Biology subjects

Rossato, M.

Publications and source records attributed to Rossato, M..

16 recordsLinked to original sources

bioRxiv2026

Functional validation of allele-specific LMNB1 silencing in patient-derived astrocytes as a therapeutic option for Autosomal Dominant Leukodystrophy

Adult-onset Autosomal Dominant Leukodystrophy (ADLD) is a rare fatal leukodystrophy caused by increased LMNB1 gene dosage, most commonly resulting from duplication of the LMNB1 locus. Because ADLD is a gene dosage disorder, selective reduction of pathological LMNB1 expression represents a rational therapeutic strategy. Although allele-specific RNA interference has previously been shown to lower LMNB1 levels in patient-derived fibroblasts and directly reprogrammed neurons, its therapeutic effects have not been evaluated in disease-relevant human glial cells or using functional efficacy endpoints. Here, we established human induced pluripotent stem cell-derived astrocytes from ADLD patients as a human glial model in which to validate allele-specific LMNB1 silencing across molecular, cellular, and functional readouts. ADLD astrocytes recapitulated increased LMNB1 expression and characteristic nuclear abnormalities and displayed transcriptional alterations affecting extracellular matrix organization, calcium homeostasis, metabolism and RNA processing. Functionally, these cells also exhibited functional phenotypes suitable for therapeutic evaluation: astrocyte-conditioned medium impaired the viability of both murine and human oligodendroglial cultures, while conditioned-medium and direct astrocyte-seeding paradigms revealed impaired post-lesion myelin recovery in lysolecithin-treated cerebellar organotypic slices. Allele-specific LMNB1 silencing restored physiological LMNB1 levels, corrected nuclear abnormalities, attenuated astrocyte-mediated oligodendroglial toxicity, improved post-lesion myelin recovery, and was associated with selective transcriptional programs associated with extracellular support and cholesterol metabolism. Together, these findings provide molecular, cellular, and functional validation of allele-specific LMNB1 dosage correction in patient-derived human astrocytes and offer key support for LMNB1-lowering strategies in disease-relevant human glial cells.

neuroscience↗

bioRxiv2026

Photoprotection comes at a cost: non-photochemical quenching shapes growthproductivity trade-offs in Haematococcus lacustris

Non-photochemical quenching (NPQ) dissipates excess absorbed light energy and protects photosynthetic organisms from photodamage, but its role in regulating the balance between growth, stress tolerance and astaxanthin accumulation in Haematococcus lacustris remains unclear. Here, we investigated how enhanced NPQ affects photosynthetic performance, stress-induced differentiation, and productivity in this astaxanthin-producing microalga. We isolated and characterized an NPQ-enhanced mutant line, A116, using cultivation assays under different stress conditions, analysis of photosynthetic parameters, pigment profiling, and whole-genome resequencing. A116 displayed stronger and faster NPQ induction, driven by increased LHCSR accumulation, resulting in decreased photosynthetic electron transport and lower photochemical efficiency under moderate-to-high light. Enhanced NPQ delayed the transition to astaxanthin-rich cysts under high light, allowing greater biomass accumulation under CO2-limiting conditions. However, under high CO2 availability, where carbon fixation relieved excitation pressure supporting efficient photosynthesis, the enhanced NPQ phenotype reduced growth and astaxanthin productivity compared with the wild type. These results show that NPQ modulates a context-dependent trade-off between photoprotection and productivity in Haematococcus lacustris. Increased energy dissipation can improve high-light tolerance under carbon limitation, but becomes detrimental when absorbed light can be efficiently used for carbon assimilation. Thus, optimal algal productivity requires tuning photoprotective capacity to environmental conditions rather than maximizing NPQ.

plant biology↗

bioRxiv2026

Dissecting the phenotypic and genetic determinants of maize-bean interactions in intercropping

O_LICereal-legume intercropping is a cornerstone of agroecological systems because interactions between species can enhance agroecosystem resilience. Yet, the mechanisms underlying these interactions remain poorly understood. To address this gap, we investigated the maize-bean association under low-input conditions. C_LIO_LIWe conducted a two-year intercropping experiment with 200 climbing bean lines grown alongside three maize landraces in France and Romania. We evaluated bean phenotypic responses above- and below-ground to 3 maize landraces, treated as distinct biotic environments (E). Direct and indirect genetic effects were assessed by mapping bean and maize phenotypic traits onto the bean genome (G), with G x E interactions tested using contrasts among maize landraces. C_LIO_LICompetitive interactions dominated, maize acting as the stronger competitor. Maize landraces created distinct biotic environments affecting bean traits. Best-performing partners varied across experimental fields, with no evidence of bean local adaptation. The most productive and balanced mixtures were obtained with the traditionally intercropped maize landrace. Genome-wide association analyses identified loci underlying direct and indirect genetic effects, including candidate genes associated with neighbor perception. C_LIO_LIThese findings reveal the genetic complexity of maize-bean interactions and highlight competitive tolerance in bean and reduced aggressiveness in maize as key traits for improving cereal-legume intercrop performance. C_LI

genetics↗

bioRxiv2026

Long-read cross-platform validation reveals novel repeat features in myotonic dystrophy type 2

The broader application of long-read sequencing (LRS) for repeat expansion characterization in myotonic dystrophy type 2 (DM2) and other repeat expansion disorders (REDs) remains limited by the lack of systematic validation and benchmarking of sequencing results and bioinformatic workflows. Here, we performed an orthogonal cross-platform validation of previously generated Oxford Nanopore Technologies (ONT) data by sequencing the same DNA samples with Pacific Biosciences (PacBio) HiFi following amplification-free targeted enrichment in a cohort of 8 DM2 patients. Despite substantial differences in sequencing chemistry and coverage, the two platforms showed high concordance in repeat size estimation, somatic mosaicism, and repeat architecture. This validation confirmed the presence of the (TCTG)n motif and enabled the identification of a previously unreported (CCCG)n motif at the 3' end of expanded alleles, further highlighting the structural complexity of the CNBP expansion. Through this analysis, we also established a bioinformatic workflow that improved ONT-based repeat characterization, addressing limitations in motif resolution and enabling more accurate analysis of CNBP expansions. Overall, this study provides a validated framework for LRS-based CNBP repeat analysis, supporting the integration of these technologies into routine molecular investigation for DM2 and other REDs.

genomics↗

bioRxiv2025

On-site microbial community analysis in rivers: integrating autonomous sampling with a portable sequencing workflow

Studies focused on bacterial diversity in rivers often face significant limitations. Traditional sampling approaches frequently overlook spatial and temporal variability at the reach scale. Molecular techniques, such as metabarcoding and metagenomics, can partially address this issue by providing deeper insights. However, this approach usually involves highly specialized laboratories and high costs, making large-scale and long-term monitoring of river networks unfeasible in most cases. Thus, we developed a methodology based on remote-controlled boats, allowing the collection of integrated samples in freshwater ecosystems. Combined with a portable laboratory, this approach enabled the development of new surface water monitoring strategies. Here, we describe the operational application of this system for in situ monitoring of bacterioplankton communities across 8 sections of the Ter River (Catalonia, Spain). [To explore its potential, we applied both 16S rRNA gene sequencing using Nanopore technology (MinION) in the field, and shotgun metagenomics using Illumina technology in the laboratory, acknowledging the intrinsic differences between sequencing targets, platforms, and analysis pipelinesMinIONMinION sequencing enabled microbiome characterization and identification of the main bacterial taxa just 72 h after sampling, offering significantly lower costs and reduced manpower requirements. Furthermore, amplification facilitated the full characterization of bacterioplankton diversity along the river, preventing the exclusion of uncommon taxa. While shotgun metagenomics is still necessary for understanding the functional activity of these organisms, our approach provides a cost-effective framework for developing efficient follow-up sampling methodologies.

microbiology↗

bioRxiv2024

Landscape genomics highlights the adaptive evolution of chickpea

Environmental heterogeneity and human-mediated dispersal have jointly shaped the genetic diversity and local adaptation of crop species. Understanding the genetic basis of these processes is essential for improving crops across diverse agro-environmental conditions. We characterized population structure and geographic patterns of genetic diversity in 532 chickpea genotypes spanning most of the cultivated range. Using redundancy analysis on 208 georeferenced landrace-derived genotypes from the Mediterranean Basin to Central Asia, we identified genotype-environment associations (GEAs) and traced adaptive variation along historical Silk Road routes. Both environmental and geographic factors significantly shaped chickpea diversity. GEA loci were enriched for genes involved in heat and drought tolerance, while key geographic regions harbored reservoirs of adaptive alleles and early-flowering genotypes, supporting flowering time as an escape strategy from terminal stress. These findings provide a genomic framework for integrating climate-adaptive alleles into chickpea breeding programs targeting drought- and heat-prone environments.

genomics↗

bioRxiv2023

Adaptive gene loss in the common bean pan-genome during range expansion and domestication

The common bean (Phaseolus vulgaris L.) is a crucial grain legume crop [1,2] whose life history offers an ideal evolutionary model to identify and study adaptive variants in wild and domestication populations [3]. Here we present the first common bean pan-genome based on five high-quality genomes and whole-genome reads representing 339 genotypes. We found [~]243 Mb of additional sequences containing 7,495 protein-coding genes missing from the reference, constituting 51% of the total presence/absence variations (PAVs). There were more putatively deleterious mutations in PAVs than core genes, probably reflecting the lower effective population size of PAVs as well as fitness advantages due to the purging effect of gene loss. Our results suggest strong pan-genome shrinkage occurred during wild range expansion from Mexico to South America, with more PAV loss per individual in Andean vs Mesoamerican populations. Selection signatures during wild spreading and domestication were also associated with PAV loss involved in important adaptive traits. Our findings provide evidence that partial or complete gene loss was a key adaptive trait leading to localized and genome-wide reductions. This novel result has major implications for the understanding of the process of plant adaptation and claims for a paradigm shift in evolutionary genetics. Moreover, the common bean pan-genome is a valuable resource for food legume research and breeding towards climate change mitigation, and sustainable agriculture.

evolutionary biology↗

bioRxiv2023

Nitrogen availability during domestication shaped the divergence of tetraploid wheat

The domestication of crops, with the development of the agroecosystems, is associated with major environmental changes and represent a model to test the role of phenotypic plasticity. Here we investigated 32 genotypes representing key stages of tetraploid wheat domestication. We developed a dedicated pipeline combining RNA-Seq data, estimates of evolvability and QST to characterize the plasticity of gene expression and identify signatures of selection under different nitrogen conditions. The analysis of gene expression diversity showed contrasting results between primary and secondary domestication in relation to nitrogen availability. Indeed, nitrogen triggered the expression of twice the number of genes in durum wheat compared to emmer and wild emmer. QST distributions and QST-FST comparisons revealed distinct selection signatures at each domestication stage. While primary domestication affected the expression of genes involved in biotic interactions, secondary domestication was associated with changes in expression of genes involved in metabolism of amino acids, particularly lysine. Selection signatures were found also in differentially expressed genes, specifically involved in nitrogen metabolism, such as glutamate dehydrogenase. Overall, our findings show that nitrogen availability had a pivotal role during the domestication and adaptive responses of a major food crop, with varying effects across different traits and growth conditions.

genetics↗

bioRxiv2023

The evolutionary history of the common bean (Phaseolus vulgaris) revealed by chloroplast and nuclear genomes.

The remarkable evolutionary history of the common bean (Phaseolus vulgaris L.) has led to the emergence of three wild main genepools corresponding to three different ecogeographic areas: Mesoamerica, the Andes and northern Peru/Ecuador. Recent works proposed novel scenarios and the northern Peru/Ecuador population has been described as a new species called P. debouckii, rekindling the debate about the origin of P. vulgaris. Here we shed light on the origin of P. vulgaris by analysing the chloroplast and nuclear genomes of a large varietal collection representing the entire geographical distribution of wild forms. We assembled 37 chloroplast genomes de novo and used them to construct a time frame for the divergence of the genotypes under investigation, revealing that the separation of the Mesoamerican and northern Peru/Ecuador genepools occurred [~]0.15 Mya. Our results clearly support a Mesoamerican origin of the common bean and reject the recent P. deboukii hypothesis. These results also imply two independent migratory events from Mesoamerica to the North and South Andes, probably facilitated by birds. Our work represents a paradigmatic example of the importance of taking into account recombination events when investigating phylogeny and of the analysis of wild forms when studying the evolutionary history of a crop species.

genetics↗

bioRxiv2022

CRISPR/Cas9-based repeat depletion for the high-throughput genotyping of complex plant genomes

High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genomewide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods due to the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR/Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-Seq data. The novel depletion method removed [~]40% of reads mapping to repeats, increasing those mapping to single-copy regions by [~]2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of [~]10-fold compared to non-depleted libraries. In the same condition, we were also able to identify [~]12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed due to low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely-related species (L. orientalis). Our results demonstrated that CRISPR/Cas9-driven repeat depletion focuses sequencing data on meaningful genomic regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.

genomics↗

bioRxiv2022

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully-expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire [~]50-kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype- phenotype correlations for the better stratification of DM2 patients in clinical trials.

genomics↗

bioRxiv2021

Nuclear receptor subfamily 4A signaling as a key disease pathway of CD1c+ dendritic cell dysregulation in systemic sclerosis

ObjectivesTo identify key disease pathways driving conventional dendritic cell (cDC) alterations in Systemic Sclerosis (SSc). MethodsTranscriptomic profiling was performed on peripheral blood CD1c+ cDCs (cDC2s) isolated from 12 healthy donors and 48 SSc patients with all major disease subtypes. Differential expression analysis comparing the different SSc subtypes and healthy donors was performed to uncover genes dysregulated in SSc. To identify biologically relevant pathways, a gene co-expression network was built using Weighted Gene Correlation Network Analysis. We validated the role of key transcriptional regulators using ChIP-sequencing and in vitro functional assays. ResultsWe identified 17 modules of co-expressed genes in cDC2s that correlated with SSc subtypes and key clinical traits including auto-antibodies, skin score, and occurrence of interstitial lung disease. A module of immune regulatory genes was markedly down regulated in patients with the diffuse SSc subtype characterized by severe fibrosis. Transcriptional regulatory network analysis performed on this module predicted NR4A (nuclear receptor 4A) subfamily (NR4A1, NR4A2, NR4A3) genes as the key transcriptional mediators of inflammation. Indeed, ChIP-sequencing analysis supported that these NR4A members target numerous differentially expressed genes in SSc cDC2s. Inclusion of NR4A receptor agonists in culture-based experiments provided functional proof that dysregulation of NR4As affects cytokine production by cDC2s and modulates downstream T-cell activation. ConclusionsNR4A1, NR4A2 and NR4A3 are important regulators of immunosuppressive and fibrosis-associated pathways in SSc cDC2s. Thus, the NR4A family represent novel potential targets to restore cDC homeostasis in SSc. KEY MESSAGESO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LICD1c+ conventional dendritic cells (cDC2s) are implicated as key players in Systemic Sclerosis (SSc), but key molecular mechanisms underlying their dysregulation were unknown. C_LI What does this study add?O_LITranscriptomic analysis and network analysis identified modules of coexpressed genes in cDC2s that correlated with SSc subtypes and key clinical traits. C_LIO_LIThe NR4A (nuclear receptor 4A) subfamily (NR4A1, NR4A2, NR4A3) genes act as master regulators of key immune regulatory genes dysregulated in SSc cDC2s, as shown by multi-omics integration analysis using transcriptomics and targeted ChIP-sequencing. C_LIO_LIPharmacological activation of NR4As inhibits pro-inflammatory cytokine production and CD4+ T-cell activation by cDC2s. C_LI How might this impact on clinical practice or future developments?O_LINR4As are attractive candidates for novel treatment options to attenuate pro-inflammatory and pro-fibrotic responses in SSc patients. C_LI

immunology↗

bioRxiv2020

Genomic structural variation in Nebbiolo grapevines at the individual, clonal and cultivar levels

Structural Variants (SVs) are a widely unexplored source of genetic variation, both due to methodological limitations and because they are generally associated to deleterious effects. However, with the advent of long-range genomic platforms, it has become easier to directly detect SVs. In the same direction, clonally propagated crops provide a unique opportunity to study SVs, offering a suitable genomic environment for their accumulation in heterozygosis. In particular, it has been reported that SVs generate drastic levels of heterozygosity in grapevines. Nebbiolo (Vitis vinifera L.) is a grapevine cultivar typical of north-western Italy, appreciated for its use in producing high-quality red wines. Here, we aimed to analyze the frequency of SVs in Nebbiolo, at three different organizational levels. For this purpose, we generated genomic data based on long-reads, linked-reads and optical mapping. We assembled a reference genome for this cultivar and compared two different clones, including V. vinifera reference genome (PN40024) in our comparisons. Our results indicate that SVs differentially occurring between Nebbiolo clones might be rare, while SVs differentiating haplotypes of the same individual are as abundant as those that occur differentially between cultivars.

genomics↗

bioRxiv2020

The P2X7 receptor localizes to the mitochondria, modulates mitochondrial energy metabolism and enhances physical performance

Basal expression of the P2X7 receptor (P2X7R) improves mitochondrial metabolism, ATP synthesis and overall fitness of immune and non-immune cells. We investigated P2X7R contribution to energy metabolism and subcellular localization in fibroblasts (mouse embryo fibroblasts and HEK293 human fibroblasts), mouse microglia (primary brain microglia and the N13 microglia cell line), and heart tissue. The P2X7R localizes to mitochondria, and its lack a) decreases basal respiratory rate, ATP-coupled respiration, maximal uncoupled respiration, resting mitochondrial potential, mitochondrial matrix Ca2+ level, b) modifies expression pattern of oxidative phosphorylation (OxPhos) enzymes, and c) severely affects cardiac performance. Hearts from P2rx7-deleted versus WT mice are larger, heart mitochondria smaller, and stroke volume (SV), ejection fraction (EF), fractional shortening (FS) and cardiac output (CO), are significantly decreased. Accordingly, physical fitness of P2X7R-null mice is severely reduced. Thus, the P2X7R is a key modulator of mitochondrial energy metabolism and a determinant of physical fitness.

cell biology↗

bioRxiv2020

Pod indehiscence in common bean is associated to the fine regulation of PvMYB26 and a non-functional abscission layer

In legumes, pod shattering occurs when mature pods dehisce along the sutures, and detachment of the valves promotes seed dispersal. In Phaseolus vulgaris, the major locus qPD5.1-Pv for pod indehiscence was identified recently. We developed a BC4/F4 introgression line population and narrowed the major locus down to a 22.5-kb region. Here, gene expression and a parallel histological analysis of dehiscent and indehiscent pods identified an AtMYB26 orthologue as the best candidate for loss of pod shattering, on a genomic region ~11 kb downstream of the highest associated peak. Based on mapping and expression data, we propose early and fine up-regulation of PvMYB26 in dehiscent pods. Detailed histological analysis establishes that pod indehiscence is associated to the lack of a functional abscission layer in the ventral sheath, and that the key anatomical modifications associated with pod shattering in common bean occur early during pod development. We finally propose that loss of pod shattering in legumes resulted from histological convergent evolution and that this is the result of selection at orthologous loci. One-sentence summaryA non-functional abscission layer determines the loss of pod shattering; mapping data, and parallel gene expression and histological analysis support PvMYB26 as the candidate gene for pod indehiscence.

genetics↗

bioRxiv2019

A rapid and accurate MinION-based workflow for tracking species biodiversity in the field

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION platform. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure reliable and robust performance under suboptimal field conditions without increasing costs. Here we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 500 reads per sample. ONTrack-derived consensus barcodes have high accuracy, ranging from 99,8% to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

genomics↗