Biology subjects

Chatterjee, S.

Publications and source records attributed to Chatterjee, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Serum Lewis fucosylation reports on survivorship of patients with septic shock upon admission to intensive care unit

Septic shock, the excessive immune response to pathogen infection, poses a major health concern accounting globally for [~]20% of all deaths. Current methods to establish disease severity are unacceptably slow, unspecific and insensitive, hindering timely and effective treatment. Aiming to identify easy-to-assay glyco-signatures that may identify and guide the clinical management of the most critically unwell patients, we applied quantitative glycomics and glycoproteomics to sera longitudinally collected from septic shock survivors (n=29) and non-survivors (n=8). Glycomics of all 134 serum samples (sampled daily until recovery/death) revealed significant N-glycome dynamics across both patient groups. Unsupervised clustering of the serum N-glycome upon ICU admission (day 1) indicated survivorship-specific glyco- signatures. We therefore employed machine learning to train a random forest model using the serum N-glycome data. The model accurately classified survivorship outcomes of 35 of 37 patients (specificity 94.6%) and correctly predicted 6 of 8 non-survivors (sensitivity 75%) based on ICU day 1 data. Further interrogation of the serum N-glycome data revealed elevated Lewis fucosylation in non-survivors relative to survivors at ICU admission, a finding recapitulated by glycoproteomics. Amongst 58 other serum proteins strongly linked to acute phase response and stress pathways, alpha-1-acid-glycoprotein was identified as a principal carrier of Lewis fucosylation with a potential to stratify septic shock survivors from non- survivors (AUC 0.935). This study lays a foundation for risk stratification of septic shock patients by uncovering easy-to-assay glyco-signatures that identify individuals with poor survival outcomes upon ICU admission, with the potential to translate to early individualised clinical care at the bedside.

biochemistry↗

bioRxiv2025

Single-molecule analysis of gap and nick binding by LIG1 and LIG3α at the final step of DNA repair

DNA ligase (LIG) 1 and LIG3 repair broken single-strand breaks in the phosphodiester backbone at the final ligation step of DNA excision repair pathways, and complement each other during nuclear replication in case of unligated Okazaki fragments. We previously reported that both ligases discriminate against nicks containing non-canonical ends and ligate gap intermediate if left unfilled by DNA polymerases. However, it remains unknown how the dynamics of DNA binding differ for gap versus nick substrates by LIG1 and LIG3 at single-molecule level. Here, using total internal reflection fluorescence (TIRF) and ligation assays, we showed that LIG3 binds less frequently but forms longer-lived complex than LIG1 for nicks containing canonical A:T, mismatch G:T, and damaged 8oxoG:A, and they exhibit subtle differences in discriminating unusual ends. Moreover, our results identified gap DNA as a new target to which LIG1 and LIG3 can bind as efficient as their preferential nick sites. We showed gap ligation and observed that more percentage of LIG1 molecules form stable long-lived complex on DNA containing one nucleotide gap, whereas LIG3 forms short-lived gap complex without any differences in the percentage of molecule forming gap-bound complex. Finally, our findings demonstrated that LIG1 can still stably bind to larger gaps with better recognition, whereas LIG3 binding becomes further infrequent and shorter-lived. Overall, our study provides single-molecule insights into intricate differences between LIG1 and LIG3 for binding to a range of mutagenic and deleterious DNA repair and replication intermediates that could be a threat for maintaining genome stability at the final step.

biochemistry↗

bioRxiv2025

PEDOT:PSS Microparticles for Extrudable and Bioencapsulating Conducting Granular Hydrogel Bioelectronics

Conducting hydrogels are promising materials for forming physiomimetic bioelectronic interfaces to monitor and stimulate biological activity. However, most developed materials are non-microporous and possess fixed shapes, both of which can limit the integration of cells and tissues with devices. In non-conducting biomaterials, materials fabrication strategies imparting microporosity and dynamic mechanical properties have been shown to support cell infiltration and support biointerfaces of various geometries. Specifically, granular hydrogels have enabled encapsulating, conformal, and injectable interfaces through these features. However, granular hydrogels remain largely unexplored as conducting biomaterials. We present methods for fabricating spherical, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) hydrogel microparticles. When densely packed, these microparticles form a conducting granular hydrogel with microporosity as well as shear-thinning and self-healing dynamic mechanical properties. The PEDOT:PSS granular hydrogel can be extruded and maintain structure post-3D printing. Modulating microparticle PSS content achieves high granular hydrogel conductivity (137 S/m), and microparticles exhibit excellent cytocompatibility (>98% viability). Finally, we demonstrate utility as bioencapsulating electrodes for electrophysiological monitoring. These results highlight the functionality of our PEDOT:PSS conducting granular hydrogel, suggesting its potential as 3D printed bioencapsulating electrodes, 3D tissue engineering scaffolds for monitoring encapsulated cells, and injectable therapies for enhanced cell recruitment and tissue regeneration combined with electronic stimulation.

bioengineering↗

bioRxiv2025

Direct observation of single intrinsically disordered proteins in solution

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) in structured proteins are integral to many biological processes including neurotransmitter regulation, microtubule regulation, and transcription. IDP/IDRs are heterogenous, existing in a conformational ensemble of various interconnected states without a definitive tertiary structure. The high dynamicity of IDPs/IDRs limits ensemble protein characterisation techniques from capturing their properties, and measurements at the single-molecule level are hampered by the necessity to label the protein or modify its microenvironment, affecting their biophysics. Consequently, our understanding of IDPs/IDRs is limited, translating to a lack of knowledge of their roles in related diseases including Alzheimers disease, Parkinsons disease, and various cancers. This work presents the first experimental observation of unmodified IDP/IDR conformational dynamics in vitro, at the single-molecule level in real time, achieved by trapping individual IDPs/IDRs in a nanoscale volume using nanoaperture optical tweezers. Our results reveal that IDPs/IDRs exhibit significantly larger conformational variations in solution compared to globular proteins of similar size, as expected. We demonstrate that phosphorylation of native tau-441 by glycogen synthase kinase 3-beta (GSK3{beta}-tau) induces compaction and reduced conformational dynamics. We further observed a disorder-to-order transition during binding of an IDR, the N-terminal region of Src-associated protein in mitosis of 68 kDa (Sam68), to G8.5 RNA. The capability of nanoaperture optical tweezers to monitor the dynamic behaviours of single, unmodified IDPs/IDRs provides a powerful approach to advance our understanding of their elusive behaviours and further decode their roles in associated diseases.

biophysics↗

bioRxiv2025

De Novo Hydration of Cryo-EM Reconstructions through Molecular Dynamics Simulations of the Excess Chemical Potential

Predicting the precise positions of water molecules at the protein interface remains a formidable challenge, fueling active research in this field. Here, we present a novel approach based on molecular dynamics simulations that utilizes statistical thermodynamic signatures of water at protein interfaces to improve the accuracy of water placement in cryo-EM maps, with apoferritin as a model benchmark system. The interaction energy of solvent with the protein is insufficient to distinguish between high- and lower-consensus water positions, consistent with reports of earlier work. Instead, we employ a detailed statistical thermodynamic analysis based on the excess chemical potential (WT) - a measure of the thermodynamic balance between the interaction energy of an interfacial water molecule with the protein and its free energy of interaction with all of the other solvent molecules. WT is proportional to the log ratio of the local density of water molecules at the protein interface to the bulk density. 85% of the top 100 water locations with the most favorable excess chemical potential values are observed in one or more high resolution Cryo-EM maps deposited in the PDB, and 70% of the top 200 water locations indexed by excess chemical potential are observed in the cryo-EM maps. This work paves the way for the development of a cryo-EM refinement tool that integrates molecular dynamics simulations with cryo-EM data for high-resolution modeling of water networks.

biochemistry↗

bioRxiv2025

Dictionary of human intestinal organoid responses to secreted niche factors at single cell resolution

The intestinal epithelium is often a site of pathology, such as in inflammatory bowel disease (IBD), and its maintenance is highly modulated by interactions with the microenvironment. However, a systematic understanding of how the myriad of niche cues impact distinct epithelial cell types in a diseased context is still lacking. To address this gap, we first benchmarked diverse human colonic organoid injury models against IBD tissue, and established a disease-relevant model of epithelial inflammation using TNF, IFN{gamma}, and IL1{beta}. Using this system, we built a dictionary of epithelial responses to 81 secreted niche factors at single cell resolution via donor-pooled, multiplexed single cell RNA-sequencing (scRNA-seq). The comprehensive nature of our atlas allowed us to map relationships between perturbations, infer the function of less well-characterized ligands, and identify cell type-specific perturbed pathways. Finally, we established the relevance of organoid-derived gene programs by mapping them to single cell and spatial atlases of human IBD tissue. Our resource offers a global view of epithelial responses to microenvironmental cues in a physiologically relevant disease context and generates new hypotheses for signaling factors that may be involved in epithelial homeostasis and repair.

bioengineering↗

bioRxiv2025

Effects of a Cancer-Associated Mutation and Multiple SerinePhosphorylation on Poly(ADP-Ribose) Polymerase 2

Poly [ADP-ribose] polymerase 2 (PARP2) plays a crucial role in DNA repair. A common single-nucleotide polymorphism (SNP) in the PARP2 gene, rs3093921, has been associated with pancreatic cancer and marginal zone lymphoma (MZL). This SNP results in a missense mutation, D235G, in the PARP2 protein. PARP2 is also reported to undergo post-translational modifications (PTMs), particularly phosphorylation at serine residues 226, 232, and 353. The C-terminal region of PARP2 includes the Trp-Gly-Arg (WGR) and ADP-ribosyl transferase (ART) domains, and the helical subdomain (HD). The latter two, spanning residues 220 to 583, comprise the catalytic region of PARP2. The DNA-induced enzymatic activation of PARP2 is regulated by local destabilization of the HD domain. We used molecular dynamics (MD) simulations to investigate the impact of these three PTMs on both the wild-type (WT) and the mutant (D235G) forms of PARP2. Our simulations suggest that, while neither the cancer-associated mutation nor PTMs on their own significantly alter the overall flexibility of residues within the HD domain, they cause notably greater deviations in the backbone structure of the HD domain compared to the WT. In addition, PTMs in the context of the mutation show reduced interactions between the mutation site and other protein regions, likely due to structural stabilization induced by the PTMs. Importantly, PTMs mitigate the structural disruption caused by the mutation, helping the mutant protein retain a WT-like conformation. Additionally, the HD domain contributes to maintaining PARP2 in its inactive state through its connection with the ART domain. However, the D235G mutation weakens this connection. While PTMs alone do not have a significant impact on this connection, the simultaneous presence of both the mutation and PTMs partially alleviates the disruptive effect of the mutation and leads to partial restoration of the connection between the HD and ART domains.

biophysics↗

bioRxiv2025

Characterization of nick binding and sealing by LIG1 Huntington's disease-asssociated K845N variant at biochemical, structural, and single-molecule levels

DNA ligase 1 (LIG1) joins broken strand breaks and discriminates against nicks containing mismatch or oxidative damage. Huntingtons disease (HD)-associated mutation K845N in LIG1 gene has been predicted to be onset delaying and suppresses CAG repeat expansion. Yet, how this mutation impacts faithful nick sealing and efficient DNA binding by LIG1 remains unknown. Here, using biochemistry, X-ray crystallography, and total internal reflection fluorescence microscopy, we comprehensively characterized the impact of LIG1 HD-associated mutation at biochemical, structural, and single-molecule levels. Our results showed a reduced ligation efficiency by LIG1 K845N variant in the presence of nick substrates containing all possible 12 mismatches, 8-oxoG, and ribonucleotides at the 3-end when compared with the wild-type enzyme. Furthermore, our structures provided an atomic insight into differences in distances between the functional groups of K/N845 and DNA ends, demonstrating similar conformation and a lack of large scale alternations at the ligase active site. Finally, our single-molecule measurements in real-time revealed that K845N mutant binds less frequently for shorter life-time to nick DNA than wild-type protein. Overall findings contribute to understand the mechanism by which LIG1 ensures fidelity and nick binding to maintain genome stability at the final ligation step in normal versus disease states.

biochemistry↗

bioRxiv2025

Characterization of betacoronavirus HKU-1 and OC43 internal proteins using a prototypic coronavirus

Coronaviruses express a repertoire of accessory proteins for evading host immune responses. A small internal (I) accessory protein is expressed by the genus Betacoronavirus. Previous studies reported that the I proteins of SARS-CoV, MERS-CoV and SARS-CoV-2 inhibit type I interferon (IFN-I) expression through distinct mechanisms and have different roles in pathogenesis. Human coronaviruses (hCoV) HKU1 and OC43 are betacoronaviruses that also encode the I protein as an accessory protein. Although hCoV-HKU1 and hCoV-OC43 predominantly cause common cold in healthy adults, susceptible individuals infected with these viruses can develop severe disease. However, the virulence factors contributing to pathogenesis after infection with common cold CoVs (CCCoVs) have not been fully characterized. In particular, the functions of the hCoV-HKU1 and hCoV-OC43 I proteins have not been previously reported. The lack of robust reverse genetic systems, tissue culture and animal models limit the study of hCoV-HKU1 and hCoV-OC43 pathogenesis. Here, we examine the role of the hCoV-HKU1 and hCoV-OC43 I proteins in pathogenesis using a prototypic coronavirus. We introduce the I proteins of hCoV-HKU1 and hCoV-OC43 independently to a neurotropic strain of mouse hepatitis virus (J2.2). J2.2 infection is well characterized with clearly defined immune responses which allows the study of I proteins in the context of authentic coronavirus infection. We show that the I protein of hCoV-HKU1, but not that of hCoV-OC43, ameliorates MHV-J2.2 pathogenesis while the I protein of MERS-CoV exacerbates disease. The presence of the hCoV-HKU1 I protein decreases virus titers and cytokine expression while the I protein of MERS-CoV leads to increased immune cell infiltration and virus titers in mice after J2.2 infection. Moreover, the I proteins of hCoV-HKU1 and hCoV-OC43 show different patterns of subcellular localization. Overall, our findings suggest that the I protein of different betacoronaviruses play unique roles in pathogenesis. Author SummaryFactors governing the differences in the pathogenicity between highly pathogenic human coronaviruses (SARS-CoV, MERS-CoV and SARSR-CoV-2) and seasonal coronaviruses (hCoV-HKU1 and hCoV-OC43) are not completely understood. These differences are at least in part contributed to by the accessory proteins encoded between these two groups of human coronaviruses. The use of a heterologous coronavirus infection model provides an isogenic background for the direct comparison of viral proteins encoded by various coronaviruses. In this study, we compare the role of one of the accessory proteins encoded by betacoronaviruses, the I protein, in mediating disease outcome using a prototypic coronavirus. We demonstrate that the I protein of the highly pathogenic MERS-CoV but not that of the seasonal coronaviruses HKU1 and OC43 contributes to enhanced disease in the context of MHV-J2.2 infection, highlighting that virus-specific functions of accessory proteins encoded by different hCoVs.

microbiology↗

bioRxiv2025

Codon-dependent regulation of mRNA translation and stability by ZC3H7A and ZC3H7B RNA-binding proteins

Decelerated translation elongation caused by non-optimal codons can reduce mRNA stability through codon optimality-mediated mRNA degradation. A key element of this process is the coupling of sensing the mRNA codon usage with the regulation of translation efficiency and stability. We report that two paralog RNA-binding proteins (ZC3H7A and ZC3H7B), which are only found in Chordates, preferentially bind to and reduce the stability and translation of mRNAs enriched in non-optimal codons with A/U at their wobble sites (A/U3 codons). ZC3H7A/B engage with ribosomes that lack elongation factors and induce mRNA degradation or block translation initiation through their interactions with the CCR4-NOT and the GIGYF2/4EHP translation repressor complex, respectively. Depletion of ZC3H7A/B or 4EHP impairs the repression of non-optimal A/U3-rich mRNAs. This study provides insights into a unique mechanism in higher eukaryotes that couples codon usage with the regulation of translation efficiency and mRNA stability.

cell biology↗

bioRxiv2025

Repurposing without Reinventing the Wheel - Ensemble Models for Differential Analysis

Inspired by ensemble models in machine learning, we propose a general framework for aggregating multiple distinct base models to enhance the power of published differential association analysis (DAA) methods. We demonstrate this approach by augmenting popular DAA models with one or more biologically motivated alternatives. This creates an ensemble that bypasses the challenge of selecting an optimal model and instead combines the strengths of complementary statistical models to achieve superior performance. Our proposed ensemble learning approach is platform-agnostic and can augment any existing DAA method, providing a general and flexible framework for various downstream modeling tasks across domains and data types. We performed extensive benchmarking across both simulated and experimental datasets spanning single-cell gene expression, bulk transcriptomics, and microbiome metagenomics, where the ensemble strategy vastly outperformed non-ensemble methods, identified more differential patterns than the competing methods, and displayed good control of false positive and false discovery rates across diversified scenarios. In addition to highlighting a substantial performance boost for state-of-the-art DAA methods, this work has practical implications for mitigating the so-called reproducibility crisis in omics data science. An open-source R package implementing the ensemble strategy is publicly available at https://github.com/himelmallick/DAssemble.

bioinformatics↗

bioRxiv2025

HuR-Driven Reversible Mitochondrial Shuttling Buffers Cytosolic miRNA Levels in Hepatic Cells

Mitochondria, the "powerhouse" of mammalian cells, also serve as key storage sites for ions, metabolites, and enzymes vital for metabolic regulation. Exploring the regulatory processes that control the activities of miRNAs, the key non-coding RNA in mammalian cells, we found a context-dependent reversible localization of specific miRNAs to the mitochondrial matrix. Our data suggests a de novo role of mitochondria as miRNA sinks in mammalian cells. miR-122 is a key hepatic miRNA regulating metabolic processes in the mammalian liver. In this study, we observed increased mitochondrial targeting of miR-122 in amino acid-starved hepatic cells. Interestingly, when cells were refed with amino acids, mitochondrial miR-122 gets relocalized and reused in the cytosol for the translational repression process. Moreover, this phenomenon is not limited to miR-122 as several mitochondrial miRNAs (mito-miRs) follow similar transient storage inside mitochondria in stressed cells. Remarkably, mitochondria-localized mito-miRs preferentially target mRNAs encoding crucial mitochondrial components related to apoptosis. Hence, hepatic cells regulate apoptosis pathways during the starvation-refeeding cycle by shuttling a specific set of miRNAs to and from mitochondria, thereby balancing cytosolic miRNA content and homeostasis. Stress response miRNA binder ELAVL1 or HuR protein was found to be both necessary and sufficient for transporting the mature mito-miRs to the mitochondrial matrix - a process also controlled by the interaction between mitochondria and the endoplasmic reticulum. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/647748v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1178f5dorg.highwire.dtl.DTLVardef@1c5578borg.highwire.dtl.DTLVardef@f68555org.highwire.dtl.DTLVardef@ab9c40_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LISpecific miRNAs (mito-miRs), including miR-122, are targeted to mitochondria for transient storage in stressed hepatic cells. C_LIO_LIMito-miRs storage in mitochondria allows the expression of apoptosis-related genes in stressed cells. C_LIO_LIMito-miRs relocalization to cytoplasm on stress reversal allows suppression of apoptosis. C_LIO_LIBinding with ELAVL1 protein HuR allows reversible shuttling of mito-miRs to and from mitochondria in hepatic cells. C_LIO_LIER-mitochondrial interaction is key for the mitochondrial localization of miRNAs. C_LI

cell biology↗

bioRxiv2025

Single-cell resolution spatial transcriptomic signature of the retrosplenial cortex during memory consolidation

The retrosplenial cortex (RSC) is a critical brain region activated during spatial memory tasks and plays an underlying role in long-term memory consolidation. The RSC comprises multiple cell types, including different classes of excitatory neurons across laminar layers. These layer-specific cells form the hub of neuronal connection between the RSC and other brain regions, including the hippocampus. Despite the established role of the RSC in spatial memory, the transcriptomic signature of the neuronal sub-types in the RSC during spatial memory consolidation remained elusive. Here we used both unbiased and targeted spatial transcriptomic approaches to illuminate the transcriptional signature of the RSC following a spatial memory task. We found that genes related to transcription regulation, protein folding, and mitogen-activated protein kinase pathways were upregulated in the RSC after spatial learning during an early time window of memory consolidation. Further, cell type and excitatory neuronal layer-specific changes in gene expression were resolved using Xenium spatial transcriptomics. The distinct signatures of memory-responsive genes were observed in excitatory neurons across the laminar layers of the RSC following learning. Finally, we observed that blocking RSC excitatory neurons during the early temporal window after learning using a chemogenetic approach impaired long-term spatial memory. Overall, our results uncover a molecular signature of the RSC after learning and demonstrate the role of RSC excitatory neurons during the early time points of memory consolidation. This study underscores the importance of the learning-induced transcriptional signature of the RSC in long-term spatial memory consolidation and reveals a cell-type specific signature of memory-responsive gene expression.

neuroscience↗

bioRxiv2025

Telomerase modRNA offers a novel RNA-based approach to treat human pulmonary fibrosis

Pulmonary Fibrosis (PF) is a life-threatening illness that is characterized by progressive scarring in the lung interstitium. There is an urgent need for new PF therapies because current treatments only slow down the progression of fibrosis and the median life expectancy post-diagnosis is only 4 to 6 years. Since PF patients frequently exhibit telomere attrition, overexpressing telomerase, the enzyme responsible for synthesizing telomeres represents a compelling therapeutic option. In this study, we in vitro transcribed human telomerase reverse transcriptase (hTERT) mRNA using modified nucleosides (modRNA). ModRNA hTERT treatment led to transient activation of telomerase activity in a dose-dependent manner in MRC-5 cells and, importantly, in primary human alveolar type II pneumocytes (ATII cells). Consequently, the proliferative capacity was increased, concomitant with reduced DNA damage and elongated telomere length. Notably, induction of cellular immune response was only detectable at the highest modRNA concentration, and returned to normal levels within 48 hours. Next, we demonstrated that circularized, exonuclease-resistant modRNA hTERT extended the transient expression profile which may be clinically advantageous. Finally, we provided therapeutic proof of concept in organotypic 3D ex vivo human precision-cut lung slices derived from end-stage PF patients. Intriguingly, a single modRNA hTERT treatment inhibited senescence as indicated by significantly lower levels of senescence-associated {beta}-galactosidase, and pro-inflammatory IL6 and IL8. Concurrently, the key fibrosis mediators TGF{beta} and COL1A1 were markedly reduced. In conclusion, the data presented herein provide initial evidence for the potential of RNA-based hTERT therapy for treating human lung fibrosis.

molecular biology↗

bioRxiv2025

Histone Lysine Crotonylation Regulates Long-Term Memory Storage

Histone post-translational modifications (PTMs), particularly lysine acetylation (Kac), are critical epigenetic regulators of transcriptional programs underlying long-term memory consolidation. Beyond Kac, several other non-acetyl acylations have been identified with the ability to regulate transcription; however, their role in memory consolidation remains unknown. Here, we identify histone lysine crotonylation (Kcr) as a molecular switch for long-term memory and glutamatergic neurotransmission. We find that spatial learning induces distinct spatiotemporal patterns of Kcr across hippocampal subregions, and that Kcr stimulates learning-induced gene expression. Through genetic and pharmacological manipulations, we show that reducing hippocampal Kcr levels impairs memory, while increasing Kcr enhances long-term memory. Single-nuclei transcriptome and chromatin accessibility analyses reveals that Kcr facilitates activation of genes regulating glutamate signaling. Cell-cell communication analysis further infers that Kcr enhancement strengthens glutamatergic signaling within principal hippocampal neurons. Real-time fluorescence imaging with genetically encoded sensors functionally validates our multiomics and computational findings--demonstrating the role of Kcr in regulating presynaptic glutamate release and neuronal activity. In summary, our findings elucidate a novel mechanism underlying long-term memory consolidation and excitatory neurotransmission, linking epigenetic events to synaptic function and behavior.

neuroscience↗

bioRxiv2025

All-trans retinoic acid and fluid transport in myopigenesis

Myopia, or near-sightedness, is rapidly growing in prevalence, with significant long-term implications for ocular health. There is thus great impetus to better understand molecular signaling pathways leading to myopia. We and others have reported that all-trans retinoic acid (atRA) is involved in myopigenic signaling, yet the understanding of how atRA is transported and exerts a myopigenic influence is poor. Here we measured the concentrations of atRA in the serum in wild-type C57BL/6 mice under control conditions and after atRA feeding, previously shown to induce myopia. We also developed a mathematical model that describes fluid fluxes and the advective-diffusive transport of atRA in choroid and sclera, including atRA synthesis in the choriocapillaris, atRA degradation by scleral cells, and binding of atRA to the carrier protein serum albumin. This model, developed for both mice and humans, showed that atRA produced in the choriocapillaris was able to permeate well into the sclera in both mice and humans at biologically-relevant concentrations, and that atRA feeding greatly increased tissue levels of atRA across both the choroid and sclera. We were also able to identify which parameters most influence atRA concentration in ocular tissues, guiding future experimental work. Our findings support atRAs role in myopigenic signaling.

bioengineering↗

bioRxiv2025

Synergistic effects of Ret coding and enhancer loss-of-function alleles cause progressive loss of inhibitory motor neurons in the enteric nervous system

Hirschsprung disease (HSCR) is a congenital enteric neuropathy caused by disrupted development of enteric neural crest-derived cells (ENCDCs). Although pathogenic coding variants in RET account for many cases, the largest genetic contribution to HSCR risk arises from a common non-coding variant (rs2435357) within a SOX10-bound RET enhancer (MCS+9.7) that reduces RET gene expression in vivo and triggers expression changes in other ENS genes in the human fetal gut. However, the ENS cell types affected by this enhancer and the mechanisms by which these transcriptional changes lead to HSCR remain unknown. Here, we investigated the role of this enhancer by generating mice carrying a deletion of the orthologous Ret mcs+9.7 enhancer ({Delta}mcs+9.7). Single-cell RNA sequencing of E14.5 embryonic gut demonstrated that enhancer deletion reduced Ret expression by 8% without altering ENS cell composition. However, reduced Ret expression was restricted to differentiating neurons and inhibitory motor neuron lineages, revealing cell type-specific enhancer activity. To determine the functional consequences of further reducing Ret dosage, we generated compound heterozygous mice carrying both the enhancer deletion and a Ret coding null allele (+/{Delta}mcs+9.7;+/CFP). These mice exhibited additive reductions in Ret expression, altered Sox10 expression, dysregulation of cell-cycle and neuronal differentiation programs, and selective depletion of developing inhibitory motor neuron lineages. These findings establish a cell type-specific role for the mcs+9.7 enhancer in modulating Ret dosage and reveal how subtle enhancer perturbations alter neural subtype specification without overt hypoganglionosis, suggesting that HSCR arises from a cascade of cellular defects triggered by >50% loss of Ret function.

genetics↗

bioRxiv2025

Studies of all-trans retinoic acid transport in myopigenesis

PurposeMyopia incidence is increasing globally. All-trans retinoic acid (atRA) is important in myopigenic retinoscleral signaling, motivating research on its ocular transport. However, atRAs weak autofluorescence limits its direct visualization in tissues. Further, atRA is hydrophobic and must bind to protein carriers for transport. We assessed a fluorescent analog of atRA (LightOxTM14, CAS:198696-03-6, referred as floRA), as an experimentally accessible atRA surrogate by: (i) evaluating its binding to carrier proteins and (ii) visualizing its distribution in ocular tissues. MethodsO_ST_ABSBindingC_ST_ABSWe assessed atRA-carrier protein binding using fluorescence quenching assays with bovine serum albumin (BSA), high density lipoprotein (HDL), apolipoprotein A-I (Apo A-I) and retinol binding protein 4 (RBP4). Direct visualizationWild-type C57BL/6J mice were euthanized, eyes enucleated, and wedges containing sclera and choroid incubated for specific durations in 50 M floRA+BSA. The wedge centers were cryo-sectioned and counterstained for nuclei. Fluorescent micrographs were acquired and analyzed using ImageJ. ResultsAssociation constants (Ka) for atRA and floRA binding to carrier proteins were similar and ranged from 2-13 x 105 M-1, indicating non-specific binding. floRA could be visualized in sclera and choroid, yet showed significant spatial heterogeneity (enhanced fluorescence often colocalizing with nuclei). ConclusionsfloRA is a reasonable surrogate for atRA binding to BSA, HDL, Apo A-I and RBP4. Considering these proteins relative serum and extravascular abundances, and their similar binding affinity to atRA, we predict that serum albumin is an important atRA carrier. Use of floRA in whole tissue tracer studies shows promise but requires further refinement.

biochemistry↗