Biology subjects

Milan, M.

Publications and source records attributed to Milan, M..

13 recordsLinked to original sources

bioRxiv2026

Extracellular vesicles from Manila clam (Ruditapes philippinarum): tailored isolation from hemolymph and insights into water-derived vesicles

Extracellular vesicles (EVs) are evolutionarily conserved mediators of intercellular communication released by cells into biological fluids and the extracellular environment. Despite their growing relevance in biomedical and veterinary research, knowledge on EVs in marine bivalves remains limited. The aim of this study was to optimize tailored protocols for EV isolation from the hemolymph of the Manila clam (Ruditapes philippinarum) based on density gradient ultracentrifugation (dgUC) or size exclusion chromatography (SEC). EV-enriched fractions were identified through nanoparticle tracking analysis, protein quantification, transmission electron microscopy, and cryo-electron microscopy. Both methods successfully isolated small EVs (<200 nm). While dgUC yielded higher-purity preparations, SEC provided a higher recovery rate and compatibility with downstream metabolomic analyses. Metabolomics performed on SEC fractions and on hemolymph, revealed that EV-enriched fractions possessed a distinct metabolic signature including enrichment in metabolites associated with nucleotide metabolism, glycolysis, redox regulation, and energy metabolism. Furthermore, we performed a pilot investigation into the presence of EVs released into conditioned water by Manila clams. Using tangential flow filtration and ultrafiltration, EVs were successfully concentrated from water samples and characterized by nanoparticle tracking analysis, CONAN assay, atomic force microscopy, and electron microscopy. Our findings demonstrate the feasibility of isolating EVs both from Manila clam hemolymph and from conditioned water, providing the first evidence of water-derived EV recovery in aquatic animals. Although further methodological refinement is needed to improve the purity of EVs isolated from water, and additional characterization studies are required to better define the molecular composition of clam-derived EVs, these results establish a foundation for future investigations into the role of EVs in bivalve biology and their potential application as minimally invasive biomarkers for aquaculture, environmental monitoring, and ecosystem health assessment.

systems biology↗

bioRxiv2026

A dual-clam species 63K SNP array for sustainable production and conservation of wild resources

Bivalves play an essential role in coastal ecosystems and their aquaculture represents an important economic sector in Europe playing a pivotal role within the EU Blue Growth Strategy. Among clam species, the Manila clam, Ruditapes philippinarum, and the grooved carpet shell, R. decussatus, are within the top five species in terms of production volume and economic value. In this study we designed and validated the first medium-density 63K single nucleotide polymorphism (SNP) array for these two commercially important species. By leveraging a new chromosome-level genome assembly for R. philippinarum and that of R. decussatus, we identified over 300 million SNPs through whole-genome resequencing and genotyping-by-sequencing strategies. After stringent filtering, we selected 49,392 high-quality SNPs for R. philippinarum and 14,193 for R. decussatus to construct a dual-species array. Array validation was carried out by genotyping 384 individuals across multiple wild populations and hatchery samples, demonstrating excellent performance, with 67.7% and 67.5% of SNPs classified as high-quality polymorphic markers for R. philippinarum and R. decussatus, respectively. Minor allele frequency, missing data rate, and inter-marker distance met stringent quality thresholds, confirming the array robustness for clam population genetics. Parentage analysis in R. philippinarum families highlighted significant power for pedigree reconstruction in breeding programs. This publicly available genomic resource provides a reliable, cost-effective genotyping platform to enable population genomics and advanced selective breeding, genome-wide association studies, and genetic monitoring, ultimately strengthening management of genetic diversity and sustainable farming for two key clam species.

genomics↗

bioRxiv2026

Ribosome Quality Control Mitigates Proteotoxic Stress in Aneuploid Cells

Aneuploidy is widespread in tumors, but how cancer cells adapt to aneuploidy-induced cellular stresses remains poorly understood. Here, we focus on the mechanisms employed to cope with proteostasis disruption, a major stress caused by aneuploidy. We show that aneuploid cells exhibit a significant accumulation of ribosomes enclosed within autophagosomes, ultimately degraded through lysosome-mediated processes. Our data also indicate that limited folding capacity of newly synthesized polypeptides leads to lysosome-mediated degradation of ribosomes. We also found that the E3 ligase ZNF598 marks these ribosomes for degradation, thus clearing translationally-impaired ribosomes. Importantly, highly aneuploid tumors display a positive correlation with ZNF598 expression, while being negatively associated with ribosomal signatures. This suggests that ribosome-associated quality control is crucial for cancer cell survival under proteotoxic stress. Our study uncovers molecular events in response to proteotoxic stress in aneuploid cells and suggests that components of ribosome-associated quality control, including ZNF598, could serve as promising targets in cancer therapy.

cell biology↗

bioRxiv2025

Multicellular Calcium Waves in Cancer-Associated Fibroblasts Regulate Neuronal Mimicry and Anisotropy Leading to Immune Exclusion

Stromal barriers exclude CD8+ T cells from accessing cancer cells and hamper immune-mediated tumour control. Through multi-pronged analysis of tumours that transition from immune inflamed to immune excluded, we reveal that the formation of stromal barriers is associated with the acquisition of neuronal gene expression programmes in cancer-associated fibroblasts (CAFs), including TUBB3 expression. This leads to neuronal mimicry, with stromal barrier formation underpinned by coordinated transient bursts of intracellular calcium release, similar to those observed in neuronal tissue. Blockade of calcium release through either pharmacological or molecular interventions, such as STC2 depletion, prevents CAF alignment and the build-up of CD8+ T cells at stromal boundaries. Nintedanib treatment prevents neuronal mimicry and restores immune-mediated tumour control. Thus, we uncover unexpected mimicry of neuronal behaviour in CAFs, document the mechanism by which it leads to immune exclusion, and identify ways to prevent the induction of neuronal mimicry and restore immune-mediated tumour control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/684281v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@938ee5org.highwire.dtl.DTLVardef@18a9c48org.highwire.dtl.DTLVardef@11a5982org.highwire.dtl.DTLVardef@ea6a79_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

The transcriptome of highly aneuploid senescent cells reveals interaction with the tumour microenvironment and a pro-survival role of Yorkie

Chromosomal instability (CIN), characterized by frequent changes in chromosome number and structure, is common in human carcinomas and often leads to aneuploidy. Drosophila has been instrumental in demonstrating that CIN can promote tumour growth and malignancy through aneuploidy-induced senescence, a state marked by cell-cycle arrest and high secretory activity. Despite extensive chromosomal heterogeneity, we show that these cells share a distinct transcriptional program, with most responses to aneuploidy and senescence regulated at the transcriptional level. Nearly 10% of the most upregulated genes encode secreted proteins of the senescence-associated secretory phenotype. Five of these proteins act additively, locally or systemically, to block proliferation and induce cell death in neighbouring tissues. This non-autonomo*us cell death feeds back to the tumour to enhance its growth, resembling super-competition and providing insight into tumour-host interactions relevant to human cancer. Teaser: Aneuploidy-induced senescent cells activate the pro-survival Hippo-Yorkie pathway and the SASP drives tumour host interactions

cancer biology↗

bioRxiv2025

A systemic role of macrophage-derived BMP2/4 homolog Dpp in regulating sterol hormone synthesis under dietary stress

A high-sugar diet (HSD), prevalent in Western countries, compromises the growth and development of children and primes their bodies for chronic diseases. Macrophages, which are immune cells found throughout the body, can sense environmental changes and influence surrounding tissues. Using Drosophila as a model, here we show that macrophages respond to an HSD by increasing the production of Decapentaplegic (Dpp), a protein related to human BMP2/4. Dpp then signals to the endocrine system, the prothoracic gland, to inhibit the synthesis of ecdysone, a crucial steroid hormone that triggers the transition from larva to pupa. By reducing ecdysone synthesis, the larval stage is prolonged. This extended developmental period provides larvae exposed to an HSD more time to reach an optimal size. Our findings highlight how different organs communicate to counteract the negative impact of an HSD during development and expand our understanding of Dpp function, showing that it acts not only as a local developmental signal but also as a systemic regulator.

developmental biology↗

bioRxiv2025

A pivotal contribution of proteostasis failure and mitochondrial dysfunction to chromosomal instability-induced microcephaly

Mosaic variegated aneuploidy (MVA), a rare human congenital disorder that causes microcephaly, is characterized by extensive abnormalities in chromosome number and results from mutations in genes involved in accurate mitotic chromosome segregation. To characterize the cellular mechanisms underlying this disease, here we generated a Drosophila model of microcephaly caused by the depletion of a single spindle assembly checkpoint (SAC) gene in the neural stem cell (NSC) compartment. We present evidence that loss of stemness - compromised identity and proliferative capacity of NSCs- is the underlying cause of MVA and results in a reduced number of neurons and glial cells. We show that loss of stemness arises from the accumulation over time of an unbalanced number of gains and losses of more than one chromosome, rather than a direct consequence of chromosomal instability-induced DNA damage or the production of simple aneuploidies. We unravel that the negative impact of complex aneuploidies on stemness, a highly energy demanding cellular state, is a result of proteostasis failure and mitochondrial dysfunction. We identify autophagy activation--either directly or through TOR depletion--, overexpression of Radical Oxygen Species scavengers, and restoration of mitochondria proteostasis as genetic interventions capable of dampening the deleterious effects of aneuploidy on NSC identity and brain development. HighlightsSAC depletion in NSCs induces microcephaly through the production of complex aneuploidies Complex aneuploidies compromise the stemness of NSCs Loss of stemness results from proteostasis failure and mitochondrial dysfunction Boosting proteostasis or mitochondria homeostasis mitigate the negative effects on NSCs eTOC BlurbDepletion of the spindle assembly checkpoint in the neural stem cell compartment leads to microcephaly. Gonzalez-Blanco et al. provide evidence that proteostasis failure and mitochondrial dysfunction play a pivotal role in causing microcephaly by compromising the stemness of highly aneuploid neural stem cells.

neuroscience↗

bioRxiv2024

Depletion of aneuploid cells is shaped by cell-to-cell interactions

Aneuploidy, the major cause of miscarriages in humans, is pervasive in early embryos but robustly dampened during development, allowing for healthy births. Later in life, aneuploidy correlates with pathological conditions including cancer. Identification of the mechanisms underlying the elimination of aneuploid cells is relevant in development and disease. Here we generated cells carrying molecularly defined segmental monosomies and trisomies and characterized their immediate impact on cellular behavior. Our data reveal signs of out-competition of cells carrying monosomies in genomic regions devoid of previously known haploinsufficient genes. Dose-dependent effects of single genes or a discrete number of genes contribute to the observed cellular behaviors. By simultaneously inducing cells carrying monosomies and trisomies of the same genomic location, we present evidence that segmental trisomies potentiate or alleviate the negative effects of the monosomy on growth, thus revealing a key role of cell interactions in defining the in vivo elimination of aneuploid cells. HighlightsSegmental monosomies cause growth impairment and are out-competed. Growth impairment relies on dose-dependent effects of single genes or a discrete number of genes. Segmental trisomies of up to 1500 genes do not have a major impact on proliferation and survival. Cell competition is modulated by the presence of cells trisomic for the same genomic region. eTOC BlurbAneuploidy has a negative impact on the growth and proliferation of all animal cells analyzed so far. Fusari et al. unravel a role of cell interactions in defining the in vivo elimination of aneuploid cells through cell competition.

developmental biology↗

bioRxiv2023

Transcriptional profiling of populations in the clam Ruditapes decussatus suggests genetically determined differentiation in gene-expression along parallel temperature gradients and between races of the Atlantic Ocean and West Mediterranean sea

Ongoing ocean warming due to climate change poses new challenges for marine life and its exploitation. We have used transcriptomics to find genetically-based responses to increased temperature in natural populations of the marine clam Ruditapes decussatus living along parallel thermal gradients in southern Europe. Clams of the Atlantic and West Mediterranean races were collected in a northern (cool) and a southern (warm) localities. The animals were kept in running seawater in the warm, Atlantic southern locality for a 15-week period, during which water temperature raised to typical southern European summer values. After this period, the expression profile was obtained for a total of 34 clams and 11,025 probes by means of an oligonucleotide microarray. We found distinct transcriptional patterns for each population based on a total of 553 differentially expressed genes (DEG), indicating innate differences which probably have a genetic basis. Race and latitude contributed significantly to gene expression differences, with very different sets of DEG. A gene ontology analysis showed that races differed mainly at genes involved in ribosomal function and protein biosynthesis, while genes related to glutathione metabolism and ATP synthesis in the mitochondria were the most outstanding as to north/south transcriptional differences.

genomics↗

bioRxiv2022

Human platelet lysate derived extracellular vesicles enhance angiogenesis through miR-126

Objectivesextracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL. Methodswe tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL-derived EVs. Resultsa subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in HUVEC and increasing the angiogenic tubularly-like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched of small RNAs and a high amount of miR-126, the most abundant angiogenic miRNA in platelets. The transfer of miR-126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as downstream target in this system. ConclusionsPL is a biological source of available EVs with angiogenic effects involving a miRNAs-based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.

cell biology↗

bioRxiv2019

Eiger/TNFα-mediated Dilp8 and ROS production coordinate intra-organ growth in Drosophila

Coordinated intra- and inter-organ growth is essential during animal development to generate individuals of proper size and shape. The Drosophila wing has been a valuable model system to reveal the existence of a stress response mechanism mediated by Drosophila p53 (Dmp53) and involved in the coordination of tissue growth between adjacent cell populations upon targeted reduction of growth rates. Here we present evidence that a two-step molecular mechanism is being used by Dmp53 to reduce in a non-autonomous manner growth and proliferation in adjacent cell populations. First, Dmp53-mediated transcriptional induction of Drosophila TNF ligand Eiger leads to cell autonomous activation of JNK. Second, two different signaling events downstream of the Eiger/JNK axis are induced in the growth depleted territory in order to independently regulate tissue size and cell number in adjacent cell populations. Whereas expression of the systemic hormone dILP8 coordinates intra-organ growth and final tissue size, induction of Reactive Oxygen Species downstream of Eiger/JNK and, as a consequence of apoptosis induction, acts non-cell-autonomously to regulate proliferation rates of adjacent epithelial cells. Our results unravel how local and systemic signals can act concertedly to coordinate growth and proliferation within an organ in order to generate well-proportioned organs and functionally integrated adults.\n\nAuthor SummaryCoordination of growth between the different parts of a given developing organ is an absolute requirement for the generation of functionally integrated structures during animal development. Although this question has fascinated biologists for centuries, the responsible molecular mechanisms have remained so far unknown. In this work, we have used the developing wing primordium of Drosophila to identify the molecular mechanisms and signaling molecules mediating communication between adjacent cell populations upon targeted reduction in growth rates. We first present evidence that activation of Drosophila p53 in the growth-depleted territory induces expression of the fly TNF ligand Eiger which cell autonomously activates the JNK stress signaling pathway. While JNK-dependent expression of the systemic hormone dILP8 reduces growth and final size of the adjacent territories, production of Reactive Oxygen Species downstream of JNK and the apoptotic machinery act locally to regulate proliferation rates in adjacent epithelial cells. Our data reveal how signals acting locally or systemically can regulate cell proliferation and growth independently to accomplish coordination in tissue size and cell number among different parts of an organ in order to give rise to well-proportioned adult structures.\n\nHIGHLIGHTS{checkmark} Dmp53-dependent Eiger expression is required to coordinate intra-organ growth\n{checkmark}Eiger acts through its receptor Grindelwald and JNK signaling to reduce growth and proliferation rates in a non-cell-autonomous manner\n{checkmark}Eiger/JNK-dependent Dilp8 expression coordinates intra-organ growth but not proliferation\n{checkmark}Eiger/JNK activation triggers ROS production\n{checkmark}ROS act non-cell-autonomously to regulate proliferation of adjacent epithelial cells.

developmental biology↗

bioRxiv2019

A high-resolution, chromosome-assigned Komodo dragon genome reveals adaptations in the cardiovascular, muscular, and chemosensory systems of monitor lizards

Monitor lizards are unique among ectothermic reptiles in that they have a high aerobic capacity and distinctive cardiovascular physiology which resembles that of endothermic mammals. We have sequenced the genome of the Komodo dragon (Varanus komodoensis), the largest extant monitor lizard, and present a high resolution de novo chromosome-assigned genome assembly for V. komodoensis, generated with a hybrid approach of long-range sequencing and single molecule physical mapping. Comparing the genome of V. komodoensis with those of related species showed evidence of positive selection in pathways related to muscle energy metabolism, cardiovascular homeostasis, and thrombosis. We also found species-specific expansions of a chemoreceptor gene family related to pheromone and kairomone sensing in V. komodoensis and several other lizard lineages. Together, these evolutionary signatures of adaptation reveal genetic underpinnings of the unique Komodo sensory, cardiovascular, and muscular systems, and suggest that selective pressure altered thrombosis genes to help Komodo dragons evade the anticoagulant effects of their own saliva. As the only sequenced monitor lizard genome, the Komodo dragon genome is an important resource for understanding the biology of this lineage and of reptiles worldwide.

genomics↗

bioRxiv2018

Ask1 and Akt act synergistically to promote ROS-dependent regeneration in Drosophila

The mechanism by which apoptotic cells release signals that induce undamaged neighbor cells to proliferate and regenerate missing parts remains elusive. Oxidative stress originated by dying or damaged cells can be propagated to neighboring cells, which then promote regeneration. We investigated the nature of the stress sensing mechanism by which neighboring cells are recruited. We found that Drosophila apoptosis signal-regulating kinase 1 (Ask1) senses reactive oxygen species (ROS) differently in stressed dying cells and unstressed neighboring cells and this differential sensing is pivotal for tissue repair. In undamaged cells, this activity is attenuated, but not abolished, by Akt1 phosphorylation, which thus acts as a survival signal that results in the tolerable levels of p38 and JNK necessary for regeneration. These observations demonstrate that the non-autonomous activation of the ROS-sensing mechanism by Ask1 and Akt1 in neighboring unstressed cells. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration.\n\nAuthor summaryOne of the early events that occur after tissue damage is oxidative stress production that signals to initiate wound healing and regeneration. Several signaling pathways, such as JNK and p38, respond to oxidative stress and are necessary for regeneration. We decided to explore the mechanism that links the oxidative stress and the activation of these pathways. We used epithelia of Drosophila to genetically direct cell death in specific zones of the tissue as means of experimentally controlled cell damage. We found that the Ask1 protein, which is sensitive to oxidative stress, is a key player in this scenario. Actually it acts as an intracellular sensor that upon damage activates those signaling pathways. However, high activity of Ask1 can be toxic for the cell. This is controlled by Akt, an enzyme dowstream the insulin pathway, with attenuates the activity of Ask1 to tolerable levels. In conclusion, Ask1 and Akt act synergistically to respond to the stress generated after tissue damage and drive regeneration. In other words, we found that the link between oxidative stress and nutrition is key for tissue regeneration.

developmental biology↗