Biology subjects

Romano, J.

Publications and source records attributed to Romano, J..

11 recordsLinked to original sources

bioRxiv2026

De novo design of RNA pseudoknots with deep learning

RNA design has been hindered by the limited accuracy of 3D structure prediction. Here, we show that intricate RNA structures can be generated with current deep learning tools through accurate de novo design of pseudoknot secondary structures. In an Eterna competition involving 57 pseudoknots, generative AI methods matched experienced human designers in solving most blind challenges, evaluated by single-nucleotide-resolution chemical mapping, compensatory mutagenesis, and cryogenic electron microscopy. AI-generated molecules with accurate secondary structures formed well-ordered 3D folds stabilized by noncanonical tertiary interactions not modeled during design. Success was guided by an RNet foundation model trained on prior chemical mapping data, suggesting that some difficult RNA design tasks may be tractable without first solving RNA 3D structure prediction.

biophysics↗

bioRxiv2026

Site-specific O-glycans influence lacritin structure and multimerization in tears

Lacritin is an abundantly expressed glycoprotein in tear fluid and plays key roles in immune response, tear secretion, and bacterial killing. These biological functions are tightly regulated through several biochemical mechanisms including multimerization, proteolysis, and alternative splicing, especially within its C-terminal domain. Given its critical role at the ocular surface, lacritin is currently under investigation as a diagnostic biomarker and therapeutic candidate for dry eye disease (DED). However, despite over three decades since its initial discovery, the functional significance of the O-glycans that comprise more than 50% of its molecular weight remain largely unknown. To address this gap, we leveraged mass spectrometry (MS)-based glycoproteomics and molecular dynamics (MD) to explore the structural role of site-specific O-glycans on C-terminal lacritin. In doing do, we identified distinct glycosylation profiles between monomeric and multimeric lacritin, particularly at glycosites located near crosslinking residues (Lys101 and Lys104) that modulate multimer formation. Building on our glycoproteomics data, we performed MD simulations on monomer and multimer glycoforms and revealed that O-glycans participate in intra-glycan-protein interactions, thereby affecting the conformational flexibility of lacritin and the spatial arrangement of Lys101 and Lys104. Finally, we quantified the solvent-accessible surface area (SASA) of Lys101 and Lys104, highlighting that proximal O-glycosylation is predicted to affect the propensity of these residues to participate in crosslinking. Taken together, these findings underscore a central role for lacritin O-glycans in affecting structural topology with implications for its downstream biological activity.

biophysics↗

bioRxiv2026

Adipose-Derived Extracellular Vesicles Mitigate Experimental Cutaneous Leishmaniasis Through an IL-10 Dependent Mechanism

Leishmaniasis, a neglected tropical disease caused by protozoa of the Leishmania genus, is characterized by an imbalanced immune response that promotes parasite survival while inducing tissue damage. We previously showed that adipose-derived mesenchymal stem cells (AD-MSCs) limited lesion progression in C57BL/6 mice infected with Leishmania amazonensis. In the current study, we examined whether extracellular vesicles released by AD-MSCs (AD-MSC-EVs) reproduce these therapeutic effects. AD-MSC-EVs were purified, expanded, and characterized, confirming their typical morphology and EV-associated surface markers. In vitro, treatment of infected macrophages with AD-MSC-EVs 4 hours post-infection reduced parasite load through the induction of reactive oxygen species (ROS), independent of nitric oxide (NO). However, treatment 24 hours post-infection failed to reproduce this effect. In vivo, intralesional administration of AD-MSC-EVs significantly reduced lesion size without altering parasite load, while also decreasing levels of specific IgM and IgG for Leishmania amazonensis antigens. AD-MSC-EV therapy also reduced proinflammatory cytokines production by CD4 and cytotoxicity CD8 T cells, while increasing IL-10 production by {gamma}{delta} T cells. Moreover, combined therapy with pentavalent antimonial further reduced lesion size but did not affect parasite load. Notably, AD-MSC-EVs failed to control lesions in IL-10-deficient mice, suggesting that their therapeutic effect is dependent on IL-10. In conclusion, these findings showed that AD-MSC-EVs modulated the host immune response to attenuate L. amazonensis-induced cutaneous pathology and may represent a promising adjunct or alternative therapy for cutaneous leishmaniasis. Significance StatementCutaneous leishmaniasis is a neglected tropical disease with limited therapeutic options and significant immunopathology. This study demonstrates that extracellular vesicles derived from adipose-derived mesenchymal stem cells (AD-MSC-EVs) can modulate host immune responses and reduce lesion severity in Leishmania amazonensis infection. Rather than directly eliminating parasites, AD-MSC-EVs attenuate tissue damage through IL-10 dependent immunoregulation, highlighting a novel host-directed therapeutic strategy. These findings advance understanding of EV-mediated immune modulation and support AD-MSC-EVs as a promising adjunct or alternative approach for treating cutaneous leishmaniasis and potentially other inflammatory infectious diseases.

immunology↗

bioRxiv2025

Mapping galectin-3 ligands in tear fluid establishes spliceoform-dependent lacritin binding

Galectin-3 (Gal-3) is a carbohydrate-binding protein which plays crucial roles in inflammation, immune response, cell migration, autophagy, and signaling. At the ocular surface, Gal-3 is also known to crosslink transmembrane mucins across the epithelial cell glycocalyx, forming lattice structures important for barrier function. However, the biological role of Gal-3 in circulating tear fluid remains largely unexplored. Similarly, whether Gal-3 engages extracellular glycoproteins in tears to affect downstream biological processes has yet to be investigated. As increased Gal-3 levels in tears are known to correlate with ocular pathologies such as dry eye disease (DED), we sought to elucidate the Gal-3 interactome in tear fluid and uncover biological insights into the function of Gal-3 beyond adhesion to the epithelial cell surface. Here, we combined ELISA, lectin affinity enrichment, mass spectrometry (MS)-based glycoproteomics, and lectin blotting to uncover Gal-3 interactors and their associated glycoepitopes. Overall, we report nearly 100 proteins enriched from tear fluid across 3 different patients, identifying proteins involved in immune response, inflammation, and antimicrobial activity. Most notably, we report lacritin as a novel ligand for Gal-3 and demonstrate that specific glycoforms of lacritin bearing core 2 O-glycans preferentially engage with Gal-3. Lastly, we show that the Gal-3-lacritin axis is spliceoform-specific and dependent on lacritin multimerization. Taken together, this study elucidates new ligands for Gal-3 in tear film and reveals mRNA splicing and multimerization as new biochemical mechanisms that fine-tune Gal-3 binding events.

biochemistry↗

bioRxiv2024

OpenASO: RNA Rescue--designing splice-modulating antisense oligonucleotides through community science

Splice-modulating antisense oligonucleotides (ASOs) are precision RNA-based drugs that are becoming an established modality to treat human disease. Previously, we reported the discovery of ASOs that target a novel, putative intronic RNA structure to rescue splicing of multiple pathogenic variants of F8 exon 16 that cause hemophilia A. However, the conventional approach to discovering splice-modulating ASOs is both laborious and expensive. Here, we describe an alternative paradigm that integrates data-driven RNA structure prediction and community science to discover splice-modulating ASOs. Using a splicing-deficient pathogenic variant of F8 exon 16 as a model, we show that 25% of the top-scoring molecules designed in the Eterna OpenASO challenge have a statistically significant impact on enhancing exon 16 splicing. Additionally, we show that a distinct combination of ASOs designed by Eterna players can additively enhance the inclusion of the splicing-deficient exon 16 variant. Together, our data suggests that crowdsourcing designs from a community of citizen scientists may accelerate the discovery of splice-modulating ASOs with potential to treat human disease.

biochemistry↗

bioRxiv2024

Ribonanza: deep learning of RNA structure through dual crowdsourcing

Prediction of RNA structure from sequence remains an unsolved problem, and progress has been slowed by a paucity of experimental data. Here, we present Ribonanza, a dataset of chemical mapping measurements on two million diverse RNA sequences collected through Eterna and other crowdsourced initiatives. Ribonanza measurements enabled solicitation, training, and prospective evaluation of diverse deep neural networks through a Kaggle challenge, followed by distillation into a single, self-contained model called RibonanzaNet. When fine tuned on auxiliary datasets, RibonanzaNet achieves state-of-the-art performance in modeling experimental sequence dropout, RNA hydrolytic degradation, and RNA secondary structure, with implications for modeling RNA tertiary structure.

biophysics↗

bioRxiv2021

Community science designed ribosomes with beneficial phenotypes

Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

synthetic biology↗

bioRxiv2021

A tyrosine-based trafficking signal in the simian immunodeficiency virus envelope cytoplasmic domain is strongly selected for in pathogenic SIV infection

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-dependent trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting of Env. Despite extensive characterization, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which the Tyr-based signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239{Delta}GY) replicates to high levels acutely in pigtail macaques (PTM) but is rapidly controlled. We previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239{Delta}GY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the {Delta}GY deletion and a nine nucleotide deletion that encodes amino acids 734-736 ({Delta}QTH) and overlaps with the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected, which generated a new signal for polarized sorting but not endocytosis. This mutation by itself was sufficient to maintain high viral loads for several months when introduced into naive PTMs. These findings reveal, for the first time, strong selection pressure for Env endocytosis and, in particular, for polarized sorting during pathogenic SIV infection in vivo.

cell biology↗

bioRxiv2021

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that "superfolder" mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

molecular biology↗

bioRxiv2020

Chemoprophylaxis vaccination with a Plasmodium liver stage autophagy mutant affords enhanced and long-lasting protection

Genetically-attenuated sporozoite vaccines can elicit long-lasting protection against malaria but pose risks of breakthrough infection. Chemoprophylaxis vaccination (CVac) has proven to be the most effective vaccine strategy against malaria. Though CVac with WT sporozoites confers better immunity, the overhanging threat of drug resistance limits its use as a vaccine. Here, we demonstrate that a liver stage-specific mutant of Plasmodium berghei when used as a vaccine under a CVac regimen provides superior long-lasting protection, in both inbred and outbred mice, as compared to WT-CVac. Uniquely, the protection elicited by this mutant is predominantly dependent on a CD8+T-cell response through an IFN-{gamma}-independent mechanism and is associated with a stable population of antigen-experienced CD8+T cells. Jointly, our findings support the benefit of liver stage mutants as vaccines over WT, under a CVac protocol. This vaccination strategy is also a powerful model to study the mechanisms of protective immunity and discover new protective antigens.

immunology↗

bioRxiv2018

VenomKB v2.0: A knowledge repository for computational toxinology

MotivationVenom peptides comprise one of the richest sources of bioactive compounds available for drug discovery. However, venom data and knowledge are fragmentary and poorly structured, and fail to capitalize on the important characteristics of venoms that make them so interesting to the biomedical community.\n\nResultsWe present VenomKB v2.0, a new open-access resource for knowledge representation and retrieval of venom bioactivities, sequences, structures, and classifications. VenomKB provides a complete infrastructure for computational toxinology, with a focus on drug discovery and effects that venoms have on the human body. VenomKB is accompanied by a suite of tools for programmatic access, and, in this article, we highlight scenarios demonstrating its usefulness and novel contributions to toxinology, pharmacology, and informatics.\n\nAvailabilityVenomKB can be accessed online at http://venomkb.org/, and the code can be found at https://github.com/tatonetti-lab/venomkb/. All code and data are available under open-source and open-access licenses.

bioinformatics↗