Biology subjects

Bruno, F.

Publications and source records attributed to Bruno, F..

11 recordsLinked to original sources

bioRxiv2026

DNA2Graph enables automated identification of non-linear DNA molecules in electron microscopy

Electron microscopy of spread and rotary-shadowed DNA molecules provides a direct readout of DNA structure and remains uniquely informative for studying replication, recombination and repair intermediates. However, quantitative EM analysis is limited by the rarity of biologically informative structures and by the time required for expert operators to inspect very large numbers of molecules. Automated image acquisition and stitching have increased the scale of EM datasets but have shifted the main bottleneck from image collection to image analysis. Standard image segmentation tools and machine-learning approaches fail to faithfully preserve molecular continuity in EM images of DNA molecules contrasted by rotary shadowing. Here we present DNA2Graph, an open-source software for segmentation of DNA molecules from EM images. Rather than treating segmentation as a purely pixel-level task, DNA2Graph represents each molecule as a spatial graph of nodes and edges. It applies dedicated graph-based error-correction algorithms that repair signal interruptions and spurious connections introduced during segmentation, while enforcing the biological priors of continuity and thinness. DNA2Graph classifies molecules as linear or non-linear, thereby converting large imaging datasets into focused lists of candidate structures for operator review. We validated DNA2Graph on two genomic DNA datasets: a structure-poor, non-enriched human sample and a structure-rich yeast sample. DNA2Graph reduced the number of molecules requiring operator review by 45-fold and 7-fold, respectively. In addition, DNA2Graph-assisted review slightly improved the operators structure-detection sensitivity compared to unaided image review. The software also enables automated length measurement of individual DNA molecules and generates machine-readable outputs for downstream quantitative and computational analyses. DNA2Graph does not require training on manually annotated data, can run on a personal computer and can be adapted across experimental preparations through interpretable parameters.

molecular biology↗

bioRxiv2026

Multilocus microsatellite typing (MLMT) reveals high genetic diversity of Leishmania infantum strains causing tegumentary leishmaniasis in northern Italy

BackgroundTegumentary leishmaniasis (TL) caused by Leishmania infantum has re-emerged in northern Italy, raising questions about the genetic diversity and population structure of circulating parasites and their potential role in shaping different clinical outcomes. Methodology/Principal findingsMultilocus microsatellite typing (MLMT) based on 15 polymorphic loci was applied to 44 L. infantum strains obtained from TL cases diagnosed between 2013 and 2024 in the Emilia-Romagna region. These strains were compared with sympatric isolates from VL cases, dogs and sand flies. MLMT revealed a considerable genetic variation among TL-associated strains, with 43 distinct multilocus genotypes identified. Population structure analyses using Bayesian clustering, multivariate approaches and phylogenetic reconstruction consistently identified three highly differentiated genetic populations (Fst >0.25). TL strains were divided into two main populations: one shared with VL-associated strains (PopB; 9/44) and a second population found exclusively among TL cases (PopC; 28/44). In contrast, the canine-associated population (PopA) showed no overlap with TL cases in this region. Populations also displayed divergent heterozygosity patterns, as indicated by positive and negative Fis values. Conclusions/SignificanceThese findings revealed previously unknown diversity within L. infantum in the study area and demonstrated that inclusion of tegumentary strains is essential to uncover hidden components of parasite population structure. The identification of a TL-associated population supports the existence of multiple evolutionary pathways and emphasises the importance of integrated One Health surveillance, which combines data from humans, animal hosts and vectors to improve understanding of the epidemiology of leishmaniasis in Italy. Author summaryLeishmania infantum is a parasite transmitted to humans through the bite of infected insect vectors. It can cause different forms of leishmaniasis, ranging from a systemic disease known as visceral leishmaniasis to a less common form that affects the skin and mucous membranes, called tegumentary leishmaniasis. Dogs are the main reservoir of the parasite and play a key role in maintaining its circulation in endemic areas. In recent years, cases of tegumentary leishmaniasis have re-emerged in northern Italy. This unexpected increase has raised questions about how the parasite is spreading and whether genetic differences among the parasites could explain these new patterns. To explore this, we examined parasitic DNA obtained from tegumentary leishmaniasis cases and compared it with DNA from patients with visceral leishmaniasis, from dogs and insect vectors from the same area. By examining multiple genetic markers, we found that parasites causing the tegumentary form of the disease are genetically diverse and belong to different groups. Notably, one parasite group was found only in cases of tegumentary leishmaniasis and not in visceral infections nor in infected dogs, suggesting that some parasite lineages may be more closely associated with skin and mucosal disease. Overall, our findings show that studying parasites from cutaneous and mucosal lesions provides important information that would otherwise remain hidden. By combining data from humans, animals and insect vectors, our study highlights the importance of integrated surveillance systems for improving our understanding of disease spread and supporting effective public health strategies.

genetics↗

bioRxiv2025

The microcephaly-associated protein YIPF5 differentially regulates ER-export

YIPF5 is a small ER-membrane protein implicated in ER-Golgi transport. Mutations in YIPF5 cause MEDS2 (microcephaly with simplified gyral pattern, epilepsy, and neonatal diabetes syndrome), a fatal disorder manifesting in early childhood. We demonstrate that YIPF5 is involved in ER export of a subset of proteins, including cargoes of the ER-export receptor SURF4, with which it directly interacts. In YIPF5 knockout cells, we observe a shift in the cell surface and secretome composition, marked by reduced neuronal adhesion molecules and increased secretion of ER chaperones influencing cell migration. YIPF5 depletion enhances cell migration in a wound-healing assay and alters SURF4 localization, causing elongated ERGIC53- and Rab1-positive tubules from COPII-labeled ER exit sites. Kinetic analysis suggests that YIPF5 negatively regulates SURF4-mediated ER export. In utero knockdown of Yipf5 in embryonic mouse brains induces premature neuronal migration and abnormal neuronal morphology. These findings suggest that YIPF5 and SURF4 coordinate ER export, and disruption may underlie cortical development defects leading to microcephaly.

cell biology↗

bioRxiv2025

A subset of Haematopoietic Stem Cells resists Plasmodium infection-induced stress by uncoupling interferon sensing and metabolic activation

Hematopoietic stem cells (HSCs) sustain lifelong haematopoiesis as their progeny differentiate into all blood cell lineages. Homeostatic HSCs are mostly quiescent and only rarely divide, however their proliferation and differentiation rates can be modulated by external factors. Acute and chronic infections from a wide range of pathogens are known to challenge HSCs at the population level, being forced to respond to inflammation-mediated organismal demand to replenish the myeloid cell pool. However, less is known about the degree of heterogeneity in the HSCs response to inflammation at the single cell level. Here, using a natural murine malaria model and an NHS-ester biotin dilution assay we identify two subsets of HSCs, BiotinLo and BiotinHi, with distinct proliferation kinetics. Using combined functional, single-cell transcriptomics and phenotypic analyses, we uncover that BiotinHi HSCs remain highly functional despite expressing strong interferon response signatures. These infection-resistant HSCs express high levels of MHC II and are metabolically distinct from the remaining HSCs as they maintain less active mitochondria. These findings demonstrate that a likely reserve pool of HSCs remains highly functional during Plasmodium infection not because cells are shielded, but because they maintain a stemness associated metabolic profile despite effectively sensing inflammation.

cell biology↗

bioRxiv2025

Drug-Resistance Biomarkers in Leishmania infantum through Nanopore-Based Detection of Aneuploidy and Gene Copy Number Variations with LeishGenApp

BackgroundDrug-resistant strains of Leishmania infantum challenge the effectiveness of treatments for clinical leishmaniosis and may lead to more frequent relapses. Copy number variation (CNV) at specific genetic loci is associated with drug resistance and virulence, but information about its prevalence in endemic regions is limited. This study examines the drug resistance and virulence status of Leishmania strains in human and canine isolates from the Mediterranean region. MethodsForty-eight Leishmania infantum isolates were whole-genome sequenced with nanopore long reads, followed by de novo assembly. We analyzed chromosomal aneuploidies and gene copy number variation in loci linked to drug resistance and virulence in Leishmania, alongside the genomic structure and rearrangements responsible for these variations. ResultsComplete genomes were de novo assembled for 35 L. infantum isolates (22 from dogs and 13 from humans), revealing significant chromosomal variability. We assessed copy number variation for 22 potential biomarkers: 15 genes related to drug resistance to first-line drugs (METK for allopurinol; LdSMT for amphotericin B; AQP1 and H-locus for antimonials; LdMT, LdRos3, and MSL for miltefosine; PPM for paramomycin) and seven genes related to virulence (lipophosphoglycan and proteophosphoglycan biosynthesis, and the Lack protein). Drug-resistance biomarkers were identified in 80% of the isolates. Canine strains primarily showed resistance to allopurinol and antimonials, while human isolates exhibited a broader resistance spectrum, especially to antimonials and paromomycin. The co-occurrence of resistance biomarkers was common, especially for allopurinol and antimonial resistance. Distinct mechanisms underlie the observed copy number variations. Virulence-associated genes were less variable among isolates. ConclusionsThe prevalence of drug-resistance biomarkers in Leishmania infantum strains from the Mediterranean region, as revealed by this study, underscores the critical need for routine resistance surveillance in managing clinical leishmaniosis. These findings not only inform current clinical practice but also pave the way for more effective management strategies in the future.

genetics↗

bioRxiv2024

Differential responses and recovery dynamics of HSPC populations following Plasmodium chabaudi infection.

Severe infections such as malaria are on the rise worldwide, driven by both climate change and increasing drug-resistance. It is therefore paramount that we better understand how the hosts organism responds to severe infection. Hematopoiesis is particularly of interest in this context because hematopoietic stem and progenitor cells (HSPCs) maintain the turnover of all blood cells, including all immune cells. Severe infections have been widely acknowledged to affecting HSPCs, however this disruption has been mainly studied during the acute phase, and the process and level of HSPC recovery remains understudied. Using a self-resolving model of natural rodent malaria, infection by Plasmodium chabaudi, here we systematically assess HSPCs acute response and recovery upon pathogen clearance. We demonstrate that during the acute phase of infection the most quiescent and functional stem cells are depleted, multipotent progenitor compartments are drastically enlarged, and oligopotent progenitors virtually disappear, underpinned by dramatic, population-specific and sometimes unexpected changes in proliferation rates. HSPC populations return to homeostatic size and proliferation rate again through specific patterns of recovery. Overall, our data demonstrate that HSPC populations adopt different responses to cope with severe infection and suggest that the ability to adjust proliferative capacity becomes more restricted as differentiation progresses.

developmental biology↗

bioRxiv2024

SIRPalpha+ PD-L1+ bone marrow macrophages aid AML growth by modulating T cell function

Acute myeloid leukemia (AML) continues to have a poor prognosis due to its ability to relapse following initial response to chemotherapy. While immunotherapies hold the promise to revolutionize cancer treatment, AML has been particularly challenging to target. It is therefore important to better understand the relationship between AML cells and immune cells within the bone marrow (BM) microenvironment, where this disease grows. Here we focus on non-malignant BM macrophages, and using a combination of intravital microscopy, flow cytometry, transcriptomics and functional analyses we identify a subpopulation of immunomodulatory BM macrophages (IMMs) with a unique profile and function during AML progression. While the majority of macrophages are already being lost at early infiltration, IMMs are locally enriched. They are capable of efferocytosis and support AML growth through inhibition of T cells. Enrichment of IMMs in the BM of patients developing early relapse indicates that future development of interventions that target IMMs development and function may improve AML patients outcome.

cancer biology↗

bioRxiv2024

IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport

Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 or the presence of the pathogenic p.L78P mutation results in the retention of specific cell-surface receptors and secreted proteins crucial for neuronal migration within the ER. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 is compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome.

cell biology↗

bioRxiv2023

Genomic and epidemiological evidence for the emergence of a putative L. donovani/L. infantum hybrid with unusual epidemiology in Northern Italy

Leishmania (L.) infantum is the main causative agent of animal and human leishmaniasis in the Mediterranean basin. Despite its clinical significance, little is known on the genetic diversity of L. infantum parasites circulating in Italy. Here, we apply a comparative genomics approach on seven L. infantum isolates from different hosts (human, dog, cat, marten) and geographic regions (Emilia-Romagna, Sicily, Sardinia) as a first attempt to explore the breadth of parasite genetic heterogeneity in Italy. We revealed important genome instability at karyotype levels, with each isolate presenting a unique aneuploidy profile. Read depth analysis further identified strain-specific changes in gene dosage, which affected important virulence factors most of which are encoded by multi-copy gene arrays, such as amastins or surface antigen-like proteins. SNP-based clustering analysis of these genomes together with over 80 publicly available L. infantum and L. donovani genomes placed the Italian isolates into three geographically distinct clusters, with two isolates grouping with Spanish strains, two isolates grouping with Tunisian strains, and three isolates clustering with putative L. infantum/L. donovani hybrids isolated in Cyprus. As judged by microsatellite profiling of 73 isolates from dogs, sand flies and VL cases, these hybrid isolates are representative of a sub-population of parasites circulating in northeastern Italy that preferentially infects humans, but not dogs. In conclusion, our data uncover a remarkable heterogeneity of L. infantum isolates that indicates different geographic origin, including a novel hybrid-like genotype associated with an unusual infection pattern, placing Italy at the crossroad of Leishmania infection in the Mediterranean region.

genomics↗

bioRxiv2022

Engineered model of t(7;12)(q36;p13) AML recapitulates patient-specific features and gene expression profiles

Acute myeloid leukaemia carrying the translocation t(7;12)(q36;p13) is an adverse-risk leukaemia uniquely observed in infants. Despite constituting up to 30% of cases in under 2-year-olds, it remains poorly understood. Known molecular features are ectopic overexpression of the MNX1 gene and generation of a fusion transcript in 50% of patients. Lack of research models has hindered understanding of t(7;12) biology, which has historically focused on MNX1 overexpression rather than the cytogenetic entity itself. Here, we employed CRISPR/Cas9 to generate t(7;12) in the human K562 cell line, and in healthy CD34+ haematopoietic progenitors where the translocation was not sustained in long-term cultures or through serial replating. In contrast, in K562 cells, t(7;12) was propagated in self-renewing clonogenic assays, with sustained myeloid bias in colony formation and baseline depletion of erythroid signatures. Nuclear localisation analysis revealed repositioning of the translocated MNX1 locus to the interior of t(7;12)-harbouring K562 nuclei - a known phenomenon in t(7;12) patients which associates with ectopic overexpression of MNX1. Crucially, the K562-t(7;12) model successfully recapitulated the transcriptional landscape of t(7;12) patient leukaemia. In summary, we engineered a clinically-relevant model of t(7;12) acute myeloid leukaemia with the potential to unravel targetable molecular mechanisms of disease.

cancer biology↗

bioRxiv2021

Antiparasitic effect of stilbene and terphenyl compounds against Trypanosoma cruzi parasites

BackgroundChagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since Nifurtimox was introduced more than fifty years ago and is considered very aggressive and may cause several adverse effects. Currently, this drug has severe limitations, including high frequency of undesirable side effects and limited efficacy and availability and the research to discover new drugs for the treatment of Chagas disease is imperative. Many drugs available in the market are natural products as found in nature or compounds designed based on the structure and activity of these natural products. Methodology/Principal FindingsThis study evaluated the in vitro antiparasitic activity in T. cruzi epimastigotes and intracellular amastigotes of a series of stilbene and terphenyl compounds previously synthesized. The action of the most selective compounds has been investigated by flow cytometry analysis to evaluate the mechanism of cell death. The ability to induce apoptosis or caspase-1 inflammasome were assayed in macrophages infected with T. cruzi after treatment comparing with Nifurtimox. Conclusions/SignificanceThe stilbene ST18 was the most potent compound of the series. It was slightly less active than Nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase of parasites positive to Annexin V and monodansylcadaverine. Moreover, ST18 induced the activation in infected macrophages of caspase-1, a conserved enzyme which plays a main role in controlling parasitemia, host survival, and the onset of adaptive immune response in Trypanosoma infection. The antiparasitic activity of ST18 together to its ability to activate caspase-1 in infected macrophages and its low toxicity on normal cells makes this compound interesting for further clinical investigations. Author SummaryChagas disease is a pathology caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since benznidazole and Nifurtimox were introduced more than fifty years ago. However, these drugs have severe limitations and the research to discover new drugs for the treatment of Chagas disease is imperative. We evaluated the in vitro antiparasitic activity in T. cruzi epimastigotes of a series of stilbene and terphenyl compounds previously synthesized. The stilbene ST18 was the most potent compound of the series. It was slightly less active than nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase of parasites positive to Annexin V and monodansylcadaverine. Moreover, this compound induced the activation in infected macrophages of caspase-1, an evolutionarily conserved enzyme which plays a main role in controlling parasitemia, host survival, and the onset of adaptive immune response in T. cruzi infection. The antiparasitic activity of ST18 together to its ability to activate caspase-1 in infected macrophages and its low toxicity on normal cells makes this compound interesting for further clinical investigations.

microbiology↗