Biology subjects

Castro, N.

Publications and source records attributed to Castro, N..

10 recordsLinked to original sources

bioRxiv2026

Biohybrid toggle switches: leveraging bistability in muscle-actuated machines

Tissue-engineered skeletal muscle offers robotics unique advantages in energy density, adaptability, and self-repair, but current biohybrid machines couple muscle directly to artificial skeletons, limiting net force output and requiring continuous stimulation maintain mechanism positions. Here, we introduce a biohybrid bistable switching mechanism that converts antagonistic muscle contractions to snap-through actuation of an elastomeric four-bar flexure, maintaining stable positions without energy consumption. We decouple passive muscle pre-tensioning from flexure operation with thin-film elastomer resistance bands, enabling fine tuning of spring stiffness and force to match muscle mechanical output envelopes. Leveraging mesoscale additive manufacturing, spin coating, and replica molding techniques, components are integrated into a millimeter-scale linear stage to axially align muscles with artificial components. Our prototype achieves reversible toggling between bistable states with a 130 ms switching time, establishing skeletal muscle actuators as viable electromechanical switches, and broadening biohybrid robotics towards novel applications such as grippers, valves, and mechanical computers.

bioengineering↗

bioRxiv2025

Biohybrid tendons enhance the power-to-weight ratio and modularity of muscle-powered robots

Biohybrid robots powered by tissue engineered skeletal muscle have historically relied on architectures in which muscle actuators are placed directly on skeletons, thus limiting the accessible design space for such machines. By contrast, native musculoskeletal architecture relies on tendons to bridge the interface between muscles and skeletons, enabling precise, space-efficient, and energy-efficient force transmission. In this study, we use a mathematical model of the muscle-tendon-skeleton interface to design a biohybrid muscle-tendon unit composed of tissue engineered muscle coupled to adhesive tough hydrogel tendons. We show how tuning tendon stiffness and pre-tension modulates actuator performance, measure fatigue characteristics of our actuators over >7000 cycles, and tune skeleton stiffness to increase force transmission muscles to skeletons by [~]29X. Furthermore, we demonstrate an [~]11X improvement in power-to-weight ratio of muscle-tendon units as compared to previous demonstrations of robots powered by muscles alone. This work validates a robust approach for designing, manufacturing, and deploying muscle-tendon actuators that promises to enhance the modularity and efficiency of biohybrid robots.

bioengineering↗

bioRxiv2024

Mitochondrial and Microtubule Defects in Exfoliation Glaucoma

Exfoliation Syndrome (XFS) is an age-related systemic condition characterized by large aggregated fibrillar material deposition in the anterior eye tissues. This aggregate formation and deposition on the aqueous humor outflow pathway are significant risk factors for developing Exfoliation Glaucoma (XFG), a secondary open-angle glaucoma. XFG is a complex, multifactorial late-onset disease that shares common features of neurodegenerative diseases, such as altered cellular processes with increased protein aggregation, impaired protein degradation, and oxidative and cellular stress. XFG patients display decreased mitochondrial membrane potential and mitochondrial DNA deletions. Here, using Tenon Capsule Fibroblasts (TFs) from Normal (No Glaucoma) and XFG patients, we found that XFG TFs have impaired mitochondrial bioenergetics and increased reactive oxygen species (ROS) accumulation. These defects are associated with mitochondrial abnormalities as XFG TFs exhibit smaller mitochondria that contain dysmorphic cristae, with an increase in mitochondrial localization to lysosomes and slowed mitophagy flux. Mitochondrial dysfunction in the XFG TFs was associated with an increase in the dynamics of the microtubule cytoskeleton, decreased acetylated tubulin, and increased HDAC6 activity. Treatment of XFG TFs with a mitophagy inducer, Urolithin A, and a mitochondrial biogenesis inducer, NAD+ precursor, Nicotinamide Ribose, improved mitochondrial bioenergetics and reduced ROS accumulation. Our results demonstrate abnormal mitochondria in XFG TFs and suggest that mitophagy inducers may represent a potential class of therapeutics for reversing mitochondrial dysfunction in XFG patients.

cell biology↗

bioRxiv2024

2.5D Actuating Substrates Enable Decoupling the Mechanical and Biochemical Effects of Muscle Exercise on Motor Neurons

Emerging in vivo evidence suggests that exercise impacts peripheral nerves, but the difficulty of isolating and studying the muscle-specific impact on motor neurons in vivo, as well as the inability to decouple the biochemical and mechanical impacts of exercise in this setting, motivate investigating this phenomenon in vitro. In this study, we show that tuning the mechanical properties of fibrin hydrogels can generate stable 2.5D motor neuron and contractile skeletal muscle cultures that enable long-term efficient secretome harvesting from exercised tissues. Motor neurons stimulated with muscle-secreted cytokines significantly upregulate neurite outgrowth and migration, with an effect size dependent on exercise intensity. Actuating magnetic microparticles embedded within 2.5D substrates enabled us to dynamically stretch motor neurons and non-invasively mimic the mechanical effects of exercise, revealing that dynamic stretch has an equally significant impact on axonogenesis. RNA sequencing revealed different transcriptomic signatures between groups, with biochemical stimulation having a significantly greater impact on cell signaling related to axon growth and development, neuron projection guidance, and neuron-muscle synapse maturation. Our study thus leverages 2.5D actuating substrates to robustly validate a hypothesized role for muscle exercise in regulating motor neuron growth and maturation through both mechanical and biochemical signaling.

bioengineering↗

bioRxiv2023

Circulating tumor extracellular vesicles to monitor metastatic prostate cancer genomics and transcriptomic evolution

Extracellular vesicles (EVs) secreted by tumors are abundant in plasma, but their potential for interrogating the molecular features of tumors through multi-omic profiling remains widely unexplored. Genomic and transcriptomic profiling of circulating EV-DNA and EV-RNA isolated from a range of in-vitro and in-vivo models of metastatic prostate cancer (mPC) revealed a high contribution of tumor material to EV-loaded DNA/RNA. Findings were validated in a cohort of longitudinal plasma samples collected from mPC patients during androgen receptor signaling inhibitor (ARSI) therapy. EV-DNA genomic features recapitulated matched-patient biopsies and associated with clinical progression. We developed a novel approach to enable the transcriptomic profiling of EV-RNA (RExCuE). We report how the transcriptomic profile in mPC EV-RNA is enriched for tumor-associated transcripts when compared to same patient blood RNA and healthy individuals EV-RNA, and reflect early on-therapy tumor adaptation changes. Altogether, we show that EV profiling enables longitudinal transcriptomic and genomic profiling of mPC in liquid biopsy.

cancer biology↗

bioRxiv2022

Comparative transcriptomics reveals human-specific cortical features

Humans have unique cognitive abilities among primates, including language, but their molecular, cellular, and circuit substrates are poorly understood. We used comparative single nucleus transcriptomics in adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets from the middle temporal gyrus (MTG) to understand human-specific features of cellular and molecular organization. Human, chimpanzee, and gorilla MTG showed highly similar cell type composition and laminar organization, and a large shift in proportions of deep layer intratelencephalic-projecting neurons compared to macaque and marmoset. Species differences in gene expression generally mirrored evolutionary distance and were seen in all cell types, although chimpanzees were more similar to gorillas than humans, consistent with faster divergence along the human lineage. Microglia, astrocytes, and oligodendrocytes showed accelerated gene expression changes compared to neurons or oligodendrocyte precursor cells, indicating either relaxed evolutionary constraints or positive selection in these cell types. Only a few hundred genes showed human-specific patterning in all or specific cell types, and were significantly enriched near human accelerated regions (HARs) and conserved deletions (hCONDELS) and in cell adhesion and intercellular signaling pathways. These results suggest that relatively few cellular and molecular changes uniquely define adult human cortical structure, particularly by affecting circuit connectivity and glial cell function.

neuroscience↗

bioRxiv2022

Daam1 negatively regulates USP10 activity

The differentiation of fibroblasts into pathological myofibroblasts during wound healing is in part characterized by increased cell surface expression of v-integrins. Our previous studies found that the deubiquitinase (DUB) USP10 removes ubiquitin from v-integrins, leading to cell surface integrin accumulation, subsequent TGF{beta}1 activation, and myofibroblast differentiation. In this study, a yeast-two hybrid screen elucidated a novel binding partner for USP10, the formin, Daam1. The USP10/Daam1 interaction was also supported by proximity ligation assay (PLA) activity. Treatment with TGF{beta}1 significantly increased USP10 and Daam1 protein expression, PLA signal, and co-localization to actin stress fibers. Furthermore, Daam1 siRNA knockdown significantly reduced a) co-precipitation of USP10 and Daam1 on purified actin stress fibers, and b) {beta}1- and {beta}5-integrin ubiquitination resulting in increased v-, {beta}1-, and {beta}5-integrin total protein levels, v integrin recycling to the cell surface, and extracellular fibronectin (FN) organization. Together, our data suggest that Daam1 negatively regulates USP10s DUB activity and subsequently maintains integrin protein homeostasis.

cell biology↗

bioRxiv2020

Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration

Intestinal organoids capture essential features of the intestinal epithelium such as folding of the crypt, spatial compartmentalization of different cell types, and cellular movements from crypt to villus-like domains. Each of these processes and their coordination in time and space requires patterned physical forces that are currently unknown. Here we map the three-dimensional cell-ECM and cell-cell forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the ECM and folds through apical constriction, whereas the transit amplifying zone pulls the ECM and elongates through basal constriction. Tension measurements establish that the transit amplifying zone isolates mechanically the stem cell compartment and the villus-like domain. A 3D vertex model shows that the shape and force distribution of the crypt can be largely explained by cell surface tensions following the measured apical and basal actomyosin density. Finally, we show that cells are pulled out of the crypt along a gradient of increasing tension, rather than pushed by a compressive stress downstream of mitotic pressure as previously assumed. Our study unveils how patterned forces enable folding and collective migration in the intestinal crypt.

cell biology↗

bioRxiv2017

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near the centrosome, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

cell biology↗

bioRxiv2016

Human pancreatic β cell lncRNAs control cell-specific regulatory networks

Recent studies have uncovered thousands of long non-coding RNAs (IncRNAs) in human pancreatic {beta} cells. {beta} cell lncRNAs are often cell type-specific, and exhibit dynamic regulation during differentiation or upon changing glucose concentrations. Although these features hint at a role of lncRNAs in {beta} cell gene regulation and diabetes, the function of {beta} cell lncRNAs remains largely unknown. In this study, we investigated the function of {beta} cell-specific lncRNAs and transcription factors using transcript knockdowns and co-expression network analysis. This revealed lncRNAs that function in concert with transcription factors to regulate {beta} cell-specific transcriptional networks. We further demonstrate that lncRNA PLUTO affects local three-dimensional chromatin structure and transcription of PDX1, encoding a key {beta} cell transcription factor, and that both PLUTO and PDX1 are downregulated in islets from donors with type 2 diabetes or impaired glucose tolerance. These results implicate lncRNAs in the regulation of {beta} cell-specific transcription factor networks.

genomics↗