Biology subjects

San Miguel, A.

Publications and source records attributed to San Miguel, A..

9 recordsLinked to original sources

bioRxiv2026

TAXISCAN, Optimizing throughput and behavioral depth in standard C. elegans chemotaxis assays

Chemosensory signaling is crucial for organisms to respond to their environment and dysfunctions in these complex pathways have been implicated in diseases such as neurological disorders. The nematode C. elegans is a useful tool for studying chemosensation as it offers a fully-mapped nervous system with relatively simple chemosensory circuits. One of the most common methods to quantify chemosensory function in worms is through chemotaxis assays, which require extensive time and manual effort. In this work, we sought to capture novel chemosensory metrics and increase the throughput of these assays by combining rapid image acquisition with bioinformatics processing. In doing so, we have developed a Technique for Automating chemotaXis Investigations using Simple-Capture Approaches in Nematodes (TAXISCAN), a rapid acquisition platform that can be used to study natural chemosensory behavior as well as to characterize behavioral and locomotion phenotypes in half the time of standard assays. HighlightsO_LIThe TAXISCAN platform provides a rapid, accurate, and inexpensive means of conducting unbiased chemotaxis assays in C. elegans C_LIO_LIIn addition to the Chemotaxis Index (CI), the TAXISCAN platform can quantify behavioral metrics such as movement speed, distance travelled, and dispersion patterns C_LIO_LIWe successfully use TAXISCAN to discriminate behavioral phenotypes in neurosensory mutants C_LI

bioengineering↗

bioRxiv2026

Looking beyond stereotyped neuron structures reveals links between beading and morphological rearrangements in aging phenotypes.

1Understanding how neuronal morphology changes during aging and acute stress is essential for elucidating mechanisms of neurodegeneration. The highly branched PVD neuron of Caenorhabditis elegans provides a powerful model for studying dendritic remodeling and degeneration-associated phenotypes such as dendritic beading. However, the complexity of this arbor presents substantial challenges for automated segmentation and quantitative analysis. In this study, we adapted a convolutional neural network (CNN)-guided region growing framework for automated dendrite tracing, coupled with two topology-based algorithms for categorizing dendritic segments by branching degree. The segmentation algorithm achieved high accuracy relative to manual tracing, with a median Dice coefficient of 0.82, while reducing analysis time by approximately tenfold. Automated dendrite categorization demonstrated strong agreement with manual annotations across branching orders, though position-based mapping performance declined with age due to progressive morphological distortion. Leveraging this platform, we investigated mechanistic differences in dendritic beading patterns observed during aging and cold shock. Consistent with prior work, aging was associated with decreased inter-bead spacing, whereas cold shock produced increased bead dispersion with stress severity. Structural analysis revealed that these trends were not driven by dendritic pruning or reduced arbor complexity. Instead, while a traditional anatomically unflexible paradigm falsely implicated lower-degree dendrites as highly vulnerable, our branching-informed framework revealed that age-dependent beading is fundamentally dictated by a segments history of successive branching events. Conversely, acute cold shock triggered systemic beading that expanded across all dendritic orders in a severity-dependent manner. Together, these findings demonstrate that chronic aging and acute stress engage distinct degenerative pathways (compartment-specific lineage vulnerability versus global architectural collapse) rather than gross morphological loss, as well as highlighting the need for paradigms that enable reliable analysis of changing morphologies.

bioinformatics↗

bioRxiv2025

Assessing PDMS Biocompatibility in Microfluidic Applications: Toxicity and Survival Outcomes in C. elegans

Polydimethylsiloxane (PDMS), often assumed to be biocompatible, is widely used in microfluidic devices and biomedical research. Here, we systematically assess the organismal effects of PDMS network components and their leachates using Caenorhabditis elegans as a whole-animal model. We demonstrate that uncrosslinked vinyl-terminated PDMS (v-PDMS) chains, which comprise the majority of a PDMS network and are known to diffuse into aqueous environments, exhibit acute, environmentally-dependent toxicity. Low-molecular-weight v-PDMS (6 kDa) caused mild lethality in nutrient-rich S-Medium but significantly higher mortality in minimal S-Buffer, showing that media composition strongly influences toxic effects. Adding cholesterol, calcium, or magnesium notably reduced v-PDMS-induced lethality, whereas trace metals increased it. Using a DAF-16::GFP reporter strain, we show that cholesterol influences organismal stress responses to v-PDMS exposures. Progeny from starved parents showed full resistance to v-PDMS, suggesting transgenerational stress memory plays a role in reducing PDMS toxicity. We also find that linear siloxanes cause modest but significant lethality, whereas cyclic siloxanes do not. The PDMS crosslinker TDSS, however, provides partial protection when present with v-PDMS, revealing diverse biological effects among PDMS network precursors. Overall, these results show that PDMS-derived components are not universally harmless and that susceptibility depends greatly on environmental conditions, sterol levels, and physiological history. Our findings emphasize the importance of carefully evaluating PDMS formulations for biomedical use and offer a framework for assessing polymer leachate toxicity in living organisms.

pharmacology and toxicology↗

bioRxiv2024

Amyloid β Induces Hormetic-Like Effects Through Major Stress Pathways in a C. elegans Model of Alzheimer's Disease

Amyloid {beta} (A{beta}) is a peptide known for its characteristic aggregates in Alzheimers Disease and its ability to induce a wide range of detrimental effects in various model systems. However, A{beta} has also been shown to induce some beneficial effects, such as antimicrobial properties against pathogens. In this work, we explore the influence of A{beta} in stress resistance in a C. elegans model of Alzheimers Disease. We found that C. elegans that express human A{beta} exhibit increased resistance to heat and hypoxia, but not to oxidative stress. This beneficial effect of A{beta} was driven from A{beta} in neurons but not muscles, and the abundance of A{beta} in neurons correlated with stress resistance levels. Transcriptomic analysis revealed that this selective stress resistance was mediated by the Heat Shock Protein (HSPs) family of genes. Furthermore, neuropeptide signaling was necessary for A{beta} to induce stress resistance, suggesting neuroendocrine signaling plays a major role in activating organismal stress response pathways. These results highlight the potential beneficial role of A{beta} in cellular function, as well as its complex effects on cellular and organismal physiology that must be considered when using C. elegans as a model for Alzheimers Disease.

physiology↗

bioRxiv2024

Derivation of human trophoblast stem cells from placentas at birth

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool for modeling the placental cytotrophoblast (CTB) in vitro. hTSCs were originally derived from CTBs of the first trimester placenta or blastocyst-stage embryos in trophoblast stem cell medium (TSCM) that contains epidermal growth factor (EGF), the glycogen synthase kinase-beta (GSK3{beta}) inhibitor CHIR99021, the transforming growth factor-beta (TGF{beta}) inhibitors A83-01 and SB431542, valproic acid (VPA), and the Rho-associated protein kinase (ROCK) inhibitor Y-27632. Here we show that hTSCs can be derived from CTBs isolated from the term placenta, using TSCM supplemented with a low concentration of mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (TUA medium). Notably, hTSCs could not be derived from term CTBs using TSCM alone, or in the absence of either UK5099 or lipid-rich albumin. Strikingly, hTSCs cultured in TUA medium for a few passages could be transitioned into TSCM and cultured thereafter in TSCM. hTSCs from term CTBs cultured in TUA medium as well as those transitioned into and cultured in TSCM thereafter could be differentiated to the extravillous trophoblast and syncytiotrophoblast lineages and exhibited high transcriptome similarity with hTSCs derived from first trimester CTBs. We anticipate that these results will enable facile derivation of hTSCs from normal and pathological placentas at birth with diverse genetic backgrounds and facilitate in vitro mechanistic studies in trophoblast biology. Significance statementHuman trophoblast stem cells (hTSCs) derived from early pregnancy have emerged as a powerful tool for modeling early human placental development in vitro. However, restrictions on derivation of cell lines from fetal tissues limit the availability and genetic diversity of hTSC lines. Also, the associated pregnancy outcome of hTSCs derived from early gestation is unknown. Here we describe the derivation and characterization of hTSCs from placentas obtained at birth. Our results will enable derivation of genetically diverse hTSCs associated with normal and pathological pregnancies, enabling research in placental biology.

developmental biology↗

bioRxiv2023

Programming Probiotics: Diet-responsive gene expression and colonization control in engineered S. boulardii

Saccharomyces boulardii (Sb) is an emerging probiotic chassis for delivering biomolecules to the mammalian gut, offering unique advantages as the only eukaryotic probiotic. However, precise control over gene expression and gut residence time in Sb have remained challenging. To address this, we developed five ligand-responsive gene expression systems and repaired galactose metabolism in Sb, enabling inducible gene expression in this strain. Engineering these systems allowed us to construct AND logic gates, control the surface display of proteins, and turn on protein production in the mouse gut in response to a dietary sugar. Additionally, repairing galactose metabolism expanded Sbs habitat within the intestines and resulted in galactose-responsive control over gut residence time. This work opens new avenues for precise dosing of therapeutics by Sb via control over its in vivo gene expression levels and localization within the gastrointestinal tract.

bioengineering↗

bioRxiv2021

Identifying C. elegans Lifespan Mutants by Screening for Early-Onset Protein Aggregation

Genetic screens have been widely used to identify genetic pathways that control specific biological functions. In C. elegans, forward genetic screens rely on the isolation of reproductively active mutants that can self-propagate clonal populations. Since aged individuals are unable to generate clonal populations, screens that target post-reproductive phenotypes, such as longevity, are challenging. In this work, we developed an approach that combines microfluidic technologies and image processing to perform a high-throughput, automated screen for mutants with shortened lifespan using protein aggregation as a marker for aging. We take advantage of microfluidics for maintaining a reproductively-active adult mutagenized population and for performing serial high-throughput analysis and sorting of animals with increased protein aggregation, using fluorescently labeled PAB-1 as a readout. We identified five mutants with increased aggregation levels, of which two exhibited a reduced lifespan. We demonstrate that lifespan mutants can be identified by screening for accelerated protein aggregation through quantitative analysis of fluorescently-labeled aggregates in populations that do not require conditional sterilization or manual separation of parental and progeny populations. We further analyzed the morphology of protein aggregates and reveal that patterns of aggregation in naturally-aging animals differ from mutants with increased aggregation, suggesting aggregate growth is time-dependent. This screening approach can be customized to other non-developmental phenotypes that appear during adulthood, as well as to other aging markers to identify additional longevity-regulating genetic pathways.

bioengineering↗

bioRxiv2021

Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions.

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we do not utilize transforming growth factor-beta inhibitors or a passage step for EVT differentiation, or forskolin for STB formation. Strikingly, under these conditions, presence of a single additional extracellular cue - lam-inin-1 - switched the terminal differentiation of hTSCs from STB to the EVT lineage. Activation of the sphingosine-1 receptor 3 receptor (S1PR3) using a chemical agonist could drive EVT differentiation of hTSCs in the absence of exogenous laminin, albeit less efficiently. To illustrate the utility of a chemically defined culture system for mechanistic studies, we examined the role of protein kinase C (PKC) signaling during hTSC differentiation to the EVT lineage. Inhibition of PKC/{beta} signaling significantly reduced HLA-G expression and the formation of HLA-G+ mesen-chymal EVTs during hTSC differentiation mediated by laminin exposure; however, it did not prevent commitment to the EVT lineage or STB differentiation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation, and will enable mechanistic studies in vitro. SignificanceDespite its importance to a healthy pregnancy, early human placental development remains poorly understood. Mechanistic studies are impeded by restrictions on research with human embryos and fetal tissues, and significant differences in placentation between humans and commonly used animal models. In this context, human trophoblast stem cells (hTSCs) have emerged as attractive in vitro models for the epithelial cytotrophoblast of the early gestation human placenta. Here we describe chemically defined culture conditions for differentiation of hTSCs to the two major differentiated cell types - extravillous trophoblast and syncytiotrophoblast. These culture conditions enable in vitro studies to reveal molecular mechanisms regulating hTSC differentiation.

developmental biology↗

bioRxiv2019

Two distinct trophectoderm lineage stem cells from human pluripotent stem cells

Trophoblasts are the principal cell type of the placenta. The use of human trophoblast stem cells (hTSCs) as a model for studies of early placental development is hampered by limited genetic diversity of existing hTSC lines, and constraints on using human fetal tissue or embryos needed to generate additional cell lines. Here we report the derivation of two distinct stem cells of the trophectoderm lineage from human pluripotent stem cells. The first is a CDX2- stem cell equivalent to primary hTSCs - they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ putative human trophectoderm stem cell (hTESC) with distinct cell culture requirements and differences in gene expression and differentiation relative to hTSCs. Derivation of hTSCs and hTESCs from pluripotent stem cells significantly enables construction of models for normal and pathological placental development.

developmental biology↗