Biology subjects

Villani, A.

Publications and source records attributed to Villani, A..

8 recordsLinked to original sources

bioRxiv2026

A phiKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases

Bacteria employ diverse DNA-targeting systems, including restriction-modification (R-M) and CRISPR-Cas, to cleave invading bacteriophage genomes. In response, phages encode counter-defense strategies that block or mitigate DNA damage. Here, we screened a panel of Pseudomonas aeruginosa phages against native and heterologous DNA-targeting systems and identified the Phikmvvirus phage genus as broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which phiKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.

microbiology↗

bioRxiv2026

Conserved role of EGFR signaling in apoptotic cell recognition and processing across different phagocytes

Efferocytosis is an essential process that clears dying cells from tissues and prevents inflammation. However, the mechanisms by which apoptotic cells recruit and activate phagocytes remain poorly understood. Here, we show that EGFR signaling is both necessary and sufficient for efficient efferocytosis in two diverse phagocytes: the gonadal sheath cells of C. elegans and the zebrafish microglia. In C. elegans, loss of LET-23 EGFR or its downstream effector MPK-1 ERK in the sheath cells impairs the recognition and degradation of apoptotic germ cells. Germ cells undergoing apoptosis secrete the EGF ligand LIN-3, which promotes their recognition and engulfment. Overexpression of EGF in non-apoptotic germ cells is sufficient to trigger their engulfment, indicating that EGF can function as an engulfment signal independently of apoptotic cell death. In zebrafish larvae, pharmacological inhibition of EGFR signaling reduces microglial motility, recognition of apoptotic neurons, and corpse clearance in the optic tectum. Similar to C. elegans, an ectopic source of EGF serves as an attractive cue that promotes microglial recruitment. Our findings suggest that EGFR signaling controls an evolutionarily conserved efferocytosis module that coordinates the recognition and processing of apoptotic corpses in different types of phagocytes.

cell biology↗

bioRxiv2025

nAPOGEE: A machine-learning platform for clinically actionable pathogenicity assessment of all mitochondrial noncoding variants

Mitochondrial noncoding variants, particularly those in tRNA and rRNA genes, pose significant challenges for clinical interpretation due to heteroplasmy, broad phenotypic heterogeneity where symptoms can overlap with other conditions, and the limited availability of well-established genotype-phenotype correlations. Despite their central role in mitochondrial translation, these variants have remained largely unexplored by the existing variant-effect predictors. Here, we present nAPOGEE, a novel machine-learning framework specifically designed to assess the pathogenicity of all possible single-nucleotide variants in human mitochondrial noncoding RNAs. nAPOGEE integrates two specialized predictors: tAPOGEE, which outperforms existing tools for tRNAs, and rAPOGEE, the first dedicated classifier for mitochondrial rRNA variants. Using curated training datasets, phylogenetic conservation metrics, secondary structure modeling, RNA-specific embeddings, and thermodynamic features, nAPOGEE provides biologically interpretable predictions and posterior probabilities aligned with the ACMG/AMP guidelines. Applied to both curated variant sets and population-scale data, nAPOGEE revealed consistent spatial correlation of the predicted pathogenicity, reflecting underlying structural and evolutionary constraints. This study addresses a longstanding gap in mitochondrial genomics and offers a clinically applicable tool for variant prioritization, reclassification, and research into mitochondrial disease mechanisms.

bioinformatics↗

bioRxiv2025

In genomes we trust: assessing genomic reliability within the family Nectriaceae

The Nectriaceae includes major plant and human pathogens, yet the genomic foundation underpinning its taxonomy remains uneven and largely unassessed. We analysed 1,530 genome sequence assemblies to quantify metadata completeness, geographic and taxonomic bias, and assembly quality across the family. One-third of the assemblies lacked essential metadata, sequencing was heavily skewed toward a few agriculturally important lineages, and sampling of many genera was limited or nonexistent. BUSCO and QUAST metrics revealed striking heterogeneity in assembly quality, with widespread fragmentation and a substantial subset of genomes falling outside the expected quality thresholds. From orthologous protein sequences of 763 single-copy genes in 576 high-quality genomes, we reconstructed a phylogenomic backbone for the Nectriaceae and quantified gene- and site-level concordance. While major clades broadly match current concepts, extensive gene-tree discordance and a polyphyletic Nisikadoi complex highlight unresolved evolutionary and taxonomic boundaries. Our study delivers the first integrated, family-wide evaluation of Nectriaceae genomic resources and outlines a framework for quality standards, curated metadata, and stable phylogenomic inference to support future taxonomic and comparative work.

genomics↗

bioRxiv2025

A scalable human-zebrafish xenotransplantation model reveals gastrosome-mediated processing of dying neurons by human microglia

Microglia engulf dying neurons through efferocytosis, a critical function in both development and disease. How microglia process the engulfed neuronal material--especially lipids--remains poorly understood, despite its central role in neurodegeneration. Thus, we developed HuZIBRA, a scalable in vivo xenotransplantation model in which human iPSC-derived microglia-like cells (iMGLs) are introduced into the developing zebrafish brain (zf-hiMG), a system characterized by high levels of neuronal cell death and amenable to precise genetic and pharmacological manipulation. We show that human microglia-like cells recognize and engulf apoptotic zebrafish neurons, indicating conserved efferocytic mechanisms. In these cells, engulfed neuronal material accumulates into a distinct, lipid-rich intracellular compartment, the gastrosome, which we also observed in iMGLs placed in a human brain-like environment. The size of the human gastrosome dynamically reflects neuronal cell death levels and is regulated by key genes, including TREM2 and SLC37A2. Pharmacological inhibition of the cholesterol transporter NPC1 induces gastrosome expansion and lipid accumulation, recapitulating pathological features of Niemann-Pick disease type C. Thus, HuZIBRA provides a powerful in vivo platform to uncover cell-autonomous adaptive responses of human microglia to apoptotic and metabolic stress, with the gastrosome emerging as a key integrator of neuronal debris processing and disease-relevant lipid metabolism.

cell biology↗

bioRxiv2024

Multigenerational Proteolytic Inactivation of Restriction Upon Subtle Genomic Hypomethylation

Restriction-modification (R-M) systems, present in most bacterial genomes, protect against phage infection by detecting and degrading invading foreign DNA. However, like many prokaryotic anti-phage systems, R-M systems pose a significant risk of auto-immunity, exacerbated by the presence of hundreds to thousands of potential cleavage sites in the bacterial genome. In Pseudomonas aeruginosa, restriction inactivation upon growth at high temperatures was previously described, however, which system is being inactivated, the underlying mechanism, as well as the timing of recovery, remain unknown. Here, we report that P. aeruginosa Type I methyltransferase (HsdMS) and restriction endonuclease (HsdR) components are degraded by two Lon-like proteases when replicating above 41 {degrees}C, which induces partial genome hypomethylation and simultaneously prevents self-targeting, respectively. Interestingly, upon return to 37 {degrees}C, methyltransferase activity returns gradually, with restriction activity not fully recovering for over 60 bacterial generations, representing the longest bacterial memory to our knowledge. Forced expression of HsdR over the first 45 generations is toxic, demonstrating the fitness benefit of HsdR inactivation. Our findings demonstrate that type I R-M is tightly regulated post-translationally with a remarkable memory effect to ensure genomic stability and emphasize the importance of mitigating auto-toxicity for bacterial defense systems.

microbiology↗

bioRxiv2024

DNA Damage Response Deficiency Enhances Neuroblastoma Progression and Sensitivity to Combination PARP and ATR Inhibition

Next generation sequencing of neuroblastoma (NB) tumors have revealed frequent somatic and germline genetic alterations in genes encoding proteins involved in DNA damage response (DDR) pathways. Despite being well-studied in many adult cancers, roles for DDR disruption in pediatric solid tumors remains poorly understood. To address this, patient-relevant loss-of-function mutations in DDR pathway components including Brca2, Atm, and Palb2 were incorporated into an established zebrafish MYCN transgenic model (Tg(dbh:EGFP-MYCN)). These mutations were found to enhance NB formation and metastasis in vivo, and result in upregulation of proliferation, cell cycle checkpoint and DNA damage repair transcriptional signatures, revealing potential molecular vulnerabilities in DDR-deficient NB. Zebrafish DDR-deficient NB and human NB cells with DDR protein knock-down were sensitive to the poly(ADP-ribose)-polymerase (PARP) inhibitor olaparib, and this effect was further enhanced by inhibition of the ataxia telangiectasia and rad3 related (ATR) kinase. Altogether, our data supports a functional role for DDR-deficiency in NB in vivo and therapeutic potential for combination PARP + ATR inhibition in NB patients with alterations in DDR genes. SignificanceThis work provides the first in vivo evidence supporting a functional role for DDR-deficiency in NB by demonstrating that alterations in certain DDR pathway genes promote NB formation and metastasis. NGS and pre-clinical drug testing also provides rationale for PARP + ATR inhibitor therapy combinations for patients with NB and pathogenic DDR pathway alterations.

cancer biology↗

bioRxiv2022

A role for the centrosome in regulating the rate of neuronal efferocytosis by microglia in vivo

During brain development, many newborn neurons undergo apoptosis and are engulfed by microglia, the tissue-resident phagocytes of the brain, in a process known as efferocytosis. A hallmark of microglia is their highly branched morphology characterised by the presence of numerous dynamic extensions that these cells use for scanning the brain parenchyma and engulfing unwanted material. The mechanisms driving branch formation and apoptotic cell engulfment in microglia are unclear. By taking a live imaging approach in zebrafish, we show that while microglia generate multiple microtubule-based branches, they only successfully engulf one apoptotic neuron at a time. Further investigation into the mechanism underlying this sequential engulfment revealed that targeted migration of the centrosome into one branch is predictive of phagosome formation and polarised vesicular trafficking. Moreover, experimentally doubling centrosomal numbers in microglia increases the rate of engulfment and even allows microglia to remove two neurons simultaneously, providing direct supporting evidence for a model where centrosomal migration is a rate-limiting step in branch-mediated efferocytosis. Conversely, light-mediated depolymerization of microtubules causes microglia to lose their typical branched morphology and switch to an alternative mode of engulfment, characterised by directed migration towards target neurons, revealing unexpected plasticity in their phagocytic ability. Finally, building on work focusing on the establishment of the immunological synapse, we identified a conserved signalling pathway underlying centrosomal movement in engulfing microglia.

cell biology↗