Elton Jose Rosas Vasconcelos

Mutations in the PRPF31 gene cause retinitis pigmentosa type 11 (RP11), a progressive blinding disease triggered by defects in the spliceosome, the cellular machinery responsible for RNA processing. Although gene replacement holds therapeutic promise, it remains unclear whether it can effectively rescue both primary target cell types across different disease stages. Here we show that viral-mediated delivery of PRPF31 to patient-derived retinal pigment epithelium (RPE) cells and 3D retinal organoids restores RNA splicing activity and reverses core cellular pathology. Gene delivery cleared toxic protein aggregates, restored essential RPE functions, including structural polarity, cilia integrity, and cellular waste clearance, and boosted light-evoked activity in photoreceptors. Notably, robust rescue was achievable even in mature cells, whereas combining gene therapy with autophagy-activating drugs offered no added benefit. These findings demonstrate that restoring splicing homeostasis alone drives functional recovery, supporting a broad clinical window for PRPF31 gene therapy in RP11.

Mutations in PRPF31 gene cause retinitis pigmentosa type 11 (RP11) through haploinsufficiency, impairing spliceosome assembly and triggering progressive retinal degeneration. While gene augmentation holds therapeutic promise, key questions remain regarding the mechanistic basis of rescue and its therapeutic efficacy across all primarily affected human retinal cell types and disease stages. Here, we utilised patient-specific induced pluripotent stem cells (iPSCs)-derived retinal pigment epithelium (RPE) cells and three-dimensional retinal organoids (ROs) to determine the therapeutic mechanism of AAV-mediated PRPF31 delivery. Using the ShH10(Y445F) serotype to ensure robust dual targeting of RPE and photoreceptors, we demonstrated that PRPF31 transduction restores nuclear localisation, reorganises SC35+ nuclear speckles and enhances p-SF3B1⁺ active spliceosome foci. Transcriptomic and proteomic profiling revealed a global rescue of splicing activity and upregulation of phagocytosis, protein aggregate clearance pathways, and mitochondrial proteins. These molecular shifts facilitated the clearance of proteotoxic cytoplasmic aggregates and reversed key functional deficits; specifically, they reinforced RPE apical-basal polarity, restored phagocytic capacity and normal ciliary morphology and incidence, and boosted light-evoked activity in photoreceptors. Combining gene therapy with rapamycin-mediated-autophagy activation conferred no additive benefit, identifying the restoration of splicing homeostasis as the critical therapeutic driver. Notably, substantial phenotypic rescue is achievable in mature RPE, supporting a broad clinical window for intervention. Collectively, these data provide a systems-level validation of ShH10(Y445F)-PRPF31 gene therapy and establish a mechanistic framework for its clinical translation in RP11.

Background/Objectives: Preclinical models that accurately reflect Ewing sarcoma (ES) will enable the prioritisation of clinically active targeted agents from bench to clinic. To expedite this process, we have established and characterised patient-derived ES cultures (PDES) in vitro. Methods: Fluorescence in situ hybridisation, RT-PCR and western blotting were used to examine expression of the pathognomonic EWSR1 fusions. Activation or repression of EWSR1 fusion downstream targets and proliferation was examined by immunofluorescence and immunohistochemistry. Using next-generation sequencing, the DNA and transcriptomic profiles of PDES and cell lines were compared. The response of PDES and cell lines to standard-of-care chemotherapeutics, ionising radiation and investigational drugs was examined. Results: All PDES contain EWSR1 fusion DNA, consistent with a diagnosis of ES. EWSR1 fusion gene RNA and protein were detected in 70% and 21% of PDES, respectively. Markers of proliferation and expression of EWSR1 fusion target genes were consistent with the tumours from which PDES were derived (R² = 0.74, p < 0.0001) and the paediatric mesenchymal lineage (SBS5 and SBS1, ID1 and ID2). In contrast, the transcriptome of PDES was significantly different from that of cell lines. PDES had a significantly increased doubling time (p < 0.00001), decreased expression of Ki67 (p < 0.0001) and increased migration (p < 0.02) compared to cell lines. Consistent with the longer doubling time, PDES were more resistant to doxorubicin, etoposide and vincristine and ionising radiation (p < 0.0001) than cell lines. PDES were sensitive to mTKIs (cabozantinib, lenvatinib, and regorafenib), and trabectedin. The response of PDES to drugs in vitro reflects the clinical experience of patients. Conclusions: Models incorporating PDES cells may positively contribute to the preclinical pipeline.

We developed a hormonally responsive organoid model of the bovine endometrium. Bovine glandular epithelial cells isolated from reproductive tracts were cultured in an extracellular matrix hydrogel (Cultrex 2) at 38.5°C, 5% CO2 (n = 3). RNA was extracted and qPCR confirmed the presence of gland markers: leukemia inhibitory factor, mucin-1, insulin-like growth factor binding protein-1, kruppel-like factor-5, and forkhead box protein-A2. Organoids were imaged at specific time-points to monitor growth. Morphologically, organoids were spherical and fast-growing at passages 0 and 1, but this declined following passage 2. Passage 0 (n = 3) organoids were treated with 1000 ng/ml recombinant ovine Interferon Tau (IFNT) or 10 μg/ml progesterone (P4) for 24 h and analysed by RNASeq to assess hormone responsiveness. 373 transcripts were upregulated (padj2) in response to P4 treatment, with downstream analysis showing significant overrepresentation (FDR < 0.05) of genes associated with positive regulation of protein localisation to plasma membrane and cell periphery. Of the 240 genes significantly downregulated by P4 these were significantly overrepresented (FDR < 0.05) in biological processes of cilium and cytoskeleton organisation. IFNT treatment resulted in significant upregulation of 414 genes and downregulation of 119 genes. The largest cluster associated with differentially expressed genes in response to IFNT is defence to virus and interferon signalling. Thirty genes were altered by both P4 treatment and IFNT treatment. Organoids were also shown to express conserved microRNAs, and it was possible to culture them in a microfluidics device—making them a useful model for a multitude of potential investigations. Bovine endometrial organoids are therefore a new tool to investigate conceptus-maternal interactions.

Pseudouridylation is a prevalent RNA modification occurring in transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small non-coding RNAs (snoRNAs), and has been most recently identified in mRNAs and lncRNAs. Emerging evidence suggests that this dynamic RNA modification is implicated in altering gene expression by regulating RNA stability, modulating translation elongation, and modifying amino acid substitution rates. However, the role of pseudouridylation in infection is poorly understood. Herein, we demonstrate that Kaposi’s sarcoma–associated herpesvirus (KSHV) manipulates the pseudouridylation pathway to enhance replication. We show that the pseudouridine synthases (PUS), PUS1, and PUS7 are essential for efficient KSHV lytic replication, supported by their redistribution to viral replication and transcription complexes. We present a comprehensive analysis of KSHV RNA pseudouridylation, revealing hundreds of modified RNAs at single-nucleotide resolution. Notably, we demonstrate that pseudouridylation of the KSHV-encoded polyadenylated nuclear RNA (PAN) plays a significant role in the expression of PAN RNA. These findings reveal an essential role of pseudouridine modification in the KSHV replication cycle.

Environmental contamination by Cr(VI) leaching from chromite ore processing residue (COPR) legacy disposal sites can pose a threat to human health. Under iron‐reducing conditions, microbial activity can convert mobile and toxic Cr(VI) to less mobile and less toxic Cr(III); however, COPR waste is a very hostile environment for microbial life. Microcosms using soil from beneath a COPR disposal site were challenged with Cr(VI) with and without acetate to stimulate microbial metabolism. Geochemistry showed that when the microbial populations were reducing iron, Cr(VI) was also reduced, and 16S rRNA gene sequencing showed that the community composition evolved over the course of the experiment. Meta‐transcriptome data revealed ~3% of transcripts were differentially regulated ( p = 0.01) between the acetate amended and unamended systems, with twice as many transcripts downregulated by acetate. Gene ontology (GO) terms for processes involving the cell wall, cell periphery, plasma membrane and encapsulating structures as well as catabolic processes, especially carbohydrate metabolism, were significantly enriched in the unamended microcosm meta‐transcriptome. Transcripts for alternative sigma (σ) factors and anti‐σ factors were prominent among the differentially regulated genes. The study provides insight into how the provision of acetate shapes metabolic processes and life history strategies in an alkaline Cr(VI) impacted environment.

Lysine acetylation plays a crucial role in cellular processes and is found across various evolutionary organisms. Recent advancements in proteomic techniques revealed the presence of acetylation in thousands of non‐histone proteins. Here, we conducted extensive meta‐analysis of 48 acetylomes spanning diverse organisms, including archaea, bacteria, fungi, protozoa, worms, plants, insects, crustacea, fish, and mammals. Our analyzes revealed a predominance of a single acetylation site in a protein detected in all studied organisms, and proteins heavily acetylated, with > 5–10 acetylated‐sites, were represented by Hsp70, histone, or transcription GTP‐biding domain. Moreover, using gene enrichment approaches we found that ATP metabolic processes, glycolysis, aminoacyl‐tRNA synthetase pathways and oxidative stress response are among the most acetylated cellular processes. Finally, to better explore the regulatory function of acetylation in glycolysis and oxidative stress we used aldolase and superoxide dismutase A (SODA) enzymes as model. For aldolase, we found that K147 acetylation, responsible to regulate human enzyme, conserved in all phylogenic clade, suggesting that this acetylation might play the same role in other species; while for SODA, we identified many lysine residues in different species present in the tunnel region, which was demonstrated for human and Trypanosoma cruzi, as negative regulator, also suggesting a conserved regulatory mechanism. In conclusion, this study provides insights into the conservation and functional significance of lysine acetylation in different organisms emphasizing its roles in cellular processes, metabolic pathways, and molecular regulation, shedding light in the extensive function of non‐histone lysine acetylation.

Translational regulation at the stage of initiation can impact the number of ribosomes translating each mRNA molecule. However, the translational activity of single 80S ribosomes (monosomes) on mRNA is less well understood, even though these 80S monosomes represent the dominant ribosomal complexes in vivo. Here, we used cryo-EM to determine the translational activity of 80S monosomes across different tissues in Drosophila melanogaster. We discovered that while head and embryo 80S monosomes are highly translationally active, testis and ovary 80S monosomes are translationally inactive. RNA-Seq analysis of head monosome- and polysome-translated mRNAs, revealed that head 80S monosomes preferentially translate mRNAs with TOP motifs, short 5′-UTRs, short ORFs and are enriched for the presence of uORFs. Overall, these findings highlight that regulation of translation initiation and protein synthesis is mostly performed by monosomes in head and embryo, while polysomes are the main source of protein production in testis and ovary.

Background: Adherence to the World Cancer Research Fund (WCRF) and American Institute for Cancer Research (AICR) cancer prevention guidelines is linked to lower cancer incidence and improved outcomes. However, the relationship between these guidelines and chemotherapy response, particularly in Triple Negative Breast Cancer, is not well understood. TNBC has the poorest survival rates among breast cancer subtypes, with only 32% of patients achieving pathological complete response after neoadjuvant chemotherapy. Predicting which patients will respond and gain survival benefits remains a challenge and identifying patients unlikely to respond would help provide more effective treatment options and reduce side-effects and hospital admissions. This study assesses the feasibility of collecting data for a clinical trial aimed at identifying factors that predict chemoresponse with particular attention on diet, nutrition, physical activity, adherence to WCRF/AICR recommendations, and tumour and circulating biomarkers. Methods: This prospective, non-randomised feasibility study will recruit, over 24 months, between 15-20 triple negative breast cancer patients undergoing neoadjuvant chemotherapy. The data collected are: body mass index, chemotherapy details, surgery type, gene expression analysis in diagnostic tumour cores, serum and plasma samples for lipid and vitamin analysis, tumour response by magnetic resonance imaging during and after treatment and pathological response after treatment. Participants will complete patient-reported outcome measures, food and physical activity questionnaires, at the start and end of treatment. Discussion: This study aims to explore the impact of dietary patterns on chemotherapy responses in TNBC patients, a subtype with poor prognosis and high relapse risk. Adherence to the WCRF/AICR cancer prevention guidelines is linked to reduced cancer incidence and better outcomes. However, the role of diet in predicting chemotherapy response remains unclear. The study seeks to gather data for a future clinical trial examining these connections, aligning with research priorities to prevent cancer relapse and provide evidence-based dietary advice. This feasibility study will inform patient recruitment, data collection, and trial design. Trial registration: This trial was prospectively registered on 12 ^th December 2022 ( ISRCTN20130557 ).

Variation in the composition of different ribosomes, termed ribosome heterogeneity, is a now well established phenomenon. However, the functional implications of this heterogeneity on the regulation of protein synthesis are only now beginning to be revealed. While there are numerous examples of heterogeneous ribosomes, there are comparatively few bona fide specialized ribosomes described. Specialization requires that compositionally distinct ribosomes, through their subtly altered structure, have a functional consequence to the translational output. Even for those examples of ribosome specialization that have been characterized, the precise mechanistic details of how changes in protein and rRNA composition enable the ribosome to regulate translation are still missing. Here, we suggest looking at the evolution of specialization across the tree of life may help reveal central principles of translation regulation. We consider functional and structural studies that have provided insight into the potential mechanisms through which ribosome heterogeneity could affect translation, including through mRNA and open reading frame selectivity, elongation dynamics and post-translational folding. Further, we highlight some of the challenges that must be addressed to show specialization and review the contribution of various models. Several studies are discussed, including recent studies that show how structural insight is starting to shed light on the molecular details of specialization. Finally, we discuss the future of ribosome specialization studies, where advances in technology will likely enable the next wave of research questions. Recent work has helped provide a more comprehensive understanding of how ribosome heterogeneity affects translational control. This article is part of the discussion meeting issue ‘Ribosome diversity and its impact on protein synthesis, development and disease’.