Biology subjects

Raghavan, S. A.

Publications and source records attributed to Raghavan, S. A..

8 recordsLinked to original sources

bioRxiv2026

Discovery and Characterization of Interleukin-4-Specific Affibodies for Affinity-Controlled Protein Release and Macrophage Polarization

Interleukin-4 (IL-4) is a key immunoregulatory cytokine that promotes type 2 inflammation, drives macrophage polarization toward an anti-inflammatory M2 phenotype, and supports tissue repair. However, clinical translation of IL-4 therapies to modulate the immune response is limited by the need for precise control over its delivery to avoid immune dysregulation. Here, we report an affinity-based strategy to modulate IL-4 delivery and bioactivity using engineered affibody proteins. A yeast surface display library was screened via magnetic- and fluorescence-activated cell sorting to identify two IL-4-specific affibodies with moderate binding affinities (dissociation constants, KD = 459 and 141 nM). Circular dichroism confirmed expected alpha-helical folding, and biolayer interferometry characterized the kinetics of IL-4 binding. Structural modeling using AlphaFold3 and RosettaDock and molecular dynamics simulations using GROMACS predicted distinct binding sites for each IL-4-specific affibody on the IL-4 protein and suggested potential interference with receptor complex formation. Bioactivity studies using murine bone marrow-derived macrophages demonstrated that IL-4 complexed with affibodies maintained Ym1 gene expression but significantly reduced Ym1 protein levels, indicating partial inhibition of IL-4 signaling. To enable controlled cytokine delivery via affinity interactions, affibodies were conjugated to polyethylene glycol maleimide (PEG-mal) hydrogels, which were loaded with IL-4. Affibody-conjugated hydrogels achieved high IL-4 loading efficiency (>90%) and exhibited sustained release over 7 days. Increasing affibody-to-IL-4 ratios significantly reduced both the rate and total amount of cytokine release. Overall, this work establishes IL-4-specific affibodies as versatile tools for tuning cytokine presentation and modulating bioactivity and provides a promising approach for regulating inflammatory responses and advancing cytokine-based therapies with improved temporal control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/723637v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@146d4c1org.highwire.dtl.DTLVardef@139dcfeorg.highwire.dtl.DTLVardef@1dbe8f3org.highwire.dtl.DTLVardef@9ac211_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Modular Albumin-Chaperoned NIR-II Nanofluorophores Enables Pan-Ovarian Cancer Imaging Across Multiscale Tumor Models

Ovarian cancer remains the most lethal gynecological malignancy, primarily due to late-stage diagnosis and the challenges of achieving complete cytoreduction. While fluorescence image-guided surgery (FIGS) offers intraoperative visualization, current clinical agents are limited by insufficient brightness, rapid photobleaching, and poor molecular selectivity, particularly in the near-infrared window. Here, we report the rational modular design of ultrabright NIR-II semiconducting polymer (SP) nanofluorophores for high-fidelity ovarian cancer imaging. By nanoconfining of a representative hydrophobic SP within a functional albumin matrix induces a "chaperone" effect that suppresses aggregation-induced quenching and shifts emission in the NIR-II window (1000-1250 nm). This platform integrates a dual-receptor targeting strategy, leveraging intrinsic albumin-receptor interactions (GP60 and SPARC) alongside folate receptor alpha (FR) functionalization. This synergistic approach enables pan-ovarian cancer imaging by ensuring high-affinity binding across diverse tumor phenotypes, regardless of heterogeneous receptor expression. Across a multiscale validation framework, the nanofluorophores demonstrate efficient receptor-mediated endocytosis in 2D cultures and deep interstitial penetration in 3D tumor spheroids. Furthermore, microfluidic tumor-on-chip models incorporating endothelial-like fenestrations confirm controlled extravasation and targeting under physiological shear stress. 3D bioprinted tumor phantoms and ex vivo porcine ovary tissues further confirm that BSA-FA@SP2 provides superior lesion delineation and signal-to-background ratios compared to indocyanine green, a clinical standard. Importantly, the nanofluorophores exhibit excellent hemocompatibility, with minimal hemolysis and negligible complement activation, indicating a non-immunogenic, stealth profile. Collectively, this work establishes albumin-shielded NIR-II nanofluorophores as a robust platform for precision intraoperative pan-ovarian imaging and advances the translational potential of nanotechnology-enabled surgical oncology.

bioengineering↗

bioRxiv2026

Oncogene-Mechanics Axis: KRAS G12C Confers Agility Enabling Malignant Mechano-responses to Peristalsis in Colorectal Cancer

Oncogene activity and mechanical forced individually and collective drive colorectal cancer, yet the integration of these signals is unknown. We used a patented peristalsis bioreactor to determine how oncogenic KRAS G12C mutations alter the cellular response to colonic peristalsis. Although both ehalthy intestinal cells and KRAS G12C cells sensed peristalsis via ERK phosphorylation, their mechano-responses diverged significantly. Peristalsis triggered a 9-fold enrichment of LGR5+ cancer stem cells in KRAS G12C cancer cells, an effect absent in healthy controls. Using Brillouin microscopy, we discovered that KRAS G12C induced a more agile and deformable mechano-phenotype by lowering intracellular viscosity, a state further amplified by peristalsis. This agility allowed KRAS G12C cancer cells to leverage, rather than resist peristalsis, resulting in LGR5 enrichment and malignant progression. Pharmacologic inhibition of KRAS G12C reverse the mechano-phenotype, while introducing KRAS G12C into healthy cells recapitulated it. Our findings identify a novel KRAS oncogene-mechanics axis, suggesting that targeting the cell's mechanical state could be a powerful complement to emerging KRAS-directed therapies.

bioengineering↗

bioRxiv2026

EMMIs: Engineered Myometrial Microtissues for Direct Quantification of Oxytocin-Induced Contractility

Forceful and coordinated contractions of the uterine myometrium are essential for successful labor, delivery, and postpartum uterine involution. Failure of the uterus to generate or sustain contractile force (uterine atony) after delivery results in postpartum hemorrhage, a leading cause of maternal mortality globally. Paradoxically, uterine atony is exacerbated by prolonged oxytocin exposure used to induce or augment labor through a process of contractile desensitization. Despite its prevalent use in obstetrics, the direct impact of oxytocin desensitization on myometrial contractile force generation remains poorly defined. Current model systems are inadequate to address this gap: ex vivo myometrial tissue strips are limited by tissue availability, donor variability, and lack of genetic tractability, while existing in vitro models provide only indirect readouts of contractility without direct force quantification. Here, we introduce engineered myometrial microtissues (EMMIs), a platform enabling the direct, isometric measurement of contractile force in response to physiological agonists like oxytocin. By embedding and molding immortalized human myometrial smooth muscle cells within a collagen hydrogel, we induced significant structural and molecular maturation over six days. Upon maturation, EMMIs were characterized by circumferential cellular alignment, sustained expression of smoothelin, upregulation of connexin-43, and a transcriptomic shift toward a contractile phenotype. Mature EMMIs generated calcium-sensitive, dose-dependent contractions to oxytocin and potassium chloride. Genetic deletion of the oxytocin receptor abolished oxytocin-induced contractility, establishing receptor specificity. Finally, we utilized EMMIs to recapitulate clinical oxytocin desensitization, providing a direct link between prolonged oxytocin exposure and diminished contractile output. Together, these findings establish engineered myometrial microtissues (EMMIs) as a genetically manipulable, and reproducible system for investigating myometrial contractile physiology to improve obstetric outcomes. TeaserEngineered 3D uterine tissues quantify how labor-inducing drugs weaken contractions and drive maternal hemorrhage

bioengineering↗

bioRxiv2026

Human breast milk extracellular vesicles from mothers with asthma differentially modulate the release of inflammatory cytokines by primary human airway smooth muscle cells in a recipient-cell specific manner

Breastfeeding provides health benefits in childhood, reducing the frequency of gastrointestinal and respiratory infections. Breastmilk (BM) is a rich source of bioactive molecules including extracellular vesicles (EVs), which exert immunomodulatory signalling in recipient cells, with cargo that is affected by maternal characteristics. Here we investigated the biophysical characteristics of BM-EVs from mothers with (asthmatic BM-EVs) or without asthma (control BM-EVs) and their effect on the release of cytokines from primary human hTERT-immortalized airway smooth muscle cells (hASMs) from asthmatic or non-asthmatic (control) donors. BM-EVs were isolated using size exclusion chromatography (N=5/group), characterized biophysically and by EV-specific protein markers. In addition, BM-EV were co-cultured (48h) with primary hASM cells from both non-asthmatic (control) and asthmatic donors to determine the effect on cytokine release. All participants were Caucasian and the BM was collected 12-15 weeks postpartum. BM-EVs showed the presence of intact and small-EVs ([~]100 nm). Asthmatic BM-EVs appeared to have a smaller average EV size (135.6 nm) vs. controls (148.3 nm, p=0.0613), but [~]5-fold higher concentration of both total (p=0.0014) and small EVs (p=0.0016). The expression of EV subtype protein expression was reduced in asthmatic BM-EVs vs. control BM-EVs: CD63 by 86% (p=0.0224), flotillin-1 by 40% (p=0.0196), CD9 by 24% (p=0.0646) and HSP70 by 69% (p=0.0873). Asthmatic BM-EVs co-cultured with hASMs from control donors decreased pro-inflammatory cytokine release: MCP-1 by 55% (p=0.0286), IL-6 by 45% (p=0.0801) and IL-2 by 32% (p=0.0970) vs. control-BM-EVs. Conversely, asthmatic BM-EVs co-cultured with hASMs from asthmatic donors increased secretion of anti-inflammatory cytokine IL-10 by 32% (p=0.0660), and IL-1Ra by 75% (p=0.0875), and pro-inflammatory IL-2 by 57% (p=0.0688) vs. control-BM-EVs. Internalization of control and asthmatic BM-EVs was confirmed by labelled EV uptake experiments. No detrimental effects on cell viability with BM-EV treatment were observed. In summary, asthmatic BM-EVs are smaller and enriched in BM, and exert differential effects on cytokine release in a BM-donor and recipient-cell specific manner. Given that BM can enter infant airways, the immunomodulatory effects of BM-EVs on hASMs warrants further investigation to delineate the under underlying mechanisms.

cell biology↗

bioRxiv2026

Modeling Inflammation-Driven Colon Hypertrophy and Motility Changes in Gulf War Illness

Gastrointestinal (GI) symptoms are a prominent feature of Gulf War Illness (GWI). Animal models attribute them to pyridostigmine bromide (PB) exposure, which induces smooth muscle hypertrophy, neuroinflammation, and motility impairment. However, animal studies only provide static snapshots of disease progression and can only partially resolve how inflammatory, neuronal, and biomechanical processes interact dynamically over time. To address this gap, we developed a computational model that couples cytokine kinetics, macrophage activation, and an excitatory-inhibitory neuronal imbalance to predict smooth muscle hypertrophy and colonic motility changes in GWI. The model was calibrated using data from mice exposed to PB under acute (7-day exposure and measurement) and chronic (7-day exposure and 30-day measurement) conditions, reproducing measured cytokine IL-6 elevations, macrophage accumulation, circular muscle thickening, and shifts in excitatory and inhibitory gene expression. Simulations captured reduced excitatory stress, and sustained loss of inhibitory relaxation, consistent with organ-bath recordings. Sensitivity analyses identified macrophage persistence as a dominant regulator of chronic inhibitory dysfunction, whereas excitatory pathways exhibited relative robustness and recovery. Thus, our model provides a systems-level view of how acute PB-induced inflammation evolves into chronic dysmotility and establishes a first step towards a virtual platform for testing hypotheses and interventions translatable to neuroimmune GI disorders. HighlightsO_LINeuroinflammation model predicts colon hypertrophy and motility in GWI C_LIO_LICalibrated to acute (day 7) and chronic (day 30) PB-exposed mouse data C_LIO_LIReproduced IL-6 rise, CD40+ persistence, colon thickening, and ChAT/Nos1 shifts C_LIO_LIPredicted excitatory stress rebound but sustained inhibitory relaxation loss C_LI

bioengineering↗

bioRxiv2025

Immune-Competent 3D Bioengineered Colons for Functional Interrogation of Neuroinflammation-Induced Colonic Dysmotility

Functional gastrointestinal disorders (FGIDs) affect [~]40% of the global population and are frequently characterized by colonic dysmotility. Symptomatic manifestations of colonic dysmotility significantly reduce quality of life in inflammatory bowel disease (IBD), diabetes, and Gulf War Illness (GWI). Current in vitro models lack the integration of functional physiology with immune and neuronal complexity required to establish causal links between neuroinflammation and dysmotility. Here, an immune-competent bioengineered colon assembloid is introduced that integrates multiple cell types of the external colonic wall, along with functional readouts of motility. Within bioengineered colon assembloids, various inflammatory insults resulted in enteric neuroinflammation, cascading to changes in colonic motility. Key mechanisms of dysmotility following inflammatory insult within the bioengineered colon assembloids included impaired neuronal regeneration, and aberrant smooth muscle remodeling. The bioengineered colon assembloid model mimicked diverse aspects of enteric neuroinflammation. Ultimately, the platform offers a physiologically relevant avenue to interrogate neuroimmune crosstalk and dissect mechanisms of colonic dysmotility, paving the way to new therapeutic strategies to improve colonic motility.

bioengineering↗

bioRxiv2024

Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

developmental biology↗