Biology subjects

Fernandez-Godino, R.

Publications and source records attributed to Fernandez-Godino, R..

7 recordsLinked to original sources

bioRxiv2026

VCP inhibition preserves photoreceptor integrity under hydroquinone-induced oxidative stress in a human iPSC-RPE/porcine neuroretina co-culture model

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in older adults. It is characterised by early retinal pigment epithelium (RPE) dysfunction followed by progressive photoreceptor degeneration. Cigarette smoking is a major environmental risk factor for AMD, and hydroquinone (HQ), a redox-active cigarette smoke component, induces oxidative stress and apoptosis in RPE cells. To analyse how RPE stress contributes to photoreceptor degeneration, we employed a retinal co-culture model composed of human induced pluripotent stem cell-derived RPE (iPSC-RPE) cells in conjunction with porcine neuroretina explants. Exposure to HQ induced oxidative stress in iPSC-RPE cells as well as retinal photoreceptors (RPR), resulting in apoptosis, executed at least in part by caspase activation. Concomitantly, HQ caused endoplasmic reticulum (ER) stress (ERAD) in RPR followed by their degeneration, evidenced by reduced outer nuclear layer (ONL) rows and shortened RPR outer segments (OS). Based on earlier results, which suggest a perturbation of proteostasis due to HQ, we tested whether ML240, a bona fide inhibitor of valosin-containing protein (VCP), would influence the degree of degenerative activities. ML240 did not prevent HQ-induced apoptosis in iPSC-RPE cells. However, it significantly preserved photoreceptor integrity, retaining OS length and cone density in HQ-stressed co-cultures. Proteomic analysis suggested that ML240 reshapes stress response patterns of the HQ-exposed neuroretina, as evidenced by a reduction in ERAD-associated markers, increased levels of antioxidant response proteins, and the preservation of cytochrome c enrichment in photoreceptor inner segments, which indicates improved mitochondrial integrity consistent with the observed preservation of photoreceptor structure. Together, these findings establish the iPSC-RPE/neuroretina co-culture as a platform to analyse pathophysiological features of AMD, dissect cell type-specific retinal responses to environmental stress and test neuroprotective pharmacological approaches to protect photoreceptors in oxidative stress-associated retinal degeneration.

cell biology↗

bioRxiv2026

BODIPY-Tagged β-Lactams as Selective Quenched Activity-Based Probes to Target Human Neutrophil Elastase

Activity-based probes are indispensable tools for interrogating protease function, and quenched fluorescent variants enable dynamic, real-time imaging of enzymatic activity. Despite these advances, very few quenched activity-based probes (qABPs) have been reported for serine proteases, which constitute the largest and most diverse mechanistic class of proteases. {beta}-Lactams have been extensively used to develop molecular tools and drugs designed to bind or be hydrolysed by serine-dependent bacterial enzymes. Here, we report the first monocyclic {beta}-lactam-containing qABPs for detecting serine proteases. Both the enzyme-triggered activation mechanism and intrinsic reactivity of the probes were highly dependent on the relative position of the BODIPY-FL fluorophore and quencher moiety at the {beta}-lactam core. qABPs displaying the most efficient turn-on mechanism were shown to selectively target human neutrophil elastase (HNE) in different human cell lysates. The most successful qABP was rapidly internalised and targeted HNE in U937 cells and human neutrophils. These results demonstrate the potential of the modular {beta}-lactam warhead to develop turn-on probes to track neutrophil serine proteases in live cells

pharmacology and toxicology↗

bioRxiv2025

Affinity-based selection of anti-Feline Leukaemia Virus p27 monoclonal antibodies for efficient lateral flow assay development

Classic diagnostic modalities--including RT-PCR and proviral DNA PCR--present certain drawbacks, highlighting the need of alternative diagnostic techniques, such as lateral flow immunoassays (LFIA), valued for their rapidity and sensitivity. Said desirable features depend on the efficiency of the ternary interaction wherein the antigen bridges immobilized capture and labelled detection antibodies. However, the cost and the throughput of current screening methods, for finding monoclonal antibody (mAbs) pairs efficient at establishing this type of ternary interactions, remains as a universal bottleneck for LFIA development. Traditional sandwich enzyme-linked immunosorbent assays (ELISA), although extensively employed in mAb screening procedures, is burdened by its high consumption of labelled mAbs and the potential alteration of antigen conformation upon plate immobilization, often yielding suboptimal surrogates for native interactions within the final LFIA setup. In this study, we developed an orthogonal approach integrating ELISA and Spectral Shift (SST) that jointly enable the minimisation of reagent consumption (mAbs, antigens, labelling reagents) to comprehensively profile a panel of mAbs against a bona fide antigen of Feline Leukaemia Virus FeLV p27. This facilitated the quick selection of two low nanomolar mAbs with distinct epitope recognition, for which we validated a ternary complex formation in a LFIA prototype. This strategy enables advanced, resource-efficient LFIA development for FeLV and other critical antigens, such as H5 Avian Influenza Virus or African Swine Fever Virus, in addition to major human pathogens including Helicobacter pylori or Mpox Virus, facilitating sensitive diagnostics and streamlined quality control.

biophysics↗

bioRxiv2024

Morphological Profiling Dataset of EU-OPENSCREEN Bioactive Compounds Over Multiple Imaging Sites and Cell Lines

Morphological profiling with the Cell Painting assay has emerged as a promising method in drug discovery research. The assay captures morphological changes across various cellular compartments enabling the rapid identification of the effect of compounds. We present a comprehensive morphological profiling dataset using the carefully curated and well-annotated EU-OPENSCREEN Bioactive Compound Set. Our profiling dataset was generated across multiple imaging sites with high-throughput confocal microscopes using the Hep G2 as well as the U2 OS cell line. We employed an extensive assay optimization process to achieve high data quality across the different imaging sites. An analysis of the four replicates validates the robustness of the generated data. We compare morphological features of the different cell lines and map the profiles to activity, toxicity, and basic compound targets to further describe the dataset as well as to demonstrate the potential of this dataset to be used for mechanism of action exploration.

cell biology↗

bioRxiv2022

Extracellular Vesicles from PRPF31+/- iPSC-Derived RPE Have a Distinct RNA Profile Compared to Those From Healthy RPE

Retinitis pigmentosa (RP), is the most common inherited retinal degeneration (IRD), leading to vision loss via dysfunction and death of photoreceptor cells and retinal pigment epithelium (RPE). Mutations in the pre-mRNA processing factor 31 (PRPF31) gene are associated with autosomal dominant RP, impairing RPE function. While adeno-associated virus (AAV)-mediated gene therapy shows promise for treating IRDs, the slow progression of these diseases often makes timely measurement of clinical efficacy challenging. Extracellular vesicles (EVs) are lipid enclosed vesicles secreted by cells and their RNA contents are being explored as circulating biomarkers of cancer and other diseases. We hypothesize that EV RNAs could serve as biomarkers of the health status of the neural retina and RPE health. To test this, we used PRPF31+/+ and PRPF31+/- human-induced pluripotent stem cell (hiPSC)-derived RPE (hi-RPE) to investigate the RNAs contained in RPE-derived EVs, and how they change in disease. We also compared the RNA contents of RPE-EVs with the RNAs contained in the hi-RPE cells themselves. We found that EVs from mutant PRPF31+/- hi-RPE cells have distinct RNA profiles compared to those from control cells, suggesting EV RNA contents change during disease and could serve as biomarkers for retinal degeneration.

molecular biology↗

bioRxiv2019

AAV-mediated gene augmentation therapy restores critical functions in mutant iPSC-derived PRPF31+/- cells.

Retinitis pigmentosa (RP) is the most common form of inherited vision loss and is characterized by degeneration of retinal photoreceptor cells and the retinal pigment epithelium (RPE). Mutations in pre-mRNA processing factor 31 (PRPF31) cause dominant RP via haploinsufficiency with incomplete penetrance. There is good evidence that the diverse severity of this disease is a result of differing levels of expression of the wild type allele among patients. Thus, we hypothesize that PRPF31-related RP will be amenable to treatment by adeno-associated virus (AAV)-mediated gene augmentation therapy. To test this hypothesis, we used induced pluripotent stem cells (iPSC) with mutations in PRPF31 and differentiated them into RPE cells. The mutant PRPF31 iPSC-RPE cells recapitulate the cellular phenotype associated with the PRPF31 pathology, including defective cell structure, diminished phagocytic function, defects in ciliogenesis, and compromised barrier function. Treatment of the mutant PRPF31 iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and cilia formation, and partially restored structure and barrier function. These results provide proof-of concept that AAV-based gene therapy can be used to treat patients with PRPF31-related RP.

neuroscience↗

bioRxiv2017

Changes in extracellular matrix cause RPE cells to make basal deposits and activate the alternative complement pathway.

The design of efficient therapies for age-related macular degeneration (AMD) is limited by our understanding of the pathogenesis of basal deposits, which form between retinal pigment epithelium (RPE) and Bruchs membrane (BrM) early in disease, and involve activation of the complement system. To investigate the roles of BrM, RPE and complement in AMD, we generated ARPE-19 cells with the p.R345W mutation in EFEMP1, which causes early-onset macular degeneration. The ARPE-19-EFEMP1R345W/R345W cells make abnormal extracellular matrix (ECM) that binds active complement C3 and causes the formation of basal deposits by normal human fetal (hf)RPE cells. hfRPE cells grown on abnormal ECM or BrM explants from AMD donors show chronic activation of the alternative complement pathway by excessive deposition of C3b. This process is exacerbated by impaired ECM turnover via increased matrix metalloproteinase-2 (MMP-2) activity. Therapies that target ECM synthesis and turnover and activation of C3 could be effective for early AMD.

cell biology↗