Biology subjects

Navarro, F.

Publications and source records attributed to Navarro, F..

15 recordsLinked to original sources

bioRxiv2025

Nanostructured lipid carriers overcome the low immunogenicity of M2e peptide via surface click chemistry conjugation, improving the anti-M2e antibody response

Influenza vaccines are considered the most effective measure for preventing influenza. The key antigen of commercial vaccines is hemagglutinin (HA), the main protein on the surface of influenza viruses. This protein is genetically variable, necessitating the annual redefinition of the vaccine formulation. Furthermore, these vaccines do not offer protection against pandemic viral strains. The M2e peptide, a small surface protein conserved among influenza type A strains, could enable cross-immunity when used as an antigen. Nevertheless, its limited immunogenicity has restricted its application in universal influenza vaccines. To enhance the immune responses to M2e, we associated it with Nanostructured Lipid Carriers (NLCs). These NLCs are stable over time and their small diameter (<100 nm) enhances interactions with antigen-presenting cells and improve lymphatic drainage, facilitating their uptake by the immune system. The M2e specific antibody response in mice was enhanced by using "click" chemistry to conjugate the M2e peptide to the NLC surface. Also, the incorporation of PADRE-specific CD4+ T cells stimulating peptide between M2e peptide and its linking region greatly enhanced the specific antibody response to M2e and generated immune response levels that could be compatible with influenza protection. This promising M2e formulation vectorized by NLC represents a potential universal influenza vaccine candidate warranting further preclinical investigations and clinical evaluation.

immunology↗

bioRxiv2024

Growth rate regulates RNA polymerase II transcription elongation in Saccharomyces cerevisiae

Cells must adapt to changing environmental conditions to maintain their fitness and to compete with other genotypes during the natural selection process. The growth rate (GR) is a determining factor in this competition, and it influences gene expression. Some genes increase mRNA levels, while others decrease with the GR. mRNA levels depend on the dynamic balance between their synthesis by RNA polymerase II and their degradation rates. RNA polymerase I and III are also influenced by the GR because they transcribe protein synthesis machinery required to make proteins that increase cell mass during growth. Although RNA levels have been extensively studied in relation to the GR in many organisms, synthesis and degradation rates have, however, been much less investigated. In a previous work, we found a positive correlation between RNA polymerase (RNA pol) II transcription and mRNA degradation with GRs in yeast in batch cultures. Here we extend our study under constant growth conditions in a chemostat and find that overall chromatin-associated RNA pol II levels increase in parallel with the GR. This increase appears to involve the accumulation of partially dephosphorylated RNA pol II with a greater tendency to backtracking, which suggests that the GR regulates RNA pol II-dependent transcription at the elongation level. RNA pol I also increases its association with chromatin with the GR, which confirms the general dependence of at least RNA pol I and II transcription on the GR.

molecular biology↗

bioRxiv2024

Open Educational Resources for distributed hands-on teaching in molecular biology

The urgent need to develop a more equitable bioeconomy has positioned biotechnology capacity building at the forefront of international priorities. However, in many educational institutions, particularly in low-and middle-income countries, this remains a major challenge due to limited access to reagents, equipment, and technical documentation. In this work, we describe Open Educational Resources (OER) composed of locally produced biological reagents, open source hardware and free software to teach fundamental techniques in biotechnology such as LAMP DNA amplification, RT-PCR RNA detection, enzyme kinetics and fluorescence imaging. The use of locally produced reagents and devices reduces costs by up to one order of magnitude. During the pandemic lockdowns, these tools were distributed nationwide to students homes as a lab-in-a-box for remote teaching of molecular biology. To test their performance, a total of 93 undergraduate students tested these resources during a biochemistry practical course. 27 out of 31 groups ([~]87%) successfully achieved the objectives of the PCR activity, while 28 out of 31 groups ([~]90%) correctly identified the target using LAMP reactions. To assess the potential application in secondary school, we organized three workshops for high school teachers from different institutions across Chile and performed an anonymous questionnaire, obtaining a strong agreement on how these OER broaden teachers perspectives on the techniques and facilitate the teaching of molecular biology topics. This effort was made possible through a close collaboration with open source technology advocates and members of DIYbio communities, whose work is paving the way for low-cost training in biology. All the protocols and design files are available under open source licenses.

molecular biology↗

bioRxiv2023

Maf1 phosphorylation is regulated through the action of prefoldin-like Bud27 on PP4 phosphatase in Saccharomyces cerevisiae

Bud27 is a prefoldin-like protein that participates in transcriptional regulation mediated by the three RNA polymerases in Saccharomyces cerevisiae. Lack of Bud27 significantly affects RNA pol III transcription, although the involved mechanisms have not been characterized. Here we show that Bud27 regulates the phosphorylation state of the RNA pol III transcriptional repressor, Maf1, influences its nuclear localization, and likely its activity. We demonstrate that Bud27 is associated with the Maf1 main phosphatase PP4 in vivo, and that this interaction is required for proper Maf1 dephosphorylation. Lack of Bud27 decreases the interaction among PP4 and Maf1, Maf1 dephosphorylation, and its nuclear entry. Our data uncover a new nuclear function of Bud27, identify PP4 as a novel Bud27 interactor and demonstrate the effect of this prefoldin-like on the posttranslational regulation of Maf1. Finally, our data reveal a broader effect of Bud27 on PP4 activity by influencing, at least, the phosphorylation of Rad53.

molecular biology↗

bioRxiv2023

The association of the RSC remodeler complex with chromatin depends on prefoldin-like Bud27 and determines nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae

The tripartite interaction between the chromatin remodeler complex RSC, RNA polymerase subunit Rpb5 and prefoldin-like Bud27 is necessary for proper RNA pol II elongation. Indeed lack of Bud27 alters this association and affects transcription elongation. This work investigates the consequences of lack of Bud27 on the chromatin association of RSC and RNA pol II, and on nucleosome positioning. Our results demonstrate that RSC binds chromatin to gene bodies and lack of Bud27 alters this association, mainly around polyA sites. This alteration impacts chromatin organization and leads to the accumulation of RNA pol II molecules around polyA sites, likely to be arrested or slower. Our data suggest that RSC is necessary to maintain chromatin organization around those sites, and any alteration of this organization results in the widespread use of alternative polyA sites. Finally, we also find a similar molecular phenotype that occurs upon TOR inhibition with rapamycin, which suggests that alternative polyadenylation observed upon TOR inhibition is likely Bud27-dependent.

molecular biology↗

bioRxiv2021

An automated microfluidic platform integrating functional vascularized organoids-on-chip

The development of vascular networks on-chip is crucial for the long-term culture of three-dimensional cell aggregates such as organoids, spheroids, tumoroids, and tissue explants. Despite the rapid advancement of microvascular network systems and organoid technology, vascularizing organoids-on-chips remains a challenge in tissue engineering. Moreover, most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical settings to operate. Considering these constraints, we developed an innovative platform to establish and monitor the formation of endothelial networks around model spheroids of mesenchymal and endothelial cells as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 15 days on-chip. Importantly, these networks were functional, demonstrating intravascular perfusion within the spheroids or vascular organoids connected to neighbouring endothelial beds. This microphysiological system thus represents a viable organ-on-chip model to vascularize biological tissues and should allow to establish perfusion into organoids using advanced microfluidics.

bioengineering↗

bioRxiv2021

Microfluidic device integrating a network of hyper-elastic valves for automated glucose stimulation and insulin secretion collection from a single pancreatic islet

Advances in microphysiological systems have prompted the need for robust and reliable cell culture devices. While microfluidic technology has made significant progress, devices often lack user-friendliness and are not designed to be industrialized on a large scale. Pancreatic islets are often being studied using microfluidic platforms in which the monitoring of fluxes is generally very limited, especially because the integration of valves to direct the flow is difficult to achieve. Considering these constraints, we present a thermoplastic manufactured microfluidic chip with an automated control of fluxes for the stimulation and secretion collection of pancreatic islet. The islet was directed toward precise locations through passive hydrodynamic trapping and both dynamic glucose stimulation and insulin harvesting were done automatically via a network of large deformation valves, directing the reagents and the pancreatic islet toward different pathways. This device we developed enables monitoring of insulin secretion from a single islet and can be adapted for the study of a wide variety of biological tissues and secretomes.

bioengineering↗

bioRxiv2021

Multi-tissue integrative analysis of personal epigenomes

Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of personal epigenomes, for [~]25 tissues and >10 assays in four donors (>1500 open-access functional genomic and proteomic datasets, in total). Each dataset is mapped to a matched, diploid personal genome, which has long-read phasing and structural variants. The mappings enable us to identify >1 million loci with allele-specific behavior. These loci exhibit coordinated epigenetic activity along haplotypes and less conservation than matched, non-allele-specific loci, in a fashion broadly paralleling tissue-specificity. Surprisingly, they can be accurately modelled just based on local nucleotide-sequence context. Combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci and enables models for transferring known eQTLs to difficult-to-profile tissues. Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.

genomics↗

bioRxiv2020

RNA polymerase II assembly and mRNA decay regulation are mediated and interconnected via CTD Ser5P phosphatase Rtr1 in Saccharomyces cerevisiae

Rtr1 is an RNA pol II CTD-phosphatase that influences gene expression by acting during the transition from transcription initiation to elongation, and during transcription termination. Rtr1 has been proposed as an RNA pol II import factor in RNA pol II biogenesis, and participating in mRNA decay by autoregulating the turnover of its own mRNA. In addition, the interaction of Rtr1 with RNA pol II depends on the phosphorylation state of CTD, which also influences Rpb4/7 dissociation during transcription. In this work, we demonstrate that Rtr1 acts in RNA pol II assembly, likely in a final cytoplasmic RNA pol II biogenesis step, and mediates the Rpb4 association with the rest of the enzyme, However, we do not rule out discard a role in the Rpb4 association with RNA pol II in the nucleus. This role of Rtr1 interplays RNA pol II biogenesis and mRNA decay regulation. In fact, RTR1 deletion alters RNA pol II assembly and leads to the chromatin association of RNA pol II lacking Rpb4, in addition to whole RNA pol II, decreasing mRNA-Rpb4 imprinting and, consequently, increasing mRNA stability. Notably, the RPB5 overexpression that overcomes RNA pol II assembly and the defect in Rpb4 binding to chromatin-associated RNA pol II partially suppresses the mRNA stability defect of rtr1{Delta} cells. Our data also indicate that Rtr1 mediates mRNA decay regulation more broadly than previously proposed in cooperation with Rpb4 and Dhh1. Interestingly, these data include new layers in the crosstalk between mRNA synthesis and decay.

molecular biology↗

bioRxiv2020

Rpb4 and Puf3 imprint and post-transcriptionally control the stability of a common set of mRNAs in yeast

Gene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, crosstalk in which mRNA decay machinery and transcription machinery respectively impact transcription and mRNA stability. Rpb4, and likely dimer Rpb4/7, seem the central components of the RNA pol II governing these processes. In this work we unravel the molecular mechanisms participated by Rpb4 that mediate the posttranscriptional events regulating mRNA imprinting and stability. By RIP-Seq, we analyzed genome-wide the association of Rpb4 with mRNAs and demonstrated that it targeted a large population of more than 1400 transcripts. A group of these mRNAs was also the target of the RNA binding protein, Puf3. We demonstrated that Rpb4 and Puf3 physically, genetically, and functionally interact and also affect mRNA stability, and likely the imprinting, of a common group of mRNAs. Furthermore, the Rpb4 and Puf3 association with mRNAs depends on one another. We also demonstrated, for the first time, that Puf3 associates with chromatin in an Rpb4-dependent manner. Our data also suggest that Rpb4 could be a key element of the RNA pol II that coordinates mRNA synthesis, imprinting and stability in cooperation with RBPs.

molecular biology↗

bioRxiv2020

Xrn1 influences RNA pol II-dependent transcription elongation rates across the yeast genome and this control is particularly relevant for late elongation of regulatory genes

mRNA homeostasis is favored by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5 exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3 was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favoring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.

genomics↗

bioRxiv2019

In vitro and in vivo efficacy of the combination of colistin and endolysins against clinical strains of Multi-Drug Resistant (MDR) pathogens

The multidrug resistance (MDR) among pathogenic bacteria is jeopardizing the worth of antimicrobials, which had previously changed medical sciences. In this study, we used bioinformatic tools to identify the endolysins ElyA1 and ElyA2 (GH108-PG3 family) present in the genome of bacteriophages Ab1051{Phi} and Ab1052{Phi}, respectively. The muralytic activity of these endolysins over MDR clinical isolates (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) was tested using the turbidity reduction assay. The minimal inhibitory concentrations (MICs) of endolysin, colistin and their combination were determined using the microdilution checkerboard method. The antimicrobial activity of the combinations was confirmed by time kill curves and in vivo assays in larvae of Galleria mellonella. Our results showed that ElyA1 displayed activity against all 25 strains of A. baumannii and P. aeruginosa tested and against 13 out of 17 strains of K. pneumoniae. No activity was detected when assays were done with endolysin ElyA2. The combined antimicrobial activity of colistin and endolysin ElyA1 yielded a reduction in the colistin MIC for all strains studied, except K. pneumoniae. These results were confirmed in vivo in G. mellonella survival assays. In conclusion, the combination of colistin with new endolysins such as ElyA1 could increase the bactericidal activity and reduce the MIC of the antibiotic, thus also reducing the associated toxicity.\n\nIMPORTANCEThe development of multiresistance by pathogen bacteria increases the necessity of the development of new antimicrobial strategies. In this work, we combined the effect of the colistin with a new endolysin, ElyA1, from a bacteriophage present in the clinical strain of Acinetobacter baumannii Ab105. ElyA1 is a lysozyme-like family (GH108-GP3), whose antimicrobial activity was described for first time in this work. Also, another endolysin, ElyA2, with the same origin and family, was characterized but in this case no activity was detected. ElyA1 presented lytic activity over a broad spectrum of strains from A. baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. When colistin was combined with ElyA1 an increase of the antimicrobial activity was observed with a reduced concentration of colistin, and this observation was also confirmed in vivo in Galleria mellonella larvae. The combination of colistin with new endolysins as ElyA1 could increase the bactericidal activity and lowering the MIC of the antibiotic, thus also reducing the associated toxicity.

microbiology↗

bioRxiv2019

TeXP: Deconvolving the effects of pervasive and autonomous transcription of transposable elements

Long interspersed nuclear element 1 (LINE-1) is a primary source of genetic variation in humans and other mammals. Despite its importance, LINE-1 activity remains difficult to study because of its highly repetitive nature. Here, we developed and validated a method called TeXP to gauge LINE-1 activity accurately. TeXP builds mappability signatures from LINE-1 subfamilies to deconvolve the effect of pervasive transcription from autonomous LINE-1 activity. In particular, it apportions the multiple reads aligned to the many LINE-1 instances in the genome into these two categories. Using our method, we evaluated well-established cell lines, cell-line compartments and healthy tissues and found that the vast majority (91.7%) of transcriptome reads overlapping LINE-1 derive from pervasive transcription. We validated TeXP by independently estimating the levels of LINE-1 autonomous transcription using ddPCR, finding high concordance. Next, we applied our method to comprehensively measure LINE-1 activity across healthy somatic cells, while backing out the effect of pervasive transcription. Unexpectedly, we found that LINE-1 activity is present in many normal somatic cells. This finding contrasts with earlier studies showing that LINE-1 has limited activity in healthy somatic tissues, except for neuroprogenitor cells. Interestingly, we found that the amount of LINE-1 activity was associated with the with the amount of cell turnover, with tissues with low cell turnover rates (e.g. the adult central nervous system) showing lower LINE-1 activity. Altogether, our results show how accounting for pervasive transcription is critical to accurately quantify the activity of highly repetitive regions of the human genome.\n\nAuthor SummaryRepetitive sequences, such as LINEs, comprise more than half of the human genome. Due to their repetitive nature, LINEs are hard to grasp. In particular, we find that pervasive transcription is a major confounding factor in transcriptome data. We observe that, on average, more than 90% of LINE signal derives from pervasive transcription. To investigate this issue, we developed and validated a new method called TeXP. TeXP accounts and removes the effects of pervasive transcription when quantifying LINE activity. Our method uses the broad distribution of LINEs to estimate the effects of pervasive transcription. Using TeXP, we processed thousands of transcriptome datasets to uniformly, and unbiasedly measure LINE-1 activity across healthy somatic cells. By removing the pervasive transcription component, we find that (1) LINE-1 is broadly expressed in healthy somatic tissues; (2) Adult brain show small levels of LINE transcription and; (3) LINE-1 transcription level is correlated with tissue cell turnover. Our method thus offers insights into how repetitive sequences and influenced by pervasive transcription. Moreover, we uncover the activity of LINE-1 in somatic tissues at an unmatched scale.

bioinformatics↗

bioRxiv2018

Sensitive information leakage from functional genomics data: Theoretical quantifications & practical file formats for privacy preservation

The generation of functional genomics datasets is surging, as they provide insight into gene regulation and organismal phenotypes (e.g., genes upregulated in cancer). The intention of functional genomics experiments is not necessarily to study genetic variants, yet they pose privacy concerns due to their use of next-generation sequencing. Moreover, there is a great incentive to share raw reads for better analyses and general research reproducibility. Thus, we need new modes of sharing beyond traditional controlled-access models. Here, we develop a data-sanitization procedure allowing raw functional genomics reads to be shared while minimizing privacy leakage, thus enabling principled privacy-utility trade-offs. It works with traditional Illumina-based assays and newer technologies such as 10x single-cell RNA-sequencing. The procedure depends on quantifying the privacy leakage in reads by statistically linking study participants to known individuals. We carried out these linkages using data from highly accurate reference genomes and more realistic environmental samples.

bioinformatics↗

bioRxiv2018

Multiple laboratory mouse reference genomes define strain specific haplotypes and novel functional loci

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like-/- mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

genomics↗