Biology subjects

Ryu, S.

Publications and source records attributed to Ryu, S..

At least 19 recordsLinked to original sources

bioRxiv2026

CRTC, not phosphorylated CREB1, drives cAMP-induced transcription across diverse cell types

Specialized cell types layer cell-type-restricted proteins, metabolites, and organelles onto a shared foundation of core cellular processes. How this ubiquitous machinery generates lineage-specific outputs is central to both cell biology and therapeutic development. G-protein-coupled receptors (GPCRs) respond to receptor-restricted ligands to drive cell-type-specific transcription through cAMP-mediated activation of protein kinase A (PKA), which activates the transcription factor CREB1 through two parallel modes: direct phosphorylation at serine 133, and inhibition of salt-inducible kinases (SIK) that restrain the CRTC coactivators. How these modes integrate and contribute to signaling across cell types has remained unresolved, obscured by genetic redundancy and essentiality. Here we combine focused genetic analyses with a cross-lineage transcriptomic survey to dissect these parallel inputs. In Creb1/Atf1/Crem triple-knockout cells, a non-phosphorylatable mutant CREB1S133A fully rescued endogenous target gene activation, while Crtc1/Crtc2/Crtc3 ablation abolished transcription even with intact CREB1 serine 133 phosphorylation. As part of this mechanism, we found the annotated repressor ICER can instead act as a positive regulator, substituting for full-length CREB1 paralogs to drive a feedforward loop. Across melanocytes, hepatocytes, osteocytes, macrophages, and neurons, SIK inhibition recapitulated cAMP-PKA-driven transcription across both shared and cell-type-specific gene expression programs, with neurons a notable exception. These results invert the canonical model, placing CRTC recruitment as the dominant driver of CREB1-mediated transcription across diverse lineages, reframing how cAMP-PKA signaling can be interpreted and therapeutically targeted.

cell biology↗

bioRxiv2026

A novel fracture lattice in spiny mouse skin facilitates tissue autotomy and regeneration

Autotomy is a unique phenotype whereby an animal sheds a body part to escape predation1-3. The timing and location of autotomy are tightly regulated by preformed planes of weakness (aka fracture planes) which facilitate tissue loss. While autotomy is often followed by regeneration, these phenotypes are rarely reported in mammals4-9. A notable exception are spiny mice (Acomys) which exhibit skin autotomy and more remarkably, complete tissue regeneration10-14. Presently, mechanisms underlying autotomy and complete regeneration in Acomys skin remain elusive. Here, we report the discovery of a honeycomb-like fracture lattice in Acomys skin whose design directs tissue destruction but also facilitates regenerative healing. Unlike the single continuous surface of a fracture plane, this fracture lattice consists of a three-dimensional array of hexagonal units whose boundaries guide tissue breakage. Moreover, we identify collagen VI as the main constituent of the fracture lattice and find that it is distinctly arranged to initiate fracturing and propagation of skin tearing. By preconditioning the tissue for autotomy, the fracture lattice dampens the damage-induced inflammatory response but also upregulates a pro-regenerative gene signature, accelerating skin appendage regeneration. Lastly, we discovered the key role of spiny hairs in fracture lattice formation, as inhibiting their development leads to abnormal pattern formation and changes in skin fracture mechanics. Our results present a novel example of a uniquely evolved structural adaptation in mammalian skin that links tissue patterning, autotomy and regeneration. We expect that the application of a modular compartment structure to artificial skin and other organ engineering may enhance resilience to injury and facilitate efficient regeneration.

developmental biology↗

bioRxiv2026

Lung endothelial niche signaling governs self-renewal and fate transitions of human alveolar stem cells

Chronic lung diseases such as pulmonary fibrosis are characterized by the irreversible loss of alveolar type 1 (AT1) cells, yet the mechanisms governing human alveolar stem cell self-renewal and differentiation remain poorly defined. Here, we identify a lung endothelial niche that sustains the self-renewal of human alveolar type 2 (AT2) stem cells through MAPK signaling, enabling robust long-term expansion while preserving stem cell fate. Although YAP activation initiates AT1 transcriptional programs, it is insufficient to complete lineage maturation. We show that MAPK inhibition together with LATS inhibition promotes nuclear translocation of YAP, enhancing AT1 differentiation. Expanded human AT2 stem cells engraft in fibrotic lungs and contribute to alveolar regeneration while undergoing directed differentiation within diseased human lung tissue. Together, our findings define a niche-controlled signaling mechanism governing human alveolar stem cell fate and advance our understanding of alveolar regeneration.

cell biology↗

bioRxiv2025

Precision at Every Scale: Efficiency in AI-Driven De Novo Antibody Design

The precise de novo design of antibodies remains a therapeutic challenge. The AI platform, GaluxDesign, was evaluated in a high-efficiency Precision-Scale Workflow by synthesizing and testing only 50 full-length IgG candidates per epitope across eight distinct epitopes from six therapeutic targets. This campaign yielded a 10.5% binder rate (estimated EC50 < 100 nM), identifying target-specific binders for seven of eight epitopes, with multiple candidates exhibiting sub-nanomolar to single-digit nanomolar dissociation constants (Kd). We further assessed the same workflow on nine shared benchmark targets selected for external comparison, where GaluxDesign identified target-specific binders for eight of nine targets, demonstrating strong target-level performance relative to previously reported de novo antibody design approaches. Together, these results establish a high-efficiency, precision-scale workflow for generating novel, high-affinity therapeutic antibodies.

bioengineering↗

bioRxiv2025

Pth4 neurons define a novel hypothalamic circuit that promotes sleep via brainstem monoaminergic neurons

Classical studies identified a critical role for the hypothalamus in regulating sleep and wake states, but few such hypothalamic neuronal populations have been identified. Here we describe a sleep-promoting population of hypothalamic neurons that expresses the neuropeptides QRFP and parathyroid hormone 4 (Pth4) in zebrafish. Optogenetic stimulation of these neurons results in a large increase in sleep that requires pth4 but not qrfp. Noradrenergic locus coeruleus (LC) neurons and serotonergic raphe neurons (RN) in the hindbrain express distinct pth receptors, and genetic epistasis and cell ablation experiments revealed that Pth4 neuron-induced sleep is suppressed in mutants that lack noradrenaline in the LC or lack the serotonergic RN. Pth4 neuron-induced sleep is also suppressed in serine/threonine kinase 32a (stk32a) mutants, possibly via stk32a-expressing neurons in the prethalamus that express pth receptors. These results identify QRFP/Pth4 neurons as a novel hypothalamic sleep-promoting population and support a model in which distinct sleep- and wake-promoting hypothalamic populations act via monoaminergic neurons in the hindbrain to control vigilance state.

neuroscience↗

bioRxiv2025

SHARP: Generating Synthesizable Molecules via Fragment-based Hierarchical Action-space Reinforcement Learning for Pareto Optimization

Designing drug-like molecules that satisfy multiple objectives--such as high binding affinity, synthesizability, and drug-likeness--poses a complex global optimization problem over an astronomically large chemical space. Existing deep learning-based molecular generative models often treat this task as distribution modeling, relying on atom-level autoregressive actions with less consideration of explicit optimization feedback. Consequently, they frequently generate invalid structures, converge to local optima, or produce synthetically infeasible candidates. Here, we introduce SHARP (Synthesizable Hierarchical Action-space Reinforcement learning for Pareto optimization), a molecular generator that addresses these limitations via a fragment-based hierarchical action space and reinforcement learning. SHARP ensures synthetic accessibility by applying action masks guided by a pretrained Synthesizability Estimation Model (SEM). The reinforcement learning (RL) policy is trained using a composite reward function integrating docking scores, pharmacophore matching, and solvent accessibility to generate functionally relevant and experimentally tractable molecules. Furthermore, across four lead optimization tasks--fragment growing, linker design, scaffold hopping, and sidechain decoration--on a diverse receptor set, SHARP consistently outperforms prior methods in producing molecules at high affinity and synthesizability. These results demonstrate that reinforcement learning with a chemically intuitive action space design can be an effective solution to the optimization challenges in AI-driven drug discovery, offering a robust framework for rational molecular design in structure-based applications.

bioinformatics↗

bioRxiv2025

Estimating Actual Striking Forces Using Attenuation Properties of Taekwondo Protectors

BackgroundProtectors attenuate impact forces significantly, however, their quantitative attenuation characteristics under varying conditions remain poorly understood. Accurate measurement of striking forces is essential for evaluation. However, direct measurement of athletes striking forces presents significant practical and safety challenges, necessitating the development of indirect estimation approaches. MethodA controllable pendulum-based impact testing apparatus was developed to evaluate force attenuation characteristics of Taekwondo body protectors. The system delivered repeatable impacts across varying magnitudes and contact durations to a mannequin equipped with a protector. Simultaneous measurements of input and transmitted forces through the protector enabled direct quantification of attenuation ratios under controlled conditions. To demonstrate practical application, five Taekwondo athletes performed standardized kicks on the instrumented protector setup, allowing estimation of their actual striking forces using the derived attenuation factor. ResultsForce attenuation ratios demonstrated high consistency across impact magnitudes ranging from 300 to 5800 Newtons, with impact duration variations showing minimal influence on attenuation performance. Linear attenuation relationships were established between input and transmitted forces (R2[≥] 0.987), enabling derivation of a predictive attenuation factor. Validation testing with the pendulum system showed that the estimated input force trajectories achieved an nRMSE of 3.5%. Averaged across the entire force range, the MAPE was 4.7% for maximum force, 1.4% for impact duration, and 3.5% for impulse. Applying the attenuation factor to standardized kicks performed by five Taekwondo athletes yielded estimated maximum striking forces ranging from approximately 1300 to 1800 Newtons, demonstrating the methods capability to quantify actual forces that would otherwise be difficult to measure directly. DiscussionThis study establishes the first quantitative characterization of impact attenuation in Taekwondo body protectors, providing a validated attenuation factor for estimating striking forces from transmitted forces. These findings demonstrate a practical methodology for quantifying striking forces that are otherwise difficult to measure directly. Author summary

bioengineering↗

bioRxiv2025

Hormetic elevation of taurine restrains inflammaging by deactivating the NLRP3 inflammasome.

Taurine, the most abundant sulfonic amino acid in humans is largely obtained from diets rich in animal proteins. However, taurine is dietary non-essential because it can be synthesized from cysteine by activation of transsulfuration pathway (TSP) when food consumption is low or if the diet is predominantly plant based. The decline of taurine was proposed as the driver of aging through an undefined mechanism. Here, we found that mild food restriction in humans for one year that resulted in 14% reduction of calorie intake elevated the hypotaurine and taurine concentration in adipose tissue. Therefore, we investigated whether elevated taurine mimics caloric-restrictions beneficial effects on inflammation, a key mechanism of aging. Interestingly, aging increased the circulating and tissue concentrations of taurine suggesting that elevated taurine may serve as a hormetic stress response metabolite that regulates mechanism of age-related inflammation. The elevated taurine protected mice against mortality from sepsis and inhibited inflammasome-driven inflammation and gasdermin-D (GSDMD) mediated pyroptosis. Mechanistically, danger signals including hypotonicity that activate NLRP3-inflammasome, caused upstream taurine efflux from macrophages, which triggered potassium (K+) release and downstream canonical NLRP3 inflammasome assembly, caspase-1 activation, GSDMD cleavage and IL-1{beta} and IL-18 secretion that was reversed by taurine restoration. Notably, taurine does not efflux from GSDMD pore and inhibited IL-1{beta} from macrophages independently of known transporters SLC6A6 and SLC36A1. Increased taurine in old mice promotes healthspan by inducing anti-inflammatory pathways previously linked to youthfulness. These findings demonstrate that taurine is an upstream metabolic sensor of cellular perturbations that control NLRP3 inflammasome and lowers age-related inflammation.

immunology↗

bioRxiv2025

Modeling Friedreich's ataxia with Bergmann glia-enriched human cerebellar organoids

The human cerebellum is a specialized brain region that is involved in various neurological and psychiatric diseases but has been challenging to study in vitro due its complex neurodevelopment and cellular diversity. Despite the progress in generating neural tissues from human induced pluripotent stem cells (iPSCs), an organoid model that recapitulates the key features of cerebellar development has not been widely established. Here, we report the generation of a 60-day method for human cerebellar organoids (hCBOs) that is characterized by induction of rhombomere 1 (R1) cellular identity followed by derivation of neuronal and glial cell types of the cerebellum. In contrast to forebrain organoids with multiple neural rosettes and inside-out neuronal migration, hCBOs develop a SOX2+ cerebellar plate on the outermost surface of organoids with outside-in neuronal migration, which is a characteristic hallmark of cerebellar histogenesis. These hCBOs produced various other cell types including granule neurons, Purkinje cells, Golgi neurons, and deep cerebellar nuclei. By using a glial induction strategy, we generate Bergmann glial cells (BGCs) within the hCBOs that not only serve as scaffolds for granule cells migration but also enhance electrophysiological response of the hCBOs. Furthermore, by generating hCBOs from patients with Friedreichs ataxia (FRDA), we revealed abnormal disease-specific phenotypes that could be reversed by histone deacetylase (HDAC) inhibitors and gene editing by CRISPR-Cas9. Taken together, our advanced hCBO model provides new opportunities to investigate the molecular and cellular mechanisms of cerebellar ontogenesis and utilize patient-derived iPSCs for translational research.

developmental biology↗

bioRxiv2025

H2O2-induced astrocytic collagen triggers neuronal death via fucosylation-dependent glial barrier formation upon ischemic stroke

The cascade of molecular and cellular events leading to neuronal death after a focal ischemic stroke remains enigmatic. Although astrocytes form glial barriers that may protect surrounding tissue, how barriers develop and contribute to neuronal death is unclear. Here, we show that H2O2 induces astrocytic type I collagen (COL1)-production via miR-29-mediated post-transcriptional and fucosylation-dependent post-translational regulations, leading to integrin activation and neuronal death. In photothrombosis (PT)-induced cortical stroke model, PT triggered H2O2-surge, astrogliosis, glial barrier formation, COL1 expression, fibrotic scarring, altered N-glycosylation, neuronal loss, and neurological deficits. Remarkably, these effects were reversed by astrocyte-specific COL1 or FUT8 gene-silencing or treatment with KDS12025, an H2O2-decomposing peroxidase enhancer. KDS12025s neuroprotective effects were also recapitulated in non-human primate stroke model. These findings delineate a previously unrecognized astrocyte-driven mechanism in which oxidative stress induces COL1 production, promoting neuronal death, and position H2O2 and astrocytic COL1 and FUT8 as promising therapeutic targets for ischemic stroke.

neuroscience↗

bioRxiv2025

ADAR1 Regulates Lipid Remodeling through MDM2 to Dictate Ferroptosis Sensitivity

Triple-negative breast cancer (TNBC), defined by the lack of estrogen, progesterone, and HER2 receptor expressions, is aggressive and lacks targeted treatment options. Adenosine deaminase acting on RNA 1 (ADAR1) has been shown to contribute to TNBC tumorigenesis by modulating an innate immune response. However, little is known about its role in the regulation of metabolic fitness of TNBC. We tested the hypothesis that ferroptosis is an ADAR1-protected vulnerability by showing that ADAR1 knockdown sensitizes TNBC cells to ferroptosis inducers. Lipidomic analyses showed that ADAR1 loss increased the abundance of phospholipids enriched with polyunsaturated fatty acids (PUFA), of which peroxidation is the main driver of ferroptosis. Transcriptomic analyses discovered that the proto-oncogene MDM2 contributes to the lipid remodeling phenotype. A ferroptosis-focused drug screen identified FDA-approved cobimetinib as a drug-repurposing candidate to synergize with ADAR1 loss. This finding supports further basic, pre-clinical, and clinical studies to develop novel therapeutic strategies for TNBC through ADAR1-mediated metabolic regulation.

cancer biology↗

bioRxiv2024

Transcriptional Regulation of Neuropeptide Receptors Decodes Complexity of Peptidergic Modulation of Behavior and Physiology.

The modulation of complex behaviors in response to environmental and physiological contexts is a fundamental aspect of animal biology, with neuropeptides (NPs) playing a crucial role in this process. This study investigates the transcriptional regulation of neuropeptide receptors (NPRs) as a mechanism for context-dependent neuropeptidergic modulation of physiology and behavior. We hypothesize that the transcriptional control of NPR genes, rather than the NPs themselves, is a critical determinant in the context-dependent modulation of behavior and physiology. Using a multi-faceted approach, including comparative genomics, transcription factor network analysis, and empirical validation in model organisms such as Drosophila melanogaster, we reveal a complex regulatory landscape where NPR expression is tightly controlled. Our findings demonstrate that NPR genes exhibit a higher number of enhancers, CTCF-binding sites, and open chromatin regions compared to NP genes, suggesting a greater susceptibility to transcriptional modulation. This regulatory architecture allows for precise control over neuropeptidergic signaling, enabling dynamic and context-specific behavioral and physiological responses. Our results highlight the importance of NPR-expressing cells by transcriptional regulation in mediating the effects of NPs on behavior and physiology. We show that this regulation is conserved across species, indicating an evolutionarily significant mechanism for fine-tuning neuropeptidergic signaling. Furthermore, our study provides insights into the distinct regulatory mechanisms underlying the multifunctionality of NPs and their receptors, offering a novel perspective on the transcriptional control of complex behaviors. In conclusion, this study advances our understanding of neuropeptidergic signaling by focusing on the transcriptional regulation of NPRs. Our findings have broad implications for the development of therapeutic strategies targeting neuropeptidergic systems in various neurological and behavioral disorders.

neuroscience↗

bioRxiv2024

Early life challenge enhances cortisol regulation in zebrafish larvae

The hypothalamic-pituitary-adrenal (HPA) axis in mammals and the hypothalamic-pituitary-interrenal (HPI) axis in fish are open systems that adapt to the environment during development. Little is known about how this adaptation begins and regulates early stress responses. We used larval zebrafish to examine the impact of prolonged forced swimming at 5 days post-fertilization (dpf), termed early-life challenge (ELC), on cortisol responses, neuropeptide expression in the nucleus preopticus (NPO), and gene transcript levels. At 6 dpf, ELC-exposed larvae showed normal baseline cortisol but reduced reactivity to an initial stressor. Conversely, they showed increased reactivity to a second stressor within the 30-minute refractory period, when cortisol responses are typically suppressed. ELC larvae had fewer corticotropin-releasing hormone (crh), arginine vasopressin (avp), and oxytocin (oxt)-positive cells in the NPO, with reduced crh and avp co-expression. Gene expression analysis revealed upregulation of genes related to cortisol metabolism (hsd11b2, cyp11c1), steroidogenesis (star), and stress modulation (crh, avp, oxt). These results suggest that early environmental challenge initiates adaptive plasticity in the HPI axis, tuning cortisol regulation to balance responsiveness and protection during repeated stress. Future studies should explore the broader physiological effects of prolonged forced swimming and its long-term impact on cortisol regulation and stress-related circuits. SummaryThis study explores how early-life challenges in zebrafish affect stress responses and hormone regulation, offering insights into developmental adaptability and stress management mechanisms.

physiology↗

bioRxiv2024

Pleiotropic cellular responses underlying antibiotic tolerance in Campylobacter jejuni

Antibiotic tolerance enables antibiotic-susceptible bacteria to withstand prolonged exposure to high concentrations of antibiotics. Although antibiotic tolerance presents a major challenge for public health, its underlying molecular mechanisms remain to be elucidated. Previously, we have demonstrated that Campylobacter jejuni develops tolerance to clinically important antibiotics, including ciprofloxacin and tetracycline. To identify cellular responses associated with antibiotic tolerance, we conducted RNA-sequencing analysis on C. jejuni following the induction of antibiotic tolerance through exposure to elevated levels of ciprofloxacin or tetracycline. Additionally, we constructed knockout mutants for genes that showed significant changes in expression levels during antibiotic tolerance. We observed a significant upregulation of genes involved in protein chaperones, bacterial motility, DNA repair system, drug efflux pump, and iron homeostasis during antibiotic tolerance. Furthermore, the viability of these knockout mutants was significantly reduced compared to the wild-type strain, indicating the critical role of these cellular responses in sustaining antibiotic tolerance. Notably, we also found that the protein chaperone mutants {Delta}dnaK, {Delta}clpB, and {Delta}groESL exhibited increased protein aggregates under antibiotic treatment, suggesting protein chaperones play a critical role in managing protein aggregation and facilitating the survival of C. jejuni during antibiotic tolerance. In conclusion, our findings demonstrate that various cellular defense mechanisms collectively contribute to sustaining antibiotic tolerance in C. jejuni, providing novel insights into the molecular mechanisms of antibiotic tolerance that enable C. jejuni to withstand the lethal effects of antibiotics.

microbiology↗

bioRxiv2024

Cortisol dynamics and GR-dependent feedback regulation in zebrafish larvae exposed to repeated stress

Zebrafish larvae show a rapid increase in cortisol in response to acute stressors, followed by a decline. While these responses are documented, both the duration of the refractory period to repeated stressors and the role of glucocorticoid receptors (GR) in specific phases of the glucocorticoid negative feedback are still being clarified. We explored these questions using water vortices as stressors, combined with GR blockage and measurements of whole-body cortisol in zebrafish larvae subjected to single and repeated stress protocols. Cortisol levels were elevated 10 minutes after stress onset and returned to baseline within 30-40 minutes, depending on the stressor strength. In response to homotypic stress, cortisol levels rose above baseline if the second stressor occurred 60 or 120 minutes after the first, but not with a 30-minute interval. This suggests a rapid cortisol-mediated feedback loop with a refractory period of at least 30 minutes. Treatment with a GR blocker delayed the return to baseline and suppressed the refractory period, indicating GR-dependent early-phase feedback regulation. These findings are consistent with mammalian models and provide a framework for further analyses of early-life cortisol responses and feedback in zebrafish larvae, ideal for non-invasive imaging and high-throughput screening. Summary statementThis study investigates cortisol responses in zebrafish larvae to acute stress, revealing a rapid feedback loop and the glucocorticoid receptors role, offering insights for future research in stress biology.

physiology↗

bioRxiv2024

Expanded Genome and Proteome Reallocation in a Novel, Robust Bacillus coagulans Capable of Utilizing Pentose and Hexose Sugars

Bacillus coagulans, a Gram-positive thermophilic bacterium, is recognized for its probiotic properties and recent development as a microbial cell factory. Despite its importance for biotechnological applications, the current understanding of B. coagulans robustness is limited, especially for undomesticated strains. To fill this knowledge gap, we characterized the metabolic capability and performed functional genomics and systems analysis of a novel, robust strain, B. coagulans B-768. Genome sequencing revealed that B-768 has the largest B. coagulans genome known to date (3.94 Mbp), about 0.63 Mbp larger than the average genome of sequenced B. coagulans strains, with expanded carbohydrate metabolism and mobilome. Functional genomics identified a well-equipped genetic portfolio for utilizing a wide range of C5 (xylose, arabinose), C6 (glucose, mannose, galactose), and C12 (cellobiose) sugars present in biomass hydrolysates, which was validated experimentally. For growth on individual xylose and glucose, the dominant sugars in biomass hydrolysates, B-768 exhibited distinct phenotypes and proteome profiles. Faster growth and glucose uptake rates resulted in lactate overflow metabolism, which makes B. coagulans a lactate overproducer; however, slower growth and xylose uptake diminished overflow metabolism due to the high energy demand for sugar assimilation. Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) made up 60-65% of the measured proteomes but were allocated differently when growing on xylose and glucose. The trade-off in proteome reallocation, with high investment in COG-C over COG-G, explains the xylose growth phenotype with significant upregulation of xylose metabolism, pyruvate metabolism, and TCA cycle. Strain B-768 tolerates and effectively utilizes inhibitory biomass hydrolysates containing mixed sugars and exhibits hierarchical sugar utilization with glucose as the preferential substrate. IMPORTANCEThe robustness of B. coagulans makes it a valuable microorganism for biotechnology applications, yet this phenotype is not well understood at the cellular level. Through phenotypic characterization and systems analysis, this study elucidates the functional genomics and robustness of a novel, undomesticated strain, B. coagulans B-768, capable of utilizing inhibitory switchgrass biomass hydrolysates. The genome of B-768, enriched with carbohydrate metabolism genes, demonstrates high regulatory capacity. The coordination of proteome reallocation in Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) is critical for effective cell growth, sugar utilization, and lactate production via overflow metabolism. Overall, B-768 is a novel, robust, and promising B. coagulans strain that can be harnessed as a microbial biomanufacturing platform to produce chemicals and fuels from biomass hydrolysates.

systems biology↗

bioRxiv2024

Scalable Hypothalamic Arcuate Neuron Differentiation from Human Pluripotent Stem Cells Suitable for Modeling Metabolic and Reproductive Disorders

The hypothalamus, composed of several nuclei, is essential for maintaining our bodys homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.

developmental biology↗

bioRxiv2024

Activation of PKR by a short-hairpin RNA

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54s potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

molecular biology↗