Biology subjects

Manfredi, M.

Publications and source records attributed to Manfredi, M..

14 recordsLinked to original sources

bioRxiv2026

Profilin 1 maintains cell cycle fidelity to prevent unscheduled genome doubling and polyploidy in cancer

Whole-genome doubling (WGD) represents a major route to genome instability and therapeutic resistance in cancer; yet, the mechanisms enabling genome duplication in p53-proficient cells remain poorly understood. Here, we identify Profilin 1 (PFN1) loss as a driver of WGD through impaired mitotic entry. Using FUCCI live-cell imaging and single-cell genomic profiling, we show that PFN1-deficient cells bypass mitosis and undergo endoreplication, generating tetraploid cells. Rather than undergoing stable arrest after mitotic bypass, these genome-doubled cells retain proliferative capacity and proceed through aberrant mitotic divisions, thereby amplifying genomic instability. Proteomic analyses reveal coordinated attenuation of late cell-cycle programs, including downregulation of key mitotic regulators such as CDK1, PLK1 and CKS2, consistent with impaired G2/M transition. Despite accumulating polyploidy, PFN1-knockout cells fail to activate an effective p53 tetraploidy checkpoint and display increased nuclear MDM2, promoting cell-cycle arrest evasion and chemotherapeutic resistance. We supported clinical relevance by an orthotopic osteosarcoma xenograft model, in which PFN1-deficient SaOS2 cells showed enhanced metastatic dissemination, and by pan-cancer TCGA analyses confirming a recurrent association between PFN1 loss and WGD. Together, these findings identify Profilin 1 as a safeguard of cell-cycle fidelity whose loss enables genome doubling, cellular plasticity and therapy tolerance.

cancer biology↗

bioRxiv2026

Rare variants alter mitochondrial lipid homeostasis and neuronal excitability in PD patient-derived dopaminergic neurons

Parkinsons disease (PD) exhibits substantial genetic heterogeneity, yet how combinations of rare variants converge on disease-relevant cellular mechanisms remains unclear. Here, we generated human induced pluripotent stem cell-derived dopaminergic neurons from PD patients carrying rare variants in recently implicated genes and performed integrated electrophysiological, proteomic, lipidomic, and genetic analyses. Patient-derived neurons showed reduced membrane capacitance and altered action potential firing, indicating impaired intrinsic excitability and synaptic dysfunction, with marked variability across genetic backgrounds. Multi-omics profiling revealed dysregulation of mitochondrial function, glycolysis, and oxidative phosphorylation, accompanied by extensive lipid remodeling, including increased fatty acids, acylcarnitines, and sphingolipids, and reduced gangliosides. These alterations were more pronounced in neurons harboring specific variant combinations in KIF21B, SLC6A3, HMOX2, TMEM175, and AIMP2. Integrative analyses uncovered coordinated protein-lipid changes linking mitochondrial dysfunction and membrane homeostasis. Notably, Calpastatin and CXCR4 were consistently dysregulated across PD neurons. Genetic association analyses in independent cohorts identified PD-associated variants in genes encoding dysregulated proteins, supporting the functional relevance of these pathways. Overall, our results define convergent and variant-specific mechanisms underlying PD and highlight candidate biomarkers and therapeutic targets.

neuroscience↗

bioRxiv2026

Prostaglandin E2 Reverses Myofibroblast Differentiation in Eosinophilic Esophagitis

Background & AimsUnchecked inflammation in Eosinophilic esophagitis (EoE) leads to esophageal fibrosis and eventual stricture. Differentiated fibroblasts, termed myofibroblasts, are the main effector cells in fibrosis, responsible for secreting extracellular matrix proteins leading to tissue stiffness. Regulating myofibroblasts has not been explored as a therapeutic possibility in the fibrostenotic esophagus. Herein, we aim to investigate the efficacy of Prostaglandin E2 (PGE2) in dedifferentiation of the EoE myofibroblast. MethodsWe evaluated the efficacy and mechanism of myofibroblast dedifferentiation using fetal esophageal fibroblasts (FEF3), patient-derived fibroblasts, and a murine model of EoE. ResultsFibrosis markers (SMA, FN1, and COL1A1) and contractility of myofibroblasts were significantly decreased by PGE2 via the cAMP pathway. PGE2 treatment decreased nuclear accumulation of phospho-Smad2/3-YAP complex and induced phospho-YAP proteasomal degradation. Transcriptome analyses of FEF3 treated with TGF{beta} or PGE2 revealed that the Integrin1 pathway, and specifically thrombospondin 1 (THBS-1), was significantly upregulated by TGF{beta} and downregulated by PGE2, as supported by pseudo-bulk single-cell RNA-seq of EoE biopsies. THBS-1 was shown to be regulated by PGE2 via the cAMP/YAP pathway, and its knockdown induced myofibroblasts dedifferentiation. In a murine model of EoE, Butaprost, agonist of the E-prostanoid G protein-coupled receptor 2, treatment significantly reduced the expression of THBS-1, SMA, and FN1 along with a decrease in YAP nuclear translocation. Additionally, collagen fiber organization in the lamina propria was markedly reduced. ConclusionPGE2 promotes dedifferentiation of myofibroblasts in EoE via the cAMP/YAP/ THBS-1 pathway. Our data suggest that PGE2 is a promising treatment strategy for EoE with stenosis. What You Need to KnowO_ST_ABSBackground and ContextC_ST_ABSIn Eosinophilic esophagitis, unchecked inflammation and tissue stiffness drives fibroblast differentiation and fibrostenosis of the esophagus, yet targeting myofibroblasts as regulators of extracellular matrix deposition in fibrostenotic disease remains clinically unexplored. New FindingsProstaglandin E2 promotes dedifferentiation of myofibroblasts in eosinophilic esophagitis via the cAMP/YAP pathway, with Thrombospondin-1 identified as a critical YAP regulated target driving fibrostenosis. LimitationsThis study focused on fibroblast-specific mechanisms. The effects of PGE2 on esophageal epithelial differentiation, barrier function, and immune cell recruitment in EoE remain to be determined. Clinical Research RelevanceThis study demonstrates proof-of-concept that pharmacological reversal of established fibrosis is achievable in EoE. PGE2 and its EP2-selective agonists represent translatable therapeutic targets for fibrostenotic EoE--a patient population that remains treatment-refractory to current immunosuppressive approaches. Basic Research RelevanceThe cAMP/YAP/THBS-1 signaling in fibroblasts emerges as a critical therapeutic target for esophageal fibrosis. Importantly, this work demonstrates that terminally differentiated myofibroblasts retain remarkable plasticity and can dedifferentiate--challenging the paradigm that fibrosis is irreversible.

cell biology↗

bioRxiv2025

Human liver organoids uncover ductular reaction in Wolman disease and inform gene therapy design

Wolman disease (WD), the severe infantile form of lysosomal acid lipase deficiency, is a rare metabolic disorder caused by inactivating mutations in the LIPA gene. Although WD is characterized by profound hepatic dysfunction, experimental human systems capable of modelling multicellular liver pathology and supporting therapeutic testing remain limited. Here, we generated an isogenic human model of WD by introducing LIPA loss-of-function mutations into induced pluripotent stem cells and differentiating them into multicellular human liver organoids (HLO). LIPA-deficient HLO preserved hepatic lineage specification while recapitulating key biochemical and cellular features of WD, including loss of LIPA activity, lysosomal expansion, lipid accumulation, and activation of inflammatory and fibrogenic programs. Single-cell RNA sequencing resolved cell-type-specific disease states across hepatocyte-, stromal-, and biliary-like populations, revealing the emergence of a reactive biliary program consistent with ductular reaction, a complex tissue response associated with chronic liver injury. Importantly, this reactive biliary phenotype was supported by targeted gene-expression analysis in WD liver organoids and independently validated in liver tissue from mouse models and WD patients. Isolated LIPA-deficient cholangiocyte organoids failed to reproduce the DR-associated program, indicating that this response depends on multicellular interactions within the hepatic microenvironment rather than on biliary cell-autonomous dysfunction alone. Consistently, hepatocyte-directed AAV-mediated restoration of LIPA expression attenuated metabolic stress, inflammatory and fibrogenic programs, and suppressed ductular reaction both in organoids and in vivo. Together, these findings establish multicellular human liver organoids as a physiologically relevant platform for modelling emergent tissue-level responses in WD and for evaluating therapeutic rescue strategies in a human context.

pathology↗

bioRxiv2024

DDGemb: predicting protein stability change upon single- and multi-point variations with embeddings and deep learning

The knowledge of protein stability upon residue variation is an important step for functional protein design and for understanding how protein variants can promote disease onset. Computational methods are important to complement experimental approaches and allow a fast screening of large datasets of variations. In this work we present DDGemb, a novel method combining protein language model embeddings and transformer architectures to predict protein {Delta}{Delta}G upon both single- and multi-point variations. DDGemb has been trained on a high-quality dataset derived from literature and tested on available benchmark datasets of single- and multi-point variations. DDGemb performs at the state of the art in both single- and multi-point variations.

bioinformatics↗

bioRxiv2024

A novel family of lncRNAs relate facioscapulohumeral muscular dystrophy to nucleolar architecture and protein synthesis rate

Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary myopathy linked to deletions of the tandemly arrayed D4Z4 macrosatellite repeats at human chromosome 4q35. These deletions accompany local chromatin changes and the anomalous expression of nearby transcripts FRG2A, DBET, and D4Z4. We discovered that FRG2A is one member of a family of long non-coding RNAs (lncRNAs) expressed at elevated levels in skeletal muscle cells with distinct amounts detected in individual patients. We found that FRG2A lncRNA preferentially associates with rDNA sequences and centromeres and promotes the three-dimensional association of centromeres with the nucleolar periphery in FSHD cells. Furthermore, we demonstrate that the elevated FRG2A expression in cells from FSHD patients reduces rDNA transcription and global protein synthesis. Our results frame an entirely unanticipated new disease model in which elevated lncRNAs levels mediated by deletions of D4Z4 macrosatellite repeats leads to a diminished protein synthesis capacity, thereby contributing to muscle wasting.

molecular biology↗

bioRxiv2024

Evaluation of enzyme activity predictions for variants of unknown significance in Arylsulfatase A

Continued advances in variant effect prediction are necessary to demonstrate the ability of machine learning methods to accurately determine the clinical impact of variants of unknown significance (VUS). Towards this goal, the ARSA Critical Assessment of Genome Interpretation (CAGI) challenge was designed to characterize progress by utilizing 219 experimentally assayed missense VUS in the Arylsulfa-tase A (ARSA) gene to assess the performance of community-submitted predictions of variant functional effects. The challenge involved 15 teams, and evaluated additional predictions from established and recently released models. Notably, a model developed by participants of a genetics and coding bootcamp, trained with standard machine-learning tools in Python, demonstrated superior performance among sub-missions. Furthermore, the study observed that state-of-the-art deep learning methods provided small but statistically significant improvement in predictive performance compared to less elaborate techniques. These findings underscore the utility of variant effect prediction, and the potential for models trained with modest resources to accurately classify VUS in genetic and clinical research.

genetics↗

bioRxiv2023

RORγ bridges cancer-driven lipid dysmetabolism and myeloid immunosuppression

Despite well-documented metabolic and hematopoietic alterations during tumor development1, the mechanisms underlying this crucial immunometabolic intersection have remained elusive. Of particular interest is the ligand-activated transcription factor retinoic acid-related orphan receptor 1 (RORC1/ROR{gamma}), whose activity is boosted by cholesterol metabolites2, acting as a modulator of cancer-related emergency myelopoiesis3, while hypercholesterolemia itself is associated with dysregulated myelopoiesis4,5. Here we show that both cancer growth and hypercholesterolemic diet can independently or cooperatively activate ROR{gamma}-dependent expansion of myeloid-derived suppressor cells (MDSCs) and M2 polarization of tumor-associated macrophages (TAMs), thereby supporting cancer spread. Moreover, we report that tumor development enhances the hepatic production of IL-1{beta} and IL-6, which in turn promote upregulation of hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, as we confirmed in models of fibrosarcoma, melanoma, colorectal (CRC), and lung cancer, as well as in CRC, non-small-cell lung cancer (NSCLC), breast (BRC), pancreatic ductal adenocarcinoma (PDAC), biliary tract carcinoma (BTC) and pancreatic neuroendocrine tumor (PNET) patients. Importantly, lowering cholesterol levels prevents MDSC expansion and M2 TAM accumulation in a ROR{gamma}-dependent manner, unleashing specific anti-tumor immunity and improving the efficacy of anti-PD-1 immunotherapy. Overall, we identify ROR{gamma} as a novel sensor of lipid disorders in cancer bearers, bridging hypercholesterolemia and pro-tumor myelopoiesis.

immunology↗

bioRxiv2022

ISPRED-SEQ: Deep neural networks and embeddings for predicting interaction sites in protein sequences.

The knowledge of protein-protein interaction sites (PPIs) is crucial for protein functional annotation. Here we address the problem focusing on the prediction of putative PPIs having as input protein sequences. The problem is important given the huge volume of sequences compared to experimental and/or computed protein structures. Taking advantage of recently developed protein language models and Deep Neural networks here we describe ISPRED-SEQ, which overpasses state-of-the-art predictors addressing the same problem. ISPRED-SEQ is freely available for testing at https://ispredws.biocomp.unibo.it.

bioinformatics↗

bioRxiv2022

Plant signals anticipate the induction of the type III secretion system in Pseudomonas syringae pv. actinidiae facilitating efficient temperature-dependent effector translocation

Disease resistance in plants depends on a molecular dialogue with microbes that involves many known chemical effectors, but the time course of the interaction and the influence of the environment are largely unknown. The outcome of host-pathogen interactions is thought to reflect the offensive and defensive capabilities of both players. When plants interact with Pseudomonas syringae, several well-characterized virulence factors contribute to early bacterial pathogenicity, including the type III secretion system (T3SS), which must be activated by signals from the plant and environment to allow the secretion of virulence effectors. The manner in which these signals regulate T3SS activity is still unclear. Here, we strengthen the paradigm of the plant-pathogen molecular dialogue by addressing overlooked details concerning the timing of interactions, specifically the role of plant signals and temperature on the regulation of bacterial virulence during the first few hours of the interaction. Whole-genome expression profiling after 1 h revealed that the perception of plant signals from kiwifruit or tomato extracts anticipates T3SS expression in P. syringae pv. actinidiae compared to apoplast-like conditions, facilitating more efficient effector transport in planta, as revealed by the induction of a temperature-dependent hypersensitive response in the non-host plant Arabidopsis thaliana Col-0. Our results show that, in the arms race between plants and bacteria, the temperature-dependent timing of bacterial virulence versus the induction of plant defenses is probably one of the fundamental parameters governing the outcome of the interaction. Abstract importancePlant diseases- their occurrence and severity- result from the impact of three factors: the host, the pathogen, and the environmental conditions, interconnected in the disease triangle. Time was further included as a fourth factor accounting for plant disease, leading to a more realistic three-dimensional disease pyramid to represent the evolution of disease over time. However, this representation still considers time only as a parameter determining when and to which extent a disease will occur, at a scale from days to months. Here, we show that time is a factor regulating the arms race between plants and pathogens, at a scale from minutes to hours, and strictly depends on environmental factors. Thus, besides the arms possessed by pathogens and plants per se, the opportunity and the timing of arms mobilization should make the difference to determine the outcome of an interaction and thus the occurrence of plant disease.

microbiology↗

bioRxiv2022

E-SNPs&GO: Embedding of protein sequence and function improves the annotation of pathogenic variants.

MotivationThe advent of massive DNA sequencing technologies is producing a huge number of human single-nucleotide polymorphisms occurring in protein-coding regions and possibly changing protein sequences. Discriminating harmful protein variations from neutral ones is one of the crucial challenges in precision medicine. Computational tools based on artificial intelligence provide models for protein sequence encoding, bypassing database searches for evolutionary information. We leverage the new encoding schemes for an efficient annotation of protein variants. ResultsE-SNPs&GO is a novel method that, given an input protein sequence and a single residue variation, can predict whether the variation is related to diseases or not. The proposed method, for the first time, adopts an input encoding completely based on protein language models and embedding techniques, specifically devised to encode protein sequences and GO functional annotations. We trained our model on a newly generated dataset of 65,888 human protein single residue variants derived from public resources. When tested on a blind set comprising 6,541 variants, our method outperforms recent approaches released in literature for the same task, reaching a MCC score of 0.71. We propose E-SNPs&GO as a suitable, efficient and accurate large-scale annotator of protein variant datasets. Contactpierluigi.martelli@unibo.it

bioinformatics↗

bioRxiv2022

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimers disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2 and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2 without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2 and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2 and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2 levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

neuroscience↗

bioRxiv2020

Deletion of calcineurin from astrocytes reproduces proteome signature of Alzheimer's disease and epilepsy and predisposes to seizures

In astrocytes, calcineurin (CaN) is involved in neuroinflammation and gliosis, while its role in healthy CNS or in early neuro-pathogenesis is poorly understood. Here we report that in astroglial CaN KO (ACN-KO) mice, at one month of age, proteome is deranged in hippocampus and cerebellum. Bioinformatic analysis reveals association with Alzheimers disease (AD) and epilepsy. We found significant overlap with the proteome of an AD mouse model and of human subjects with drug-resistant epilepsy. In Barnes maze ACN-KO mice learned the task but adopted serial search strategy. Strikingly, from five months of age ACN-KO mice develop spontaneous seizures with an inflammatory signature of epileptic brains. These results suggest that astroglial CaN KO impairs hippocampal connectivity, produces proteome features of neurological disorders and predisposes mice to seizures. We suggest that astroglial CaN may serve as a novel Ca2+-sensitive switch which regulates protein expression and homeostasis in the CNS.

neuroscience↗

bioRxiv2020

Limited Environmental Serine Confers Sensitivity to PHGDH Inhibition in Brain Metastasis

A hallmark of metastasis is the adaptation of tumor cells to new environments. Although it is well established that the metabolic milieu of the brain is severely deprived of nutrients, particularly the amino acids serine and its catabolite glycine, how brain metastases rewire their metabolism to survive in the nutrient-limited environment of the brain is poorly understood. Here we demonstrate that cell-intrinsic de novo serine synthesis is a major determinant of brain metastasis. Whole proteome comparison of triple-negative breast cancer (TNBC) cells that differ in their capacity to colonize the brain reveals that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is the most significantly upregulated protein in cells that efficiently metastasize to the brain. Genetic silencing or pharmacological inhibition of PHGDH attenuated brain metastasis and improved overall survival in mice, whereas expression of catalytically active PHGDH in a non-brain trophic cell line promoted brain metastasis. Collectively, these findings indicate that nutrient availability determines serine synthesis pathway dependence in brain metastasis, and suggest that PHGDH inhibitors may be useful in the treatment of patients with cancers that have spread to the brain. Statement of SignificanceOur study highlights how limited serine and glycine availability within the brain microenvironment potentiates tumor cell sensitivity to serine synthesis inhibition. This finding underscores the importance of studying cancer metabolism in physiologically-relevant contexts, and provides a rationale for using PHGDH inhibitors to treat brain metastasis.

cancer biology↗