Life Sciences
All scientific publications in Area Science Park
Scalable, fast and accurate differential gene expression testing from millions of cells of multiple patients
Abstract Since the development of DNA microarrays and later RNA bulk sequencing, testing with statistically independent samples has been the standard method for detecting genes with different transcription patterns. Single-cell assays challenge these assumptions because individual cells are statistically dependent, and all proposed methodologies present mathematical limitations or computational bottlenecks that prevent a seamless integration of data from many cells and patients simultaneously. In this work, we solve this crucial limitation by introducing a Bayesian framework that retrieves the independence structure at the level of individual patients, separating differences across individuals from actual transcriptional differences. Leveraging multi-GPU and variational inference, our approach excels across different experimental designs and scales to analyse over 10 million cells. This framework enables single-cell differential expression analysis that can finally integrate datasets from large clinical cohorts, atlas projects, or drug-response screens with thousands of samples and millions of cells. Authors Giovanni Santacaterina, Niccolò Tosato, Salvatore Milite, Katsiaryna Davydzenka, Edoardo Insaghi, Guido Sanguinetti, Stefano Cozzini, Leonardo Egidi, Giulio Caravagna Journal Nature Communications 2026 Publication Date 19/06/2026 Consult the publication
Decoding the Grammar of Protein-Protein Interaction Interfaces with Multimodal Representations
Abstract Protein-protein interactions govern essential cellular processes, making the identification of interacting sites a central challenge in structural biology, with important implications for protein engineering and the development of targeted therapeutics. Existing prediction algorithms include sequence-based methods, which lack structural information, or structure-based approaches, which often struggle to effectively integrate evolutionary context. Here, we present ESM3-PPISites, a supervised model for residue-level classification of interfaces, leveraging the multimodal representations of the ESM3 Protein Language Model. To ensure a bias-free evaluation, a stringent redundancy filtering protocol is adopted, systematically eliminating latent homology between the training data and a curated benchmark set in both sequence and structural space. ESM3-PPISites achieves unprecedented accuracy, vastly outperforming current approaches. Our findings demonstrate that while ESM3 largest proprietary version yields the highest predictive power, targeted fine-tuning of its small open-weight counterpart significantly narrows the performance gap. We also show the practical impact of these predictions by integrating them as spatial restraints within the HADDOCK docking platform. When evaluated on an independent subset of 12 complexes from the Docking Benchmark v5, the prediction-guided pipeline strongly enhances the identification of near-native binding poses over blind docking, while reducing computational runtime by an order of magnitude. This framework establishes a scalable paradigm for high-throughput structural characterization of protein–protein interactions. Authors Yuri Gardinazzi, Edith Natalia Villegas Garcia, Sergio Senci, Davide Di Vora, Antonio Feltrin, Francesca Cuturello Journal Preprint Publication Date 09/06/2026 Consult the publication
Chirality Unlocks Sub-Terahertz Molecular Dynamics in Peptide Nanotubes
Abstract Low-frequency modes in the picosecond range play a key role in molecular recognition and biomolecular function. Here, we investigate how these dynamics at the interfaces of self-assembled peptide biomaterials relate to fine structural details of the supramolecular assembly. We resolve terahertz and mid-infrared modes in individual diphenylalanine nanotubes by nanoscale microspectroscopy, uncovering a direct correlation between supramolecular chirality, molecular arrangement, and picosecond dynamics at the nanotube-environment boundary. By combining THz and mid-IR nanoscopy with a multiscale strategy spanning single nanotubes and ensemble measurements, supported by density functional theory calculations, we access the intrinsic picosecond response of peptide assemblies beyond the limits of conventional microscopy and ensemble averaging. Heterochiral (D-L) nanotubes display sharp resonances, whereas homochiral (L-L) nanotubes exhibit a comparatively featureless sub-THz profile, revealing a clear chirality-dependent contrast. Mode assignments show that the heterochiral features are dominated by localized torsional and bending motions of phenyl rings relative to the peptide backbone. Overall, chirality-dependent THz fingerprints emerge as sensitive descriptors of peptide nanotube architecture, while low-frequency nanospectroscopy provides a route to interrogate biomaterial interfaces under biologically relevant conditions. Authors Rajat Kumar, Erica Scarel, Andrea Perucchi, Lisa Vaccari, Prasanta Kumar Datta, Francesco D’amico, Paola Di Pietro, Silvia Marchesan, Federica Piccirilli Journal ChemrXiv Publication date 11/03/2026 Consult the publication
Ultrafast Intermolecular Dynamics of Nanoconfined Water in Swollen Lipid Cubic Mesophases
Abstract Understanding the structure and dynamics of the hydrogen-bond network ofwater in topologically distinct swollen lipidic mesophases, is fundamental fortheir application in biomedical, pharmaceutical, and food science fields. Here,a positive and non-linear correlation between water hydrogen-bond dynamicsand interfacial water population is uncovered in inverse bicontinuous swollenmesophases across an extended temperature range (298–340 K). Particularly,small-angle X-ray scattering determines the mesophase’s structural features,uncovering a temperature-driven re-entrant phenomenon (reappearance) ofPn̄ 3m phase upon heating. This topologically rich environment, however, hasno detectable impact on the temperature dependence of the intermolecularmodes of water, as revealed by terahertz absorption spectroscopy. Specifically,these modes show distinct dynamics: the stretching mode exhibits longerlifetimes than the libration mode, yet with a higher temperature-dependence,with approximately two-fold lower Arrhenius activation energies. In contrast,both stretching and libration modes exhibit a monotonic decrease in lifetimewith increasing temperature, due to the increasing disruption of thehydrogen-bond network. Atomistic molecular dynamics simulations enablethe quantification of interfacial water population, which shows a positivecorrelation with intermolecular lifetimes in a nonlinear manner, revealing anon-additive coupling between interfacial water population and waterhydrogen-bond network dynamics within these systems. Authors Eva Zunzunegui-Bru, Serena Rosa Alfarano, Patrick Züblin, Laura Baraldi,Hendrik Vondracek, Federica Piccirilli, Lisa Vaccari, and Raffaele Mezzenga Journal Small Publication date 03/10/2025 Consult the publication
Evolutionary Constraints Guide AlphaFold2 in Predicting Alternative Conformations and Inform Rational Mutation Design
Abstract Investigating structural variability is essential for understanding protein biological functions. Although AlphaFold2 accurately predicts static structures, it fails to capture the full spectrum of functional states. Recent methods have used AlphaFold2 to generate diverse structural ensembles, but they offer limited interpretability and overlook the evolutionary signals underlying the predictions. In this work, we enhance the generation of conformational ensembles and identify sequence patterns that influence the alternative fold predictions for several protein families. Building on prior research that clustered multiple sequence alignments to predict fold-switching states, we introduce a refined clustering strategy that integrates protein language model representations with hierarchical clustering, overcoming limitations of density-based methods. Our strategy effectively identifies high-confidence alternative conformations and generates abundant sequence ensembles, providing a robust framework for applying direct coupling analysis (DCA). Through DCA, we uncover key coevolutionary signals within the clustered alignments, leveraging them to design mutations that stabilize specific conformations, which we validate using alchemical free energy calculations from molecular dynamics. Notably, our method extends beyond fold-switching, effectively capturing a variety of conformational changes. Authors Valerio Piomponi, Alberto Cazzaniga, Francesca Cuturello Journal Journal of Chemical Information and Modelling Publication Date 03/09/2025 Consult the publication
Rational optimization of D3R/GSK-3β dual target-directed ligands as potential treatment for bipolar disorder: Design, synthesis, X-ray crystallography, molecular dynamics simulations, in vitro ADME, and in vivo pharmacokinetic studies
Abstract Bipolar disorder is a complex neuropsychiatric condition with a significant unmet medical need, as current treatments lack disease-modifying properties and multimodal therapeutic effects. To overcome the limitations of single-target drugs, we designed dual-target ligands that combine partial agonism at the dopamine D3 receptor (D3R) with inhibition of glycogen synthase kinase-3β (GSK-3β). We previously identified ARN24161 (1) as a promising prototype, demonstrating partial agonism at D3R (EC50 = 10.1 nM, % Eff. = 26.3) and GSK-3β inhibition (IC50 = 561 nM). However, its drug-like properties remained suboptimal. To optimize this compound, we initiated a multidisciplinary refinement campaign, leveraging computational modeling and crystallographic data to fine-tune the balance between D3R and GSK-3β activity, reduce P-glycoprotein (P-gp) affinity, and improve the pharmacokinetic profile. This effort led to the identification of ARN25297 (5), a moderately balanced dual-target ligand that exhibits partial agonism at D3R (EC50 = 13.1 nM, % Eff. = 17.1) and potent GSK-3β inhibition (IC50 = 47.0 nM). Notably, ARN25657 (16) emerged as the most well-balanced candidate, demonstrating enhanced D3R partial agonism (EC50 = 15.2 nM, % Eff. = 37.7) alongside strong GSK-3β inhibition (IC50 = 19.3 nM). Compound 16 also exhibited the lowest P-gp inhibition and significant improvements in in vitro ADME properties compared to prototype 1, while maintaining a balanced dual target profile. Although the PK profile of 16 remained comparable to that of prototype 1, these findings lay the groundwork for further lead optimization and structural refinement, driving future in vivo proof-of-concept toward innovative therapeutic strategies for bipolar disorder and related neuropsychiatric conditions. Authors RMC Di Martino, D Russo, I Penna, Andrea Dalle Vedove, R Spabnuolo, G Ottonello, M Summa, J Desantis, A Valeri, L Pruccoli, SK Tripathi, A Tarozzi, Paola Storici, S Girotto, R Bertorelli, A Armirotti, G Cruciani, T Bandiera, A Cavalli, G Bottegoni Journal European Journal of Medicinal Chemistry Publication date 25/06/2025 Consult the publication
FL30: an epidermal growth factor kinase inhibitor overcoming T790M and C797S mutations through unique conformational modulation mechanism
Abstract Tyrosine kinase inhibitors (TKIs) targeting the oncogene Epidermal Growth Factor Receptor (EGFR) are widely used in the treatment of non-small cell lung cancer (NSCLC). In this context, the introduction of fourth-generation TKIs has significantly advanced targeted therapy for T790M and C797S EGFR mutations. Current therapeutic strategies are increasingly focusing on the design of orthoallosteric TKIs, which have shown promise in stabilizing the inactive conformation of mutated EGFR. In this context, we report the discovery of FL30, a small molecule with a flavone core that exhibits nanomolar potency against the EGFR-L858R/T790M mutation, even in the presence of the C797S mutation. The IC50 comparable to the Osimertinib – one of the most renowned EGFR-TKIs – emphasizes the remarkable success of the design approach. In NSCLC models, FL30 effectively inhibits cancer growth and EGFR phosphorylation selectively in cells with the EGFR mutations. Kinetic studies, molecular modeling, and Plasmon Internal Reflection Surface-Enhanced Infrared Absorption (PIR-SEIRA) microscopy suggests that FL30 binds to the orthosteric site while inducing the transition of the mutant EGFR toward an inactive-like state. These findings highlight FL30’s potential for further optimization and propose a novel approach for developing targeted therapies that combine orthosteric binding with allosteric modulation. Authors Elena Romagnoli, Emiliano Laudadio, Giovanna Mobbili, Leonardo Sorci, Giovanni Birarda, Federica Piccirilli, Lisa Vaccari, Hendrik Vondracek, Brenad Romaldi, Massimo Marcaccio, Paola Storici, Marta Semrau, Roberta Galeazzi, Andrea Toma, Vincenzo Aglieri, Pierluigi Stipa, Tatiana Armeni, Cristina Minnelli Journal International Journal of Biological Macromolecules Publication date 21/06/2025 Consult the publication
Heterologous prime–boost Zika virus vaccination induces comprehensive humoral and cellular immunity in mouse models
Abstract Zika virus (ZIKV) remained poorly studied until an outbreak in 2015 linked the virus to severe neurological disorders and congenital malformations. Currently, there are no antiviral drugs or vaccines available. We have previously demonstrated that a simian adenovirus vector vaccine (ChAdOx1 prMEΔTM) and a virus-like particle-based vaccine bearing E proteins locked in covalent dimers (VLP-cvD) are effective against ZIKV infection in animal challenge models. In this study, we further explored the efficacy of these vaccines, either individually or in combination, using a heterologous prime and boost vaccination strategy in mouse challenge models. Although the individual vaccines provided good protection levels, the heterologous prime–boost vaccination regimen (ChAdOx1 prMEΔTM followed by VLP-cvD) offered the most effective protection. This regimen elicited a strong cellular response and high levels of neutralising antibodies, which were attributed to ChAdOx1 prMEΔTM and VLP-cvD, respectively. Our findings support the use of combined vaccine technologies and offer valuable insights into the multifactorial protection achievable through heterologous vaccination. These results have important implications for the development of effective vaccination strategies against ZIKV and other emerging viruses. Authors Giuditta De Lorenzo, Rapeepat Tandavanitj, Lorena Preciado-Llanes, Ricardo Sanchez-Velazquez, Raissa Prado Rocha, Young Chan Kim, Arturo Reyes-Sandoval, Arvind H Patel. Journal Frontiers in Immunology Publication Date 25/04/2025 Consult the publication
A supported lipid bilayer to model solid-ordered membrane domains
Abstract Membrane models are widely used to mimic the behaviour of native plasma membranes and to simulate interactions occurring at their interface. Such models can be built up with different molecular compositions, ranging from single phospholipids to more complex, heterogeneous mixtures of phospho- and sphingo-lipids, possibly enriched with cholesterol and proteins. In particular, mixing different lipids and cholesterol is instrumental to promote the formation of phase-separated, ordered domains, which resemble the structure of lipid rafts, specialized functional domains of real membranes. According to the specific lipid composition, physical characteristics of the rafts can be tuned, such as fluidity, strongly related to membrane biological activity. Here, we introduce a novel three-component membrane model constituted by the mixing of a saturated phospholipid, 1,2-dimyristoyl-sn–glycero-3-phosphocholine (DMPC), sphingomyelin and cholesterol to mimic the presence of solid ordered rafts and to study their behaviour. Differential scanning calorimetry, neutron reflectometry, and atomic force microscopy were synergistically applied to gain information on the membrane’s transverse and lateral organization, as well as on its thermotropic behaviour. The membrane model benefits from the use of DMPC, a lipid (i) characterized by an accessible transition temperature; (ii) saturated; (iii) fluid at physiological temperature and (iv) commercially available in both protiated and deuterated forms. The proposed model, along with the wide range of biophysical techniques employed, constitutes an ideal system to study the molecular mechanisms and the physical properties that govern membrane functions, such as molecular signalling and membrane trafficking. Authors Sally Helmy, Paola Brocca, Alexandros Koutsioubas, Stephen C.L. Hall, Luca Puricelli, Pietro Parisse, Loredana Casalis, Valeria Rondelli Journal Journal of Colloid and Interface Science Publication Date 14/03/2025 Consult the publication
Interpreting and Steering Protein Language Models through Sparse Autoencoders
Abstract The rapid advancements in transformer-based language models have revolutionized natural language processing, yet understanding the internal mechanisms of these models remains a significant challenge. This paper explores the application of sparse autoencoders (SAE) to interpret the internal representations of protein language models, specifically focusing on the ESM-2 8M parameter model. By performing a statistical analysis on each latent component’s relevance to distinct protein annotations, we identify potential interpretations linked to various protein characteristics, including transmembrane regions, binding sites, and specialized motifs. We then leverage these insights to guide sequence generation, shortlisting the relevant latent components that can steer the model towards desired targets such as zinc finger domains. This work contributes to the emerging field of mechanistic interpretability in biological sequence models, offering new perspectives on model steering for sequence design. Authors Edith Natialia Villegas Garcia, Alessio Ansuini Journal Workshop on Generative and Experimental Perspectives for Biomolecular Design @ ICLR 2025 Publication Date 13/02/2025 Consult the publication
Emergence of SARS-CoV-2 subgenomic RNAs that enhance viral fitness and immune evasion
Abstract Coronaviruses express their structural and accessory genes via a set of subgenomic RNAs, whose synthesis is directed by transcription regulatory sequences (TRSs) in the 5′ genomic leader and upstream of each body open reading frame. In SARS-CoV-2, the TRS has the consensus AAACGAAC; upon searching for emergence of this motif in the global SARS-CoV-2 sequences, we find that it evolves frequently, especially in the 3′ end of the genome. We show well-supported examples upstream of the Spike gene—within the nsp16 coding region of ORF1b—which is expressed during human infection, and upstream of the canonical Envelope gene TRS, both of which have evolved convergently in multiple lineages. The most frequent neo-TRS is within the coding region of the Nucleocapsid gene, and is present in virtually all viruses from the B.1.1 lineage, including the variants of concern Alpha, Gamma, Omicron and descendants thereof. Here, we demonstrate that this TRS leads to the expression of a novel subgenomic mRNA encoding a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production and contributes to viral fitness during infection. We observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level. Authors Harriet V Mears, George R Young, Theo Sanderson, Ruth Harvey, Jamie Barrett-Rodger, Rebecca Penn, Vanessa Cowton, Wilhelm Furnon, Giuditta De Lorenzo, Margaret Crawford, Daniel M Snell, Ashley S Fowler, Anob M Chakrabarti, Saira Hussain, Ciarán Gilbride, Edward Emmott, Katja Finsterbusch, Jakub Luptak, Thomas P Peacock, Jérôme Nicod, Arvind H Patel, Massimo Palmarini, Emma Wall, Bryan Williams, Sonia Gandhi, Charles Swanton, David LV Bauer Journal PLoS biology Publication Date 21/01/2025 Consult the publication
Ultrasensitive detection and quantification of bovine Deltapapillomavirus in the semen of healthy horses.
Abstract BPV1, BPV2, BPV13, and BPV14 are all genotypes of bovine delta papillomaviruses (δPV), of which the first three cause infections in horses and are associated with equine sarcoids. However, BPV14 infection has never been reported in equine species. In this study, we examined 58 fresh and thawed commercial semen samples from healthy stallions. In 34 (58.6%), bovine δPV DNA was detected and quantified using droplet digital polymerase chain reaction (ddPCR). Real time quantitative PCR (qPCR) was able to identify bovine δPV DNA in 5 samples (8.6%). Of the BPV-infected semen samples, 15 were positive for BPV2 (~ 44.1%) on ddPCR and 4 (~ 11.7%) on qPCR; 12 (~ 35.3%) for BPV14 on ddPCR and 1 (~ 3%) by qPCR; 4 (~ 11.7%) for BPV1 on ddPCR, whereas qPCR failed to reveal this infection; 3 (~ 8.8%) for BPV13 on ddPCR; and BPV13 infection was not detected by qPCR. Our study showed for the first time that BPV14 is an additional infectious agent potentially responsible for infection in horses, as its transcripts were detected and quantified in some semen samples. Large-scale BPV14 screening is necessary to provide substantial data on the molecular epidemiology for a better understanding of the geographical divergence of BPV14 prevalence in different areas and how widespread BPV14 is among equids. Authors Anna Cutarelli, Francesca De Falco, Francesco Serpe, Simona Izzo, Giovanna Fusco, Cornel Catoi, Sante Roperto Journal Scientific Report Publication date 04/01/2025 Consult the publication