Biology subjects

Cisneros, A.

Publications and source records attributed to Cisneros, A..

7 recordsLinked to original sources

bioRxiv2025

Incubator-Free Organoid Culture in a Sealed Recirculatory System

Discovery in human biology is pivoting toward high-dimensional computational analysis of 3D in vitro models, but this progress is limited by reliance on conventional cell culture techniques. Realism and data collection are hindered by the environmental instabilities and accessibility constraints of standard incubators. We introduce an automated, sealed recirculatory system that eliminates these barriers, enabling unconstrained instrument integration and infrastructure-independent scalability. By employing gas-tight sealing, a liquid-phase gas buffer and a non-porous plastic gas exchanger, our technology maintains biological stability without the compromises of open-air vessels. This design eliminates the need for CO2 incubators and prevents the evaporative drift that typically plagues conventional open-culture vessels. Operating on the benchtop outside the cell culture suite, we demonstrate that our system supports continuous, multi-week live imaging of vascular organoids while maintaining metabolic viability, structural fidelity and electrophysiological activity in brain organoids comparable to traditional in-incubator cultures. TeaserA sealed benchtop device enables data-rich organoid culture beyond the constraints of the cell culture laboratory.

bioengineering↗

bioRxiv2025

Paralog interference preserves genetic redundancy

Models of gene duplication often assume that loss-of-function mutations neutrally promote the return to the ancestral singleton state. They thus ignore the potential functional interference between duplicated proteins stemming from their physical interactions. Here, we show that for heteromerizing paralogs, such interference potentiates negative selection on loss-of-function mutations. This effect maintains genetic redundancy over longer timescales depending on the rate and severity of loss-of-function mutations. We experimentally estimate that around 6% of deleterious amino acid substitutions for a representative tetrameric protein interfere with a second copy. Interfering substitutions typically disrupt either catalysis or the final step of protein complex assembly, with varying degrees of severity. Our work shows that paralog interference renders the negative effects of loss-of-function mutations visible to purifying selection, contributing to the preservation of genetic redundancy.

evolutionary biology↗

bioRxiv2025

Gene Silencing in Plants by Artificial Small RNAs Derived from Minimal Precursors and Expressed via Tobacco Rattle Virus

Highly specific, second-generation RNA interference tools are based on artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). Recent progress includes the use of minimal-length precursors to express art-sRNAs in plants. These minimal precursors retain the minimal structural elements for recognition and efficient processing by host enzymes. They yield high amounts of art-sRNAs and remain stable when incorporated into potato virus X-based viral vectors for art-sRNA-mediated virus-induced gene silencing (art-sRNA-VIGS). However, further adaptation to new viral vector systems with reduced symptomatology is needed to improve the versatility of art-sRNA-VIGS. Here, we developed a novel platform based on tobacco rattle virus (TRV) -a widely used viral vector inducing minimal or no symptoms- for the delivery of art-sRNAs into plants. TRV was engineered to express authentic amiRNAs and syn-tasiRNAs from minimal precursors in Nicotiana benthamiana, resulting in robust and highly specific silencing of endogenous genes. Notably, the expression of syn-tasiRNAs through TRV conferred strong resistance against tomato spotted wilt virus, an economically important pathogen. Furthermore, we established a transgene-free approach by applying TRV-containing crude extracts through foliar spraying, eliminating the need for stable genetic transformation. In summary, our results highlight the unique advantages of minimal precursors and extend the application of art-sRNA-VIGS beyond previously established viral vector systems, providing a scalable, rapid and highly specific tool for gene silencing. Key messageWe developed a novel tobacco rattle virus-based platform for the transgene-free expression of both artificial microRNAs and synthetic trans-acting small interfering RNAs for efficient gene silencing in plants.

plant biology↗

bioRxiv2024

Network-community analysis of cellular senescence

Most cellular phenotypes are genetically complex. Identifying the set of genes that are most closely associated with a specific cellular state is still an open question in many cases. Here we study the transcriptional profile of cellular senescence using a combination of network-based approaches, which include eigenvector centrality feature selection and community detection. We apply our method to cell-type-resolved RNA sequencing data obtained from injured muscle tissue in mice. The analysis identifies some genetic markers consistent with previous findings, and other previously unidentified ones, which are validated with previously published single-cell RNA sequencing data in a different type of tissue. The key identified genes, both those previously known and the newly identified ones, are transcriptional targets of factors known to be associated with established hallmarks of senescence, and can thus be interpreted as molecular correlates of such hallmarks. The method proposed here could be applied to any complex cellular phenotype even when only bulk RNA sequencing is available, provided the data is resolved by cell type.

systems biology↗

bioRxiv2024

Rbm45 Phylogenetics, Protein Domain Conservation, and Gene Architecture in Clade Metazoa

Mammalian Rbm45 is predominately expressed in neuronal tissue and is integral in brain development and neuronal differentiation under physiological conditions. Dysregulation of Rbm45 has been strongly associated with neurodegenerative disorders in humans and can drive hepatocellular carcinoma through reprogramming lipid metabolism. Intriguingly, Rbm45 is an ancient protein, evolutionarily conserved throughout metazoans, including in sponges which lack a nervous system. Curiously, the evolution of Rbm45 gene structure and protein domain conservation across kingdom Animalia is largely unknown. We performed phylogenetic analysis of Rbm45 nucleotide and amino acid sequences from 36 species representing 9 phyla: Porifera, Cnidaria, Priapulida, Mollusca, Brachiopoda, Arthropoda, Echinodermata, Hemichordata, and Chordata. While the tree from Rbm45 nucleotide sequence data resulted in clades Protostomia and Deuterostomia showing paraphyly, the phylogeny derived from Rbm45 amino acid sequence largely recapitulated known monophyletic relationships among metazoans. Human RBM45 protein structure includes three RNA-binding domains (RBD), a homo-oligomerization association (HOA) domain, a nuclear localization sequence (NLS), and a nuclear export sequence (NES). Multiple sequence alignment across the same 36 taxa used for phylogenetic analysis revealed conservation of all three RBDs, the HOA, and NLS; in contrast the NES was only detected in clade Craniata and not in clades Ambulacraria and Protostomia. Rbm45 gene structure analysis revealed increasing gene complexity concomitant with increasing evolutionary complexity. Rbm45 from non-bilaterian taxa had from 2 to 4 large exons, while bilaterian taxa had between 6 to17 small exons. These findings demonstrate that Rbm45 is an ancient, highly conserved gene among metazoans suggesting a function in a breadth of neural/sensory systems.

evolutionary biology↗

bioRxiv2019

The BUBR1 pseudokinase domain promotes efficient kinetochore PP2A-B56 recruitment to regulate spindle checkpoint silencing and chromosome alignment

The balance of phospho-signalling at outer-kinetochores during mitosis is critical for the accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. In humans, a major player in determining this balance is the PP2A-B56 phosphatase which is recruited to the Kinase Attachment Regulatory Domain (KARD) of the Spindle Assembly Checkpoint protein Budding Uninhibited by Benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This event unleashes a rapid, switch-like phosphatase relay that reverses phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase entry. Here, we conclusively demonstrate that the pseudokinase domain of human BUBR1 lacks phosphotransfer activity and that it was maintained in vertebrates because it allosterically promotes KARD phosphorylation. Mutation or removal of this domain results in decreased PP2A-B56 recruitment to the outer kinetochore, attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. We demonstrate that the functions of the BUBR1 pseudokinase and the BUB1 kinase domains are intertwined, providing an explanation for retention of the pseudokinase domain in certain eukaryotes.

cell biology↗

bioRxiv2019

The role of structural pleiotropy and regulatory evolution in the retention of heteromers of paralogs

Many paralogs derive from the duplication of genes encoding homomeric proteins. Such duplication events lead to the formation of homomers and heteromers, thus creating new structures from a single event. We exhaustively characterize this phenomenon using the budding yeast protein-protein interaction network. We observe that paralogs that heteromerize are very frequent and less functionally diverged than those that lost this property, raising the possibility that heteromerization prevents functional divergence. Using in silico evolution, we show that for homomers and heteromers that share binding interfaces, mutations on one complex have pleiotropic effects on the other complex, resulting in highly correlated responses to selection. As a result, heteromerization could be preserved indirectly due to selection for the maintenance of homomers. By integrating data on gene expression and protein localization, we find that regulatory evolution could play a role in overcoming these structural pleiotropic effects and in allowing paralog functional divergence.

evolutionary biology↗