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Salinas, G.

Publications and source records attributed to Salinas, G..

15 recordsLinked to original sources

Combining transcriptomic resolutions and machine learning strategies uncovers new OXPHOS genes in Caenorhabditis elegans

Assigning biological functions to genes remains a major challenge in genomics because reliable functional annotations are often scarce and unevenly distributed. Although transcriptomic datasets capture rich information about gene activity and coordination, exploiting these data for gene function prediction is difficult when only a small number of genes have high-confidence functional assignments and the remaining labels are uncertain. Here, we developed a confidence-aware learning framework that integrates complementary transcriptomic landscapes while explicitly incorporating uncertainty in functional annotations. The framework combines supervised learning from time-resolved bulk RNA-seq data with co-expression analysis of embryonic and adult single-cell transcriptomes. Supervised learning uses a two-round training scheme in which genes supported by limited functional evidence are incorporated only after an initial model has been established using high-confidence annotations. To reduce module-specific biases, we implemented an informed bagging strategy in which genes from individual functional modules are systematically withheld during training and predictions are integrated by consensus across models. We applied this framework to identify genes involved in oxidative phosphorylation (OXPHOS) in Caenorhabditis elegans. Integrating supervised and co-expression evidence prioritized a small set of high-confidence candidate genes with strong predictive performance on an independent test set. Experimental validation showed that disruption of the top-ranked candidate, ril-1, produces phenotypes consistent with impaired OXPHOS function. Our results demonstrate that transcriptomic landscapes can be systematically exploited for gene function prediction under incomplete functional annotation, providing a framework that may be transferable to other biological processes and organisms where reliable annotations remain limited.

bioinformatics↗

Natural aging drives a subclinical cardiovascular phenotype in a non-human primate

Aging is an inevitable risk factor for cardiovascular disease. Profound understanding of mechanisms underlying the early stages of cardiovascular aging is essential for the development of novel therapeutics. Therefore, animal models which closely reflect the human condition are highly sought after. Here, we investigated natural cardiovascular aging in a non-human primate, comparing healthy young-adult and aged common marmosets (Callithrix jacchus). Despite preservation of most cardiac functional parameters in aged animals, significant histological alterations were found including fibrosis and microvascular rarefaction. Molecular phenotyping by single-nuclei RNA-sequencing revealed activation of cardiac stress, pro-inflammatory and fibrotic gene programs in aged hearts. Importantly, proteomic analysis of cardiac extracellular vesicles revealed a cardioprotective cargo in young animals while functionally demonstrating pro-angiogenic properties on human cardiac microvascular endothelial cells. Finally, large vessel atherosclerosis was strikingly evident in aged animals and elucidated by bulk RNA-sequencing. Overall, the aging marmoset offers a great potential for translational cardiovascular research.

physiology↗

Gas5A, a putative glucanosyltransferase from Botrytis, functions as cell death inducing protein in plants

The necrotrophic fungus Botrytis cinerea, releases numerous phytotoxic, cell death inducing proteins (CDIPs) during infection. The precise role of these proteins and their molecular function, however, is still unknown. Here, we report on the identification of a previously unknown CDIP, the glucanosyltransferase Gas5A. Functional characterization revealed that the C-terminal 60 aa of Gas5A are sufficient to induce cell death, independent from its putative enzymatic function. Gas5A localization and functional dependence on the receptor-associated kinase suppressor of BIR1-1 (SOBIR1) and the plant defense regulator ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) indicate recognition as a pathogen-associated molecular pattern (PAMP) at the plant plasma membrane, but it is toxic also when delivered inside plant cells. Generation of a CRISPR/Cas9-assisted Botrytis knockout strain did not indicate any impact of Gas5A on virulence. Taken together, Gas5A represents a novel PAMP-like CDIP with additional intracellular phytotoxic activity.

plant biology↗

SMAD4 loss drives cancer chromosomal instability through transcription-replication conflict-induced replication stress

Cancer chromosomal instability (CIN) drives tumor evolution through generating structural and numerical chromosome aberrations. However, its molecular determinants remain poorly characterized. Here, we uncover a yet unrecognized role for the tumor suppressor SMAD4 as a key regulator of genome stability. Mechanistically, loss of SMAD4 induces replication stress by promoting transcription-replication conflicts (TRCs), which induces chromosomal breaks and subsequent mitotic errors, leading to the concomitant emergence of structural and numerical CIN. While the tumor suppressor function of SMAD4 has been predominantly attributed to TGF-{beta} signaling, we find that its role in genome maintenance operates via BMP signaling and its transcriptional target gene ID3. Consequently, inhibition of BMP signaling mimics the loss of SMAD4 whereas re-expression of SMAD4 or ID3 suppresses TRCs, replication stress and mitotic errors in SMAD4-deficient cancer cells. Our findings reveal a TRC- and replication stress-driven mechanism by which loss of BMP-SMAD4 signaling causes cancer chromosomal instability.

cancer biology↗

The temporal refinement of Dach1 is a key step in the functional maturation of primary somatosensory neurons

During somatosensory neurogenesis, neurons are born in an unspecialized transcriptional state, with several transcription factors following a broad-to-restricted expression dynamic as development proceeds, supporting neuron subtype identities. The relevance of this temporal refinement remains however unclear, these broad-to-restricted transcription factors being selectively involved in neurons in which they are ultimately maintained. Here we found that Dach1 encodes for a bona fide broad-to-restricted transcription factor retained and required in tactile somatosensory neurons. Within developing nociceptors, we demonstrate that Prdm12 contributes to Dach1 extinction. Using genetic approaches to prevent its temporal restriction during somatosensory development, we reveal that Dach1 refinement is a prerequisite for the appropriate transcriptional maturation of somatosensory subtypes from which it becomes ultimately excluded. These findings highlight the essential role played by Dach1 during somatosensory neuron development. They further demonstrate that the broad-to-restricted temporal pattern followed by several transcription factors is physiologically relevant to achieve appropriate transcriptional maturation of somatosensory neurons.

neuroscience↗

Rhodoquinone-dependent electron transport chain is essential for C. elegans survival in hydrogen sulfide environments

Hydrogen sulfide (H2S) has traditionally been considered as an environmental toxin for animal lineages; yet, it plays a signaling role in various processes at low concentrations. Mechanisms controlling H2S in animals, especially in sulfide-rich environments, are not fully understood. The main detoxification pathway involves the conversion of H2S into less harmful forms, through a mitochondrial oxidation pathway. The first step of this pathway oxidizes sulfide and reduces ubiquinone (UQ) through sulfide-quinone oxidoreductase (SQRD/SQOR). Because H2S inhibits cytochrome oxidase and hence UQ regeneration, this pathway becomes compromised at high H2S concentrations. The free-living nematode C. elegans feeds on bacteria and can face high sulfide concentrations in its natural environment. This organism has an alternative ETC that uses rhodoquinone (RQ) as the lipidic electron transporter and fumarate as the final electron acceptor. In this study, we demonstrate that RQ is essential for survival in sulfide. RQ-less animals (kynu-1 and coq-2e KO) cannot survive high H2S concentrations, while UQ-less animals (clk-1 and coq-2a KO) exhibit recovery, even when provided with a UQ-deficient diet. Our findings highlight that sqrd-1 uses both benzoquinones and that RQ-dependent ETC confers a key advantage (RQ regeneration) over UQ in sulfide conditions. C. elegans also faces cyanide, another cytochrome oxidase inhibitor, whose detoxification leads to H2S production, via cysl-2. Our study reveals that RQ delays killing by the HCN-producing bacteria Pseudomonas aeruginosa PAO1. These results underscore the fundamental role that RQ-dependent ETC serves as a biochemical adaptation to H2S environments, and to pathogenic bacteria producing cyanide and H2S toxins.

biochemistry↗

A single-cell strategy for the identification of intronic variants related to mis-splicing in pancreatic cancer

Most clinical diagnostic and genomic research setups focus almost exclusively on coding regions and essential splice sites, thereby overlooking other non-coding variants. As a result, intronic variants that can promote mis-splicing events across a range of diseases, including cancer, are yet to be systematically investigated. Such investigations would require both genomic and transcriptomic data, but there currently exist very few datasets that satisfy these requirements. We address this by developing a single-nucleus full-length RNA-sequencing approach that allows for the detection of potentially pathogenic intronic variants. We exemplify the potency of our approach by applying pancreatic cancer tumor and tumor-derived specimens and linking intronic variants to splicing dysregulation. We specifically find that prominent intron retention and pseudo-exon activation events are shared by the tumors and affect genes encoding key transcriptional regulators. Our work paves the way for the assessment and exploitation of intronic mutations as powerful prognostic markers and potential therapeutic targets in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/539836v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@18ddba7org.highwire.dtl.DTLVardef@4fc53aorg.highwire.dtl.DTLVardef@11d8d0dorg.highwire.dtl.DTLVardef@1178b86_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

PSC niche develops into immune-responsive blood cells capable of transdifferentiating into lamellocytes in Drosophila

Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. Here, we combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified precursor and effector hemocytes with distinct molecular signatures and cellular functions clearly distinct from other stages of hematopoiesis. Strikingly, we identified that PSC cells, which function as lymph gland niche, are highly migratory and immune responsive cells in the pupa. PSC cells can transdifferentiate to lamellocytes triggered by wasp infection. Altogether, our data highlight a remarkable cell heterogeneity, and identifies a cell population that acts not only as a stem cell niche in larval hematopoiesis, but functions as cell reservoir to pupal and adult blood cells.

cell biology↗

A novel single-cell RNA-sequencing platform and its applicability connecting genotype to phenotype in ageing-disease

Advances in single cell multi-omics analysis such as the ability of a genotype to show phenotypic diversity lead to a comprehensive understanding of cellular events in human diseases. The cardinal feature to access this information is the technology used for single-cell isolation, barcoding, and sequencing. While numerous platforms have been reported for single-cell RNA-sequencing, technical enhancements are needed in terms of highly purified single cell selection, cell documentation or limitations in cell sizing and chemistry. In this study, we present a novel high-throughput platform using a full-length RNA-sequencing approach that offers substantial technical improvements of all parameters. We applied this platform to dermal fibroblasts derived from six patients with different segmental progeria syndromes and defined phenotype-associated pathway signatures and variant-associated expression modifiers. These results validate the applicability of our platform to highlight genotype-expression relationships for molecular phenotyping in ageing disorders.

genomics↗

Human engineered skeletal muscle of hypaxial origin from pluripotent stem cells with advanced function and regenerative capacity

Human pluripotent stem cell derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for derivation of skeletal muscle cells and their utility in three-dimensional skeletal muscle organoid formation as well as skeletal muscle tissue engineering. Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors of hypaxial origin followed by primary and secondary fetal myogenesis into hypaxial muscle with development of a satellite cell pool and evidence for innervation in vitro. Skeletal muscle organoids faithfully recapitulate all steps of embryonic myogenesis in 3D Tissue engineered muscle exhibits organotypic maturation and function, advanced by thyroid hormone. Regenerative competence was demonstrated in a cardiotoxin injury model with evidence of satellite cell activation as underlying mechanism. Collectively, we introduce a hypaxial muscle model with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease, and repair.

cell biology↗

Comparative multi-tissue profiling reveals extensive tissue-specificity in transcriptome reprogramming upon cold exposure

Cold exposure is an extensively used intervention for enhancing thermogenic and mitochondrial activity in adipose tissues. As such, it has been suggested as a potential lifestyle intervention for body weight maintenance. The metabolic consequences of cold acclimation are not limited to the adipose tissues, however the impact on rest of the tissues in context of their gene expression profile remains unclear. Here we provide a systematic characterization of cold exposure-mediated effects in a comparative multi-tissue RNA sequencing approach using wide range of organs including spleen, bone marrow, spinal cord, brain, hypothalamus, ileum, liver, subcutaneous-, visceral- and brown adipose tissues. Our findings highlight that transcriptional responses to cold exposure exhibit high degree of tissue-specificity both at the gene level and at GO enrichment gene sets, which is not directed by the basal gene expression pattern exhibited by the various organs. Our study places the cold adaptation of individual tissues in a whole-organism framework and provides an integrative transcriptional analysis necessary for understanding the cold exposure-mediated biological reprograming.

physiology↗

The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models

The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 M, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) as well as Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 300 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus. SIGNIFICANCEO_LIMTX is one of the earliest cancer drugs to be developed, giving rise to seven decades of clinical experience. It is on the World Health Organizations List of Essential Medicines, can be administered orally or parenterally, and its costs are at single digit {euro} or $ amounts/day for standard treatment. In case of its successful further preclinical evaluation for treating SARS-CoV-2 infections, its repurposing to treat COVID-19 would thus be feasible, especially under low-resource conditions. C_LIO_LIAdditional drugs exist to interfere with the synthesis of nucleotides, e.g. additional folate antagonists, inhibitors of GMP synthetase, or inhibitors of dihydroorotate dehydrogenase (DHODH). Such inhibitors have been approved as drugs for different purposes and might represent further therapeutic options against infections with SARS-CoV-2 C_LIO_LIRemdesivir is currently the most established drug for treating COVID-19. Our results argue that MTX and remdesivir, even at moderate concentrations, can act in a synergistic fashion to repress virus replication to a considerably greater extent than either drug alone. C_LIO_LICOVID-19, in its severe forms, is characterized by pneumonia and acute respiratory distress syndrome, and additional organ involvements. These manifestations are not necessarily a direct consequence of virus replication and cytopathic effects, but rather a result of an uncontrolled inflammatory and immune response. Anti-inflammatory drugs such as glucocorticoids are thus being evaluated for treating COVID-19. However, this bears the risk of re-activating virus spread by suppressing a sufficient and specific immune response. In this situation, it is tempting to speculate that MTX might suppress both excessive inflammation as well as virus replication at the same time, thus limiting both the pathogenesis of pneumonia and also the spread of virus within a patient. C_LI

molecular biology↗

Alternative splicing of COQ-2 determines the choice between ubiquinone and rhodoquinone biosynthesis in helminths

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In aerobic environments they use ubiquinone (UQ) but in anaerobic environments inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. The switch to RQ synthesis is driven by substrate selection by the polyprenyltransferase COQ-2 but the mechanisms underlying this substrate choice are unknown. We found that helminths make two coq-2 isoforms, coq-2a and coq-2e, by alternative splicing. COQ-2a is homologous to COQ2 from other eukaryotes but the COQ-2e-specific exon is only found in species that make RQ and its inclusion changes the enzyme core. We show COQ-2e is required for RQ synthesis and for survival in cyanide in C. elegans. Crucially, we see a switch from COQ-2a to COQ-2e as parasites transition into anaerobic environments. We conclude that under anaerobic conditions helminths switch from UQ to RQ synthesis via alternative splicing of coq-2.

molecular biology↗

The kynurenine pathway is essential for rhodoquinone biosynthesis in Caenorhabditis elegans

A key metabolic adaptation for some species that face hypoxia as part of their life-cycle involves an alternative electron transport chain in which rhodoquinone (RQ) is required for fumarate reduction and ATP production. RQ biosynthesis in bacteria and protists requires ubiquinone (Q) as a precursor. In contrast, Q is not a precursor for RQ biosynthesis in animals such as parasitic helminths, and this pathway has remained elusive. We used Caenorhabditis elegans as a model animal to elucidate several key steps in RQ biosynthesis. Through RNA interference and a series of mutants, we found that arylamine metabolites from the kynurenine pathway are essential precursors for RQ biosynthesis de novo. Deletion of kynu-1, which encodes a kynureninase that converts L-kynurenine (KYN) into anthranilic acid (AA), and 3-hydroxykynurenine (HKYN) into 3-hydroxyanthranilic acid (3HAA), completely abolishes RQ biosynthesis, but does not affect Q levels. Deletion of kmo-1, which encodes a kynurenine 3-monooxygenase that converts KYN to HKYN, drastically reduces RQ, but not Q levels. Knockdown of the Q biosynthetic genes, coq-5 and coq-6, affects both Q and RQ levels demonstrating that common enzymes are used in both biosynthetic pathways. Our study reveals that two pathways for RQ biosynthesis have independently evolved. In contrast to bacteria, where amination is the last step in RQ biosynthesis, worms begin with the arylamine precursor, AA or 3HAA. Since RQ is absent in mammalian hosts of helminths, inhibition of RQ biosynthesis may have broad implications for targeting parasitic infections which cause important neglected tropical diseases.

biochemistry↗

Use of selenocysteine, the 21st amino acid, in the fungal kingdom

Selenoproteins are a diverse class of proteins containing selenocysteine (Sec), the 21st amino acid, incorporated into proteins during translation through a unique recoding mechanism. Selenoproteins fulfil essential roles in several lineages, including vertebrates; yet, they are not ubiquitous across the tree of life. In particular, the fungal kingdom was thought to lack selenoproteins, a paradigm that we defy here. We show that the genetic machinery for Sec utilization is present in the genomes of five species belonging to diverse early-branching fungal phyla (Chytridiomycota, Zoopagomycota, and Mucoromycota). We identified several selenoprotein genes in each of these genomes, and we detected canonical Sec insertion RNA structures (SECIS elements) for some of them. The identified selenoproteins are known or predicted oxidoreductases, some of which are conserved in mammals. Phylogenetic analyses support a scenario of vertical inheritance of the Sec trait within eukaryotes and fungi. Sec was then lost in numerous independent events in various fungal lineages, even within Sec-containing phyla. Notably, Sec was lost at the base of Dikarya, resulting in the absence of this trait in Saccharomyces cerevisiae and other well studied fungi. Our results indicate that, despite scattered occurrence, selenoproteins are found in all kingdoms of life.

genomics↗