Biology subjects

Lal, N.

Publications and source records attributed to Lal, N..

7 recordsLinked to original sources

bioRxiv2026

A low-matrix organoid-T cell co-culture platform for functional evaluation of T cell engagers in patient-derived colorectal cancer organoids

Background Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, and emerging T cell engager (TCE) immunotherapies require predictive preclinical models that capture patient-specific tumour biology and tumour-immune interactions. Although patient-derived organoid (PDO)-immune co-culture systems show promise for evaluating immunotherapy responses, many rely on matrix-embedded cultures that limit scalability and reproducibility. We therefore developed and validated a low-matrix organoid-T cell co-culture platform for functional assessment of TCE activity in patient-derived CRC models. Methods Patient-derived CRC organoids representing diverse molecular and genetic backgrounds were co-cultured with activated allogeneic CD3+ T cells in a suspension low-matrix format. Matrix concentration, medium composition, T cell activation status, and assay duration were optimized. Tumour killing, T cells activation, cytokine secretion, apoptosis, and motility were assessed using flow cytometry, live-cell imaging, cytokine profiling, and immunofluorescence. The platform was evaluated using EGFR- and HER2-targeting bispecific T cell engagers across 21 CRC organoid models and multiple healthy donor-derived T cell populations. Results Optimization identified 1% matrix and a 1:1 organoid: T cell medium that preserved organoid integrity while maintaining T cell viability, activation, and motility. The final workflow enabled reproducible co-culture of 5-day mature organoids with 7-day activated T cells for 72 hours. EGFR-targeting TCEs induced tumour killing, T cell activation, and IFN-{gamma} secretion across donor-organoid combinations. Screening of 21 CRC organoid models revealed substantial inter-patient heterogeneity, with 12 models maintaining [≥]50% baseline viability and supporting functional TCE evaluation. EGFR- and HER2-targeting TCEs produced potent dose-dependent cytotoxicity, with IC50 values ranging from 0.0188 to 54.77 nM across responsive models and up to 75%-85% tumour killing in the most sensitive organoids. These responses were accompanied by increased effector cytokine secretion. Real-time imaging confirmed dynamic T cell engagement and apoptosis-driven organoid destruction following TCE treatment. Conclusions We establish a robust, scalable, human-relevant low-matrix organoid-T cell co-culture platform for functional screening of T cell engagers in patient-derived CRC models. By enabling integrated assessment of tumour killing, immune activation, cytokine responses, and tumour-immune interactions while preserving inter-patient heterogeneity, this system provides a translational framework for immunotherapy development. The platform may support candidate prioritisation, biomarker discovery, patient stratification, and preclinical evaluation of immune-engaging therapeutics.

cancer biology↗

bioRxiv2026

Top-down regulation of ingestive behavior fragmentation

AbstractIn natural environments, animals rarely feed continuously to satiation; instead, feeding occurs in brief bouts separated by pauses. This fragmentation is thought to balance internal drives with external demands, yet its underlying neural mechanisms remain unclear. By combining bidirectional neural activity mapping and behavioral phenotyping, we identify a projection from the dorsal subiculum (dSub) of the hippocampus to the mammillary body (MB) as a key regulator of this fragmentation. Activity along the dSub-MB pathway tracks and gates the duration of individual feeding bouts, independent of homeostatic state. A simple bistable attractor model captures both dSub-MB neural dynamics and associated behaviors across optogenetic and behavioral perturbations. Together, these findings identify a top-down circuit mechanism that implements action selection in a naturalistic setting.

neuroscience↗

bioRxiv2025

KIF5A binds RNA to orchestrate synaptic mRNA localization and stress granules in ALS

Neuronal health depends on the precise transport and local translation of mRNAs to maintain synaptic function across highly polarized cellular architecture. While kinesin motor proteins are known to mediate mRNA transport, the specificity and direct involvement of individual kinesins as RNA-binding proteins (RBPs) remain unclear. Here, we demonstrate that KIF5A, a neuron-specific kinesin implicated in amyotrophic lateral sclerosis (ALS), functions as an RBP. We show that KIF5A directly binds mRNAs encoding synaptic ribosomal proteins and is required for their synaptic localization and for maintaining normal synaptic composition and function. Additionally, we show ALS-linked KIF5A mutations confer gain-of-function properties, enhancing mRNA binding, increasing synaptic ribosomal protein accumulation, inducing neuronal hyperexcitability, and impairing stress responses. These findings reveal a previously unrecognized mechanism by which mutant KIF5A disrupts synaptic homeostasis. Our work positions a kinesin motor protein as an RBP with critical roles in mRNA transport, local translation, and stress response. HighlightsO_LIKIF5A interacts with mRNA encoding synaptic ribosomal proteins C_LIO_LIKIF5A is required for normal synaptic composition and function C_LIO_LIKIF5A binds to G3BP1 and G3BP1 stress granule associated proteins C_LIO_LIKIF5A mutant ALS patient-derived motor neurons have abnormal synaptic function and stress response C_LI

neuroscience↗

bioRxiv2025

Sphingomyelinase Licensing of Mesenchymal Stromal Cells Alters Lipid and Protein Metabolites for Immunomodulation

Mesenchymal stromal cells (MSCs) are widely studied for their immunomodulatory and tissue reparative capabilities, but clinical translation has been hampered by inconsistent efficacy and limited standardization in manufacturing. While cytokine-based priming methods, such as interferon-gamma (IFN-{gamma}) stimulation, have shown promise in enhancing MSC potency, alternative approaches targeting distinct biological metabolism integral to secretome and membrane architecture have not been explored in MSCs. In this study, we investigate sphingomyelinase (SMase), an enzyme that generates ceramide from sphingomyelin, as a novel lipid-based priming strategy to modulate MSC function. Here, human MSCs were treated with SMase and high-content imaging and morphological profiling revealed that SMase-treated cells adopted a phenotype overlapping with IFN-{gamma}-licensed MSCs, including increased cell compactness and solidity. Lipidomic analysis showed broad alterations in sphingolipid species, and dynamic flux estimation (DFE) modeling predicted distinct metabolic shifts in SMase-treated cells compared to untreated controls. These changes were sustained up to 35 hours post-stimulation, indicating stable metabolic reprogramming. SMase priming also altered the MSC secretome, enriching for factors implicated in immune regulation. Functionally, SMase-primed MSCs retained the ability to suppress T-cell activation and promote anti-inflammatory macrophage phenotypes. Collectively, these findings demonstrate that SMase stimulation induces a durable, immunomodulatory-like state in MSCs through coordinated changes in lipid metabolism and secretory activity. This lipid-centric priming approach represents a promising alternative to cytokine-based licensing strategies and may support therapeutic MSC products.

bioengineering↗

bioRxiv2025

Resolving vs. Non-resolving Sphingolipid Dynamics During Macrophage Activation: A Time-resolved Metabolic Analysis

Sphingolipids are increasingly recognized as critical regulators of inflammation and cell fate decisions, with metabolites such as ceramide and sphingosine 1{square}phosphate exerting contrasting effects on cell survival and proliferation. In macrophages, this balance is especially important, given their central role in host defense, pathogenesis and wound healing. Here, we present a time{square}resolved model of sphingolipid metabolism in RAW 264.7 macrophages stimulated with KdO{square}-Lipid A. By integrating measured metabolite concentrations with dynamic flux estimation and established enzyme kinetics, we systematically map dynamic changes in the sphingolipid network during inflammation. Our results reveal a three{square}phase pattern of sphingolipid remodeling that correlates with distinct functional states of the cell. Moreover, metabolites can be classified into "resolving" or "non{square}resolving" lipids based on whether they return to basal levels or remain dysregulated through the later phases of the inflammatory response. This partitioning suggests that targeted modulation of specific metabolic nodes may influence the resolution of inflammation. Importantly, our computational approach can assist in the rational design of experimental studies by pinpointing putative drug targets with maximal impact on sphingolipid homeostasis. Such targeted interventions may prevent the pathological amplification of inflammatory signals without globally suppressing essential sphingolipid functions. These findings highlight the utility of an integrative systems{square}level analysis for elucidating sphingolipid dynamics in macrophages and underscore its potential to guide therapeutic strategies against conditions involving dysregulated inflammation.

immunology↗

bioRxiv2023

Phosphorylation of pyruvate dehydrogenase marks the inhibition of in vivo neuronal activity

For decades, the expression of immediate early genes (IEGs) such as c-fos has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity (i.e., inhibition). Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected neuronal inhibition across the brain induced by a wide range of factors including general anesthesia, sensory experiences, and natural behaviors. Thus, as an in vivo marker for neuronal inhibition, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

neuroscience↗

bioRxiv2023

Intralimb locomotor coordination in rats walking on asymmetric pegway

Complex movements such as walking or reaching are generated by a sequence of muscle actions. How these coordinated actions subserve complex movements and their recovery after disruption remains unknown. The use of high throughput recording-stimulation systems with microelectrode access to structures along the neuraxis may complement the neurological models in rodents. To this purpose, we have trained rats to perform the precise foot placement locomotor task that allows us to assess skilled locomotor movements. Animals were pretrained on the peg walkway task, which was configured to impose either symmetric or asymmetric (with overstepping) locomotor stepping at preferred stride length. Selected forelimb muscles were implanted with intramuscular differential electrodes. After a week of recovery, we collected electromyography from the implanted muscles and ground reaction forces from the array of force sensors embedded into walkway pegs. The temporal relationship between muscle bursts was measured for each intralimb set of muscles (n=13) in symmetric and asymmetric stepping. The sequence corresponded to the progression of muscle actions responsible for limb lift, flexion and transport, overground clearance, and preparation for ground contact. The stereotyped spatiotemporal sequence of muscle activity was persistent and mirroring across the asymmetric tasks. These patterns are similar to those observed in cats during locomotion with and without obstacles and reaching movements. These findings support the hypothesis that the profiles of muscle activations are qualitatively similar across quadrupeds during precise locomotor tasks. New and NoteworthyWe characterize for the first time the spatiotemporal muscle activation in rat forelimb during precise asymmetric stepping on asymmetrically placed rungs. Similar to cats, the intralimb pattern of muscle activation in rats was stereotypical. The elements of this pattern were changing in a lateralized fashion based on the direction of the imposed asymmetry. The similarity of pattern to that of cats supports the idea of similarity of neural control across cat and rat species.

neuroscience↗