Biology subjects

Fernandez, J.

Publications and source records attributed to Fernandez, J..

30 records · Page 2Linked to original sources

bioRxiv2022

Peptaibol Production and Characterization from Trichoderma asperellum and their Action as Biofungicide.

Peptaibols (Paib), are a class of biologically active peptides isolated from soil, fungi and molds, which have interesting properties as antimicrobial agents. Paib production was optimized in flasks by adding sucrose as a carbon source, Aib as an additive amino acid, and F. oxysporum cell debris as an elicitor. Paib were purified, sequenced and identified by HPLC coupled to mass spectrometry. Afterward, a Paib prototype was prepared with the extracts obtained from the optimized fermentations. The biological activity of these prototypes was evaluated using in vitro and in vivo methods. The prototype inhibited the growth of specific plant pathogens, and it showed inhibition rates similar to those from commercially available fungicides. Growth inhibition rates were 92.2, 74.2, 58.4 and 36.2% against Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria alternata and Fusarium oxysporum, respectively. Furthermore, the antifungal activity was tested in tomatoes inoculated with A. alternata, the incidence of the disease in tomatoes treated with the prototype was 0%, while the untreated fruit showed a 92.5% of infection. Scanning electron microscopy images showed structural differences between fungi treated with or without Paib. The most visual alterations were sunk and shriveled morphology in spores, while the hyphae appeared to be fractured, rough, and dehydrated.

microbiology↗

bioRxiv2022

C-di-GMP signalling links biofilm formation and Mn(II) oxidation in Pseudomonas resinovorans.

Bioaugmentation of biological sand filters with Mn(II)-oxidizing bacteria (MOB) is used to increase Mn removal efficiencies from groundwater. While the biofilm-forming ability of MOB is important to achieve optimal Mn filtration, the regulatory link between biofilm formation and Mn(II) oxidation remains unclear. Here, the environmental isolate P. resinovorans strain MOB-513 was used as a model to investigate the role of c-di-GMP, a second messenger crucially involved in the regulation of biofilm formation by Pseudomonas, in the oxidation of Mn(II). A novel role for c-di-GMP in the up-regulation of Mn(II) oxidation through induction of the expression of Manganese-Oxidizing Peroxidase (MOP) enzymes was revealed. MOB-513 macrocolony biofilms showed a strikingly stratified pattern of Mn oxides (BMnOx) accumulation in a localized top layer. Remarkably, elevated cellular levels of c-di-GMP correlated not only with increased accumulation of BMnOx in the same top layer, but also with the appearance of a second BMnOx stratum in the bottom region of macrocolony biofilms and the expression of mop genes correlated with this pattern. Proteomic analysis under Mn(II) conditions revealed the up-regulation of a GGDEF/EAL-domain protein and a PilZ-domain protein, providing a molecular link between c-di-GMP signalling and Mn(II) oxidation. Finally, we considered the biotechnological relevance of understanding the role of c-di-GMP in MOB-513 and observed that high c-di-GMP levels are correlated with higher lyophilisation efficiencies and higher groundwater Mn(II) oxidation capacity of lyophiles. Advancing understanding of these mechanisms is essential to improve the biotechnological application of bacterial inocula designed for removing Mn in biological filter systems. IMPORTANCEThe presence of Mn(II) in groundwater - a common source of drinking water-is a cause of water quality impairment, interfering with its disinfection, causing operation problems and affecting human health. Purification of groundwater containing Mn(II) plays an important role in environmental and social safety. The typical method for Mn(II) removal is based on bacterial oxidation of metals to form insoluble oxides that can be filtered out of the water. Evidence of reducing the start-up periods and enhancing Mn removal efficiencies through bioaugmentation with appropriate biofilm-forming and MOB has emerged. As preliminary data suggest a link between these two phenotypes in Pseudomonas strains, the need to investigate the underlying regulatory mechanisms is apparent. The significance of our research lies in determining the role of c-di-GMP for increased biofilm-formation and Mn(II)-oxidizing capabilities in MOBs, which will allow the generation of super biofilm-elaborating and Mn-oxidizing strains, enabling their implementation in biotechnological applications.

microbiology↗

bioRxiv2021

One Health or Three? Transmission modelling of Klebsiella isolates reveals ecological barriers to transmission between humans, animals and the environment

The Klebsiella group is highly diverse both genetically and ecologically, being commonly recovered from humans, livestock, plants, soil, water, and wild animals. Many species are opportunistic pathogens, and can harbour diverse classes of antimicrobial resistance (AMR) genes. K. pneumoniae is responsible for a high public-health burden, due in part to the rapid spread of health-care associated clones that are non-susceptible to carbapenems. Klebsiella thus represents a highly pertinent taxon for assessing the risk to public health posed by animal and environmental reservoirs. Here we report an analysis of 6548 samples and 3,482 genome sequences representing 15 Klebsiella species sampled over a 15-month period from a wide range of clinical, community, animal and environmental settings in and around the city of Pavia, in the northern Italian region of Lombardy. Despite carbapenem-resistant clones circulating at a high frequency in the hospitals, we find no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside of the clinical environment. The non-random distribution of species and strains across sources point to ecological barriers that are likely to limit AMR transmission. Although we find evidence for occasional transmission between settings, hierarchical modelling and intervention analysis suggests that direct transmission from the multiple non-human (animal and environmental) sources included in our sample accounts for less than 1% of hospital disease, with the vast majority of clinical cases originating from other humans.

microbiology↗

bioRxiv2021

Identical Sequences, Different Behaviors: ProteinDiversity Captured at the Single-Molecule Level

The classical "one sequence, one structure, one function" paradigm has shaped much of our intuition of how proteins work inside the cell. Partially due to the insight provided by bulk biochemical assays, individual biomolecules are assumed to behave as identical entities, and their characterization relies on ensemble averages that flatten any conformational diversity into a unique phenotype. While the emergence of single-molecule techniques opened the gates to interrogating individual molecules, technical shortcomings typically limit the duration of these measurements to a few minutes, which prevents to completely characterize a protein individual and, hence, to capture the heterogeneity among molecular populations. Here, we introduce a magnetic tweezers design, which showcases enhanced stability and resolution that allows us to measure the folding dynamics of a single protein during several uninterrupted days with a high temporal and spatial resolution. Thanks to this instrumental development, we do a complete characterization of two proteins with a very different force-response: the talin R3IVVI domain and protein L. Days-long recordings on the same single molecule accumulate several thousands of folding transitions sampled with sub-ms resolution, which allows us to reconstruct their free energy landscapes and describe how they evolve with force. By mapping the nanomechanical identity of many different protein individuals, we directly capture their molecular diversity as a quantifiable dispersion on their force response and folding kinetics. Our instrumental development offers a new tool for profiling individual molecules, opening the gates to the characterization of biomolecular heterogeneity.

biophysics↗

bioRxiv2020

Role of two metacaspases in development and pathogenicity of the Rice Blast fungus, Magnaporthe oryzae

Rice blast disease caused by Magnaporthe oryzae is a devastating disease of cultivated rice worldwide. Infections by this fungus lead to a significant reduction in rice yields and threats to food security. To gain better insight into growth and cell death in M. oryzae during infection, we characterized two predicted M. oryzae metacaspase proteins, MoMca1 and MoMca2. These proteins appear to be functionally redundant and are able to complement the yeast Yca1 homologue. Biochemical analysis revealed that M. oryzae metacaspases exhibited Ca2+ dependent caspase activity in vitro. Deletion of both MoMca1 and MoMca2 in M. oryzae resulted in reduced sporulation, delay in conidial germination and attenuation of disease severity. In addition, the double {Delta}Momca1mca2 mutant strain showed increased radial growth in the presence of oxidative stress. Interestingly, the {Delta}Momca1mca2 strain showed an increase accumulation of insoluble aggregates compared to the wild-type strain during vegetative growth. Our findings suggest that MoMca1 and MoMca2 promote the clearance of insoluble aggregates in M. oryzae, demonstrating the important role these metacaspases have in fungal protein homeostasis. Furthermore, these metacaspase proteins may play additional roles, like in regulating stress responses, that would help maintain the fitness of fungal cells required for host infection. IMPORTANCEMagnaporthe oryzae causes rice blast disease that threatens global food security by resulting in the severe loss of rice production every year. A tightly regulated life cycle allows M. oryzae to disarm the host plant immune system during its biotrophic stage before triggering plant cell death in its necrotrophic stage. The ways M. oryzae navigates its complex life cycle remains unclear. This work characterizes two metacaspase proteins with peptidase activity in M. oryzae that are shown to be involved in the regulation of fungal growth and development prior to infection by potentially helping maintain fungal fitness. This study provides new insight into the role of metacaspase proteins in filamentous fungi by illustrating the delays in M. oryzae morphogenesis in the absence of these proteins. Understanding the mechanisms by which M. oryzae morphology and development promote its devastating pathogenicity may lead to the emergence of proper methods for disease control.

microbiology↗

bioRxiv2020

Cross-Subject EEG-Based Emotion Recognition through Neural Networks with Stratified Normalization

Due to a large number of potential applications, a good deal of effort has been recently made towards creating machine learning models that can recognize evoked emotions from ones physiological recordings. In particular, researchers are investigating the use of EEG as a low-cost, non-invasive method. However, the poor homogeneity of the EEG activity across participants hinders the implementation of any such system by a time-consuming calibration stage. In this study, we introduce a new participant-based feature normalization method, so-called stratified normalization, for training deep neural networks in task of cross-subject emotion classification from EEG signals. The new method is able to subtract inter-participant variability while maintaining the emotion information in the data. We carried out our analysis on the SEED dataset, which contains 62-channel EEG recordings collected from 15 participants while watching film clips. Results demonstrate that networks trained with stratified normalization outperformed standard training with batch normalization significantly. In addition, the highest model performance was achieved when extracting EEG features with the multitaper method, reaching a classification accuracy of 91.6% for two emotion categories (positive and negative) and 79.6% for three (also neutral). This analysis provides us with great insight into the potential benefits that stratified normalization can have when developing any cross-subject model based on EEG.

neuroscience↗

bioRxiv2020

Structures of an intact yeast V-ATPase alone and in complex with bacterial effector VopQ

Vacuolar-type ATPase (V-ATPase) is a rotary protein pump involved in proton translocation across various cellular membranes using the energy of ATP hydrolysis. Despite previous studies on bacterial and eukaryotic V-ATPases, information on the intact structure of a eukaryotic V-ATPase is missing. Here we report cryo-EM structures of the intact yeast V-ATPase and this complex bound to a bacterial effector. We reveal the interaction of the elusive regulatory subunit H with its neighboring subunits. Insight for the catalysis mechanism is gained by determining conformations of the catalytic subunits either empty or bound with nucleotides.

biophysics↗

bioRxiv2020

Swine viral detection by adapted Next-Generation Sequencing (NGS) for RNA and DNA species reveals first detection of porcine circovirus type 3 (PCV3) in Chile

The outbreak and propagation of COVID-19 have posed a significant challenge to our society, highlighting the relevance of epidemiological monitoring of animal pathogens closely related to humans due to potential zoonosis. To find previously undetected viruses inside Chilean swine farms, we collected non-invasive samples from animals suspected to undergo any viral disease. After screening by Next Generation Sequencing (NGS) adapted to identify viral species with RNA and DNA genomes simultaneously, different viral species were successfully detected. Among viruses with an RNA genome, Porcine Rotavirus A (RVA), Porcine Astrovirus type 5 (PAstV-5) and Porcine Feces-Associated IASV-like virus (PfaIV) were identified; whereas among viruses with DNA genome, Porcine Parvovirus 1 (PPV1), Porcine Circovirus type 1 (PCV1) and Porcine Circovirus type 3 (PCV3) were found. PCV3, a recently described pathogenic virus, was detected for the first time in Chile in different samples from mummified tissue of stillbirths. The whole genome sequence of PCV3 was completed using conventional PCR and Sanger sequencing and compared with previously reported genomes. To the best of our knowledge, this is the first time that an adapted NGS system is used for the screening of DNA and RNA viral species simultaneously inside the Chilean swine industry. This adapted NGS system might be useful for the detection of swine viral emerging diseases worldwide.

genomics↗

bioRxiv2020

HUMAN PANCREATIC CANCER CELLS UNDERGO PROFOUND METABOLIC REPROGRAMMING TOWARDS CELLULAR STEMNESS AS ADAPTATION TO INHIBITION OF THE AKT PATHWAY

Cancer cells acquire resistance to cytotoxic therapies targeting major survival pathways by adapting their metabolism. The AKT pathway is a major regulator of human pancreatic adenocarcinoma progression. The mechanisms of adaptation to long-term silencing of AKT isoforms of pancreatic cancer cells were studied. Following silencing, cancer cells remained quiescent for long periods of time, after which they recovered proliferative capacities. Adaptation caused profound proteomic changes largely affecting mitochondrial biogenesis, energy metabolism, and acquisition of a number of distinct cancer stem cell (CSC) characteristics depending on the AKT isoform that was silenced. The adaptation to AKT1 silencing drove most de-differentiation and acquisition of stemness through C-MYC down-modulation and NANOG up-regulation, which were required for survival of adapted CSCs. The changes associated to adaptation sensitized cancer cells to inhibitors targeting regulators of oxidative respiration and mitochondrial biogenesis. In vivo pharmacological co-inhibition of AKT and mitochondrial metabolism effectively controlled pancreatic adenocarcinoma growth in pre-clinical models.

cancer biology↗

bioRxiv2019

Exploiting evolutionary herding to control drug resistance in cancer

Drug resistance mediated by clonal evolution is arguably the biggest problem in cancer therapy today. However, evolving resistance to one drug may come at a cost of decreased growth rate or increased sensitivity to another drug due to evolutionary trade-offs. This weakness can be exploited in the clinic using an approach called evolutionary herding that aims at controlling the tumour cell population to delay or prevent resistance. However, recapitulating cancer evolutionary dynamics experimentally remains challenging. Here we present a novel approach for evolutionary herding based on a combination of single-cell barcoding, very large populations of 108-109 cells grown without re-plating, longitudinal non-destructive monitoring of cancer clones, and mathematical modelling of tumour evolution. We demonstrate evolutionary herding in non-small cell lung cancer, showing that herding allows shifting the clonal composition of a tumour in our favour, leading to collateral drug sensitivity and proliferative fitness costs. Through genomic analysis and single-cell sequencing, we were also able to determine the mechanisms that drive such evolved sensitivity. Our approach allows modelling evolutionary trade-offs experimentally to test patient-specific evolutionary herding strategies that can potentially be translated into the clinic to control treatment resistance.

cancer biology↗

bioRxiv2017

RES complex is associated with intron definition and required for zebrafish early embryogenesis

Pre-mRNA splicing is a critical step of gene expression in eukaryotes. Transcriptome-wide splicing patterns are complex and primarily regulated by a diverse set of recognition elements and associated RNA-binding proteins. The retention and splicing (RES) complex is formed by three different proteins (Bud13p, Pml1p and Snu17p) and is involved in splicing in yeast. However, the importance of the RES complex for vertebrate splicing, the intronic features associated with its activity, and its role in development are unknown. In this study, we have generated loss-of-function mutants for the three components of the RES complex in zebrafish and showed that they are required during early development. The mutants showed a marked neural phenotype with increased cell death in the brain and a decrease in differentiated neurons. Transcriptomic analysis of bud13, snip1 (pml1) and rbmx2 (snu17) mutants revealed a global defect in intron splicing, with strong mis-splicing of a subset of introns. We found these RES-dependent introns were short, rich in GC and flanked by GC depleted exons, all of which are features associated with intron definition. Using these features we developed a predictive model that classifies RES dependent introns. Altogether, our study uncovers the essential role of the RES complex during vertebrate development and provides new insights into its function during splicing.

genomics↗

bioRxiv2017

Nucleolin internalizes Bothrops asper Lys49 phospholipase A2 forming cell surface amyloid-like assemblies

Phospholipases A2 (PLA2s) are a major component of snake venoms. Some of them cause severe muscle necrosis through a still unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA2s are not directly connected. In addition, viperid venoms contain PLA2-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. Nucleolin, a main component of the nucleolus, is a disordered protein involved in many protein assembly and phase separation phenomena. In some circumstances nucleolin is exposed on the cell surface from where it is involved in the internalization of many ligands.\n\nIn this work we demonstrate that Bothrops asper myotoxin II (Mt-II), a Lys49 PLA2-like toxin, interacts with, and is internalized in cells by nucleolin. The internalization process is functional to the toxicity of the protein, as both an antibody and an aptamer specific for nucleolin protect cells from intoxication. We identified central RRM and the C-terminal R/F-GG domain of nucleolin as the regions involved in the interaction with Mt-II. Finally we observed that Mt-II forms, on the cell surface, amyloid-like assemblies that colocalize with nucleolin and that can be involved in the activation of the internalization process. The presence, in the three dimensional structure of Mt-II and related PLA2 homologues, of four exposed loops enriched in prion-like amino acid sequences reinforces this hypothesis.\n\nPhospholipases A2 | Lys49 myotoxins | nucleolin | amyloid-like | molecular assemblies\n\nSIGNIFICANCEThe main finding of this work, the role of nucleolin as Bothrops asper Mt-II receptor, is a remarkable step forward in understanding the mechanism of action of cytotoxic PLA2s. It may suggest new strategies for anti-venom therapies and explain the anti-tumoral and anti-viral pharmacological action of snake PLA2s, since nucleolin is a receptor for many growth factors and virus.\n\nThe proposed internalization mechanism, via formation of molecular assemblies among Mt-II amyloid-like structures and other proteins, including nucleolin, can be of general validity. Cell surface molecular assemblies couldbepointsofselectionandconcentrationnotonlyofsnake,butalsoofmammaliansecretedPLA2s, proteins involved in different pathologies, and trigger the internalization pathway only when their molarity exceeds a threshold dose.

biochemistry↗