Nuclear speckles (NS) are membraneless nuclear organelles that act as critical hubs for pre-messenger RNA splicing. Defects in splicing are linked to several human diseases, including cancer, Alzheimer's disease, and dystrophies. While CLK kinases regulate the mobilization of splicing factors from NS, the molecular mechanisms underlying NS assembly and dissolution remain unclear. Using an adaptation of the Biotinylation by Antibody Recognition technique, we identified polyphosphate (polyP) as a novel and essential regulator of NS dynamics. Polyphosphate, a highly conserved polyanion composed of a chain of phosphate molecules, is involved in several functions in mammalian cells. Here, we show that polyP interacts with the NS core component SRRM2, and its depletion disrupts NS organization releasing splicing factors into the nucleoplasm. RNA-seq analysis reveals that polyP depletion increases exon exclusion, particularly in transcripts with multiple isoforms, highlighting its role in splicing regulation. Mechanistically, we demonstrate that polyP acts as a physiological inhibitor of CLK3 kinase, preventing the phosphorylation of SR proteins and thereby maintaining NS stability. Our findings not only expand our understanding of NS biology but also provide new insights into the polyP involvement in splicing-related diseases.

Polyphosphate (polyP), a biopolymer composed of phosphates, impacts a wide range of biological functions and pathological conditions in all organisms. However, polyP’s intricate physiology and structure in human cells have remained elusive, largely due to the lack of a reliable quantification method including its extraction. In this study, we assess critical points in the whole process: extraction, purification, and quantification polyP from human cell lines. We developed a highly efficient method that extracts between 3 and 100 times more polyP than previously achieved. Supported by Nuclear Magnetic Resonance (NMR), our approach confirms that mammalian polyP is primarily a linear unbranched polymer. We applied the optimized method to commonly used human cell lines, uncovering important variations of intracellular polyP that correlate with the expression levels of specific polyP converting enzymes. This study underscores the importance of employing several techniques for polyP characterization in parallel and provides a valuable and standardized tool for further exploration in this field.

CLIL Content and Language Integrated Learning, as we know, is an umbrella dual-focused pedagogical approach (e.g., Dalton-Puffer, 2011) that is more commonly associated within contexts of primary and secondary education. Pedagogical strategies and materials are designed to, on the one hand, progressively work towards higher-order thinking and production abilities in areas of content learning in a foreign language, often English, while simultaneously attending to the students’ need to develop the relevant linguistic competencies in the target language. To achieve these, and related goals, materials and teaching strategies centre on the principles of integration (Nikula et al., 2016) and high-quality classroom interaction (Walsh, 2006). But what of the EMI context? Specifically, how is content and language integration achieved (or at least, aimed at) in tertiary education? To allay concerns to some degree, language, in any context, packages knowledge, and knowledge is packaged in language. In other words, language and knowledge co-constitute each other (Airey & Linder, 2009; Lemke, 1990). So, although EMI as such does not hold to explicit language-related goals, studies have shown that teachers do hold themselves and students accountable for the correct use of conventionalised disciplinary language in the field (Clua Serrano 2021; Clua1 & Evnitskaya, forthcoming, Escobar Urmeneta, 2017; Mancho-Barés, G., & AguilarPérez, M., 2020) as well as deal with language aspects, often beyond the purely technical range (e.g., Andjelkov, 2022). However, a significant proportion of faculty rejects certain socio-constructivist-based CLIL methodologies that are often used in compulsory-level education (e.g., Kletzenbauer et al., 2022, this issue), believing these approaches should be reserved for university students with low English language proficiency (Aguilar, 2017; Airey, 2016). This not only speaks of the additional investment required of teachers in the EMI enterprise (Doiz et al. 2011), but also of the reported identities at play in teachers as content, and not language, experts (Macaro, 2018; Mancho-Barés, G., & Aguilar-Pérez, M., 2020). All in all, research continues to explore teacher (and student) practices in, and beliefs about, EMI to find ways to optimise teaching and learning in this context. The interview presented here serves to get a glimpse into pedagogical praxis and identity through the experience of one EMI teacher. Javier Jimenez Jimenez is a tenured Associate Professor in the Department of Basic Science at the Universitat Internacional de Catalunya (UIC Barcelona). He is an experienced lecturer, and through this interview, he reflects on his experience as an EMI teacher of Immunology and Microbiology between the years 2016-2019. He taught this subject in English in the English-track Dentistry programme, and in Spanish in the Spanish-track programme, back-toback. https://doi.org/10.5565/rev/clil.95 eISSN: 2604-5613 Print ISSN: 2605-5893

Polyphosphate (polyP) is an evolutionarily conserved polymer of phosphates that is difficult to study in human cells because of its low concentration and high lability. First, we described how to express and purify Xpress-tagged PPBD (Ppx1 PolyP Binding Domain). We describe the detection and quantification of nuclear polyP in HEK293T cells using Xpress-PPBD, Xpress antibody, and Alexa-conjugated secondary antibodies. We have also used this protocol in SHSY5Y HeLa and HEK293 cells.For complete details on the use and execution of this protocol, please refer to Samper-Marti & PRIME;n et al. (2021).

Polyphosphate (polyP) is a polymer of hundreds of phosphate residues present in all organisms. In mammals, polyP is involved in crucial physiological processes, including coagulation, inflammation, and stress response. However, after decades of research, the metabolic enzymes are still unknown. Here, we purify and identify Nudt3, a NUDIX family member, as the enzyme responsible for polyP phosphatase activity in mammalian cells. We show that Nudt3 shifts its substrate specificity depending on the cation; specifically, Nudt3 is active on polyP when Zn2+ is present. Nudt3 has in vivo polyP phosphatase activity in human cells, and importantly, we show that cells with altered polyP levels by modifying Nudt3 protein amount present reduced viability upon oxidative stress and increased DNA damage, suggesting that polyP and Nudt3 play a role in oxidative stress protection. Finally, we show that Nudt3 is involved in the early stages of embryo

During evolution, cells have developed a plethora of mechanisms to optimize survival in a changing and unpredictable environment. In this regard, they have evolved networks that include environmental sensors, signaling transduction molecules and response mechanisms. Hog1 (yeast) and p38 (mammals) stress-activated protein kinases (SAPKs) are activated upon stress and they drive a full collection of cell adaptive responses aimed to maximize survival. SAPKs are extensively used to learn about the mechanisms through which cells adapt to changing environments. In addition to regulating gene expression and metabolism, SAPKs control cell cycle progression. In this review, we will discuss the latest findings related to the SAPK-driven regulation of mitosis upon osmostress in yeast.

There are two major pathways for repairing DNA double-strand breaks (DSBs): homologous directed recombination (HDR) and non-homologous end-joining (NHEJ). While NHEJ functions throughout the cell cycle, HDR is only possible during S/G2 phases, suggesting that there are cell cycle-specific mechanisms regulating the balance between the two repair systems. The regulation exerted by CDKs on HDR has been extensively demonstrated, and here we present evidence that the CDK Pho85, in association with the G1 cyclin Pcl1, phosphorylates Yku80 on Ser 623 to regulate NHEJ activity. Cells bearing a non-phosphorylatable version of Yku80 show increased NHEJ and reduced HDR activity. Accordingly, yku80S623A cells present diminished viability upon treatment with the DSB-producer bleomycin, specifically in the G2 phase of the cell cycle. Interestingly, the mutation of the equivalent residue in human Ku80 increases sensitivity to bleomycin in several cancer cell lines, suggesting that this mechanism is conserved in humans. Altogether, our results reveal a new mechanism whereby G1-CDKs mediate the choice between HDR and NHEJ repair pathways, putting the error prone NHEJ on a leash and enabling error free HDR in G2 when homologous sequences are available.

Adaptation to environmental changes is crucial for cell fitness. In Saccharomyces cerevisiae, variations in external osmolarity trigger the activation of the stress-activated protein kinase Hog1 (high-osmolarity glycerol 1), which regulates gene expression, metabolism, and cell-cycle progression. The activation of this kinase leads to the regulation of G1, S, and G2 phases of the cell cycle to prevent genome instability and promote cell survival. Here we show that Hog1 delays mitotic exit when cells are stressed during metaphase. Hog1 phosphorylates the nucleolar protein Net1, altering its affinity for the phosphatase Cdc14, whose activity is essential for mitotic exit and completion of the cell cycle. The untimely release of Cdc14 from the nucleolus upon activation of Hog1 is linked to a defect in ribosomal DNA (rDNA) and telomere segregation, and it ultimately delays cell division. A mutant of Net1 that cannot be phosphorylated by Hog1 displays reduced viability upon osmostress. Thus, Hog1 contributes to maximizing cell survival upon stress by regulating mitotic exit.

MOTIVATION:Short bioactive peptides encoded by small open reading frames (sORFs) play important roles in eukaryotes. Bioinformatics prediction of ORFs is an early step in a genome sequence analysis, but sORFs encoding short peptides, often using non-AUG initiation codons, are not easily discriminated from false ORFs occurring by chance.RESULTS:AnABlast is a computational tool designed to highlight putative protein-coding regions in genomic DNA sequences. This protein-coding finder is independent of ORF length and reading frame shifts, thus making of AnABlast a potentially useful tool to predict sORFs. Using this algorithm, here, we report the identification of 82 putative new intergenic sORFs in the Caenorhabditis elegans genome. Sequence similarity, motif presence, expression data and RNA interference experiments support that the underlined sORFs likely encode functional peptides, encouraging the use of AnABlast as a new approach for the accurate prediction of intergenic sORFs in annotated eukaryotic genomes.AVAILABILITY AND IMPLEMENTATION:AnABlast is freely available at http://www.bioinfocabd.upo.es/ab/. The C.elegans genome browser with AnABlast results, annotated genes and all data used in this study is available at http://www.bioinfocabd.upo.es/celegans.SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

Resumen Los clickers o Audience Response Systems (ARS) permiten a los alumnos responder preguntas planteadas por el profesor a través de dispositivos móviles y sus respuestas aparecen instantáneamente en la pantalla del aula, de manera que se pueden discutir y analizar en grupo facilitando en gran medida un aprendizaje socrático y dirigido por iguales (peer instruction). En la Facultad de Medicina de la UIC hemos empleado uno de los mencionados ARS, Socrative, para analizar el conocimiento previo, comprobar la adquisición autónoma de conocimiento, la adquisición de conocimientos después de una clase magistral y como repaso antes de la evaluación. Una encuesta anónima entre los estudiantes valora este tipo de sistemas en comparación con una metodología basada en la lectura de clase magistral. Los resultados no muestran diferencias estadísticamente significativas entre el método tradicional y el Socrative en cuanto al grado de interés ni a los conocimientos adquiridos. Sin embargo, las calificaciones obtenidas por los estudiantes con Socrative fueron significativamente superiores a las obtenidas mediante metodología tradicional. Además el 89% de los estudiantes opina que Socrative es fácil de usar, el 86% que mejora la participación en clase, el 85% que la ameniza, el 81% que facilita la autoevaluación; sin embargo, solo un 54% opina que Socrative le ayudó a obtener mejores resultados en la evaluación. Abstract The clickers or Audience Response Systems (ARS) allow students to answer questions posed by the teacher through mobile devices and their answers appear instantly on the classroom screen, so that they can be discussed and analysed as a group, greatly facilitating Socratic and peer-directed learning. In the Faculty of Medicine of the UIC, we have used one of the aforementioned ARS, — Socrative— to analyse the following aspects: prior knowledge, the autonomous acquisition of knowledge, the acquisition of knowledge after a master class and, finally, as a way to produce a review before the evaluation. We carried out an anonymous survey among our students to compare lectures where Socrative was used with classical lectures based on the professor delivery of the content. The results show no statistically significant differences between the traditional method and the Socrative in terms of the degree of interest or the acquired knowledge. However, the grades obtained by students with Socrative were significantly higher than those obtained when traditional methodology was used. In addition, 89% of students think that Socrative is easy to use, 86% that improves participation in class, 85% that enlivens it and 81% that facilitates self-evaluation; however, only 54% think that Socrative helped them to improve their marks.

In eukaryotes, the cell cycle is driven by the actions of several cyclin dependent kinases (CDKs) and an array of regulatory proteins called cyclins, due to the cyclical expression patterns of the latter. In yeast, the accepted pattern of cyclin waves is based on qualitative studies performed by different laboratories using different strain backgrounds, different growing conditions and media, and different kinds of genetic manipulation. Additionally, only the subset of cyclins regulating Cdc28 was included, while the Pho85 cyclins were excluded. We describe a comprehensive, quantitative and accurate blueprint of G1 cyclins in the yeast Saccharomyces cerevisiae that, in addition to validating previous conclusions, yields new findings and establishes an accurate G1 cyclin blueprint. For the purposes of this research, we produced a collection of strains with all G1 cyclins identically tagged using the same and most respectful procedure possible. We report the contribution of each G1 cyclin for a broad array of growing and stress conditions, describe an unknown role for Pcl2 in heat-stress conditions and demonstrate the importance of maintaining the 3’UTR sequence of cyclins untouched during the tagging process.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets.

Colorectal cancer (CRC) is one of the most common cancers worldwide, with 8–10% of these tumours presenting a BRAF (V600E) mutation. Cyclins are known oncogenes deregulated in many cancers, but the role of the new subfamily of atypical cyclins remains elusive. Here we have performed a systematic analysis of the protein expression levels of eight atypical cyclins in human CRC tumours and several cell lines, and found that CNTD2 is significantly upregulated in CRC tissue compared to the adjacent normal one. CNTD2 overexpression in CRC cell lines increases their proliferation capacity and migration, as well as spheroid formation capacity and anchorage-independent growth. Moreover, CNTD2 increases tumour growth in vivo on xenograft models of CRC with wild-type BRAF . Accordingly, CNTD2 downregulation significantly diminished the proliferation of wild-type BRAF CRC cells, suggesting that CNTD2 may represent a new prognostic factor and a promising drug target in the management of CRC.

Phosphate is one of the essential elements supporting life. Cells accumulate phosphate in the form of a molecule called polyphosphate (polyP), which carries many functions in the physiology of cells that have not been wholly elucidated. Polyphosphate is present in all the types of cells from bacteria to mammals. It consists of a linear polymer constructed with anywhere from a few to hundreds of inorganic phosphate (Pi) molecules linked by phosphoanhydride bonds. Although polyP was described many years ago, difficulties in the study of its roles, most likely due to the many processes polyP is involved in and incomplete information obtained from multiple models and organisms relegate polyP into oblivion. But now, several interesting pieces of evidence are resurrecting the polyP as a key molecule in processes, such as protein folding, carbon metabolism, cell cycle progression, dNTP synthesis, and genomic stability. In this contribution, in addition to briefly summarize the polyP history and roles, we discuss its involvement in supporting cell cycle progression and genomic stability as well as the implications for the truthful replication of genomes.

As lung cancer has increased to the most common cause of cancer death worldwide, prognostic biomarkers and effective targeted treatments remain lacking despite advances based on patients' stratification. Multiple core cyclins, best known as drivers of cell proliferation, are commonly deregulated in lung cancer where they may serve as oncogenes. The recent expansion of the cyclin family raises the question whether new members might play oncogenic roles as well. Here, we investigated the protein levels of eight atypical cyclins in lung cancer cell lines and formalin-fixed and paraffin-embedded (FFPE) human tumors, as well as their functional role in lung cancer cells. Of the new cyclins evaluated, CNTD2 was significantly overexpressed in lung cancer compared to adjacent normal tissue, and exhibited a predominant nuclear location. CNTD2 overexpression increased lung cancer cell viability, Ki-67 intensity and clonogenicity and promoted lung cancer cell migration. Accordingly, CNTD2 enhanced tumor growth in vivo on A549 xenograft models. Finally, the analysis of gene expression data revealed a high correlation between elevated levels of CNTD2 and decreased overall survival in lung cancer patients. Our results reveal CNTD2 as a new oncogenic driver in lung cancer, suggesting value as a prognostic biomarker and therapeutic target in this disease.

Deciphering the molecular mechanisms that connect cell cycle progression and nucleocytoplasmic transport is of particular interest: this intertwined relationship, once understood, may provide useful insight on the diseases resulting from the malfunction of these processes. In the present study we report on findings that indicate a biochemical connection between the cell cycle regulator CDK Pho85 and Ran-GTPase Gsp1, an essential nucleocytoplasmic transport component. When Gsp1 cannot be phosphorylated by Pho85, the cell cycle progression is impaired. Accordingly, a nonphosphorylatable version of Gsp1 abnormally localizes to the nucleus, which impairs the nuclear transport of molecules, including key components of cell cycle progression. Furthermore, our results suggest that the physical interaction of Gsp1 and the Kap95 karyopherin, essential to the release of nuclear cargoes, is altered. Altogether, the present findings point to the involvement of a biochemical mechanism in the interlocked regulation of the cell cycle and nuclear transport.

Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention.

The judgment of December 21 of the Court of Justice of the European Union imposed on European banks the retrospective return to their customers of what was collected by virtue of the very extensive coverage of interest rate risk known as the "mortgage clause floor" that fixed a minimum interest to be paid by the mortgage client with a variable interest loan, if the rates decreased in the market. In general, this "floor" interest has hovered around 2.5% per year in the contracts, which has resulted in high losses for customers, considering that the reference European banking interest rate or Euribor is around an average of 0.06% in the last biennium, becoming negative; thus, on February 24, 2017 it was -0.11%.