMPASS complex, required to methylate H3K4, solely abolishes stress resistance in Δ ubp16 . In contrast, cells lacking Ubp8 display independence from SAGA or COMPASS for full transcriptional activation, suggesting an essential and sufficient role of H2Bub in Pol II promoter escape. Conclusions: This study provides insights into the complex histone crosstalk regulating gene expression and underscores the pivotal role of histone ubiquitination in promoting efficient nucleosome dynamics during Pol II transcription.

Entry into the cell cycle requires activation of G1 cyclin-dependent kinases (CDKs) and the G1/S transcriptional program. In fission yeast, the MBF complex is the main transcription factor driving early cell-cycle gene expression. MBF-dependent transcription is activated in metaphase and repressed at the end of S phase by a feedback loop involving the cyclin Cig2 and co-repressors Nrm1 and Yox1. While replicative stress inactivates Yox1 via phosphorylation, the mechanism that activates MBF during an unperturbed cell cycle remains unclear. Here, we identify Nrm1 as the key target of cell cycle regulation in a two-step control mechanism. First, CDK1 phosphorylates Nrm1 in metaphase, leading to its release—along with Yox1—from chromatin. Second, unphosphorylated Nrm1, generated either by dephosphorylation or de novo synthesis, is degraded during anaphase, preventing its re-association with MBF until the end of the next S phase. Together, these parallel pathways create a precisely timed window of MBF activation, ensuring proper cell cycle progression and preserving genomic stability.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a reliable technique, is rapid and precise in the identification of pathogens, and is essential in clinical microbiology laboratories. The aim of this project is to evaluate the identification performance of the MALDI-TOF MS Smart MS 5020 system (Zhuhai DL Biotech) by comparing the identification results with those of the MALDI-TOF MS Biotyper Microflex LT system (Bruker Daltonics). A total of 612 clinical isolates were analyzed in parallel: 494 routine species and 118 species not commonly isolated in routine practice, previously isolated and frozen in our hospital’s strain collection. This collection included gram-negative bacteria, gram-positive bacteria, anaerobes, and fungi. Concordance at genus and species level identification was evaluated, and 16S rRNA gene sequencing was performed for strains unidentified or with discordant results. Both systems, Smart MS 5020 and Biotyper Microflex LT, achieved high identification rates (100% and 98.9%, respectively). The concordance between the systems was 98.9% at the genus level and 97.2% at the species level. Correct species-level identification was 96.9% for the Smart MS 5020 and 96.6% for the Biotyper Microflex LT. The MALDI-TOF MS Smart MS 5020 and Biotyper Microflex LT systems offer high efficacy in identifying a wide variety of clinical isolates, both demonstrating excellent performance. IMPORTANCE: Accurate identification of microorganisms in clinical microbiology laboratories is essential for the effective diagnosis and treatment of patients. This study evaluates the performance of the Smart MS 5020 matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) system in comparison to the widely used Biotyper Microflex LT system. As a new entrant to the market, independent validation of the Smart MS 5020 system is crucial for laboratories considering its implementation. By analyzing 612 clinical isolates, including both routine and rare species, this study provides valuable data on the concordance rates between the two systems. The results confirm the Smart MS 5020 high accuracy and reinforce the reliability of MALDI-TOF MS technology. The high concordance rate between the two systems establishes the Smart MS 5020 as a promising, cost-effective alternative for routine diagnostics. These results expand the evidence base on MALDI-TOF MS platforms and can inform procurement decisions in clinical laboratories worldwide.