Centro Nacional de Biotecnología

Giant viruses are extraordinary members of the virosphere due to their structural complexity and high diversity in gene content. Haptophytes are ecologically important primary producers in the ocean, and all known viruses that infect haptophytes are giant viruses. Our in-depth electron microscopic, phylogenomic and virion proteomic analyses of two haptophyte-infecting giant viruses, Haptolina ericina virus RF02 (HeV RF02) and Prymnesium kappa virus RF02 (PkV RF02), unravel their large capacity for host manipulation and arsenals that functions during the infection cycle from virus entry to release. The virus infection induces significant morphological changes of host cell that are manipulated to build a virus proliferation factory. Both viruses’ genomes encode a putative nucleoprotein (dinoflagellate/viral nucleoprotein; DVNP), which was also found in the virion proteome of PkV RF02. Phylogenetic analysis suggests that DVNPs are widespread in marine giant metaviromes. Furthermore, the analysis shows that the dinoflagellate homologues were possibly acquired from viruses of the order Imitervirales.

Examples of long-range gene regulation in bacteria are rare and generally thought to involve DNA looping. Here, using a combination of biophysical approaches including X-ray crystallography and single-molecule analysis for the KorB–KorA system in Escherichia coli, we show that long-range gene silencing on the plasmid RK2, a source of multi-drug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We show that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters up to 1.5 kb away. We resolved the tripartite crystal structure of a KorB–KorA–DNA co-complex, revealing that KorA latches KorB into a closed clamp state. DNA-bound KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Together, our findings explain the mechanistic basis for KorB role switching from a DNA-sliding clamp to a co-repressor and provide an alternative mechanism for long-range regulation of gene expression in bacteria.

In triple‐negative breast cancer (TNBC), pro‐tumoral macrophages promote metastasis and suppress the immune response. To target these cells, a previously identified CD206 (mannose receptor)‐binding peptide, mUNO was engineered to enhance its affinity and proteolytic stability. The new rationally designed peptide, MACTIDE, includes a trypsin inhibitor loop, from the Sunflower Trypsin Inhibitor‐I. Binding studies to recombinant CD206 revealed a 15‐fold lower KD for MACTIDE compared to parental mUNO. Mass spectrometry further demonstrated a 5‐fold increase in MACTIDE's half‐life in tumor lysates compared to mUNO. Homing studies in TNBC‐bearing mice shows that fluorescein (FAM)‐MACTIDE precisely targeted CD206⁺ tumor‐associated macrophages (TAM) upon intravenous, intraperitoneal, and even oral administration, with minimal liver accumulation. MACTIDE was conjugated to Verteporfin, an FDA‐approved photosensitizer and YAP/TAZ pathway inhibitor to create the conjugate MACTIDE‐V. In the orthotopic 4T1 TNBC mouse model, non‐irradiated MACTIDE‐V‐treated mice exhibited anti‐tumoral effects comparable to those treated with irradiated MACTIDE‐V, with fewer signs of toxicity, prompting further investigation into the laser‐independent activity of the conjugate. In vitro studies using bone marrow‐derived mouse macrophages showed that MACTIDE‐V excluded YAP from the nucleus, increased phagocytic activity, and upregulated several genes associated with cytotoxic anti‐tumoral macrophages. In mouse models of TNBC, MACTIDE‐V slowed primary tumor growth, suppressed lung metastases, and increased markers of phagocytosis and antigen presentation in TAM and monocytes, increasing the tumor infiltration of several lymphocyte subsets. MACTIDE‐V is proposed as a promising peptide‐drug conjugate for modulating macrophage function in breast cancer immunotherapy.

Laminins (LMs) are a family of heterotrimeric glycoproteins that form the structural foundation of basement membranes (BM). By acting as molecular bridges between cells and the extracellular matrix (ECM) through integrins and other surface receptors, they regulate key cellular signals that influence cell behavior and tissue architecture. Despite their physiological importance, our understanding of the role of LMs in cancer pathobiology remains fragmented. In this article, we review the diverse functions of LMs in promoting cancer cell proliferation, adhesion, and migration—critical steps in cancer metastasis. Beyond their direct effects on tumor cells, LMs influence stromal interactions and modulate tumor microenvironment dynamics, affecting processes such as angiogenesis, immune cell infiltration, cancer-associated fibroblast activation, and immune evasion. Understanding the complex roles of LMs in cancer biology, as well as their differential expression patterns in malignancies, could provide new diagnostic tools for predicting disease outcomes and pave the way for innovative therapeutic strategies, such as targeting LM-receptor interactions or modulating ECM dynamics to impede tumor growth and metastasis.

We investigated whether antibody concentrations measured in plasma using the Roche Elecsys® Anti‐SARS‐CoV‐2 S assay (targeting the receptor binding domain, RBD) could estimate levels of Wuhan‐Hu‐1 and Omicron XBB.1.5 spike‐directed antibodies with neutralizing ability (NtAb) or those mediating NK‐cell activity. We analyzed 135 plasma samples from 39 vaccinated elderly nursing home residents. A strong correlation was found for NtAb against both Wuhan‐Hu‐1 (Rho = 0.73, p < 0.001) and Omicron XBB.1.5 (sub)variants (Rho = 0.73, p < 0.001). Moderate positive correlations were observed for NK‐cell activity, based on lysosome‐associated membrane protein 1 (LAMP1)‐producing NK cells stimulated with Wuhan‐Hu‐1 (Rho = 0.43, p < 0.001) and Omicron XBB.1.5 spike proteins (Rho = 0.50, p < 0.001). Similarly, interferon‐gamma (IFN‐γ)‐producing NK‐cell frequencies showed moderate correlations (Wuhan‐Hu‐1: Rho = 0.43, p < 0.001; Omicron XBB.1.5: Rho = 0.50, p < 0.001). Random Forest models accurately predicted NtAb levels against Wuhan‐Hu‐1 ( R ² = 0.72), though models for Omicron XBB.1.5 were less robust. Anti‐RBD antibody concentrations of 4.73 and 5.02 log 10 BAU/mL predicted high NtAb levels for Wuhan‐Hu‐1 and Omicron XBB.1.5, respectively. Antibody thresholds for predicting functional NK cell‐mediated responses were 4.73 log 10 and 4.54 log 10 BAU/mL for Wuhan‐Hu‐1 and Omicron XBB.1.5, respectively. For LAMP1‐producing NK cells, the thresholds were 4.94 and 4.75 log 10 BAU/mL for Wuhan‐Hu‐1 and Omicron XBB.1.5, respectively. In summary, total anti‐RBD antibody levels measured by the Roche assay may allow inference of NtAb levels and, to a lesser extent, Fc‐mediated NK‐cell responses against Omicron XBB.1.5.

Allergic reactions to foods are primarily driven by allergen‐binding immunoglobulin (Ig)E antibodies. IgE‐expressing cells can be generated through direct switching from IgM to IgE or a sequential class switching pathway where activated B cells first switch to an intermediary isotype, most frequently IgG1, and then to IgE. It has been proposed that sequential class switch recombination is involved in augmenting the severity of allergic reactions, generating high affinity IgE, differentiation of IgE plasma cells, and in holding the memory of IgE responses. We directly tested these possibilities by comparing the allergic immunity of wild‐type and IgG1‐deficient (hMT) mice. We found that sequential switching through IgG1 was not required to maintain the binding capacity of IgE nor for its ability to promote degranulation and elicit anaphylaxis against bona fide food allergens. Furthermore, the absence of sequential switching modestly impacted IgE affinity and clinical reactivity against hapten antigens, suggesting that the nature of the antigen impacts the requirement for sequential switching. At a cellular level, the capacity to undergo sequential switching through IgG1 provided no competitive advantage for subsequent IgE expression among germinal center B cells or plasma cells. Furthermore, the recall of allergic immunity at memory timepoints was preserved in the absence of sequential switching through IgG1, a finding that corresponded with intact type 2 memory B cell polarization. Together, these data demonstrate that sequential switching through IgG1 is redundant in sensitization, anaphylaxis, and the persistence of allergy, ultimately revealing that IgE derived from any switching source should be targeted by novel therapeutics seeking to ameliorate allergic diseases.

Background About half of the patients suffering from Alzheimer’s disease (AD) display sleeping disorders. Disruptions in the central circadian clock (CC), located in the brain, accelerate AD pathogenesis, making the CC a promising target. In preclinical trials, this strategy have shown efficacy but clinical results are inconsistent. The presence of the blood‐brain‐barrier (BBB) and the poor pharmacokinetic profile of candidate drugs are an explanation of their poor clinical translation. Their nanoencapsulation is a potential solution to enhance brain penetration. Nanoparticles (NP) can further be formulated in pulsatile drug delivery systems (PDDS), delivering the drug at a specific time of the day to restore the circadian rhythmicity. Method Melatonin was encapsulated in stealth polymeric NP of chitosan and PLGA, proteic NP of BSA and nanostructured lipid carriers (NLC), functionalized with brain penetrating retroenantiomer peptides, angiopep‐2 and transferrin, and labelled by a fluorophore. The NP were characterized by dynamic light scattering (DLS), zetametry, encapsulation efficiency and release profile. The best NP were selected based on their potential for passive brain permeation, using a parallel artificial membrane permeability assay (PAMPA) with brain polar lipid extract, and tracked by DLS and fluorescence. Their neuroprotective effect and toxicity were evaluated on SH‐SY5Y cell line. Result A repeatable library of NP was obtained with a size ranging from 50 to 150 nm and a zeta potential from ‐30 to 10 mV. No toxicity was observed at concentration above clinical brain concentrations. The PAMPA highlighted a better crossing ability of some NP over other types. Conclusion This study allowed us to rationally select different types of nanoparticles over others which will be further optimized using industrial methods. A validation of the active transport triggering will be made on a BBB‐on‐a‐chip model. After validation, the NP effect on CC rhythmicity will be evaluated on organotypic brain culture and be formulated in dissolving microneedles.

Knowing which residues of a protein are important for its function is of paramount importance for understanding the molecular basis of this function and devising ways of modifying it for medical or biotechnological applications. Due to the difficulty in detecting these residues experimentally, prediction methods are essential to cope with the sequence deluge that is filling databases with uncharacterized protein sequences. Deep learning approaches are especially well suited for this task due to the large amounts of protein sequences for training them, the trivial codification of this sequence data to feed into these systems, and the intrinsic sequential nature of the data that makes them suitable for language models. As a consequence, deep learning-based approaches are being applied to the prediction of different types of functional sites and regions in proteins. This review aims to give an overview of the current landscape of methodologies so that interested users can have an idea of which kind of approaches are available for their proteins of interest. We also try to give an idea of how these systems work, as well as explain their limitations and high dependence on the training set so that users are aware of the quality of expected results.

Sharka disease, caused by the plum pox virus (PPV), negatively impacts stone fruit production, resulting in economic losses. It has been demonstrated that grafting the almond (Prunus dulcis (Miller) D.A. Webb) variety ‘Garrigues’ into susceptible peach (Prunus persica (L.) Batsch) rootstocks can result in PPV resistance. The molecular circuits related to grafting in Prunus species, however, have not been fully investigated. In this study, susceptible peach rootstocks ‘GF305’ were either heterografted with ‘Garrigues’ almond or homografted with the same cultivar. Peach samples were collected at two stages of scion development, with ungrafted plants utilized as controls. Profiles of transcripts, small RNAs (sRNAs), and DNA methylation were obtained and analyzed on a genome-wide scale. Homografting and heterografting significantly altered the transcriptome and methylome of peach rootstocks, with these modifications being more pronounced during the early stages of scion development. The profiles of sRNAs were significantly more impacted when almonds were used as a scion as opposed to peaches, likely due to the transmission of PPV-unrelated viral sequences. Gene expression differences resulting from DNA methylation alterations are more thoroughly documented at the promoter sequences of genes than within their bodies. This study suggests that the ‘Garrigues’ almond variety triggers a complex defense response in the peach rootstock, potentially involving the interplay of epigenetic modifications and small RNA-mediated priming of antiviral defenses, which ultimately may contribute to PPV resistance.

Correct host cell recognition is important in the replication cycle for any virus, including bacterial viruses. This essential step should occur before the bacteriophage commits to transferring its genomic material into the target bacterium. In this chapter, we will discuss the mechanisms and proteins bacteriophages use for receptor recognition (just before full commitment to infection) and nucleic acid injection, which occurs just after commitment. Some bacteriophages use proteins of the capsid proper for host cell recognition, others use specialised spikes or fibres. Usually, several identical recognition events take place, and the information that a suitable host cell has been encountered is somehow transferred to the part of the bacteriophage capsid involved in nucleic acid transfer. The main part of the capsids of bacteriophages stays on the cell surface after transferring their genome, although a few specialised proteins move with the DNA, either forming a conduit, protecting the nucleic acids after transfer and/or functioning in the process of transcription and translation.

Viral genomes are transported between cells using various structural solutions such as spherical or filamentous protein cages, alone or in combination with lipid envelopes, in assemblies of varying complexity. Morphogenesis of the new infectious particles (virions) encompasses capsid assembly from individual components (proteins, and membranes when required), genome packaging, and maturation. This final step is crucial for full infectivity. During maturation, structural and physical changes prepare the viral particles for delivering their genome into cells at the right time and location. The virion must be stabilized for travel across harsh extracellular conditions, while enabling disassembly for genome exposure to replication and translation machineries. That is, maturation has to produce metastable particles. Common maturation strategies include structural reordering, controlled proteolysis, or posttranslational modifications. Here we outline the maturation process in representative members of the six realms proposed by the latest virus taxonomic classification.

Viral particles consist essentially of a proteinaceous capsid that protects the genome and is also involved in many functions during the virus life cycle. In structurally simple viruses, the capsid consists of a number of copies of the same, or a few different proteins organized into a symmetric oligomer. Structurally complex viruses present a larger variety of components in their capsids than simple viruses. They may contain accessory proteins with specific architectural or functional roles, or incorporate non-proteic elements such as lipids. They present a range of geometrical variability, from slight deviations from the icosahedral symmetry to complete asymmetry or even pleomorphism. Putting together the many different elements in the virion requires an extra effort to achieve correct assembly, and thus complex viruses require sophisticated mechanisms to regulate morphogenesis. This chapter provides a general view of the structure and assembly of complex viruses.

Type III protein secretion systems (T3SSs) function as multiprotein devices that span the envelope of Gram-negative bacteria using the peptidoglycan (PG) layer as scaffold. This spatial arrangement explains why modifications in PG structure can alter T3SS activity. In Salmonella, incorporation of non-canonical D-amino acids in the PG was shown to decrease the activity of the T3SS encoded by the pathogenicity island-1 (SPI-1) without affecting other T3SS, like the flagellum apparatus. Enigmatically, following invasion of host cell Salmonella enterica serovar Typhimurium modifies PG synthesis by upregulating two pathogen-specific enzymes, the penicillin-binding proteins PBP2SAL and PBP3SAL, with roles in cell elongation and division, respectively. In the mouse typhoid model, the amount of PBP2SAL and PBP3SAL produced by the pathogen exceeds by large those of the canonical enzymes PBP2 and PBP3. This change responds to acidity and high osmolarity, the same cues that intra-phagosomal S. Typhimurium perceives to switch the SPI-1 T3SS by that encoded in SPI-2. Using isogenic mutants lacking each of the four morphogenetic PBPs, we tested whether their activities and those of the T3SS encoded by SPI-1 and SPI-2, are interconnected. Our data show that PBP2 is required for proper function of SPI-1 T3SS but dispensable for motility, whereas the lack of any of the morphogenetic PBPs increases SPI-2 T3SS activity. The positive control exerted by PBP2 on SPI-1 takes place via the SPI-1-specific regulators HilA and InvF. To our knowledge, these findings provide the first evidence linking morphogenetic enzymes that synthesize PG with T3SS associated to virulence.

Conjugative plasmids promote the dissemination and evolution of antimicrobial resistance in bacterial pathogens. However, plasmid acquisition can produce physiological alterations in the bacterial host, leading to potential fitness costs that determine the clinical success of bacteria-plasmid associations. In this study, we use a transcriptomic approach to characterize the interactions between a globally disseminated carbapenem resistance plasmid, pOXA-48, and a diverse collection of multidrug resistant (MDR) enterobacteria. Although pOXA-48 produces mostly strain-specific transcriptional alterations, it also leads to the common overexpression of a small chromosomal operon present in Klebsiella spp. and Citrobacter freundii strains. This operon includes two genes coding for a pirin and an isochorismatase family proteins (pfp and ifp), and shows evidence of horizontal mobilization across Proteobacteria species. Combining genetic engineering, transcriptomics, and CRISPRi gene silencing, we show that a pOXA-48-encoded LysR regulator is responsible for the plasmid-chromosome crosstalk. Crucially, the operon overexpression produces a fitness benefit in a pOXA-48-carrying MDR K. pneumoniae strain, suggesting that this crosstalk promotes the dissemination of carbapenem resistance in clinical settings.

TDP-43 has emerged as a key protein in neurodegenerative diseases. Discovered as a prominent part of protein aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTLD) postmortem brains, its role in other neurodegenerative pathologies has been revealed. In fact, these discoveries resulted in a new classification of dementia, limbic-predominant age-related TDP-43 encephalopathy (LATE) which shares common symptomatology with Alzheimer’s disease (AD). Here we will review TDP-43 discovery and function, its role in neurodegenerative diseases, and the different in vitro assays to study its role in pathogenesis and to identify compounds able to modulate this key protein in neurodegeneration.

Metal–Organic Frameworks (MOFs) attract attention for their intrinsic porosity, large surface area, and functional versatility. To fully utilize their potential in applications requiring precise control at smaller scales, it is essential to overcome challenges associated with their bulk form. This is particularly difficult for 3D MOFs with spin crossover (SCO) behavior, which undergo a reversible transition between high‐spin and low‐spin states in response to external stimuli. Maintaining their structural integrity and SCO properties at the nanoscale remains a significant challenge, yet these properties make them ideal candidates for sensors, data storage, and molecular switch applications. This study reports the synthesis of nanocrystals of the well‐known SCO MOF [Fe2(H0.67bdt)3]·xH2O (1, x = 0–10, bdt²⁻ = 1,4‐benzeneditetrazolate), which exhibits both magnetic and charge transport properties. The nanocrystals are obtained through sonication of macrocrystals, and the preservation of their crystalline structure at the nanoscale is explored using Microcrystal Electron Diffraction (MicroED). A comparison between macro‐ and nanocrystals highlights the structural integrity and the preservation of charge‐transport properties, underlining the potential for further miniaturization of MOFs for advanced technological applications.

Background Severe Acute Respiratory syndrome coronavirus 2 (SARS-CoV-2) and Influenza A viruses (IAVs) are among the most important causes of viral respiratory tract infections, causing similar symptoms. IAV and SARS-CoV-2 infections can provoke mild symptoms like fever, cough, sore throat, loss of taste or smell, or they may cause more severe consequences leading to pneumonia, acute respiratory distress syndrome or even death. While treatments for IAV and SARS-CoV-2 infection are available, IAV antivirals often target viral proteins facilitating the emergence of drug-resistant viral variants. Hence, universal treatments against coronaviruses and IAVs are hard to obtain due to genus differences (in the case of coronavirus) or subtypes (in the case of IAV), highlighting the need for novel antiviral therapies. Interestingly, iron oxide nanoparticles (IONPs) with a 10 nm core size and coated with the biocompatible dimercaptosuccinic acid (DMSA: DMSA-IONP-10) display antiviral activity against SARS-CoV-2 in vitro. Methods We analyzed the antiviral activity of DMSA-IONP-10 against SARS-CoV-2 infection in vivo, and against IAV infection in vitro and in vivo. Results DMSA-IONP-10 treatment of mice after SARS-CoV-2 infection impaired virus replication in the lungs and led to a mildly reduced pro-inflammatory cytokine induction after infection, indicating that these IONPs can serve as COVID-19 therapeutic agents. These IONPs also had a prophylactic and therapeutic effect against IAV in tissue cultured cells at non-cytotoxic doses, and a therapeutic effect in IAV-infected-mice, inhibiting viral replication and slightly dampening the inflammatory response after viral infection. As an exacerbated inflammatory response to IAVs and SARS-CoV-2 is detrimental to the host, weakening this response in mice through IONP treatment may reduce disease severity. Interestingly, our data suggest that IONP treatment affects oxidative stress and iron metabolism in cells, which may influence IAV production. Conclusion This study highlights the antiviral activity of DMSA-IONP-10 against important human respiratory viruses.