Biology subjects

Carvalho, L. S.

Publications and source records attributed to Carvalho, L. S..

7 recordsLinked to original sources

bioRxiv2026

Characterising retinal function with optomotor visual performance in P23H rodent models of retinitis pigmentosa

Rhodopsin (RHO) P23H is one of the most common mutations causing autosomal dominant retinitis pigmentosa (adRP), yet the relationship between retinal electrophysiology, structure and visually guided behaviour in rodent models remains unclear. We characterised changes in heterozygous P23H (Sakami line) mice and P23H line 3 (P23H-3) rats using full-field electroretinography (ERG), optomotor response (OMR) assays and, in rats, optical coherence tomography (OCT). ERG assessed rod- and cone-mediated responses relative to wild-type controls, whereas OMR under scotopic and photopic conditions quantified contrast sensitivity and visual acuity. In P23H mice, scotopic ERG responses were significantly reduced from postnatal day 16 and declined further from 4 months. Scotopic OMR contrast sensitivity remained largely preserved until 2 months, and photopic acuity was comparable to wild-type up to 6 months. In 13-week-old P23H-3 rats, ERG amplitudes were significantly reduced, and OCT revealed retinal thinning. OMR showed a decline in contrast sensitivity at 7 and 15 weeks, whereas photopic acuity was maintained. Thus, in both models, electrophysiological and structural abnormalities precede detectable OMR deficits, with implications for the selection of outcome measures in preclinical studies. Summary StatementThis study compares electrical and behavioural measures of vision in rodent models of inherited blindness, revealing that retinal dysfunction appears well before measurable vision loss.

animal behavior and cognition↗

bioRxiv2025

Pre-clinical validation of a novel AAV-mediated gene therapy for KCNV2 retinopathy improves visual function in a mouse model and expression in patient organoids

Voltage-gated (Kv) potassium channels are critical for neuronal physiology, and their dysfunction can lead to serious consequences. For example, mutations in the silent modulatory Kv8.2 subunit are known to cause irreversible inherited blindness (KCNV2 retinopathy). This is a currently incurable condition that causes lifelong visual loss, reduced visual acuity, photoaversion, night blindness and abnormal colour vision, alongside a distinctive supernormal electrophysiological (ERG) retinal response to light. In this study, we demonstrate that AAV-mediated gene replacement therapy delivering a codon-optimised human KCNV2 gene subretinally into Kv8.2 knock-out mice significantly restores retinal function. Treated mice exhibited improved ERG responses and correct expression of KCNV2 and its encoded Kv8.2 protein in photoreceptors. Recovery of visually guided scotopic and photopic optomotor responses to wildtype levels was achieved at lower vector doses, highlighting dose-dependent efficacy. Furthermore, treatment of human retinal organoids derived from a KCNV2 patient iPSC line resulted in substantial Kv8.2 protein rescue. This work provides the first preclinical proof-of-concept for the safety and therapeutic potential of gene therapy for KCNV2 retinopathy, laying a strong foundation for future clinical trials.

neuroscience↗

bioRxiv2025

Engineering single-AAV CRISPR-Cas13 RNA base editors for treatment of inherited retinal diseases

Site-directed RNA editing, especially RNA base editing, allows for specific manipulation of RNA sequences, making it a useful approach for the correction of pathogenic mutations. Correction of RNA transcripts allows therapeutic gene editing in a safe and reversible manner and avoids permanent alterations in the genome. RNA-targeting CRISPR-Cas nucleases (e.g., CRISPR-Cas13) enable delivery within a single adeno-associated virus (AAV) vector for RNA base editing, making the approach clinically feasible. Here, we used the inactive CRISPR-Cas13bt3 (also known as Cas13X.1) fused to the ADAR2 deaminase domain (ADAR2DD) for targeted correction of inherited retinal disease (IRD) mutations. First, we show in vitro that dCas13bt3-ADAR2DD can efficiently correct a pathogenic nonsense mutation (c.130C>T [p.R44X]) found in the mouse Rpe65 gene and recover protein expression in retinal pigment epithelium cells (RPEs). Across clinically reported RPE65 mutations, we observed editing efficiencies ranging from 0% to 60%. In the Rpe65-deficient mouse model of retinal degeneration (rd12), we observed that RNA base editing can recover Rpe65 expression in RPEs and rescue retinal function with no observable adverse effects. We further employed our RNA base editor against the large USH2A gene to assess the promise of RNA base editing for addressing untreatable IRDs caused by genes too large for AAV gene delivery. Against the human USH2A in vitro, we observed up to 60% on-target efficiency. We further found that gRNA mismatches, domain-inlaid ADAR2DD design and nucleocytoplasmic shuttling of the RNA base editor optimised on-target and bystander editing for a highly precise base editor. Against the mouse Ush2a in vitro, we similarly observed up to 60% on-target editing in mammalian cells, while in the Ush2aW3947X mice, we observed [~]12% on-target editing, with no impact on retinal structure or function, or transcriptome-wide editing. Overall, our findings demonstrate dCas13bt3-ADAR2DD as a potent tool for gene therapy against IRDs, addressing a significant unmet clinical need in ophthalmology.

bioengineering↗

bioRxiv2023

Complete phylogeny of Micrathena spiders suggests multiple dispersal events among Neotropical rainforests, islands, and landmasses, and indicates Andean orogeny promotes speciation

The Neotropical region is the most diverse on the planet, largely due to its mosaic of tropical rainforests. Multiple tectonic and climatic processes have been hypothesized to play a role in generating this diversity; these include Andean orogeny, closure of the Panama Isthmus, the hypothesized GAARlandia land bridge, and putative historical connections among currently isolated forests. Micrathena spiny spiders (Araneidae) include [~]120 species distributed mostly in forests from Mexico to Argentina, including the Antilles. Here, we use it as a model to study the biogeographic history of Neotropical rainforests by estimating a complete, dated phylogeny using morphological data for all species and molecular data for a subsample of 79 species. This resulted in a phylogeny that is mostly robust and supports most of the previously recognized species groups, although with uncertainty in the phylogenetic position of some species, especially those lacking sequences. The genus began diversifying about 25 million years ago. We use an event-based approach and biogeographic stochastic mapping to estimate ancestral distributions and the timing and direction of dispersal events, and to identify areas where diversity was generated, while accounting for phylogenetic uncertainty. Andean cloud forests generated the majority of species through in-situ speciation, but the Amazon was the major source of species for adjacent areas through dispersal; on the other extreme, the Dry Diagonal received species from other areas but generated very little diversity. Species exchange between Central and South America was intense, with [~]23 dispersal events beginning at least 20 million years ago, indicating that Micrathena dispersed between these continents before closure of the Isthmus of Panama. We also inferred [~]4 over-water dispersal events from Central and North America to the Antilles, which happened in the last 20 million years, and thus much after the proposed age of the GAARlandia land bridge. We identified an important species exchange route between the Amazon and the Atlantic forests. Sampling all species of the genus was fundamental to some of the conclusions above, especially in identifying the Andes as the area that generated the majority of species. This study highlights the importance of a solid and complete taxonomic sampling in biogeographic studies.

evolutionary biology↗

bioRxiv2022

The rediscovery of a relict unlocks the first global phylogeny of whip spiders (Amblypygi)

Asymmetrical rates of cladogenesis and extinction abound in the Tree of Life, resulting in numerous minute clades that are dwarfed by larger sister groups. Such taxa are commonly regarded as phylogenetic relicts or "living fossils" when they exhibit an ancient first appearance in the fossil record and prolonged external morphological stasis, particularly in comparison to their more diversified sister groups. Due to their special status, various phylogenetic relicts tend to be well-studied and prioritized for conservation. A notable exception to this trend is found within Amblypygi ("whip spiders"), a visually striking order of functionally hexapodous arachnids that are notable for their antenniform first walking leg pair (the eponymous "whips"). Paleoamblypygi, the putative sister group to the remaining Amblypygi, is known from Late Carboniferous and Eocene deposits, but is survived by a single living species, Paracharon caecus Hansen, 1921, that was last collected in 1899. Due to the absence of genomic sequence-grade tissue for this vital taxon, there is no global molecular phylogeny for Amblypygi to date, nor a fossil-calibrated estimation of divergences within the group. Here, we report several individuals of a previously unknown species of Paleoamblypygi from a cave site in Colombia. Capitalizing upon this discovery, we generated the first molecular phylogeny of Amblypygi, integrating ultraconserved element sequencing with legacy Sanger datasets and including described extant genera. To quantify the impact of sampling Paleoamblypygi on divergence time estimation, we performed in silico experiments with pruning of Paracharon. We demonstrate that the omission of relicts has a significant impact on the accuracy of node dating approaches that outweighs the impact of excluding ingroup fossils. Our results underscore the imperative for biodiversity discovery efforts in elucidating the phylogenetic relationships of "dark taxa", and especially phylogenetic relicts in tropical and subtropical habitats.

genomics↗

bioRxiv2022

Increased H3K27 trimethylation contributes to cone survival in a mouse model of cone dystrophy

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD, to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in the development of a broad class of novel therapies to slow cone degeneration.

neuroscience↗

bioRxiv2018

Education can Reduce Health Disparities Related to Genetic Risk of Obesity: Evidence from a British Reform

This paper investigates whether genetic makeup moderates the effects of education on health. Low statistical power and endogenous measures of environment have been obstacles to the credible estimation of such gene-by-environment interactions. We overcome these obstacles by combining a natural experiment that generated variation in secondary education with polygenic scores for a quarter million individuals. The additional schooling affected body size, lung function, and blood pressure in middle age. The improvements in body size and lung function were larger for individuals with high genetic predisposition to obesity. As a result, education reduced the gap in unhealthy body size between those with high and low genetic risk of obesity from 20 to 6 percentage points.

epidemiology↗