Biology subjects

Castelli, M.

Publications and source records attributed to Castelli, M..

At least 19 recordsLinked to original sources

bioRxiv2026

SYMBIO-SCAN, a full-length 16S rRNA gene-based workflow optimized for bacterial symbiont detection and discovery from long read sequencing

Bacterial endosymbionts are often unculturable, making sequencing-based approaches essential for their discovery and characterization. Here we developed SYMBIO-SCAN, a dedicated workflow for recovering high-quality nearly full-length 16S rRNA gene sequences of symbionts from long reads, which provided an adequate discriminatory power for identification and for accurate phylogenetic analyses. After validation on a mock community, we applied this workflow to 30 beetle (Coleoptera) specimens from 10 species. Beetles were selected since they host a wide variety of bacterial symbionts, able to influence host physiology, ecology, population dynamics, and evolution. Despite their importance, most of these symbionts remain completely unknown or poorly characterized. By the novel approach, we obtained 80 high-quality sequences belonging to diverse bacterial lineages, mostly Rickettsiales and Enterobacterales. Putative reproductive manipulators were among the most frequently detected symbionts, including Wolbachia, Rickettsia, "Candidatus Lariskella", Arsenophonus, and Spiroplasma. Moreover, three putative novel genera and one additional putative novel species were identified, including a possible Enterobacterales nutritional symbiont of the weevil Holotrichapion pisi. Overall, our results support that full-length 16S rRNA gene amplicon sequencing combined with tailored bioinformatic analyses provide a quick, cost-effective, and robust approach for investigating the diversity of host-associated bacterial symbionts, also allowing the discovery of entirely novel lineages.

bioinformatics↗

bioRxiv2026

A shared manifold for scalable temporal representations in self-paced timing

Adaptive behaviour often relies on tracking the passage of time, yet how distinct brain regions generate coherent temporal representations remains unclear. Using a self-paced interval timing task in mice, we show that heterogeneous single-neuron temporal firing profiles distributed across regions are organized within a shared ring manifold. Within this low-dimensional space, population activity evolves along a common trajectory across different intervals and encodes elapsed time in a relative reference frame. Different durations are not represented by separate neural states, but by modulation of traversal speed. These scalable dynamics arise from coordinated population-wide co-scaling of single-neuron activity and support trial-by-trial adjustments in timing behaviour. A cross-regional assembly of start neurons predicts, at interval onset, upcoming waiting duration and behavioural adjustments, linking initial population states to trajectory evolution. Together, these findings identify population traversal of a shared activity manifold as a mechanism for scalable temporal representation across brain regions.

neuroscience↗

bioRxiv2026

Environmental DNA reveals long-term persistence of a Midichloria-like bacterium in a rainbow trout aquaculture and links Ichthyopthirius multifiliis with the red mark syndrome

Red Mark Syndrome (RMS) is a widespread skin disease affecting rainbow trout (Onchorhynchus mykiss). It provokes substantial economic losses in aquaculture, and is putatively caused by a Rickettsiales bacterium named Midichloria-like organism (RMS-MLO), which is strongly associated with RMS lesions. However, RMS-MLO ecology and epidemiology in aquaculture systems remain poorly understood. In this study, we analysed environmental DNA to monitor the presence of RMS-MLO and its putative vector Ichthyophthirius multifiliis in a trout farm in Northern Italy over one year. Water and sediment samples were monthly collected from multiple water tanks. RMS-MLO was consistently detected by PCR throughout the study in all trout-containing tanks, both in water and sediment samples, but never in the trout-free inflow tank. We did not observe an increase in RMS-MLO abundance during the single RMS outbreak recorded nor in relation with the co-occurrence of I. multifiliis. Our findings indicate a long-term persistence of RMS-MLO in the aquaculture, possibly as a consequence of infections with low prevalence or abundance, rather than its entry from the external environment at the time of RMS outbreaks. Additionally, hints were recorded for a potential role of free-living aquatic microeukaryotes as additional occasional reservoirs. In contrast, I. multifiliis was negatively related with RMS-MLO, while it significantly increased in abundance during the RMS outbreak, particularly in the inflow tank. This supports that, rather than a stable reservoir, I. multifiliis may act as a facilitator of RMS outbreaks, which might indeed be triggered by the entry of this parasite in trout farms.

microbiology↗

bioRxiv2026

An introgressed galectin-like protein is a candidate driver of the human tropism in the intestinal parasite Cryptosporidium

Cryptosporidium spp. are protozoan parasites responsible for diarrheal diseases. In humans, cryptosporidiosis is predominantly caused by the human-specific Cryptosporidium hominis and by Cryptosporidium parvum. This second species has been classically reported as zoonotic, with a host preference for ruminants. However, the recently described subspecies C. parvum anthroponosum has been found to be restricted to humans. Here, we generated novel whole genome sequences from West African samples of C. p. anthroponosum, and analyzed them together with all those already available, originating from East Africa, Europe, North America and Asia. Phylogenomics showed that all C. p. anthroponosum isolates are strongly clustered together, forming the sister clade of the zoonotic C. parvum representatives. The phylogenetic variations within C. p. anthroponosum did not present a clear geographic structure, consistent with C. hominis, primarily transmitted in humans. To elucidate the evolution of host species adaptation in C. p. anthroponosum, we then investigated genetic exchanges with C. hominis, detecting an ancestral introgression present in all C. p. anthroponosum isolates. This introgression involved a single gene, encoding for an extracellular galectin-like protein, which we predicted with high confidence to form a protein complex with the human insulin-degrading enzyme, a key metabolic regulator. Considering the role of host insulin metabolism in the proliferation of parasites as well as its known intrinsic differences between humans and ruminants, this molecular interaction could represent a plausible mechanism for an important role of the galectin-like protein in host-parasite interactions and in the host specificity of C. p. anthroponosum.

genomics↗

bioRxiv2025

Evolution and comparative genomics of tick-associated endosymbionts: insights into metabolic pathways and historical biogeographic patterns

AbstractTicks (Ixodida) are the second most important vectors of infectious diseases in vertebrates, after mosquitoes. They also maintain mutualistic relationships with bacteria, such as endosymbionts that provide essential B vitamins absent in their blood diet. The most studied endosymbionts belong to the genera Coxiella, Midichloria, and Francisella. Francisella includes endosymbionts (FE), pathogens (FP), putative intermediates (FI), and free-living (FL) strains, making them valuable for evolutionary and comparative genomics. In this study, total DNA from six adult female ticks of the genera Hyalomma and Amblyomma was sequenced to obtain new FE genomes. Additionally, two deep metagenomes from public data were assembled, resulting in a dataset of 22 Francisella strains. This dataset was used to reconstruct a phylogenomic framework and compare vitamin biosynthesis and virulence pathways. An MLST-based dense phylogeny was also built to explore biogeographic patterns. The resulting phylogenomic tree shows FE form a monophyletic group derived from FP, possibly due to historical biogeography or recent horizontal transfers. Comparative analyses reveal that FE retain key metabolic pathways while losing nonessential ones, reflecting a selective genome reduction. These results advance our understanding of symbiont evolution in a changing world, revealing molecular adaptations that underpin tick- symbiont relationships and offering genomic insights with potential applications for disease control.

evolutionary biology↗

bioRxiv2025

Hippocampal Ripple Diversity organises Neuronal Reactivation Dynamics in the Offline Brain

SO_SCPLOWUMMARYC_SCPLOWHippocampal ripples are highly synchronized neuronal population patterns reactivating past waking experiences in the offline brain. Whether the level, structure, and content of ripple-nested activity are consistent across consecutive events or are tuned in each event remains unclear. By profiling individual ripples using laminar currents in the mouse hippocampus during sleep/rest, we identified Radsink and LMsink ripples featuring current sinks in stratum radiatum versus stratum lacunosum-moleculare, respectively. These two ripple profiles recruit neurons differently. Radsink ripples integrate recent motifs of waking coactivity, combining superficial and deep CA1 principal cells into denser, higher-dimensional patterns that undergo hour-long stable reactivation. In contrast, LMsink ripples contain core motifs of prior coactivity, engaging deep cells into sparser, lower-dimensional patterns that undergo a reactivation drift, gradually updating their pre-structured content for recent wakefulness. We propose that ripple-by-ripple diversity instantiates parallel reactivation channels for stable integration of recent wakefulness and flexible updating of prior internal representations.

neuroscience↗

bioRxiv2025

Deciphering the dynamics of Cyanobacteria-Phage in a natural lake: Insights from a decade-long investigation

Phages - viruses that infect bacteria - are often seen as key players in bacterial community dynamics and the ecosystem services those communities support. However, much of our understanding of phage-bacteria interactions comes from in vitro studies, which provide limited insights into how these interactions occur in natural environments. In this study, we used cyanobacterial blooms as a model microbial community to evaluate the potential role of phages in driving cyanobacterial populations. These blooms are characterized by massive and usually rapid accumulation of cyanobacterial biomass and occur worldwide, threatening aquatic systems. The frequency and intensity of cyanobacterial blooms are increasing over time, likely due to human activities, and, until now, the role of predators like phages has remained unclear. We used a deep amplicon sequencing approach targeting the g20 capsid gene to profile the cyanophage community in eutrophic Lake Champlain over time, comparing their dynamics and diversity with bacterial communities and examining their associations. We evaluated whether phages simply followed bacterial dynamics, with limited impact on bacterial composition, or instead whether they played an active role in drving bacterial community structure. We found that phages exhibited similar dynamics to their potential bacterial hosts and shared environmental niches. However, we also observed strong differences specific to the phage community, such as inter-annual variation and an increase in Shannon diversity over time. Phage-bacteria and phage-cyanobacteria co-variance uncovered potential interactions that resulted in strongly modular networks. However, while the network structure remained modular, the composition of the modules differed significantly between environmental and temporal conditions. Lastly, viral phylogeny partially explained phage-bacteria co-variance, but only for a small proportion of bacterial ASVs. Our results indicate that phage-bacterial interactions are partly genetically structured and vary within modules across environmental conditions.

ecology↗

bioRxiv2024

Disulfide-mediated tetramerization of TRAP1 fosters its antioxidant and pro-neoplastic activities.

The mitochondrial chaperone TRAP1 exerts a protective function in cells exposed to diverse stress conditions in both physiological and pathological contexts. In cancer cells, it contributes to neoplastic progression ensuing metabolic rewiring and protection from oxidative insults. TRAP1 works as a homodimer, but recent evidence has indicated that it can form tetramers whose functional effects remain elusive. Here, we find that TRAP1 forms redox-sensitive tetramers via disulfide bonds involving two critical cysteine residues, C261 and C573. TRAP1 tetramerization is elicited by oxidative stress and abrogated upon expression of the double C261S/C573R mutant. In cancer contexts, the expression of the TRAP1 C261S/C573R mutant is unable to inhibit the activity of its client succinate dehydrogenase and to confer protection against oxidative insults, and it hampers invasiveness of aggressive sarcoma cells. Our data indicate that TRAP1 undergoes tetramerization in response to oxidative stress and identify C261 and C573 as critical for TRAP1 structural rearrangement and for functions.

cell biology↗

bioRxiv2023

Offline hippocampal reactivation during dentate spikes supports flexible memory

Stabilizing new memories requires coordinated neuronal spiking activity during sleep. Hippocampal sharp-wave ripples (SWRs) in the Cornu Ammonis (CA) region, and dentate spikes (DSs) in the dentate gyrus (DG) are prime candidate network events supporting this offline process. SWRs have been studied extensively but the contribution of DSs remains unclear. By combining triple-(DG-CA3-CA1) ensemble recordings and closed-loop optogenetics in mice we show that, like SWRs, DSs synchronize spiking across DG and CA principal cells to reactivate population-level patterns of neuronal coactivity expressed during prior waking experience. Notably, the population coactivity structure in DSs is more diverse and higher-dimensional than that seen during SWRs. Importantly, suppressing DG granule cell spiking selectively during DSs impairs subsequent flexible memory performance in a multi-object recognition task and associated hippocampal patterns of neuronal coactivity. We conclude that DSs constitute a second offline network event central to hippocampal population dynamics serving memory-guided behavior.

neuroscience↗

bioRxiv2023

Mitochondrial complex I deficiency stratifies idiopathic Parkinson's disease

Idiopathic Parkinsons disease (iPD) is believed to have a heterogeneous pathophysiology, but molecular disease subtypes have not been identified. Here, we show that iPD can be stratified according to the severity of neuronal respiratory complex I (CI) deficiency, and identify two emerging disease subtypes with distinct molecular and clinical profiles. The CI deficient (CI-PD) subtype accounts for approximately a fourth of all cases, and is characterized by anatomically widespread neuronal CI deficiency, a distinct cell type-specific gene expression profile, increased load of neuronal mtDNA deletions, and a predilection for non-tremor dominant motor phenotypes. In contrast, the non-CI deficient (nCI-PD) subtype exhibits no evidence of mitochondrial impairment outside the dopaminergic substantia nigra and has a predilection for a tremor dominant phenotype. These findings constitute a step towards resolving the biological heterogeneity of iPD with implications for both mechanistic understanding and treatment strategies.

neuroscience↗

bioRxiv2023

Comparative genomics reveals the emergence of an outbreak-associated Cryptosporidium parvum population in Europe and its spread to the USA

The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g. IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water-and food-borne outbreaks. In this study, we generated new whole genome sequence (WGS) data from 123 human-and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China and the USA (n=72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analysed a dataset from 141 isolates from the zoonotic groups IIa (n=119) and IId (n=22). Based on 28,047 high quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: isolates from China (IId) and Egypt (IIa and IId) formed population 1, a minority of European isolates (IIa and IId) formed population 2, while the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the USA (IIa) clustered in population 3. Based on analyses of the population structure, population genetics and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the UK, reach the USA where it also expanded. In addition, genetic exchanges between different populations led to the formation of mosaic genomes. The reason(s) for the successful spread of population 3 remained elusive, although genes under selective pressure uniquely in this population were identified.

genomics↗

bioRxiv2023

Decrypting the languages of allostery in membrane-bound K-Ras4B using four complementary in silico approaches

Proteins are evolved molecular machines whose diverse biochemical functions can be dynamically regulated by allostery, through which even faraway protein residues can conformationally communicate. Allostery can express itself in different ways, akin to different "languages": allosteric control pathways predominating in an unperturbed protein are superseded by others as soon as a perturbation arises--e.g., a mutation--that alters its function (pathologically or not). Accurately modeling these often-unintuitive phenomena could therefore help explain functional changes in a specific protein whenever they are unclear. Unbiased molecular dynamics (MD) simulations are a possibility; however, since allostery can operate at longer timescales than those accessible by MD, simulations require integration with a reliable method able to, e.g., detect regions of incipient allosteric change or likely perturbation pathways. Several methods exist but are typically applied singularly: we argue their joint application, in a "multilingual" approach, could significantly enrich this picture. To prove this, we perform unbiased MD simulations ([~]100 {micro}s) of the widely studied, allosterically active oncotarget K-Ras4B, solvated and embedded in a phospholipid membrane, proceeding to decrypt its allostery using four showcase "languages": Distance Fluctuation analysis and the Shortest Path Map capture allosteric communication hotspots at equilibrium; Anisotropic Thermal Diffusion and Dynamical Non-Equilibrium MD simulations assess them once the GTP that oncogenically activates K-Ras4B is, respectively, either superheated or hydrolyzed. "Languages" provide a uniquely articulate, mutually coherent, experimentally consistent picture of allostery in K-Ras4B. At equilibrium, pathways stretch from the membrane-embedded hypervariable region all the way to the active site, touching known flexible allosteric "switches" and proposed pockets. Upon GTP cleavage/perturbation, known to inactivate K-Ras4B, allosteric signals most reverberate on switches and interfaces that recruit effector proteins. Our work highlights the benefits of integrating different allostery detection methods with unbiased MD.

biochemistry↗

bioRxiv2022

How Aberrant N-Glycosylation Can Alter Protein Functionality and Ligand Binding: an Atomistic View

Protein assembly defects due to enrichment of aberrant conformational variants of proteins are emerging as a new frontier in therapeutics design. Understanding, atomistically, structural elements that remodel the energy landscape of proteins, with the consequence of rewiring the conformational dynamics of proteins and pathologically perturbing functionally-oriented ensembles, is key for development of inhibitors. This is particularly relevant for molecular chaperones, hub proteins for the assembly of large multiprotein complexes, where enrichment of aberrant conformers can have a large impact on the cellular proteome, and in turn, on phenotypes. Here, we integrate computational and experimental tools to unveil how N-glycosylation of specific residues in glucose-regulated protein 94 (GRP94) modulates internal dynamics and alters the conformational fitness of regions fundamental for interaction with the nucleotide and synthetic ligands, and impacts substructures dedicated to recognition of interacting proteins. We show how N-glycosylation plays an active role in modulating the energy landscape of the protein, with specific glycosylation patterns determining specific functionally-oriented dynamic signatures. Our results provide support for leveraging the structural-dynamics knowledge on distinct glycosylation variants to design molecules targeting GRP94 disease-associated conformational states and assemblies. Since glycosylation is the most abundant form of post-translational modification, our results and mechanistic models can readily be transferred to other targets and contexts for cancers and other diseases.

biochemistry↗

bioRxiv2022

Host association and intracellularity evolved multiple times independently in the Rickettsiales

The order Rickettsiales (Alphaproteobacteria) encompasses multiple diverse lineages of host-associated bacteria, including pathogens, reproductive manipulators, and mutualists. In order to understand how intracellularity and host association originated in this order, and whether they are ancestral or convergently evolved characteristics, we built an unprecedentedly large and phylogenetically-balanced dataset that includes de novo sequenced genomes and an accurate selection of published genomic and metagenomic assemblies. We performed detailed functional reconstructions that clearly indicated "late" and parallel evolution of obligate host-association and intracellularity in different Rickettsiales lineages. According to the depicted scenario, multiple independent series of horizontal acquisitions of transporters led to the progressive loss of biosynthesis of nucleotides, amino acids and other metabolites, producing distinct conditions of host-dependence. Coherently, each clade experienced a different pattern of evolution of the ancestral arsenal of interaction apparatuses, including development of specialised effectors involved in the lineage-specific mechanisms of host cell adhesion/invasion and intracellularity.

microbiology↗

bioRxiv2022

Discovering host protein interactions specific for SARS-CoV-2 RNA genome

SARS-CoV-2, a positive single-stranded RNA virus, interacts with host cell proteins throughout its life cycle. These interactions are necessary for the host to recognize and hinder the replication of SARS-CoV-2. For the virus, to translate, transcribe and replicate its genetic material. However, many details of these interactions are still missing. We focused on the proteins binding to the highly structured 5 and 3 end regions of SARS-CoV-2 RNA that were predicted by the catRAPID algorithm to attract numerous proteins, exploiting RNA-Protein Interaction Detection coupled with Mass Spectrometry (RaPID-MS) technology. The validated interactors, which agreed with our predictions, include pseudouridine synthase PUS7 that binds to both ends of the viral RNA. Nanopore direct-RNA sequencing confirmed that the RNA virus is heavily modified, and PUS7 consensus regions were found in both SARS-CoV-2 RNA end regions. Notably, a modified site was detected in the viral Transcription Regulatory Sequence - Leader (TRS-L) and can influence the viral RNA structure and interaction propensity. Overall, our data map host protein interactions within SARS-CoV-2 UTR regions, pinpointing to a potential role of pseudouridine synthases and post-transcriptional modifications in the viral life cycle. These findings contribute to understanding virus-host dynamics and may guide the development of targeted therapies.

molecular biology↗

bioRxiv2022

The origin and evolution of mitochondrial tropism in Midichloria bacteria

Midichloria are intracellular bacterial symbionts of ticks. Some representatives of this genus have the unique capability to colonize mitochondria in the cells of their hosts. Hypotheses on the nature of this interaction have proven difficult to test, partly due to a lack of data. Indeed, until now, mitochondrial tropism information and genomes were available only for symbionts of three and two tick host species, respectively. Here we analyzed the mitochondrial tropism of three additional Midichloria and sequenced nine novel genomes, showing that the tropism is pnon-monophyletic, either due to losses of the trait or multiple parallel acquisitions. Comparative genome analyses support the first hypothesis, as the genomes of non-mitochondrial symbionts appear to be reduced subsets of those capable of colonizing the organelles. We detect genomic signatures of mitochondrial tropism, showing a set of candidate genes characteristic of the strains capable of mitochondrial colonization. These include the type IV secretion system and the flagellum, which could allow the secretion of unique effectors, direct interaction with, or invasion of the mitochondria. Other genes, including putative adhesion molecules, proteins possibly involved in actin polymerization, cell wall and outer membrane proteins, are only present in mitochondrial symbionts. The bacteria could use these to manipulate host structures, including mitochondrial membranes, in order to fuse with the organelles or manipulate the mitochondrial network.

evolutionary biology↗

bioRxiv2021

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

genomics↗

bioRxiv2021

SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 - the closest bat relative - spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.

microbiology↗