Biology subjects

Barone, F.

Publications and source records attributed to Barone, F..

9 recordsLinked to original sources

bioRxiv2025

RHO1-2 meganuclease gene editing targets human P23H rhodopsin-induced retinitis pigmentosa to rejuvenate rods and maintain cones

Autosomal dominant retinitis pigmentosa (adRP) is an inherited retinal dystrophy characterized by progressive vision loss and eventual blindness. The P23H mutation (proline to histidine substitution at codon 23) in the rhodopsin (RHO) gene represents the most common form of adRP in North Americans. Currently, there is no cure for P23H adRP. Genome editing targeting the mutant RHO allele, leaving a functional wildtype (WT) allele, is an attractive approach for P23H adRP, as only one copy of RHO is needed for normal retinal function. We re-engineered an I-Cre meganuclease, called RHO1-2, to target a 22bp recognition sequence encompassing the mutation responsible for the p.P23H RHO mutation. In vitro, RHO1-2, cuts human P23H RHO but not WT RHO. In vivo, we delivered scAAV5:GRK1:RHO1-2 via subretinal injection in early-stage degeneration using the only large animal model of human p.P23H RHO adRP (TgP23H pigs). We tested RHO1-2 efficacy and durability, on retinal function using full-field electroretinograms and on retinal structure using spectral domain optical coherence tomography and immunohistochemistry. We observe that RHO1-2 treatment: arrests rod photoreceptor degeneration, resurrects rod-driven retinal function that does not exist in untreated TgP23H pigs, restores mislocalized rhodopsin expression and rebuilds rod inner and outer segments (IS/OS). Rod rescue maintains cones. A year after RHO1-2 treatment, we show that TgP23H pigs use rod-driven vision to navigate a maze. Our results demonstrate that genome editing via RHO1-2 meganuclease is a viable treatment to cure human p.P23H RHO adRP. They also suggest that meganuclease-based editors can be effective for other IRDs. One Sentence SummaryEngineered meganuclease, RHO1-2 is a safe and promising therapeutic genome editing approach to cure human p.P23H RHO adRP.

neuroscience↗

bioRxiv2025

Dyslipidemic SPTLC3 Integrates Bile Acid-FXR Signaling with Sphingolipid Remodeling in MASLD

Molecular mechanisms driving metabolic disease pathogenesis remain poorly understood. Genetic and functional studies implicate SPTLC3 with dyslipidemia. SPTLC3 synthesizes atypical long-chain bases (LCB) precursors for sphingolipid production. We demonstrate significant SPTLC3 expression in human liver. ORMDL1-3 regulate SPTLC3 post-translationally in hepatic and non-hepatic cells. Independently, farnesoid X receptor (FXR) represses hepatic SPTLC3 transcription via a negative promoter element. In mice, high-fat diet (HFD) induced whereas bile acids normalized hepatic SPTLC3 transcription. SPTLC3 derived LCBs in plasma originate from the liver and are elevated in HFD-fed mice and in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Cross-species comparison revealed marked differences in LCB composition between mice and humans. Notably, omega-3-methylsphingosine (meC18SO) was significantly associated with MASLD in humans but undetectable in mice. In Huh7 cells, meC18SO enhanced complex II and IV activity, oxygen consumption, and mitochondrial ROS content. FXR-SPTLC3 axis and presented findings have potential implications for future translational research.

biochemistry↗

bioRxiv2024

Unsupervised domain classification of AlphaFold2-predicted protein structures

AO_SCPLOWBSTRACTC_SCPLOWThe release of the AlphaFold database, which contains 214 million predicted protein structures, represents a major leap forward for proteomics and its applications. However, lack of comprehensive protein annotation limits its accessibility and usability. Here, we present DPCstruct, an unsupervised clustering algorithm designed to provide domain-level classification of protein structures. Using structural predictions from AlphaFold2 and comprehensive all-against-all local alignments from Foldseek, DPCstruct identifies and groups recurrent structural motifs into domain clusters. When applied to the Foldseek Cluster database, a representative set of proteins from the AlphaFoldDB, DPCstruct successfully recovers the majority of protein folds catalogued in established databases such as SCOP and CATH. Out of the 28,246 clusters identified by DPCstruct, 24% have no structural or sequence similarity to known protein families. Supported by a modular and efficient implementation, classifying 15 million entries in less than 48 hours, DPCstruct is well suited for large-scale proteomics and metagenomics applications. It also facilitates the rapid incorporation of updates from the latest structural prediction tools, ensuring that the classification remains up-to-date. The DPCstruct pipeline and associated database are freely available in a dedicated repository, enhancing the navigation of the AlphaFoldDB through domain annotations and enabling rapid classification of other protein datasets.

bioinformatics↗

bioRxiv2023

Protein family annotation for the Unified Human Gastrointestinal Proteome by DPCfam clustering

Technological advances in massively parallel sequencing have led to an exponential growth in the number of known protein sequences. Much of this growth originates from metagenomic projects producing new sequences from environmental and clinical samples. The Unified Human Gastrointestinal Proteome (UHGP) catalogue is one of the most relevant metagenomic datasets with applications ranging from medicine to biology. However, the lack of sequence annotation impairs its usability. This work aims to produce a family classification of UHGP sequences to facilitate downstream structural and functional annotation. This is achieved through the release of the DPCfam-UHGP50 dataset containing 10,778 putative protein families generated using DPCfam clustering, an unsupervised pipeline grouping sequences into multi-domain architectures. DPCfam-UHGP50 considerably improves family coverage at protein and residue levels compared to the manually curated repository Pfam. It is our hope that DPCfam-UHGP50 will foster future discoveries in the field of metagenomics of the human gut by the release of a FAIR-compliant database easily accessible via a searchable web server and Zenodo repository.

bioinformatics↗

bioRxiv2022

Salmonella infection induces the reorganisation of follicular dendritic cell networks concomitant with the failure to generate germinal centres

Germinal centres (GCs) are sites where plasma and memory B cells form to generate high-affinity, Ig class switched antibodies. Specialised stromal cells called follicular dendritic cells (FDCs) are essential for GC formation. During systemic Salmonella Typhimurium (STm) infection GCs are absent, whereas extensive extrafollicular switched antibody responses are maintained. The mechanisms that underpin the absence of GC formation are incompletely understood. Here, we show that STm-induces a reversible disruption of niches within the splenic microenvironment, including the T and B cell compartments and the marginal zone. Alongside to these effects post-infection, mature FDC networks are strikingly absent, whereas immature FDC precursors, including marginal sinus pre-FDCs (MadCAM-1+) and perivascular Pre-FDCs (PDGFR{beta}+) are enriched. As normal FDC networks re-establish, extensive GCs become detectable throughout the spleen. Therefore, the reorganisation of FDC networks and the loss of GC responses are key, parallel features of systemic STm infections. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/519997v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1917f1forg.highwire.dtl.DTLVardef@130ce0dorg.highwire.dtl.DTLVardef@127daf8org.highwire.dtl.DTLVardef@198d373_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

A cellular and spatial map of salivary glands at single cell resolution reveals the functional basis of tertiary lymphoid structure formation in Sjogren's syndrome

The key role of tertiary lymphoid structures in autoimmune and non-autoimmune conditions has been recently appreciated. While many of the molecular mechanisms involved in tertiary lymphoid structure (TLS) formation have been identified, their cellular sources and their temporal and spatial relationship to each other during the development of TLS remain unknown. Here we have constructed a cellular and functional map of key components involved in the formation of TLS in the minor salivary glands (SG) in humans. We have confirmed the presence of an immunofibroblast cell state and identified an undescribed immunopericyte cell state with potential immunological functions within TLS. The identification of TLS cellular and functional properties and their relevant modulators provided by this analysis provides key therapeutic cues for TLS associated conditions in autoimmunity and cancer.

immunology↗

bioRxiv2021

Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases

Pro-inflammatory fibroblasts are critical to pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjogrens syndrome, and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited the understanding of which pathways are shared by multiple diseases. To investigate, we profiled patient-derived fibroblasts from inflamed and non-inflamed synovium, intestine, lung, and salivary glands with single-cell RNA-sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts were expanded in all inflamed tissues and additionally mapped to dermal analogues in a public atopic dermatitis atlas. We further confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. This work represents the first cross-tissue, single-cell fibroblast atlas revealing shared pathogenic activation states across four chronic inflammatory diseases.

genomics↗

bioRxiv2019

Salivary gland macrophages assist tissue-resident CD8+ T cell immune surveillance

Tissue macrophages and tissue resident memory CD8+ T cells (TRM) play important roles for pathogen sensing and rapid protection of barrier tissues. To date, it is incompletely understood how these two cell types cooperate for efficient organ surveillance during homeostasis. Here, we used intravital imaging to show that TRM dynamically crawled along tissue macrophages in murine submandibular salivary glands (SMG) during the memory phase following a viral infection. Ex vivo confined SMG TRM integrated an unexpectedly wide range of migration modes: in addition to chemokine-and adhesion receptor-driven motility, SMG TRM displayed a remarkable capacity of autonomous motility in the absence of chemoattractants and adhesive ligands. This unique intrinsic SMG TRM motility was transmitted by friction and adaptation to microenvironmental topography through protrusion insertion into permissive gaps. Analysis of extracellular space in SMG using super-resolution shadow imaging showed discontinuous attachment of tissue macrophages to neighboring epithelial cells, offering paths of least resistance for patrolling TRM. Upon tissue macrophage depletion, intraepithelial SMG TRM showed decreased motility and reduced epithelial crossing events, and failed to cluster in response to local inflammatory chemokine stimuli. In sum, our data uncover a continuum of SMG TRM migration modes and identify a new accessory function of tissue macrophages to facilitate TRM patrolling of the complex exocrine gland architecture.\n\nOne sentence summaryCombined in vitro and in vivo imaging of salivary gland-resident tissue memory CD8+ T cells (TRM) uncovers their unique migratory behavior and describes a novel accessory function of tissue macrophages to assist TRM surveillance.

immunology↗

bioRxiv2018

Pathologically distinct fibroblast subsets drive inflammation and tissue damage in arthritis

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune mediated inflammatory diseases (IMIDs). However it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue driven processes observed in IMIDs such as inflammation and damage. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of FAP+ synovial cells suppressed both inflammation and bone erosions in murine models of resolving and persistent arthritis. Single cell transcriptional analysis identified two distinct fibroblast subsets: FAP+ THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAP+ THY1- destructive fibroblasts restricted to the synovial lining. When adoptively transferred into the joint, FAP + THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation whereas transfer of FAP + THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell based therapies aimed at modulating inflammation and tissue damage.

immunology↗