Biology subjects

Jimenez, A.

Publications and source records attributed to Jimenez, A..

28 records · Page 2Linked to original sources

bioRxiv2021

The Alzheimer's disease risk factor APOE4 drives pro-inflammation in human astrocytes via HDAC-dependent repression of TAGLN3

The Apolipoprotein E4 (APOE4) is the major allelic risk factor for late-onset Alzheimers disease (AD). APOE4 associates with a pro-inflammatory phenotype increasingly considered as critical in AD initiation and progression. Yet, the mechanisms driving an APOE4-dependent neuroinflammation remain unelucidated. Leveraging patient specific human induced Pluripotent Stem Cells (iPSCs) we demonstrate inflammatory chronicity and hyperactivated responses upon cytokines in human APOE4 astrocytes via a novel mechanism. We uncovered that APOE4 represses Transgelin 3 (TAGLN3), a new interacting partner of I{kappa}B, thus increasing the NF-kB activity. The transcriptional repression of TAGLN3 was shown to result from an APOE4-dependent histone deacetylase (HDAC) activity. The functional relevance of TAGLN3 was demonstrated by the attenuation of APOE4-driven neuroinflammation after TAGLN3 supplementation. Importantly, TAGLN3 downregulation was confirmed in the brain of AD patients. Our findings highlight the APOE4-TAGLN3 axis as a new pathogenic pathway that paves the way for the development of therapeutics to prevent maladaptive inflammatory responses in APOE4 carriers, while placing TAGLN3 downregulation as a potential biomarker of AD. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=98 HEIGHT=200 SRC="FIGDIR/small/440108v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1036557org.highwire.dtl.DTLVardef@1f245bdorg.highwire.dtl.DTLVardef@c07c4aorg.highwire.dtl.DTLVardef@1200396_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

Race specific differences in DNA damage repair dysregulation in breast cancer and association with outcome

IMPORTANCEAfrican American (AA) breast cancer patients have worse outcomes than Caucasian Americans (CAs). DNA damage repair (DDR) genes drive poor outcome in CA estrogen receptor (ER)+ breast cancer patients. Whether DDR genes similarly impact survival in AAs is unknown. Identifying AA-specific patterns of DDR dysregulation could change how we tailor predictive/prognostic biomarkers. OBJECTIVETo characterize DDR dysregulation in ER+ AA patient tumors and test associations with clinical outcome. DESIGN SETTINGS AND PARTICIPANTSThree independent tumor, and two normal breast datasets were analyzed. Tumor datasets: (1) GSE78958 (2) GSE18229 (3) The Cancer Genome Atlas (TCGA). Normal datasets: (4) GSE43973 (5) GSE50939. MAIN OUTCOME AND MEASURESUp/down-regulation of 104 DDR genes was assessed in AA samples vs CAs. Survival associations were assessed for genes dysregulated in multiple datasets. RESULTSOverall, RNA levels of single strand break repair (SSBR) genes were downregulated in AA tumors and double strand break repair (DSBR) genes were upregulated compared to CAs. While SSBR downregulation was mainly detected in tumors, DSBR upregulation was detectable in both tumor and normal breast AA samples. Seven specific DDR genes identified as dysregulated in AAs vs CAs in multiple datasets associated with poor survival. A subset of tumors with simultaneous dysregulation of homologous recombination and single strand break repair genes was enriched in AAs and had associated consistently with poor survival. CONCLUSION AND RELEVANCEOverall, these results constitute the first systematic analysis of differences in DDR regulation in AA ER+ tumors and normal tissue vs CAs. We identify a profile of DDR dysregulation enriched in AA patients, which associates with poor outcome. These results suggest a distinct molecular mechanism of DDR regulation in AAs that lays the groundwork for refining biomarker profiles by race and improving precision medicine for underserved populations.

cancer biology↗

bioRxiv2020

Single-Cell Transcriptomic Attributes and Unbiased Computational Modeling for the Prediction of Immunomodulatory Potency of Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are currently being tested in numerous clinical trials as potential cell therapies for the treatment of various diseases and due to their potential immunomodulatory, pro-angiogenic, and regenerative properties. However, variabilities in tissue sources, donors, and manufacturing processes and the lack of defined critical quality attributes (CQAs) and clinically relevant mechanism of action (MoA) pose significant challenges to identify MSC cell therapy products with a predictable therapeutic outcome. This also hinders regulatory considerations and broad clinical translation of MSCs. MSC products are often administered to the patient immediately after thawing from cryopreserved vials (out-of-thaw). However, the qualifying quality-control assays are either performed before cryopreservation, or after culturing the post-thaw cells for 24-48 hours (culture-rescued), none of which represent the out-of-thaw product administered to patients. In this study, we performed a broad functional characterization of out-of-thaw and culture-rescue MSCs from bone marrow (BM-MSCs) and cord tissue (CT-MSCs) using macrophage activation and T cell proliferation-based in vitro potency assays and deep phenotypic characterization using single-cell RNA-sequencing. Using this data, we developed unbiased computational models, specifically symbolic regression (SR) and canonical correlation analysis (CCA) models to predict the immunomodulatory potency of MSCs. Overall, our results suggest that manufacturing conditions (OOT vs. CR) have a strong effect on MSC-function on MSC interactions with macrophages and T cells. Furthermore, single-cell RNA-seq analyses of out-of-thaw BM and CT-MSCs indicate a tissue of origin-dependent variability and heterogeneity in the transcriptome profile. Using symbolic regression modeling we identified specific single-cell transcriptomic attributes of MSCs that predict their immunomodulatory potency. In addition, CCA modeling predicted MSC donors with high or low immunomodulatory potency from their transcriptome profiles. Taken together, our results provide a broad framework for identifying predictive CQAs of MSCs that could ultimately help in better understanding of their MOAs and improved reproducibility and manufacturing control of MSCs.

immunology↗

bioRxiv2020

Highly sensitive and specific multiplex antibody assays to quantify immunoglobulins M, A and G against SARS-CoV-2 antigens

Reliable serological tests are required to determine the prevalence of antibodies against SARS-CoV-2 antigens and to characterise immunity to the disease in order to address key knowledge gaps in the context of the COVID-19 pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and ELISA with their higher precision, dynamic range, throughput, miniaturization, cost-efficacy and multiplexing capacity. We developed three qSAT assays to detect IgM, IgA and IgG to a panel of eight SARS-CoV-2 antigens including spike (S), nucleoprotein (N) and membrane (M) protein constructs. The assays were optimized to minimize processing time and maximize signal to noise ratio. We evaluated the performance of the assays using 128 plasmas obtained before the COVID-19 pandemic (negative controls) and 115 plasmas from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 8 were asymptomatic, 58 had mild symptoms and 49 were hospitalized. Pre-existing IgG antibodies recognizing N, M and S2 proteins were detected in negative controls suggestive of cross-reactive to common cold coronaviruses. The best performing antibody isotype/antigen signatures had specificities of 100% and sensitivities of 94.94% at [≥]14 days since the onset of symptoms and 96.08% at [≥]21 days since the onset of symptoms, with AUC of 0.992 and 0.999, respectively. Combining multiple antibody markers as assessed by qSAT assays has the highest efficiency, breadth and versatility to accurately detect low-level antibody responses for obtaining reliable data on prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity required for vaccine development to combat pandemics like the COVID-19.

immunology↗

bioRxiv2019

Multiplex genomic edition in Ashbya gossypii using CRISPR-Cpf1

The CRISPR/Cas technologies constitute an essential tool for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables the efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained to the existence of an specific 5-NGG-3 PAM sequence in the target site.\n\nHere we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows to employ a T-rich PAM sequence (5-TTTN-3) and facilitates the implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions into five different auxotrophic marker genes (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system was demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may significantly affect to the edition efficiency of the system.

genomics↗

bioRxiv2019

Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma

BackgroundMalaria diagnostics by rapid diagnostic tests (RDTs) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium sp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory based assays are needed for evaluating RDTs performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of clinical samples (whole blood, plasma and dried blood spots) from different endemic countries.\n\nResultsThe qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum (Pf-LDH). The assay detected P. vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r=0.59 and 0.75, respectively) as well as microscopy (Spearman r=0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density.\n\nConclusionThis immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDTs performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.

microbiology↗

bioRxiv2019

Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings

Recent super-resolution microscopy studies have unveiled a periodic scaffold of actin rings regularly spaced by spectrins under the plasma membrane of axons. However, ultrastructural details are unknown, limiting a molecular and mechanistic understanding of these enigmatic structures. Here, we combine platinum-replica electron and optical super-resolution microscopy to investigate the cortical cytoskeleton of axons at the ultrastructural level. Immunogold labeling and correlative super-resolution/electron microscopy allow us to unambiguously resolve actin rings as braids made of two long, intertwined actin filaments connected by a dense mesh of aligned spectrins. This molecular arrangement contrasts with the currently assumed model of actin rings made of short, capped actin filaments. Along the proximal axon, we resolved the presence of phospho-myosin light chain and the scaffold connection with microtubules via ankyrin G. We propose that braided rings explain the observed stability of the actin-spectrin scaffold and ultimately participate in preserving the axon integrity.

cell biology↗

bioRxiv2019

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

cell biology↗

bioRxiv2018

Automatic local resolution-based sharpening of cryo-EM maps.

Recent technological advances and computational developments, have allowed the reconstruction of cryo-EM maps at near-atomic resolution structures. Cryo-EM maps benefit significantly of a \"postprocessing\" step, normally referred to as \"sharpening\", that tends to increase signal at medium/high resolution. Here, we propose a new method for local sharpening of volumes generated by cryo-EM. The algorithm (LocalDeblur) is based on a local resolution-guided Wiener restoration approach, does not need any prior atomic model and it avoids artificial structure 1 factor corrections. LocalDeblur is fully automatic and parameter free. We show that the new method significantly and quantitatively improving map quality and interpretability, especially in cases of broad local resolution changes (as is often the case of membrane proteins).

biophysics↗

bioRxiv2017

Mechanistic causes of constrained phenotypic variation revealed by synthetic gene regulatory networks

Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such restrictions are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most of the evidence for this is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in E. coli that produce a gene expression stripe - a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.

systems biology↗