Félix Recillas-targa - Academia.edu (original) (raw)

Papers by Félix Recillas-targa

ABSTRACTMassive testing is a cornerstone in efforts to effectively track infections and stop COVI... more ABSTRACTMassive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places where good vaccination coverage has been achieved. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing implementation in resource-limited settings. It is therefore important to develop testing protocols that facilitate implementation and are inexpensive, fast, and sufficiently sensitive. In this work, we optimized the composition of a buffer (PKTP) containing a protease, a detergent, and an RNase inhibitor, that is compatible with the RT-qPCR chemistry, allowing for direct testing of SARS-CoV-2 from saliva in an RNA extraction-independent manner. This buffer is compatible with heat-inactivation reducing the biohazard risk of handling the samples. We assessed the PKTP buffer performance in comparison to the RNA-e...

Proceedings of the National Academy of Sciences, 2021

Significance Low phosphate (Pi) availability is a major constraint for plant productivity in both... more Significance Low phosphate (Pi) availability is a major constraint for plant productivity in both natural and agricultural environments. Over the course of evolution, plants acquired a complex cascade of transcriptional responses that enhance their capacity to survive and reproduce in phosphate-poor soils. However, it is unclear to what extent chromatin architecture is modified to activate or repress gene expression in response to Pi starvation. Here, we report the effects of Pi starvation on chromatin accessibility and its association with gene expression. We determined that major remodeling in chromatin accessibility takes place in response to Pi starvation and that this response is activated by the PHR1 family of transcription factors.

Oncogene, 2021

Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant ... more Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant tumors. Epigenetic modifications such as DNA methylation and histone modification regulate tumor initiation and progression. However, the contribution of histone variants in PDAC is unknown. Here, we demonstrated that the histone variant H2A.Z is highly expressed in PDAC cell lines and PDAC patients and that its overexpression correlates with poor prognosis. Moreover, all three H2A.Z isoforms (H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2) are highly expressed in PDAC cell lines and PDAC patients. Knockdown of these H2A.Z isoforms in PDAC cell lines induces a senescent phenotype, cell cycle arrest in phase G2/M, increased expression of cyclin-dependent kinase inhibitor CDKN2A/p16, SA-β-galactosidase activity and interleukin 8 production. Transcriptome analysis of H2A.Z-depleted PDAC cells showed altered gene expression in fatty acid biosynthesis pathways and those that regulate cell cycle and DNA da...

Frontiers in immunology, 2017

Regulatory T cells (Tregs) are considered key players in the prevention of allograft rejection in... more Regulatory T cells (Tregs) are considered key players in the prevention of allograft rejection in transplanted patients. Belatacept (BLT) is an effective alternative to calcineurin inhibitors that appears to preserve graft survival and function; however, the impact of this drug in the homeostasis of Tregs in transplanted patients remains controversial. Here, we analyzed the phenotype, function, and the epigenetic status of the Treg-specific demethylated region (TSDR) in FOXP3 of circulating Tregs from long-term kidney transplant patients under BLT or Cyclosporine A treatment. We found a significant reduction in the proportion of CD4(+)CD25(hi)CD127(lo/-)FOXP3(+) T cells in all patients compared to healthy individual (controls). Interestingly, only BLT-treated patients displayed an enrichment of the CD45RA(+) "naïve" Tregs, while the expression of Helios, a marker used to identify stable FOXP3(+) thymic Tregs remained unaffected. Functional analysis demonstrated that Tregs ...

Nucleus, 2016

Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is invo... more Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is involved in both structural support and the regulation of gene expression. Lamin A is initially expressed as prelamin A, which contains an extended carboxyl terminus that undergoes a series of post-translational modifications and subsequent cleavage by the endopeptidase ZMPSTE24 to generate lamin A. To facilitate investigations of the role of this cleavage in normal and disease states, we developed a monoclonal antibody (PL-1C7) that specifically recognizes prelamin A at the intact ZMPSTE24 cleavage site, ensuring prelamin A detection exclusively. Importantly, PL-1C7 can be used to determine prelamin A localization and accumulation in cells where lamin A is highly expressed without the use of exogenous fusion proteins. Our results show that unlike mature lamin A, prelamin A accumulates as discrete and localized foci at the nuclear periphery. Furthermore, whereas treatment with farnesylation inhibitors of cells overexpressing a GFP-prelamin A fusion protein results in the formation of large nucleoplasmic clumps, these aggregates are not observed upon similar treatment of cells expressing endogenous prelamin A or in cells lacking ZMPSTE24 expression and/or activity. Finally, we show that specific laminopathy-associated mutations exhibit both positive and negative effects on prelamin A accumulation, indicating that these mutations affect prelamin A processing efficiency in different manners.

Biochimica et biophysica acta, 2014

Gene promoters are frequently insufficient to drive the spatiotemporal patterns of gene expressio... more Gene promoters are frequently insufficient to drive the spatiotemporal patterns of gene expression during cell differentiation and organism development. Enhancers convey these properties through diverse mechanisms, including long-distance interactions with target promoters via their association with specific transcription factors. Despite unprecedented progress in the knowledge of enhancer mechanisms of action, there are still many unanswered questions. In particular, the contribution of an enhancer's local chromatin configuration to its mechanism of action is not completely understood. Here we describe a novel regulatory element, the Upstream Enhancer Element (UEE), which modulates the activity of the chicken α-globin 3' enhancer by regulating its chromatin structure, specifically by positioning a nucleosome upstream of the core enhancer. This element binds nuclear factors and confers a more restricted activation on the α-globin 3' enhancer, suggesting a progressive/rhe...

Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 2000

Hair follicle growth cycle proceeds through a series of stages in which strict control of cell pr... more Hair follicle growth cycle proceeds through a series of stages in which strict control of cell proliferation, differentiation, and cell death occurs. Transgenic mice expressing human papillomavirus type 16 E6/E7 papillomavirus oncogenes in the outer root sheath (ORS) display a fur phenotype characterized by lower hair density and the ability to regenerate hair much faster than wild-type mice. Regenerating hair follicles of transgenic mice show a longer growth phase (anagen), and although bulb regression (catagen) occurs, rest at telogen was not observed. No abnormalities were detected during the first cycle of hair follicle growth, but by the second cycle, initiation of catagen was delayed, and rest at telogen was again not attained, even in the presence of estradiol, a telogen resting signal. In conclusion, expression of E6/E7 in the ORS delays entrance to catagen and makes cells of the ORS insensitive to telogen resting signals bearing to a continuous hair follicle cycling in tran...

Recombinant Gene Expression

Chromosomal position effects are the cause of variegation of transgene expression relating to the... more Chromosomal position effects are the cause of variegation of transgene expression relating to the eukaryotic genome environment. These effects modify heterologous transgene expression, presumably due to dominant epigenetic marks that cause variegated or gradual silencing of transgene expression. As a solution, chromosomal insulators have arisen as a way to protect transgene expression against repressive marks (position effects), allowing homogeneous and consistent expression. Sustainable expression enables us to gain insight into in vivo protein function and protein stability, or to study position effects. Here, we show that the chicken cHS4 beta-globin insulator is able to protect transgene expression in cell lines, demonstrating its potential as a tool to improve the heterologous expression of any desired transgene. In addition, we show that green fluorescent protein (GFP) can be used as a reporter protein in the study of chromosomal position effects.

Briefings in Functional Genomics, 2011

Essential Methods, 2010

LAB 15 GENE EXPRESSION IN MAMMALIAN CELLS STUDENT GUIDE Review use of the Biological Safety Cabin... more LAB 15 GENE EXPRESSION IN MAMMALIAN CELLS STUDENT GUIDE Review use of the Biological Safety Cabinet (BSC) and sterile technique for cell culture. MATERIALS Antibiotic media 10 ml Penicillin-Streptomycin 1 Liter F12 K media 110 mL Fetal Bovine Serum (FBS) Filter sterilize Trypsin-EDTA (1x) 1x Phosphate Buffered Saline (PBS without MgCl, without CaCl) BioSafety I Epithelial cell culture at ≥ 80% confluence 24 well cell culture cluster plates with flat bottom and lid (Corning Inc. Costar #3524) Sterile P-1000 pipet and tips (place in BSC during UV light sterilization) Sterile 15 ml Corning orange capped tubes Part I. Growing Cells in 24-cell plates 1. Heat the media to 37ºC in a water bath.

Molecular and Biochemical Parasitology, 2005

Nucleic Acids Research, 2003

Molecular Immunology, 2009

Class-I MHC-restricted T-cell associated molecule (CRTAM) is a member of the Nectin-like adhesion... more Class-I MHC-restricted T-cell associated molecule (CRTAM) is a member of the Nectin-like adhesion molecule family. It is rapidly induced in NK, NKT and CD8 + T cells. Interaction with its ligand Nectin-like 2 results in increased secretion of IFN-␥ by activated CD8 + T lymphocytes. Through sequential bioinformatic analyses of the upstream region of the human CRTAM gene, we detected cis-elements potentially important for CRTAM gene transcription. Analyzing 2 kb upstream from the ATG translation codon by mutation analysis in conjunction with luciferase reporter assays, electrophoretic mobility shify assay (EMSA) and supershift assays, we identified an AP-1 binding site, located at 1.4 kb from the ATG translation codon of CRTAM gene as an essential element for CRTAM expression in activated but not resting human CD8 + T cells. CRTAM expression was reduced in activated CD8 + T cells treated with the JNK inhibitor SP600125, indicating that CRTAM expression is driven by the JNK-AP-1 signaling pathway. This study represents the first CRTAM gene promoter analysis in human T cells and indicates that AP-1 is a positive transcriptional regulator of this gene, a likely important finding because CRTAM has recently been shown to play a role in IFN-␥ and IL-17 production and T cell proliferation.

Molecular Biotechnology, 2006

Manipulation of the eukaryotic genome has contributed to the progress in our knowledge of multice... more Manipulation of the eukaryotic genome has contributed to the progress in our knowledge of multicellular organisms but has also ameliorated our experimental strategies. Biological questions can now be addressed with more efficiency and reproducibility. There are new and varied strategies for gene transfer and sequence manipulation with improved methodologies that facilitate the acquisition of results. Cellular systems and transgenic animals have demonstrated their invaluable benefits. In this review, I present an overview of the methods of gene transfer with particular attention to cultured cell lines and large-scale sequence vectors, like artificial chromosomes, with the possibility of their manipulation based on homologous recombination strategies. Alternative strategies of gene transfer, including retroviral vectors, are also described and the applications of such methods are discussed. Finally, several comments are made about the influence of chromatin structure on gene expression. Recent experimental data have shown that for convenient stable transgene expression, the influence of chromatin structure should be seriously taken into account. Novel chromatin regulatory and structural elements are proposed as an alternative for proper and sustained gene expression. These chromatin elements are facing a new era in transgenesis and we are probably beginning a new generation of gene and cancer therapy vectors.

Molecular Biology of the Cell, 2007

Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation... more Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation of gene expression and cell proliferation. Here, we have studied the role of zona occludens (ZO)-2, a TJ peripheral protein, in the regulation of cyclin D1 transcription. We found that ZO-2 down-regulates cyclin D1 transcription in a dose-dependent manner. To understand how ZO-2 represses cyclin D1 promoter activity, we used deletion analyses and found that ZO-2 negatively regulates cyclin D1 transcription via an E box and that it diminishes cell proliferation. Because ZO-2 does not associate directly with DNA, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay were used to identify the transcription factors mediating the ZO-2–repressive effect. c-Myc was found to bind the E box present in the cyclin D1 promoter, and the overexpression of c-Myc augmented the inhibition generated by ZO-2 transfection. The presence of ZO-2 and c-Myc in the same complex was...

Journal of Molecular Biology, 2004

We have studied the physical properties of a segment of condensed chromatin that lies upstream of... more We have studied the physical properties of a segment of condensed chromatin that lies upstream of the chicken beta-globin locus. This segment can be excised from an avian erythroleukemia cell line by restriction enzyme digestion and released from the nucleus as an essentially homogeneous fragment about 15.5 kbp long. Because of this homogeneity we could measure its sedimentation coefficient quite accurately by a combination of sucrose gradient and analytical ultracentrifugation. By measuring additionally the buoyant density of the cross-linked particle in CsCl we could deduce the total mass of the particle, hence its frictional coefficient, f, directly related to its shape. The measured value of f is consistent with a rod-like particle of the approximate length and diameter proposed earlier for the 30 nm chromatin fiber. The method is generally applicable to homogeneous particles of unique sequence at genomic abundance.

Journal of Cellular Biochemistry, 2009

Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional r... more Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken α‐globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of α‐globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans‐activation of the embryonic α‐globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic π gene in definitive erythrocytes. However, drug‐mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of c...

Journal of Biological Chemistry, 2004

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2007

At the present time research situates differential regulation of gene expression in an increasing... more At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.