Biology subjects

Garcia, E.

Publications and source records attributed to Garcia, E..

47 records · Page 3Linked to original sources

bioRxiv2021

A protective broadly cross-reactive human antibody defines a conserved site of vulnerability on beta-coronavirus spikes

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses ({beta}-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 [A] resolution and found that the peptide adopted a mainly helical structure. Conserved residues in {beta}-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on {beta}-CoV spike proteins for protective antibodies that may facilitate the development of pan-{beta}-CoV vaccines. SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-{beta}-CoV vaccine design strategies

immunology↗

bioRxiv2021

Robust optical autofocus system utilizing neural networks trained for extended range and time-course and automated multiwell plate imaging including single molecule localization microscopy

We present a robust, long-range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, e.g. due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition. To address the impact of (thermal or mechanical) fluctuations over time in the optical autofocus system itself, we utilise a convolutional neural network (CNN) that is trained over multiple days to account for such fluctuations. To address the trade-off between axial precision and range of the autofocus, we implement orthogonal optical readouts with separate CNN training data, thereby achieving an accuracy well within the 600 nm depth of field of our 1.3 numerical aperture objective lens over a defocus range of up to approximately +/- 100 m. We characterise the performance of this autofocus system and demonstrate its application to automated multiwell plate single molecule localisation microscopy.

bioengineering↗

bioRxiv2021

Sequence-function relationships in phage-encoded bacterial cell wall lytic enzymes and their implications for phage-derived products design

Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a landscape view of lysins structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G-) (higher net charge, amphipathic helices) are responsible for an improved interaction with G- envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecules design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G- surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages host bacteria and sequence-predicted physicochemical properties. Based on such classifiers, an investigation on the differential appearance of certain features was conducted. Such analyses revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. Particularly, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment and aliphatic index) were associated to G- phage lysins, appearing specifically at their C-terminal end. Evidences on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts have been provided, specifically supporting the nowadays common hypothesis that lysins from G- usually contain AMP-like regions. IMPORTANCEPhage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The lysins potential as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance, but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysins diversity landscape by examining a set of phage lysin genes. This way, we have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures, and, thus, also the reach of their specialization regarding cell wall structures. These results provide clarity and evidences to sustain some of the common hypothesis in current literature, as well as make available an updated and characterized database of lysins sequences for further developments.

microbiology↗

bioRxiv2020

RTP801 Is A Key Modulator For Motor Learning

RTP801/REDD1 is a stress-regulated protein whose upregulation is necessary and sufficient to trigger neuronal death in in vitro and in vivo models of Parkinsons and Huntingtons diseases and is up regulated in compromised neurons in human postmortem brains of both neurodegenerative disorders. Indeed, in both Parkinsons and Huntingtons disease mouse models, RTP801 knockdown alleviates motor-learning deficits. Here, we investigated the physiological role of RTP801 in neuronal plasticity. RTP801 is found in rat, mouse and human synapses. The absence of RTP801 enhanced excitatory synaptic transmission in both neuronal cultures and brain slices from RTP801 knock-out (KO) mice. Indeed, RTP801 KO mice showed improved motor learning, which correlated with lower spine density but increased basal filopodia and mushroom spines in the motor cortex layer V. This paralleled with higher levels of synaptosomal GluA1 and TrkB receptors in homogenates derived from KO mice motor cortex, proteins that are associated with synaptic strengthening. Altogether, these results indicate that RTP801 has an important role modulating neuronal plasticity in motor learning.

neuroscience↗

bioRxiv2020

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

immunology↗

bioRxiv2019

The ciliary membrane of polarized epithelial cells stems from a midbody remnant-associated membrane patch with condensed nanodomains

The primary cilium is a specialized plasma membrane protrusion that harbors receptors involved in important signaling pathways. Despite its central role in regulating cellular behavior, the biogenesis of the primary cilium is not fully understood. In fact, the source of the ciliary membrane remains a mystery in cell types that assemble their primary cilium entirely at the cell surface, such as polarized renal epithelial cells. After cytokinesis, the remnant of the midbody of these cells moves to the center of the apical surface, where it licenses the centrosome for ciliogenesis through an unidentified mechanism. Here, to investigate the origin of the ciliary membrane and the role of the midbody remnant, we analyzed membrane compaction and lipid dynamics at the microscale and nanoscale in living renal epithelial MDCK cells. We found that a specialized patch made of condensed membranes with restricted lipid lateral mobility surrounds the midbody remnant. This patch accompanies the remnant on its journey towards the centrosome and, once the two structures have met, the remnant delivers part of membranes of the patch to build the ciliary membrane. In this way, we have determined the origin of the ciliary membrane and the contribution of the midbody remnant to primary cilium formation in cells whose primary cilium is assembled at the plasma membrane.

cell biology↗

bioRxiv2019

Epigenetic bookmarking of H2S exposure in Caenorhabditis elegans

Physiological memories of environmental stress can serve to predict future environmental changes, allowing the organism to initiate protective mechanisms and survive. Although physiological memories, or bookmarks, of environmental stress have been described in a wide range of organisms, from bacteria to plants to humans, the mechanism by which these memories persist in the absence of stress is still largely unknown. We have discovered that C. elegans transiently exposed to low doses of hydrogen sulfide (H2S) survive subsequent exposure to otherwise lethal H2S concentrations and induce H2S-responsive transcripts more robustly than naive controls. H2S bookmarking can occur at any developmental stage and persists through cell divisions and development but is erased by fasting. We show that maintenance of the H2S bookmark requires the SET-2 histone methyltransferase and the CoREST-like demethylase complex. We propose a model in which exposure to low doses of H2S generates a long-lasting, epigenetic memory by modulating H3K4me2 modifications at specific promoters. Understanding the fundamental aspects of H2S bookmarking in this tractable system can provide mechanistic insight into how environmental exposures are translated into the epigenetic landscape in animals.

genetics↗

bioRxiv2018

Transition from a meiotic to a somatic-like DNA damage response during the pachytene stage in mouse meiosis

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of {gamma}H2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid pachytene to diplotene, {gamma}H2AX appeared as large discrete foci. This late repair pattern was mediated first by NHEJ, involving Ku70/80 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early \"meiotic\" repair pathway that acts by default at the beginning of meiosis is replaced from mid pachytene onwards by a \"somatic-like\" repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, expression of a new gene profile and acquisition of competence to proceed to metaphase.

genetics↗

bioRxiv2018

Autolysin-Independent DNA Release in Streptococcus pneumoniae in vitro Biofilms

Biofilms are defined as layers of cells of microorganisms adhered to the surface of a substrate and embedded in an extracellular matrix and provide an appropriate environment for increased genetic exchange. Extracellular DNA (eDNA) is an essential component of the extracellular matrix of microbial biofilms, but the pathway(s) responsible for DNA release are largely unknown. Autolysis (either spontaneous or phage-induced) has been proposed the major event leading to the appearance of eDNA. The suicidal tendency of Streptococcus pneumoniae is well-known, with lysis mainly caused by the triggering of LytA, the major autolytic amidase. However, the LytC lysozyme and CbpD (a possible murein hydrolase) have also been shown involved. The present work examines the relationship between eDNA, autolysins, and the formation and maintenance of in vitro pneumococcal biofilms, via fluorescent labelling combined with confocal laser scanning microscopy, plus genetic transformation experiments. Bacterial DNA release mechanisms other than those entailing lytic enzymes were shown to be involved by demonstrating that horizontal gene transfer in biofilms takes place even in the absence of detectable autolytic activity. It had been previously suggested that the quorum sensing systems ComABCDE and LuxS/AI-2 are involved in the production of eDNA as a response to the accumulation of quorum sensing signals, although our immunofluorescence results do not support this hypothesis. Evidence that the release of DNA is somehow linked to the production of extracellular vesicles by S. pneumoniae is provided.

microbiology↗

bioRxiv2017

Dissecting single cell molecular spatiotemporal mobility and clustering at Focal Adhesions in polarised cells by fluorescence fluctuation spectroscopy methods

Quantitative fluorescence fluctuation spectroscopy from optical microscopy datasets is a very powerful tool to resolve multiple spatiotemporal cellular and subcellular processes at the molecular level. In particular, raster image correlation spectroscopy (RICS) and number and brightness analyses (N&B) yield molecular mobility and clustering dynamic information extracted from real-time cellular processes. This quantitative information can be inferred in a highly flexibly and detailed manner, i.e. 1) at the localisation level: from full-frame datasets and multiple regions of interest within; and 2) at the temporal level: not only from full-frame and multiple regions, but also intermediate temporal events. Here we build on previous research in deciphering the molecular dynamics of paxillin, a main component of focal adhesions. Cells use focal adhesions to attach to the extracellular matrix and interact with their local environment. Through focal adhesions and other adhesion structures, cells sense their local environment and respond accordingly; due to this continuous communication, these structures can be highly dynamic depending on the extracellular characteristics. By using a previously well-characterised model like paxillin, we examine powerful sensitivity characteristics and some limitations of RICS and N&B analyses. We show that cells upon contact to different surfaces show differential self-assembly dynamics in terms of molecular diffusion and oligomerisation. In addition, single-cell studies show that these dynamics change gradually following an antero-posterior gradient.

biophysics↗

bioRxiv2017

Polarity sensitive probes for super resolution STED microscopy

The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles show the high potential of these dyes for probing nanoscale membrane heterogeneity.

biophysics↗