Biology subjects

Rocha, S.

Publications and source records attributed to Rocha, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Linking Molecular Tension and Cellular Tractions: A Multiscale Approach to Focal Adhesion Mechanics

Focal adhesions (FAs) are mechanosensitive structures that mediate force transmission between cells and the extracellular matrix. While Traction Force Microscopy (TFM) quantifies cellular tractions exerted on deformable substrates, Forster Resonance Energy Transfer (FRET)-based tension probes, such as Vinculin Tension Sensors (VinTS), measure molecular-scale forces within FA proteins. Despite their potential synergy, these methods have rarely been combined to explore the interplay between molecular tension and cellular tractions. Here, we introduce a framework integrating TFM and VinTS to investigate FA mechanics across scales. At cell level, tractions and vinculin tension increased with substrate stiffness. At FA level, vinculin tension correlated with vinculin density, while tractions scaled with FA area, total vinculin content and vinculin density. Direct comparison of tractions to tension revealed a complex, heterogenous relationship between these forces, possibly linked to diverse cell and FA maturation states. Sub-FA analysis revealed conserved spatial patterns, with tension and traction increasing towards the cell periphery. This multiscale approach provides insights into the multiscale dynamics of FA mechanotransduction, bridging the gap between molecular forces and cellular mechanics.j

biophysics↗

bioRxiv2025

NF-κB is a Central Regulator of Hypoxia-Induced Gene Expression

Hypoxia is both a physiological and pathological signal in cells. Changes in gene expression play a critical role in the cellular response to hypoxia, enabling cells to adapt to reduced oxygen availability. These changes are primarily mediated by the HIF family of transcription factors, however other transcription factors such as NF-{kappa}B, are also activated by hypoxia. Although NF-{kappa}B is known to be activated by hypoxia, the extent to which NF-{kappa}B contributes to the hypoxic response remains poorly understood. Here, we analysed hypoxia-induced, NF-{kappa}B-dependent gene expression, to define the NF-{kappa}B-dependent hypoxic signature. Our analysis reveals that most genes downregulated by hypoxia require NF-{kappa}B for their repression. We show that although the NF-{kappa}B-mediated hypoxic response may vary between cell types, a core subset of hypoxia-inducible genes requires NF-{kappa}B across multiple cell backgrounds. We demonstrate that NF-{kappa}B is critical for reactive oxygen species (ROS) generation and regulation of genes involved in oxidative phosphorylation under hypoxia. This work highlights NF-{kappa}Bs central role in the hypoxia response and offering new insights into gene expression regulation by hypoxia and NF-{kappa}B.

molecular biology↗

bioRxiv2024

Nanoparticle Accumulation and Penetration in 3D Tumor Models: the Effect of Size, Shape, and Surface Charge

Preclinical studies have demonstrated that nanoparticles (NPs) hold significant potential for advancing cancer therapy by enhancing therapeutic efficacy while reducing side effects. Their effectiveness in solid tumors is, however, often constrained by insufficient accumulation and penetration. Understanding how the physicochemical properties of NPs - such as size, shape, and surface charge - influence their interaction with cells within the tumor is critical for optimizing NP design. In this study, we addressed the challenge of inconsistent NP behavior by systematically evaluating NP uptake in both 2D and 3D tumor models, and NP penetration in spheroids. Our results showed that larger NPs exhibited higher internalization rates in 2D models but limited penetration in 3D spheroids. Furthermore, negatively charged NPs consistently achieved superior accumulation and deeper penetration than neutral and positively charged NPs. Spherical NPs outperformed rod-shaped NPs in tumor accumulation and penetration. These findings underscore the importance of carefully tailoring NP properties to the complex tumor microenvironment for improved therapeutic outcomes in real tumors.

cancer biology↗

bioRxiv2024

Deciphering stiffness-driven changes in colorectal cancer by proteomics

Tumor stiffening plays a pivotal role in cancer progression. Increased tumor stiffness, resulting from interactions between cancer cells and their surrounding microenvironment, alters the tumors mechanical properties and significantly impacts cancer growth and metastasis, the primary cause of cancer-related deaths. Despite the importance of tumor stiffness, systematic studies exploring its effect on proteomic profiles are limited. In this study, focused on colorectal cancer, we show that matrix stiffness significantly alters the expression of secreted proteins, while intracellular protein levels remain largely unaffected. Functional assays reveal that the changes in the secretome, driven by matrix stiffness, enhance cell migration, angiogenesis, and matrix remodeling, which collectively contribute to a more aggressive cancer phenotype. Our findings emphasize the critical role of matrix stiffness in driving colorectal cancer progression through changes in the secretome, offering valuable insights for the development of biomechanical cancer therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/618701v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@fb6b58org.highwire.dtl.DTLVardef@446a31org.highwire.dtl.DTLVardef@197e22forg.highwire.dtl.DTLVardef@82a896_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Synthesis and mechanical characterization of polyacrylamide (PAAm) hydrogels with different stiffnesses for large-batch cell culture applications

The impact of mechanical cues on cell behavior is increasingly being recognized, rendering hydrogel platforms that mimic the extracellular matrix indispensable in in vitro cell biology research. Here, we present a step-by-step protocol for synthesis and rheological characterization of polyacrylamide (PAAm) hydrogels with varying stiffnesses, produced as large circular unattached gels customizable in shape and size. We outline methods for their use in cell culture and downstream applications involving secretome or cell analysis, and protein visualization by fluorescence microscopy. This protocol is based on the recent work of Shi & Janmey who describe a novel and straightforward method for the production of large PAAm hydrogels for bulk cell culture and mechanobiology studies.1 Their procedure results in one large gel that is not attached to a supporting surface and therefore can be transferred and/or stamped to generate PAAm gels of custom shapes and sizes. The aim of this step-by-step procedure is therefore not to improve the reported protocol, but to create a clearly outlined and repeatable protocol that enables a smooth implementation in any lab for a diverse audience. In addition, our protocol describes besides harvesting of cells also the collection of secretome for downstream biochemical analyses, as well as immunofluorescence labeling using antibodies that can readily be multiplexed for optimization of labeling conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/613503v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@cefcc3org.highwire.dtl.DTLVardef@1348c93org.highwire.dtl.DTLVardef@f935acorg.highwire.dtl.DTLVardef@132dbd0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Biocompatible Neodymium-Doped Nanocrystals as Probes for Diffraction-limited, in vitro Temperature Sensing

Localized hyperthermia is a promising approach to cancer therapy. However, its clinical potential is limited by heterogeneous heat distribution within tumors, and advanced methods to measure temperature at the sub-micron level are therefore required. To address this challenge, luminescent nanothermometers, such as lanthanide-doped nanocrystals (Ln-NC) operating in the near-infrared (NIR), have been investigated for accurate spatiotemporal thermal monitoring. In this study, the synthesis of neodymium-doped, sodium yttrium fluoride nanocrystals (Nd-NCs) was optimized to achieve high photoluminescence (PL) intensity by adjusting the dopant concentration and by shelling with inert layers. Standard curves for luminescence-based temperature readout were developed using ratiometric analysis of the temperature-dependent PL spectra in the 850-920 nm biological window, showing excellent linearity and high thermal sensitivity. A silica shell was added to the particles and shown to confer excellent aqueous stability and biocompatibility in A549 lung cancer cells. Finally, luminescent thermal readout was demonstrated in vitro in A549 cells by spectrally resolving the diffraction-limited luminescence spots at a single-particle scale over a clinically relevant temperature range from 20-50 {degrees}C. The application of the developed nanothermometer as preclinical tools for NP-HT characterization could provide crucial information on the therapeutic temperature achieved in and around the tumor area. This could be key to optimizing NP properties and therapeutic parameters, for the development of viable hyperthermal cancer treatments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/579538v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1b06abdorg.highwire.dtl.DTLVardef@12fa23corg.highwire.dtl.DTLVardef@4439c1org.highwire.dtl.DTLVardef@12bcedd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Systematic characterization of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry

We have developed a robust workflow for the identification of proline hydroxylation sites in proteins, using a combination of hydrophilic interaction chromatography (HILIC) enrichment and high-resolution nano-Liquid Chromatography-Mass Spectrometry (LC-MS) together with refining and filtering parameters during data analysis. Using this approach, we have combined data from cell lines treated with either the prolyl hydroxylase (PHD) inhibitor, Roxadustat (FG-4592), or with the proteasome inhibitor MG-132, or with a DMSO control, to identify a total of 4,993 and 3,247 proline hydroxylation sites, respectively, in HEK293 and RCC4 cells. A subset of 1,954 (HEK293) and 1,253 (RCC4) non-collagen proline hydroxylation sites with high confidence were inhibited by FG-4592 treatment. Features characteristic of proline hydroxylated peptides were identified, which were consistent between the HEK293 and RCC4 datasets. The more hydrophilic HILIC fractions were enriched in peptides containing hydroxylated proline residues, which showed characteristic differences in charge and mass distribution, as compared with either unmodified, or oxidised peptides. Furthermore, we discovered that the intensity of the diagnostic hydroxyproline immonium ion was dependent upon parameters including the MS collision energy setting, parent peptide concentration and the sequence of amino acids adjacent to the modified proline. We show using synthetic peptides that a combination of retention time in LC and optimised MS parameter settings allows reliable identification of proline hydroxylation sites in peptides, even when multiple prolines residues are present. Focussing on proteins in which newly identified proline hydroxylation sites were inhibited by the pan-PHD inhibitor, FG-4592, showed enrichment for proteins involved in metabolism of RNA, mRNA splicing and cell cycle regulation, including the protein phosphatase 1 regulatory subunit, Repo-Man (CDCA2). We show that Repo-Man is hydroxylated at P604 and in the accompanying study by Druker et al. 1, we present a combination of cellular and biochemical evidence that hydroxylation of Repo-Man at P604 is important for its function in controlling mitotic progression.

biochemistry↗

bioRxiv2023

tRNASer overexpression induces adaptive mutations in NSCLC tumors

tRNAs are a driving force of genome evolution in Yeast and Bacteria. Their deregulation is frequently observed in tumors with Serine tRNAs being often overexpressed. This has important functional consequences, such as increased metabolism and tumor growth. In yeast, time and chemical stimulus boost alterations in the genome driven by tRNA deregulation. Therefore, we hypothesized that tRNA deregulation may contribute to the increased genome instability observed in tumors. To study the effect of tRNA deregulation in tumors, we overexpressed tRNA-Ser-AGA-2-1 in a NSCLC cell line, H460. This cell line and a Mock (control) were xenografted in nude mice and collected at 3 timepoints: T1-Naive; T2-Treated once with cisplatin/vehicle and; T3) treated twice with cisplatin/vehicle. These tumors were characterized by WES, RNAseq and Mass Spectrometry and the data obtained was integrated. The tumor mutation burden was increased in T3 tRNASerOE tumors, regardless of treatment. Although in T1 Mock and tRNASer tumors have a similar number of variants, in T2&3, tRNASerOE tumors display two times more variants than Mock tumors regardless of treatment. Interestingly, tRNASerOE exclusive variants favor proliferation and therapy resistance, which is in line with the phenotypes observed and supported by RNAseq and proteomics data. In conclusion, tRNASerOE increases the tumor mutation burden and the variants detected favor tumor growth, proving tRNA deregulation is enough to induce adaptive mutations in the genome.

cancer biology↗

bioRxiv2023

Regulatory T Cell-like Response to SARS-CoV-2 in Jamaican Fruit Bats Transduced with Human ACE2

Insectivorous Old World horseshoe bats (Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats (Rousettus aegyptiacus) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats (Eptesicus fuscus) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats (Artibeus jamaicensis) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyers patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies. CD4+ helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptide library and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor-{beta}, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are poorly susceptibility to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insight of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease. Author SummaryBats are reservoir hosts of many viruses that infect humans, yet little is known about how they host these viruses, principally because of a lack of relevant and susceptible bat experimental infection models. Although SARS-CoV-2 originated in bats, no robust infection models of bats have been established. We determined that Jamaican fruit bats are poorly susceptible to SARS-CoV-2; however, their lungs can be transduced with human ACE2, which renders them susceptible to SARS-CoV-2. Despite robust infection of the lungs and diminishment of pulmonary cellularity, the bats showed no overt signs of disease and cleared the infection after two weeks. Despite clearance of infection, only low-titer antibody responses occurred and only a single bat made neutralizing antibody. Assessment of the CD4+ helper T cell response showed that activated cells expressed the regulatory T cell cytokines IL-10 and TGF{beta} that may have tempered pulmonary inflammation.

microbiology↗

bioRxiv2023

Flat clathrin lattices are linked to metastatic potential in colorectal cancer

Clathrin assembles at the cells plasma membrane in a multitude of clathrin-coated structures (CCSs). Among these are flat clathrin lattices (FCLs), alternative clathrin structures that have been found in specific cell types, including cancer cells. Here we show that these structures are also present in different colorectal cancer (CRC) cell lines, and that they are extremely stable with lifetimes longer than 8 hours. By combining cell models representative of CRC metastasis with advanced fluorescence imaging and analysis, we discovered that the metastatic potential of CRC is associated with an aberrant membranous clathrin distribution, resulting in a higher prevalence of FCLs in cells with a higher metastatic potential. These findings suggest that clathrin organization might play an important yet unexplored role in cancer metastasis.

cancer biology↗

bioRxiv2022

Disruption of HIF1A translational control attenuates the HIF-dependent hypoxic response and solid tumour formation in vivo

Adaptation to reduced oxygen availability is mediated by the hypoxia-inducible factor (HIF) family of transcription factors. The activity and availability of HIF proteins is primarily driven by the stability of the HIF alpha subunits. However, it is becoming increasingly apparent that preferential translation of HIF1 mRNA is also necessary for full activation of the HIF1-dependent hypoxic response. Consequently, the mechanisms controlling HIF1 translation are of equivalent importance to the proline hydroxylase-dependent degradation pathways. Here we investigate the role of the 5UTR of the HIF1 mRNA in controlling preferential translation of endogenous HIF1 in hypoxic cells. CRISPR/Cas9-mediated genetic deletion of the 5 UTR of HIF1 results in reduced HIF1 levels following hypoxia, without alteration in mRNA or protein stability. HIF1 mRNA lacking the 5UTR was efficiently translated in adequately oxygenated cells but this was inhibited during hypoxia, consistent with the global block on protein synthesis. The HIF1 translational defect observed in cells missing the 5UTR led to reduced viability in hypoxic conditions in vitro and an impaired ability to form solid tumours in murine xenografts. Prevention of preferential HIF1 translation limits the duration and intensity of the HIF-dependent hypoxic response and disrupts the formation of solid tumours. Together these results demonstrate the importance of translation control over HIF1 and suggest that strategies to inhibit preferential HIF1 protein translation in hypoxic cancer cells will be an effective strategy to limit the growth of solid hypoxic tumours.

cancer biology↗

bioRxiv2022

Infant low-frequency EEG cortical power, cortical tracking and phase-amplitude coupling predicts language a year later.

Cortical signals have been shown to track acoustic and linguistic properties of continuous speech. This phenomenon has been measured in both children and adults, reflecting speech understanding by adults as well as cognitive functions such as attention and prediction. Furthermore, atypical low-frequency cortical tracking of speech is found in children with phonological difficulties (developmental dyslexia). Accordingly, low-frequency cortical signals may play a critical role in language acquisition. A recent investigation with infants Attaheri et al., 2022 (1) probed cortical tracking mechanisms at the ages of 4, 7 and 11 months as participants listened to sung speech. Results from temporal response function (TRF), phase-amplitude coupling (PAC) and dynamic theta-delta power (PSD) analyses indicated speech envelope tracking and stimulus-related power (PSD) for delta and theta neural signals. Furthermore, delta- and theta-driven PAC was found at all ages, with theta phases displaying stronger PAC with high-frequency amplitudes than delta. The present study tests whether these previous findings replicate in the second half of the full cohort of infants (N = 122) who were participating in this longitudinal study (first half: N=61, (1); second half: N=61). In addition to demonstrating good replication, we investigate whether cortical tracking in the first year of life predicts later language acquisition for the full cohort (122 infants recruited, 113 retained) using both infant-led and parent-estimated measures and multivariate and univariate analyses. Increased delta cortical tracking in the univariate analyses, increased [~]2Hz PSD power and stronger theta-gamma PAC in both multivariate and univariate analyses were related to better language outcomes using both infant-led and parent-estimated measures. By contrast, increased [~]4Hz PSD power in the multi-variate analyses, increased delta-beta PAC and a higher theta/delta power ratio in the multi-variate analyses were related to worse language outcomes. The data are interpreted within a "Temporal Sampling" framework for developmental language trajectories.

neuroscience↗

bioRxiv2022

Emergence of the cortical encoding of phonetic features in the first year of life

Even prior to producing their first words, infants are developing a sophisticated speech processing system, with robust word recognition present by 4-6 months of age. These emergent linguistic skills, observed with behavioural investigations, are likely to rely on increasingly sophisticated neural underpinnings. The infant brain is known to robustly track the speech envelope, however to date no cortical tracking study could investigate the emergence of phonetic feature encoding. Here we utilise temporal response functions computed from electrophysiological responses to nursery rhymes to investigate the cortical encoding of phonetic features in a longitudinal cohort of infants when aged 4, 7 and 11 months, as well as adults. The analyses reveal an increasingly detailed and acoustically-invariant phonetic encoding over the first year of life, providing the first direct evidence that the pre-verbal human cortex learns phonetic categories. By 11 months of age, however, infants still did not exhibit adult-like encoding.

neuroscience↗

bioRxiv2022

Endosomal removal and disposal of dysfunctional, immunostimulatory mitochondrial DNA

Maternally inherited mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation1. This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts2. Cells experiencing mtDNA stress due to depletion of the mtDNA-packaging protein, Transcription Factor A, Mitochondrial (TFAM), or HSV-1 infection exhibit elongated mitochondria, mtDNA depletion, enlargement of nucleoids (mtDNA-protein complexes), and activation of cGAS/STING innate immune signaling via mtDNA released into the cytoplasm3. However, the relationships between altered mitochondrial dynamics and mtDNA-mediated activation of the cGAS-STING pathway remain unclear. Here, we show that entire enlarged nucleoids are released from mitochondria that remain bound to TFAM and colocalize with cGAS. These nucleoids arise at sites of mtDNA replication due to a block in mitochondrial fission at a stage when endoplasmic reticulum (ER) actin polymerization would normally commence, which we propose is a fission checkpoint to ensure that mtDNA has completed replication and is competent for segregation into daughter mitochondria. Released nucleoids also colocalize with the early endosomal marker RAB5 as well as the late endosomal marker RAB7 in TFAM-deficient cells and in response to mtDNA stress caused by the HSV-1 UL12.5 protein. Loss of RAB7 increases interferon stimulated gene (ISG) expression. Thus, we propose that defects in mtDNA replication and/or segregation enact a late mitochondrial fission checkpoint that, if persistent, leads to selective removal of dysfunctional nucleoids by a mitochondrial-endosomal pathway. Early steps in this pathway are prone to mtDNA release and cGAS-STING activation, but the immunostimulatory mtDNA is ultimately disposed of through a mechanism involving RAB7-containing late endosomes to prevent excessive innate immune signaling. This mtDNA quality control pathway might represent a therapeutic target to prevent mtDNA-mediated inflammation and associated pathology.

cell biology↗

bioRxiv2022

Synthetic fibrous hydrogels as a platform to decipher cell-matrix mechanical interactions

The interactions between cells and their direct environment are crucial for cell fate but biochemically and mechanically highly complex, and therefore, poorly understood. Despite recent advances that exposed the impact of a range of different factors, real progress remains challenging, since appropriate controllable matrices and quantitative analysis techniques that cover a range of time and length scales are unavailable. Here, we use a synthetic fibrous hydrogel with nonlinear mechanics to mimic and tailor the bi-directional cell-matrix interactions. Using advanced microscopy-based approaches, we acquire a comprehensive picture of how cellular traction forces, fiber remodeling, matrix stiffening, matrix properties and cellular behavior interact, highlighting for instance, the importance of a fibrous architecture and nonlinear mechanics of the matrix. Complete mapping of cell-matrix interactions at the cellular length scale provides indispensable information for the rational design of biomimetic materials to recreate realistic in vitro cell environments.

biophysics↗

bioRxiv2022

Estimating sequence diversity of prion protein gene (PRNP) in Portuguese populations of two cervid species: red deer and fallow deer.

Among the transmissible spongiform encephalopathies (TSEs), chronic wasting disease (CWD) in cervids is now a rising concern in wildlife within Europe, after the detection of the first case in Norway in 2016, in a wild reindeer and until June 2022 a total of 34 cases were described in Norway, Sweden and Finland. The definite diagnosis is postmortem, performed in target areas of the brain and lymph nodes. Samples are first screened using a rapid test and, if positive, confirmed by immunohistochemistry and Western immunoblotting. The study of the genetics of the prion protein gene, PRNP, has been proved to be a valuable tool for determining the relative susceptibility to TSEs. In the present study, the exon 3 of PRNP gene of 143 samples from red deer (Cervus elaphus) and fallow deer (Dama dama) of Portugal was analyzed. Three single nucleotide polymorphisms (SNPs) were found in red deer - codon A136A, codon T98A, codon Q226E - and no sequence variation was detected in fallow deer. The low genetic diversity found in our samples is compatible with previous studies in Europe. The comparison with results from North America, suggests that the free-ranging deer from our study may present susceptibility to CWD, although lack of experimental data and the necessity of extensive survey are necessary to evaluate these populations.

genetics↗

bioRxiv2022

The evolution of two transmissible leukaemias colonizing the coasts of Europe

Transmissible cancers are malignant cell clones that spread among individuals through transfer of living cancer cells. Several such cancers, collectively known as bivalve transmissible neoplasia (BTN), are known to infect and cause leukaemia in marine bivalve molluscs. This is the case of BTN clones affecting the common cockle, Cerastoderma edule, which inhabits the Atlantic coasts of Europe and north-west Africa. To investigate the origin and evolution of contagious cancers in common cockles, we collected 6,854 C. edule specimens and diagnosed 390 cases of BTN. We then generated a reference genome for the species and assessed genomic variation in the genomes of 61 BTN tumours. Analysis of tumour-specific variants confirmed the existence of two cockle BTN lineages with independent clonal origins, and gene expression patterns supported their status as haemocyte-derived marine leukaemias. Examination of mitochondrial DNA sequences revealed several mitochondrial capture events in BTN, as well as co-infection of cockles by different tumour lineages. Mutational analyses identified two lineage-specific mutational signatures, one of which resembles a signature associated with DNA alkylation. Karyotypic and copy number analyses uncovered genomes marked by pervasive instability and polyploidy. Whole-genome duplication, amplification of oncogenes CCND3 and MDM2, and deletion of the DNA alkylation repair gene MGMT, are likely drivers of BTN evolution. Characterization of satellite DNA identified elements with vast expansions in the cockle germ line, yet absent from BTN tumours, suggesting ancient clonal origins. Our study illuminates the evolution of contagious cancers under the sea, and reveals long-term tolerance of extreme instability in neoplastic genomes.

evolutionary biology↗

bioRxiv2022

Transcriptome landscape of kleptoplastic sea slug Elysia viridis

Certain sacoglossan sea slugs can sequester and maintain photosynthetically active chloroplasts through algae feeding, a phenomenon called kleptoplasty. The period while these plastids remain active inside the slugs body is species- and environment-dependent and can span from a few days to more than three weeks. Here we report for the first time the transcriptome of sea slug Elysia viridis (Montagu, 1804), which can maintain kleptoplasts for more than two weeks and is distributed along all the Atlantic European coastline. The obtained transcriptome of E. viridis comprised 12,884 protein-coding sequences (CDS). The shortest one was 261bp, and the longest 8,766bp; the whole transcriptome has a total length of 9.3Mb (Table S4 and Fig. S2). Analysing these CDS, we identified 9,422 different proteins, with best hits mainly from two genera: Elysia (87.2%), and Plakobranchus (11.0%) (Fig. S2); the other 2.3% corresponded to multiple genera of sea slugs and snails (Tectipleura) (Kano et al., 2016). We got the functional annotation (Gene Ontologies, GO) corresponding to 9,333 CDS: 4,755 CDS associated with 2,583 Biological Process (BP); 5,466 CDS linked to 683 Cellular Components (CC); and 6,693 related to 1,606 Molecular Functions (MF). We identified 201 CDS related to response to stress (GO:0006950) and 10 CDS associated with the regulation of response to stress (GO:0080134). Focussing on the ROS-quenching toolkit, we found 24 CDS related to oxidoreductase complex (GO:1990204) and 560 annotated with oxidoreductase activity (GO:0016491) acting in a large number of donors, e.g., CH-OH, CH=O, C=O, CH and CH2. In addition, we found 39 CDS with antioxidant activity (GO:00162099) and other CDS with ROS-quenching function: superoxide dismutase (GO:0004784), peroxidase (GO:0004601), glutathione oxidoreductase (GO:0097573) and peroxidase (GO:0004602); and thioredoxin peroxidase (GO:0008379) activity. Furthermore, we found 8 CDS related to the symbiont response (GO:0140546) and nine related to the pattern recognition receptor signalling pathway (GO:0002221).

evolutionary biology↗