Biology subjects

Novo, M.

Publications and source records attributed to Novo, M..

8 recordsLinked to original sources

bioRxiv2025

Critical role of Cas9 aggregation on in vitro DNA cleavage

The CRISPR/Cas9 system is a powerful genome-editing tool widely used in molecular biology and gene therapy, whose efficiency strongly depends on the physicochemical properties of the Cas9 ribonucleoprotein complex. Optimizing Cas9 activity remains essential for reliable genome- editing applications, yet the factors limiting its in vitro cleavage efficiency are not fully understood. Among these, protein aggregation has been suggested to critically impair Cas9 functionality, although its role has not been systematically analysed. Here, we investigate Cas9 aggregation under different environmental conditions and evaluate its impact on in vitro DNA cleavage efficiency. Using fluorescently labelled Cas9 and single-molecule fluorescence techniques, we quantify aggregation as a function of buffer composition, ionic strength, salt concentration and sgRNA presence, and relate these properties to cleavage activity. Our results show that Cas9 aggregation significantly reduces DNA cleavage efficiency, with higher aggregation levels consistently correlating with lower activity. In contrast, buffers with higher ionic strength or stabilizing components reduce aggregation and enhance Cas9 performance. Overall, this study demonstrates that Cas9 aggregation plays a critical role in determining in vitro cleavage efficiency and highlights the importance of controlling protein aggregation to optimize CRISPR/Cas9-based genome-editing applications and delivery strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/696176v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@14dbd87org.highwire.dtl.DTLVardef@d5f812org.highwire.dtl.DTLVardef@1916c53org.highwire.dtl.DTLVardef@5ed798_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Coping with extremes: How Epigenetic and Molecular Adaptations Enable Earthworms to Thrive in Volcanic Soils

Earthworms thriving in naturally occurring geothermal soils offer rare insight into rapid adaptation to environmental extremes. Here, we show that the pantropical earthworm Amynthas gracilis survives and flourishes in soils of the Furnas Volcano (Sao Miguel Island, Azores), where conditions include elevated temperatures (up to 40 {degrees}C), high CO2 (88.6%), low O2 (10%), toxic metals, and mildly acidic pH. In a reciprocal-transplant, mesocosm-based experiment between soils overlying areas of active degassing volcanic gassing (hereafter active degassing soils) and reference soils, convergence of the epidermal thickness of the transplanted earthworms to the resident-soil phenotype (24 {+/-} 3.9 {micro}m active degassing soil, 43.8 {+/-} 8 {micro}m reference soil), was observed within 31 days. Combining RNA-Seq, DNA (5-cytosine) methylation mapping, and microRNA profiling, this phenotypic change results from coordinated transcriptional and epigenetic reprogramming. While gene-body methylation occurred at [~]98 % of loci, levels varied, and differentially methylated regions were enriched ffor genes with altered expression under volcanic stress. Multi-omics network analysis identified epithelial morphogenesis, circulatory system formation, and neural development as regulatory hubs, highlighted by a set of 41 epithelial-morphogenesis genes showing consistent methylation and miRNA patterns. Additional modules governing ion transport and signal transduction complemented the adaptive response. Collectively these findings demonstrate that A. gracilis employs dynamic DNA methylation and microRNA regulation alongside transcriptional reprogramming to generate a persistent phenotypic adjustment to a volcanic stress. This work advances our understanding of extremophile resilience and provides a scalable model for predicting organismal adaptive capacity in the face of environmental extremes.

molecular biology↗

bioRxiv2024

A punctuated burst of massive genomic rearrangements by chromosome shattering and the origin of non-marine annelids

The genomic basis of cladogenesis and adaptive evolutionary change has intrigued biologists for decades. Here, we show that the tectonics of genome evolution in clitellates, a clade composed of most freshwater and all terrestrial species of the phylum Annelida, is characterised by extensive genome-wide scrambling that resulted in a massive loss of macrosynteny between marine annelids and clitellates. These massive rearrangements included the formation of putative neocentromeres with newly acquired transposable elements and preceded a further period of genome-wide reshaping events, potentially triggered by the loss of genes involved in genome stability and homeostasis of cell division. Notably, while these rearrangements broke short-range interactions observed between Hox genes in marine annelids, they were reformed as long-range interactions in clitellates. Our findings reveal extensive genomic reshaping in clitellates at both the linear (2D) and three-dimensional (3D) levels, suggesting that, unlike in other animal lineages where synteny conservation constrains structural evolution, clitellates exhibit a remarkable tolerance for chromosomal rearrangements. Our study thus suggests that the genomic landscape of Clitellata resulted from a rare burst of genomic changes that ended a long period of stability that persists across large phylogenetic distances.

evolutionary biology↗

bioRxiv2024

The nunatak and tabula rasa hypotheses may be compatible: the European phylogeography of a riparian earthworm.

The tabula rasa hypothesis of postglacial immigration supports the notion that species now found in northern European areas must have been recently recolonized from historical refugia. Until the 1960s, however, there was almost complete consensus that disjunctions and endemism in the North Atlantic region of Europe could not be explained without in situ survival during glacial periods (the nunatak hypothesis). Although some earthworms can survive in permafrost and tolerate cold conditions, it is generally believed that most earthworms were eradicated from northern latitudes during the Last Glacial Maximum. To test which hypothesis explains the phylogeography of the riparian and parthenogenetic earthworm Eiseniella tetraedra, we collected 1,640 specimens from 19 different countries in Europe. We examined three molecular markers (COI, 16S and 28S) and their morphology. Eleven lineages were found, nested in five clades. Clade I was more prevalent in cold biogeographical regions such as the continental, the Atlantic or even the Arctic, while clade II was prevalent in Mediterranean regions. We investigated their potential niches through Species Distribution Models, which agreed with the distribution trends. The presence of restricted clades in the Iberian and Scandinavian peninsulas, as well as in Eastern Europe, suggests that these three regions served as refugia during the Last Glacial Maximum. Thus, both hypotheses were necessary to explain the actual distribution of this shore-dwelling earthworm.

zoology↗

bioRxiv2019

Local adaptation fuels cryptic speciation in terrestrial annelids

Uncovering the genetic and evolutionary basis of cryptic speciation is a major focus of evolutionary biology. Next Generation Sequencing (NGS) allows the identification of genome-wide local adaptation signatures, but has rarely been applied to cryptic complexes - particularly in the soil milieu - as is the case with integrative taxonomy. The earthworm genus Carpetania, comprising six previously suggested putative cryptic lineages, is a promising model to study the evolutionary phenomena shaping cryptic speciation in soil-dwelling lineages. Genotyping-By-Sequencing (GBS) was used to provide genome-wide information about genetic variability between seventeen populations, and geometric morphometrics analyses of genital chaetae were performed to investigate unexplored cryptic morphological evolution. Genomic analyses revealed the existence of three cryptic species, with half of the previously-identified potential cryptic lineages clustering within them. Local adaptation was detected in more than 800 genes putatively involved in a plethora of biological functions (most notably reproduction, metabolism, immunological response and morphogenesis). Several genes with selection signatures showed shared mutations for each of the cryptic species, and genes under selection were enriched in functions related to regulation of transcription, including SNPs located in UTR regions. Finally, geometric morphometrics approaches partially confirmed the phylogenetic signal of relevant morphological characters such as genital chaetae. Our study therefore unveils that local adaptation and regulatory divergence are key evolutionary forces orchestrating genome evolution in soil fauna.

evolutionary biology↗

bioRxiv2019

Genome-informed integrative taxonomic description of three cryptic species in the earthworm genus Carpetania (Oligochaeta, Hormogastridae)

Research on cryptic species complexes has reached a consensus on the necessity of integrating multiple sources of evidence. Low-coverage genomic scan techniques like Genotyping-by-Sequencing (GBS) have proven useful to study these groups. Both integrative taxonomy and genome-wide single nucleotide polymorphism (SNP) data remain to be widely applied to earthworms, an animal group with widespread presence of cryptic diversity. The genus Carpetania (formerly the Hormogaster elisae species complex) was found to contain six deeply divergent genetic lineages and some inconspicuous morphological differentiation based in a handful of Sanger-sequenced markers. Marchan et al. (submitted) delimited three well supported species-level clades on the basis of a genomewide SNP dataset and geometric morphometric analyses, highlighting the necessity of a formal taxonomic description of these taxa. In this work, further analyses are applied to the SNP data and a thorough morphological study is performed in order to provide an integrative description of two new species and to redescribe Carpetania elisae. Species-specific SNPs are identified and used as diagnostic characters, and genome-wide and cytochrome oxidase C subunit 1 (COI) genetic distances are compared finding a strong correlation between them. The taxonomic description of these three cryptic species provides a useful tool to include them effectively in ecological studies and biodiversity conservation actions.

zoology↗

bioRxiv2019

Unveiling the multi-step solubilization mechanism of single vesicles by detergents.

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Forster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

biophysics↗

bioRxiv2018

Delegating Sex: differential gene expression in stolonizing syllids uncover the hormonal control of reproduction in Annelida

Stolonization in syllid annelids is a unique mode of reproduction among animals. During the breeding season, a structure resembling the adult but containing only gametes, called stolon, is formed at the posterior end of the animal. When the stolons mature, they detach from the adult and the gametes are released into the water column. The process is synchronized within each species, and it has been reported to be under environmental and endogenous control, probably via endocrine regulation. To further understand the reproduction in syllids and to elucidate the molecular toolkit underlying stolonization, we generated Illumina RNA-seq data from different tissues of reproductive and non-reproductive individuals of Syllis magdalena, and characterized gene expression during the stolonization process. Several genes involved in gametogenesis (ovochymase, vitellogenin, testis-specific serine/threonine-kinase), immune response (complement receptor 2), neuronal development (tyrosine-protein kinase Src42A), cell proliferation (alpha-1D adrenergic receptor), and steroid metabolism (hydroxysteroid dehydrogenase 2) were found differentially expressed in the different tissues and conditions analyzed. In addition, our findings suggest that several neurohormones, such as methyl farnesoate, dopamine and serotonin, might trigger the stolon formation, the correct maturation of gametes and the detachment of stolons when gametogenesis is complete. The process seems to be under circadian control, as indicated by the expression patterns of r-opsins. Overall, our results shed light into the genes that orchestrate the onset of gamete formation, and improve our understanding of how some hormones, previously reported to be involved in reproduction and metamorphosis processes in other invertebrates, seem to also regulate reproduction via stolonization.

molecular biology↗