Biology subjects

Fernandez, E. A.

Publications and source records attributed to Fernandez, E. A..

5 recordsLinked to original sources

bioRxiv2024

Identification of microRNAs regulated by E2F transcription factors in human pluripotent stem cells

Human pluripotent stem cells (hPSCs), which include embryonic and induced pluripotent stem cells (hESCs and hiPSCs, respectively), have an unusual cell cycle structure which consists of a short G1 phase and the absence of the G1/S checkpoint regulation. E2F transcription factors (E2Fs) play an important role in the G1/S transition. G1 duration contributes to hPSC fate determination, and microRNAs (miRNAs) play critical roles in this commitment. As little is known about the interplay between E2Fs and miRNAs in hPSCs, we aimed to identify miRNAs that are regulated by E2Fs in these cells. We first found that mRNA expression levels of canonical E2F repressors were more expressed than most E2F activators in G1-arrested hPSCs. Moreover, we observed higher mRNA and protein expression levels of canonical E2F2, E2F3A, and E2F5 in G1 synchronized hPSCs compared to human fibroblasts (HF). However, E2F1 and E2F4 protein expression levels were higher in HF. We next found that E2F inhibition with HLM006474 induced an increase in the G1 cell population without affecting hPSC viability, concomitantly with a decrease in OCT-4 mRNA levels and the percentage of OCT-4+ hPSCs. Next, by RNA-seq analysis we found 52 differentially expressed (DEGs) miRNAs in HLM006474-treated hESCs. RT-qPCR validation of some of the DEGs let us conclude that miR-19a-3p, miR-19b-3p, miR-4454, miR-1260a, miR-1260b, miR-454-3p and miR-301a-3p are regulated by E2Fs in hPSCs. Interestingly, gene target and ontology analysis of these miRNAs revealed a possible implication in proliferation and cell cycle regulation, development, and neural differentiation.

developmental biology↗

bioRxiv2022

Antibodies to protozoan variable surface antigens induce antigenic variation

The genomes of most protozoa encode families of variant surface antigens, whose mutually exclusive changes in expression allow parasitic microorganisms to evade the host immune response1,2. It is widely assumed that antigenic variation in protozoan parasites is accomplished by the spontaneous appearance within the population of cells expressing antigenic variants that escape antibody-mediated cytotoxicity1,2. Here we show, both in vitro and in animal infections, that antibodies to Variant-specific Surface Proteins (VSPs) of the intestinal parasite Giardia lamblia are not cytotoxic, inducing instead VSP clustering into liquid-ordered phase membrane microdomains that trigger a massive release of microvesicles carrying the original VSP and switch in expression to different VSPs by a calcium-dependent mechanism. Surface microvesiculization and antigenic switching are also stimulated when Trypanosoma brucei and Tetrahymena thermophila are confronted to antibodies directed to their GPI-anchored variable surface glycoproteins. This novel mechanism of surface antigen clearance throughout its release into microvesicles coupled to the stochastic induction of new phenotypic variants not only changes the current paradigm of spontaneous antigenic switching but also provides a new framework for understanding the course of protozoan infections as a host/parasite adaptive process.

microbiology↗

bioRxiv2022

MHC-I binding affinity derived metrics fail to predict tumor specific neoantigen immunogenicity

Tumor-specific antigens emerging through somatic genomic rearrangements, known as neoantigens, play a critical role in current anticancer immunotherapy. They may or may not elicit an immune response when presented on the tumor cell surface bound to the MHC-I molecule, whose strength has been assumed as an indicator of immunogenicity. Several in silico peptide-MHC-I binding affinity predictors are used to prioritize putative immunogenic neoantigens to be experimentally and clinically explored either as biomarkers or targets for anticancer vaccines. This claims for a fair evaluation of such predictors, making essential the development of appropriate databases with experimentally validated, immunogenic/non-immunogenic neoantigens. Thus far, such a database is lacking. We herein present ITSNdb, a new and curated immunogenic neoantigen database and use it to benchmark current neoantigen immunogenicity predictors. Benchmark results failed to support the application of the predicted peptide- MHC-I binding affinity or its derived metrics as a tool to estimate neoantigen immunogenicity and the tumor neoantigen burden as an immunotherapy response biomarker. Moreover, binding affinity based immunogenicity definition leads to identifying wild-type peptide counterparts as predictors of immunotherapy response. We demonstrate that MHC-I binding affinity is insufficient to define neoantigen immunogenicity, despite being necessary for neoantigen tumor cell presentation suggesting that a paradigm shift for the emergence of new rules to identify immunogenic neoantigens is required.

bioinformatics↗

bioRxiv2022

Characterization of Cysteine-rich protein families of Giardia lamblia and their role during antigenic variation.

Giardia lamblia encode several families of cysteine-rich proteins. Among these families are the Variant-specific Surface Proteins (VSPs), which are involved in the process of antigenic variation. In addition to VSPs, other Cys-rich proteins have been described, such as High Cysteine Membrane Proteins (HCMPs), High Cysteine Proteins (HCPs) and Tenascin-like Proteins (TLPs). However, these proteins are less characterized and there is no consensus on their subcellular localization, function, expression, and relationship with the VSPs. Although numerous efforts have been made to determine the distinctive characteristics of VSPs and the number of VSP genes present in the genome of Giardia, a clear profile of the VSP repertoire is still lacking. Here, we performed an exhaustive analysis of the Cys-rich families in the recently updated version of the Giardia genome, including their organization, characteristic features, evolution and levels of expression, by combining simple pattern searches and predictions with massive sequencing techniques, integrating and reanalyzing as much omics data as possible. We propose a new classification for the Cys-rich protein-encoding genes and pseudogenes that better describes their involvement in the parasite biology and define unique characteristics of the VSPs that include, besides their known features, an Initiator element/Kozak-like sequence, an extended polyadenylation signal and a unique pattern of mutually exclusive transcript accumulation. Our findings also imply that the HCMPs (now named Cys-Rich Membrane Proteins, CRMPs) are upregulated under stress conditions and might protect the parasite during VSP switching. These results contribute to a better understanding of the process of antigenic variation in this pathogen. Author SummaryThe most common cause of parasite-induced diarrhea is Giardia lamblia. This intestinal parasite causes 180 million cases of symptomatic disease (giardiasis) yearly but also many asymptomatic infections, resulting in that more than 0.5 billion people are currently colonized by the parasite. One key virulence mechanism of G. lamblia, resulting in long-term infections and frequent re-infections, is antigenic variation of Variant-specific Surface Proteins (VSPs). The cysteine-rich VSP proteins are encoded by a multi-gene family but until now the number of genes and how they are regulated has been unclear. Here a recently assembled Giardia reference genome was analyzed using different bioinformatic analyses and this revealed that there are 136 VSP genes in the Giardia genome. The analyses revealed that there are additional cysteine rich proteins in gene families with fewer members that are related to VSPs but with other roles than antigenic variation. A large number of incomplete VSP genes were also identified and they can function as a sequence reservoir for generation of VSP variability. The VSP genes have unique regulatory elements in the upstream and downstream regions, suggesting a role in regulation. Several gene expression data sets were re-analyzed and it showed that one major VSP is expressed per cell. This study is the first to reveal the organization of VSPs in Giardia and it will be the basis for further studies of the mechanism of antigenic variation in this important intestinal parasite.

microbiology↗

bioRxiv2017

A benchmarking of workflows for detecting differential splicing and differential expression at isoform level in human RNA-seq studies

Over the last few years, RNA-seq has been used to study alterations in alternative splicing related to several diseases. Bioinformatics workflows used to perform these studies can be divided into two groups, those finding changes in the absolute isoform expression and those studying differential splicing. Many computational methods for transcriptomics analysis have been developed, evaluated and compared; however, there are not enough reports of systematic and objective assessment of processing pipelines as a whole. Moreover, comparative studies have been performed considering separately the changes in absolute or relative isoform expression levels. Consequently, no consensus exists about the best practices and appropriate workflows to analyse alternative and differential splicing. To assist the adequate pipeline choice, we present here a benchmarking of nine commonly used workflows to detect differential isoform expression and splicing. We evaluated the workflows performance over three different experimental scenarios where changes in absolute and relative isoform expression occurred simultaneously. In addition, the effect of the number of isoforms per gene, and the magnitude of the expression change over pipeline performances were also evaluated. Our results suggest that workflow performance is influenced by the number of replicates per condition and the conditions heterogeneity. In general, workflows based on DESeq, DEXSeq, Limma and NOISeq performed well over a wide range of transcriptomics experiments. In particular, we suggest the use of workflows based on Limma when high precision is required, and DESeq2 and DEXseq pipelines to prioritize sensitivity. When several replicates per condition are available, NOISeq and Limma pipelines are indicated.

bioinformatics↗