Biology subjects

Roy, N.

Publications and source records attributed to Roy, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Fly Viral Atlas: A single-nucleus transcriptomic atlas of RNA viruses and transposable elements (TEs) in Drosophila melanogaster

Drosophila RNA viruses often persist in wild and lab populations, yet their tissue and cellular tropism is poorly understood. In the Fly Cell Atlas (a comprehensive Drosophila single-nucleus transcriptome) data, we detected four RNA virus infections: Nora virus, Drosophila A virus, Drosophila C virus, and Newfield virus. Nora and Drosophila A virus were the most abundant and widespread across tissues and cell types, while Drosophila C virus and Newfield virus RNA transcript were only found in oenocyte and fat body tissues. We found transcriptional changes associated with viral infection in canonical viral immunity genes (e.g. Vago, vir-1). Additionally, we observed that during persistent viral infections, transposable element (TE) transcripts were upregulated in somatic cells. TEs are traditionally associated with the germline, but recent studies and our data suggest they are also expressed in somatic cells. Using the Fly Cell Atlas data, we found that distinct somatic cell types express specific TE subtypes, indicating regulated and cell-type specific TE activity often overlooked in transcriptomic studies. We present Fly Viral Atlas (https://flyviralatlas.shinyapps.io/home/), a single-nucleus level atlas of RNA viruses and TE expressions in Drosophila, providing new insights into viral tropism and TE dynamics across cell types and tissues.

genomics↗

bioRxiv2026

Molecular basis of promiscuous chemokine-engagement by the Duffy antigen receptor

The Duffy blood group antigen, encoded by a seven transmembrane protein known as Duffy Antigen Receptor for Chemokines (DARC), or Atypical Chemokine Receptor 1 (ACKR1), serves as a key receptor for the malarial parasite, Plasmodium vivax, on erythrocytes. DARC exhibits remarkable functional divergence compared to prototypical chemokine receptors and other G protein-coupled receptors (GPCRs) as it does not engage canonical signal-transducers such as G-proteins, GPCR regulatory kinases (GRKs), and {beta}-arrestins. DARC is a highly promiscuous receptor interacting with several homeostatic and inflammatory C-C and C-X-C subtype chemokines, and similar to other chemokine receptors, these interactions are modulated by post-translational tyrosine sulfation in the N-terminus. A single nucleotide polymorphism in DARC leading to Gly42Asp substitution forms the basis for Fya vs. Fyb Duffy blood group antigen classification with differential impact on Plasmodium vivax infection and cancer progression. However, the molecular basis of promiscuous chemokine-binding by DARC and the modulation by receptor sulfation and Fya/Fyb allelic variation remains unclear. Here, we design a sortase-mediated chemical-ligation strategy to generate purified DARC with naturally-occurring tyrosine sulfation at the N-terminus, and determine high-resolution cryo-EM structures in complex with a C-C type chemokine, CCL7, and a C-X-C type chemokine, CXCL8. We observe that similar to CCL7, CXCL8 engages with DARC primarily through the N-terminus of the receptor, which is in stark contrast with the two-site binding mechanism displayed by other chemokine receptors. Interestingly, the E-L-R motif in CXCL8 engages a pseudo-R-D motif in DARC leading to superficial engagement unlike CXCR2, a prototypical chemokine receptor sharing the same agonist. Moreover, tyrosine sulfation and Fyb allelic variation leads to a repositioning of the receptor N-terminus on the core domain of CXCL8, resulting in distinct interaction network imparting greater binding affinity. Taken together, our study presents novel insights into promiscuous chemokine-binding to DARC, tyrosine sulfation-mediated fine-tuning of chemokine engagement, and a generalizable sortase-mediated chemical-ligation platform applicable to other chemokine receptors.

biochemistry↗

bioRxiv2026

Persistent viral infection in the Drosophila fat body is associated with immune activation at the single-cell level

BackgroundViruses are ubiquitous and can spread in two main ways: vertically, which involves transmission through or associated with gametes, and horizontally, which occurs through direct contact, airborne transmission, or indirect contact, such as through ingestion. Vertically transmitted, low virulence viruses can go undetected by both the immune system and researchers, and cause chronic, asymptomatic infections. In many Drosophila studies, researchers are unaware or ambivalent about the fact that the flies used may be infected with persistent viruses. Although they often have minimal or no observable fitness costs in laboratory fly samples, recent studies suggest that an increase in viral titer is associated with a decrease in lifespan. ResultsIn this study, we explored changes in expression occurring during these cryptic virus infections. To achieve this, we utilized publicly accessible single-nuclear RNA sequencing (snRNA-seq) data of the Drosophila fat body, where we detected persistent infections of Nora and Drosophila A virus. We observed that Nora virus and Drosophila A virus exhibit broad cell-type tropism in the Drosophila fat body, and when coinfected, Drosophila A virus showed higher viral titer and cell infection rate. Transcriptomic analysis revealed substantial immune pathway alterations: Nora virus is broadly associated with upregulation of immune pathways (IMD, Toll), whereas Drosophila A virus is associated with downregulation of specific Toll pathway effector genes. Additionally, the expression of somatic transposable element (TE) transcripts was associated with viral infection, showing mating status-dependent patterns with downregulation in Nora virus-infected virgin flies and upregulation in mated flies for both viruses. ConclusionsOverall, our results indicate that cryptic and persistent viral infections in Drosophila elicit transcriptional changes in the fat body, including activation of immune responses, and are associated with dysregulation of TE activity in somatic fat body cells.

genomics↗

bioRxiv2025

Potential probiotic application of a novel commensal E. coli with antagonistic activity against different enteric pathogens

Diarrheal diseases remain a leading cause of global morbidity and mortality, and treatment options are increasingly compromised by the global rise in multidrug-resistant (MDR) pathogens. Furthermore, the absence of effective vaccines against many causative agents renders large populations vulnerable. This critical gap necessitates the development of novel alternatives that can function both prophylactically to prevent infection and therapeutically to treat established diseases. Here, we investigated the commensal Escherichia coli strain HK220822 (HK5), which was isolated from a healthy human, as a potential live biotherapeutic. Comprehensive genomic and phenotypic characterization established a robust safety profile, confirming the absence of key virulence genes while demonstrating essential probiotic traits, including high tolerance to gastrointestinal stressors, such as acid and bile. The functional efficacy of Escherichia coli HK5 has been validated in murine models of infection. Prophylactic administration before pathogen exposure significantly inhibited intestinal colonization by Salmonella Typhimurium, Shigella flexneri 2a, and Vibrio cholerae (El Tor O1 strain). Moreover, HK5 demonstrated therapeutic potential by significantly reducing pathogen shedding when administered to previously infected animals. These findings establish commensal E. coli (HK5) as a potent candidate with dual preventative and therapeutic efficacy, offering a promising non-antibiotic strategy to combat diarrheal diseases.

microbiology↗

bioRxiv2025

Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

The conceptual framework of biased signaling has revolutionized our understanding of GPCR signaling and regulatory paradigms, and greatly impacted the efforts focused on the discovery of GPCR-targeted therapeutics. However, the mechanistic basis of biased signaling remains primarily defined based on synthetic ligands and receptor mutants with relatively limited progress in understanding naturally-encoded signaling-bias. Here, we present fundamental molecular and structural insights into naturally-encoded signaling-bias at the complement anaphylatoxin C5a receptors namely, C5aR1 and C5aR2. We first discover that C5a-d-Arg, the naturally-occurring version of C5a lacking the terminal arginine, exhibits robust G-protein signaling-bias at C5aR1, characterised by attenuated {beta}arr recruitment. This signaling-bias manifests in both cytokine release from primary human immune cells, and in vivo, during neutrophil mobilization. We combine the cryo-EM structures of C5a/C5a-d-Arg-C5aR1 complexes with MD simulation, site-directed mutagenesis, and cellular experiments to elucidate that the G-protein-bias exhibited by C5a-d-Arg results from a distinct orientation of TM7 and helix 8 in C5aR1 leading to inefficient GRK recruitment and receptor phosphorylation. Next, we determine the first cryo-EM structures of C5aR2, a naturally-encoded {beta}-arrestin-biased receptor, in an apo state, complexed with the natural agonists C5a and C5a-d-Arg, and three peptide agonists including a first-in-class, newly discovered C5aR2-selective agonist, R8Y. These structural snapshots reveal key differences between the binding of C5a and C5a- d-Arg to C5aR1 and C5aR2, and provide a molecular basis of functional specialization at these two receptors. Moreover, the structural insights also allow us to decipher the molecular basis of naturally-encoded signaling-bias at C5aR2 originating from a shallower cytoplasmic interface with hydrophobic interior pocket that is not permissive to efficient G-protein-coupling and activation. Finally, we also engineer and characterize loss-of-function and gain-of-function variants of C5aR1 and C5aR2, which in turn corroborate and validate the structural observations presented here. Collectively, our findings offer crucial insights into previously lacking molecular mechanisms of the naturally-encoded signaling-bias at GPCRs, which have broad implications not only for the general framework of biased-signaling, but also for novel therapeutic design.

biochemistry↗

bioRxiv2025

Atypical activation and molecular glue-like dimerization mechanism of an intrinsically-biased chemokine receptor

CXCR7, also known as atypical chemokine receptor 3 (ACKR3), is a naturally-biased, {beta}-arrestin-coupled seven transmembrane receptor (7TMR) that lacks productive coupling with heterotrimeric G-proteins. Despite a critical involvement in cancer metastasis, cardiovascular pathophysiology, and inflammatory disorders, the molecular basis of non-canonical activation and functional divergence of CXCR7 remains elusive. Here, we present a complete landscape of CXCR7 activation using a series of cryo-EM structures, and discover an atypical activation mechanism that is distinct from prototypical GPCRs. CXCR7 is maintained in a basal conformation by a unique tripartite ionic-lock involving TM5-TM6, in contrast to a broadly conserved TM3-TM6 ionic-lock in GPCRs, which is disrupted upon receptor activation. Importantly, activation of CXCR7 results in a constricted pocket and distinct surface topology on the intracellular side compared to prototypical GPCRs. Serendipitously, we capture novel dimeric arrangements of CXCR7 with an inter-protomer stitching by a native phospholipid serving as a molecular glue, and identify previously unanticipated extrahelical allosteric sites on the receptor. Surprisingly, in an intermediate state structure of CXCR7, the second extracellular loop (ECL2) displays a self-blocking conformation, in stark contrast to ECL2-mediated self-activating mechanism reported recently for some orphan GPCRs. Finally, we unequivocally establish CXCR7 as an atypical opioid receptor via a large peptide library screening and structure elucidation in complex with distinct opioid peptides imparting full receptor activation. In summary, our study elucidates an atypical mechanism of CXCR7 activation, and establishes it as an alternative, non-canonical opioid receptor target with potential for novel pain therapeutics.

biochemistry↗

bioRxiv2025

Arginine methyltransferase PRMT1 equipoises trophoblast development to prevent early pregnancy loss

Abstract1-2% of all human pregnancies suffer from idiopathic recurrent pregnancy loss (RPL) and underlying molecular causes are poorly understood. Here we show that defective Protein Arginine Methyltransferase 1 (PRMT1) function in trophoblast progenitors is a molecular cause for early pregnancy failure. PRMT1 is conserved in trophoblast progenitors and conditional deletion of PRMT1 in mouse trophoblast progenitors arrests placenta and embryonic development leading to lethality [~]E7.5. Remarkably, a subset of idiopathic RPL is associated with loss of PRMT1 in cytotrophoblast progenitors (CTBs). Experiments with human trophoblast stem cells (hTSCs), derived from these RPL-patients as well as PRMT1-depleted hTSCs revealed that PRMT1 is crucial for trophoblast progenitors self-renewal. Employing RNA-seq and CUT&RUN-sequencing in hTSCs, CTBs and primary mouse trophoblast progenitors we discover that PRMT1 promotes transcription of trophoblast stem-state regulators, like TEAD4 and MYBL2, by directly enriching histone H4 arginine 3 asymmetric di-methylation (H4R3Me2a) at their chromatin loci. PRMT1 is also essential for extravillous trophoblast (EVT) development during human placentation, while loss of PRMT1 in hTSCs spontaneously promotes syncytiotrophoblast (STB) differentiation. Our findings indicate that PRMT1 is an epigenetic governor that orchestrates mammalian trophoblast development and implicate the therapeutic potential of targeting the PRMT1-H4R3Me2a axis to mitigate early pregnancy loss.

developmental biology↗

bioRxiv2025

Engineering Antibody-Drug Conjugates targeting an Adhesion GPCR, CD97

Adhesion G protein-coupled receptors (aGPCRs) are key cell-adhesion molecules involved in many cellular functions and contribute to human diseases, including cancer. aGPCRs are characterized by large extracellular regions that could serve as readily accessible antigens. However, the potential of aGPCRs as targets for biologic therapeutics has not been extensively explored. CD97, also known as ADGRE5, is an aGPCR that is upregulated in various cancer types, including acute myeloid leukemia (AML) and glioblastoma (GBM), and their respective cancer stem cells. Here, we developed antibody-drug conjugates (ADCs) targeting CD97 and assessed their efficacy against AML and GBM cells. We generated a panel of synthetic human antibodies targeting distinct epitopes of CD97, from which we identified an antibody that was efficiently internalized. This antibody binds to all isoforms of human CD97 but not to its close homolog, EMR2. Structure determination by single-particle cryo-electron microscopy revealed that this antibody targets the CD97 GPCR autoproteolysis-inducing (GAIN) domain, whose presence is conserved in aGPCRs, through an unconventional binding mode where it extensively utilizes the light chain framework for antigen recognition. Screening of conjugation methods and payloads resulted in a stable ADC that effectively killed AML and GBM cell lines, as well as patient-derived GBM stem cells, with minimal cytotoxicity against peripheral blood mononuclear cells from healthy donors. Our study demonstrates the therapeutic potential of targeting CD97, as well as the aGPCR GAIN domain in general, and broadens our mechanistic understanding of antibody-antigen interactions.

biochemistry↗

bioRxiv2025

Exposure to third generation cephalosporin induces L-form transition in Shigella sonnei, potentially acting as a bacterial reservoir for persistent infection

Shigellosis remains a major global health burden, and the increasing prevalence of multidrug-resistant (MDR) Shigella strains is complicating effective antibiotic therapy. Bacteria may survive antibiotics by transitioning into cell wall-deficient L-forms, which are intrinsically resistant to {beta}-lactams and can revert to a virulent state, potentially causing relapsing infections. Here we characterized a clinical MDR isolate Shigella sonnei HK8, a.k.a. PD552A, whose genome contains key resistance (gyrA, PBP3) and virulence (icsA) genes. Exposure to ceftriaxone induced a transition into a viable L-form state that was hyper-adhesive to macrophages in vitro. However, this survival adaptation was linked to a profound loss of pathogenicity. Using murine and guinea pig models, the L-form variant was shown to be profoundly attenuated, failing to cause the keratoconjunctivitis, diarrheal disease, or significant histopathology characteristic of the wild-type strain. These findings reveal a critical virulence-survival trade-off, positioning the L-form as a "stealth" phenotype that enables bacterial persistence at the expense of acute virulence. This offers a potential mechanism for asymptomatic carriage and recurrent infections, highlighting a previously underappreciated mechanism by which antibiotic treatment may resolve acute symptoms while permitting the persistence of a cryptic bacterial reservoir capable of driving recurrent infection. ImportanceOur research provides critical insight into the challenge of antibiotic treatment failure in shigellosis. By integrating experimental validation of reversible L-form transitions with mathematical modelling, we uncover a crucial virulence-survival trade-off. We show that MDR S. sonnei survives ceftriaxone by adopting a "stealth" phenotype, quantified by a high Stealth Index - maintaining bacterial burden while evading host inflammatory detection. These findings imply that standard antibiotics may resolve acute symptoms while inadvertently selecting for a cryptic, persistent reservoir poised for relapse. This work challenges the conventional view of therapeutic success and highlights an urgent need to develop novel diagnostic and therapeutic strategies capable of identifying and eliminating these resilient, "stealth" L-form persisters to achieve true bacterial clearance and prevent chronic infections.

microbiology↗

bioRxiv2025

Molecular fingerprints of a convergent mechanism orchestrating diverse ligand recognition and species-specific pharmacology at the complement anaphylatoxin receptors

Complement anaphylatoxin receptors (C3aR and C5aR1) are prototypical G protein-coupled receptors (GPCRs) playing crucial physiological roles in innate immunity by combating pathogenic infections and orchestrating inflammatory responses. They continue to be important therapeutic targets for multiple disorders including autoimmune diseases, acute and chronic inflammation, and allergy-related conditions. Recent structural coverage has provided important insights into their activation and signaling, however, confounding observations in the literature related to ligand efficacy and functional responses, especially in different model systems, present a major challenge for drug discovery efforts. Here, we systematically and comprehensively profile a broad set of natural and synthetic ligands at C3aR and C5aR1 and discover a previously unanticipated level of functional specialization in terms of species-specific pharmacology and receptor activation. Taking a lead from this, we determine seventeen cryo-EM structures of different ligand-receptor-G-protein complexes and uncover distinct orientation of agonists between the human and mouse receptors despite an overlapping positioning in the orthosteric binding pocket. Combined with extensive mutagenesis and functional assays, these structural snapshots allow us to decode and validate a convergent molecular mechanism involving a "Five-Point-Switch" in these receptors that orchestrates the recognition and efficacy of diverse agonists. We also identify species-specific differences at the level of phosphorylation patterns encoded in the carboxyl-terminus of these receptors and directly visualize their impact on {beta}arr binding and activation using cryo-EM structures. Interestingly, we observe that {beta}arrs engage with the mouse C5aR1 using a variation of previously discovered P-X-P-P phosphorylation motif via a "Sliding-Mechanism" and also exhibit distinct oligomeric state for the human vs. mouse receptors. Taken together, this study elucidates functional specialization at the complement anaphylatoxin receptors and underlying molecular mechanisms, offering a previously lacking framework with direct and immediate implications for the development of novel therapeutics.

biochemistry↗

bioRxiv2024

Covalent inhibitors of the RAS binding domain of PI3Ka impair tumor growth driven by RAS and HER2

Genetic disruption of the RAS binding domain (RBD) of PI 3-kinase (PI3K) prevents the growth of mutant RAS driven tumors in mice and does not impact PI3Ks role in insulin mediated control of glucose homeostasis. Selectively blocking the RAS-PI3K interaction may represent an attractive strategy for treating RAS-dependent cancers as it would avoid the toxicity associated with inhibitors of PI3K lipid kinase activity such as alpelisib. Here we report compounds that bind covalently to cysteine 242 in the RBD of PI3K p110 and block the ability of RAS to activate PI3K activity. These inhibitors have a profound impact on the growth of RAS mutant and also HER2 over-expressing tumors, particularly when combined with other inhibitors of the RAS/MAPK pathway, without causing hyperglycemia.

cancer biology↗

bioRxiv2024

Suppression of NRF2-dependent cancer growth by a covalent allosteric molecular glue

The NRF2 transcription factor is constitutively active in cancer where it functions to maintain oxidative homeostasis and reprogram cellular metabolism. NRF2-active tumors exhibit NRF2-dependency and resistance to chemo/radiotherapy. Here we characterize VVD-065, a first-in-class NRF2 inhibitor that acts via an unprecedented allosteric molecular glue mechanism. In the absence of stress or mutation, NRF2 is rapidly degraded by the KEAP1-CUL3 ubiquitin-ligase complex. VVD-065 specifically and covalently engages C151 on KEAP1, which in turn promotes KEAP1-CUL3 complex formation, leading to enhancement of NRF2 degradation. Previously reported C151-directed compounds decrease KEAP1-CUL3 interactions and stabilize NRF2, thus establishing KEAP1_C151 as a tunable regulator of the KEAP1-CUL3 complex and NRF2 stability. VVD-065 inhibited NRF2-dependent tumor growth and sensitized cancers to chemo/radiotherapy, supporting an open Phase I clinical trial (NCT05954312).

cancer biology↗

bioRxiv2024

Majority-LCL: Towards Malaria Cell Detection using Label Contrastive Learning and Majority voting Ensembling

Malaria is a contagious disease caused by Plasmodium, a group of single-celled parasites, and is most commonly transmitted by an infected female Anopheles mosquito. More than 40 percent of the global population is at risk, with approximately 219 million reported cases and around 435000 deaths recorded in 2017 alone. Despite the availability of several advanced diagnostic tools, accurate malaria diagnosis remains challenging in resource-constrained settings, where microscopists often struggle to improve diagnostic accuracy. Deep learning-based cell image classification helps reduce incorrect diagnostic conclusions by enabling automated analysis. This research aims to improve diagnostic accuracy by classifying malaria-infected cells using a majority voting ensemble framework combined with triplet loss aided label contrastive learning. Experimental results demonstrate the effectiveness of the proposed method on microscopic cell images in terms of accuracy, precision, recall, and other evaluation metrics.

bioinformatics↗

bioRxiv2024

RAF2Net: Automated grading of Renal cell Carcinoma utilizing Attention-enhanced deep learning models through Feature Fusion

It is anticipated that the number of instances of kidney cancer will continue to rise globally, which motivates changes to the current diagnostic framework in order to address emerging issues. Renal cell carcinoma (RCC) accounts for 80-85% of all renal tumors and is the most common kind of kidney cancer. Based on kidney histopathology images, this study presented a completely automated, robust, and computationally efficient Renal Cell Carcinoma Grading Network (RAF 2Net). Our suggested model incorporates 3 different Mobilenet backbones with intelligent feature fusion. Moreover, the attention blocks help us give more importance to the important pixels, which are majorly responsible for classification. For comparison purposes, Similar tests were conducted using transfer learning methods with pre-trained ImageNet weights and deep learning models created from scratch. To show the efficacy of the suggested method, we have computed evaluation parameters like Accuracy, Precision, F score, Recall, Confusion Matrix, and TSNE. Based on the provided KMC dataset, the experimental result demonstrates that the proposed RAF 2Net outperforms the nine most recent classification methods regarding prediction Accuracy, Recall, Precision, and F score with a value greater than 92%.

bioinformatics↗

bioRxiv2024

A Single Trophoblast Layer Acts as the Gatekeeper at the Endothelial-Hematopoietic Crossroad in the Placenta

During embryonic development the placental vasculature acts as a major hematopoietic niche, where endothelial to hematopoietic transition ensures emergence of hematopoietic stem cells (HSCs). However, the molecular mechanisms that regulate the placental hematoendothelial niche are poorly understood. Using a parietal trophoblast giant cell (TGC)-specific knockout mouse model and single-cell RNA-sequencing, we show that the paracrine factors secreted by the TGCs are critical in the development of this niche. Disruptions in the TGC-specific paracrine signaling leads to the loss of HSC population and the concomitant expansion of a KDR+/DLL4+/PROM1+ hematoendothelial cell-population in the placenta. Combining single- cell transcriptomics and receptor-ligand pair analyses, we also define the parietal TGC- dependent paracrine signaling network and identify Integrin signaling as a fundamental regulator of this process. Our study elucidates novel mechanisms by which non-autonomous signaling from the primary parietal TGCs maintain the delicate placental hematopoietic- angiogenic balance and ensures embryonic and extraembryonic development.

developmental biology↗

bioRxiv2024

METTL3 shapes m6A epitranscriptomic landscape for successful human placentation

Methyltransferase-like 3 (METTL3), the catalytic enzyme of methyltransferase complex for m6A methylation of RNA, is essential for mammalian development. However, the importance of METTL3 in human placentation remains largely unexplored. Here, we show that a fine balance of METTL3 function in trophoblast cells is essential for successful human placentation. Both loss-of and gain-in METTL3 functions are associated with adverse human pregnancies. A subset of recurrent pregnancy losses and preterm pregnancies are often associated with loss of METTL3 expression in trophoblast progenitors. In contrast, METTL3 is induced in pregnancies associated with fetal growth restriction (FGR). Our loss of function analyses showed that METTL3 is essential for the maintenance of human TSC self-renewal and their differentiation to extravillous trophoblast cells (EVTs). In contrast, loss of METTL3 in human TSCs promotes syncytiotrophoblast (STB) development. Global analyses of RNA m6A modification and METTL3-RNA interaction in human TSCs showed that METTL3 regulates m6A modifications on the mRNA molecules of critical trophoblast regulators, including GATA2, GATA3, TEAD1, TEAD4, WWTR1, YAP1, TFAP2C and ASCL2, and loss of METTL3 leads to depletion of mRNA molecules of these critical regulators. Importantly, conditional deletion of Mettl3 in trophoblast progenitors of an early post-implantation mouse embryo also leads to arrested self-renewal. Hence, our findings indicate that METLL3 is a conserved epitranscriptomic governor in trophoblast progenitors and ensures successful placentation by regulating their self-renewal and dictating their differentiation fate.

developmental biology↗

bioRxiv2024

Cell Targeting and Adjuvant Activity of Dietary Titanium Dioxide

Food-grade titanium dioxide (fgTiO2) is a bio-persistent particle under intense regulatory scrutiny. Paradoxically, meaningful in vivo cellular accumulation has never been demonstrated: the only known cell reservoirs for fgTiO2 are graveyard intestinal pigment cells which are metabolically and immunologically quiescent. Here we identify major new immunocompetent cell targets of fgTiO2 in humans, most notably in the subepithelial dome region of intestinal Peyers patches. Using multimodal microscopy techniques with single-particle detection and per-cell / vesicle image analysis we achieved correlative dosimetry, quantitatively recapitulating human cellular exposures in the terminal ileum of mice fed a fgTiO2-containing diet. Epithelial microfold cells selectively funneled fgTiO2 into LysoMac and LysoDC cells with ensuing accumulation. Notwithstanding, proximity extension analyses for 92 protein targets revealed no measureable perturbation of cell signalling pathways. When chased with oral {Delta}aroA-Salmonella, pro-inflammatory signalling was confirmed, but no augmentation by fgTiO2 was revealed despite marked same-cell loading. Interestingly, Salmonella caused the fgTiO2-recipient cells to migrate basolaterally in the patch and, sporadically, to the lamina propria, thereby fully recreating the intestinal tissue distribution of fgTiO2 in humans. Immunocompetent cells that accumulate fgTiO2 in vivo are now identified and we demonstrate a mouse model that finally enables human-relevant risk assessments of ingested, bio-persistent (nano)particles.

pharmacology and toxicology↗

bioRxiv2024

A genetically-encoded nanobody sensor reveals conformational diversity in beta-arrestins orchestrated by distinct seven transmembrane receptors

Agonist-induced interaction of G protein-coupled receptors (GPCRs) with {beta}-arrestins ({beta}arrs) is a critical mechanism that regulates the spatio-temporal pattern of receptor localization and downstream signaling. While the underlying mechanism governing GPCR-{beta}arr interaction is primarily conserved and involves receptor activation and phosphorylation, there are several examples of receptor-specific fine-tuning of {beta}arr-mediated functional outcomes. Considering the key contribution of conformational plasticity of {beta}arrs in driving receptor-specific functional responses, it is important to develop and characterize novel sensors capable of reporting distinct {beta}arr conformations in cellular context. Here, we design an intrabody version of a {beta}arr-recognizing nanobody (nanobody32), referred to as intrabody32 (Ib32), in NanoLuc enzyme complementation assay format, and measure its ability to recognize {beta}arr1 and 2 in live cells upon activation of a broad set of GPCRs. We discover that Ib32 robustly recognizes activated {beta}arr1 and 2 in the plasma membrane as well as in the endosomes, and effectively mirrors {beta}arr recruitment profile upon stimulation of GPCRs. We also design an Ib32 sensor for single-photon polarization microscopy with a change in linear dichroism as readout and demonstrate its utility for monitoring {beta}arr activation upon stimulation of angiotensin receptor by its natural and biased agonists. Interestingly, when used side-by-side with a previously described sensor of {beta}arr1 conformation known as Ib30, Ib32 uncovers distinct conformational signatures imparted on {beta}arrs by different GPCRs, which is further corroborated using an orthogonal limited proteolysis assay. Taken together, our study presents Ib32 as a novel sensor to monitor {beta}arr activation and leverages it to uncover conformational diversity encoded in the GPCR-{beta}arr system with direct implications for improving the current understanding of GPCR signaling and regulatory paradigms.

biochemistry↗