Biology subjects

Raj, A.

Publications and source records attributed to Raj, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Single-cell spatial transcriptomics of inherited retinal degeneration reveals broad and focal organization of disease responses

Inherited retinal degenerations are largely monogenic disorders characterized by progressive photoreceptor dysfunction and death. Although the transcriptomic responses to inherited degeneration are well-characterized, how these responses are spatially organized across the retinal axis has not been mapped. Here, we used seqFISH to spatially profile 173 cell identity and degeneration-associated genes with single-cell, single-transcript resolution in male wild-type and LCA5gt/gtmouse retinas at early, intermediate, and advanced disease stages. In early disease, rod injury and stress response markers were enriched in the superior retina, while rod function and identity markers were enriched in the inferior retina. Nested within the broader superior-inferior organization, a coordinated rod injury response comprising Edn2, Fgf2, Socs3, and Stat3 was organized into focal regions on the order of 100 m in width, with high-response regions directly adjacent to low-response regions. These focal regions were locally enriched for markers of Muller reactive gliosis and rod stress responses. Our single-cell spatiotemporal map reveals that transcriptional responses to degeneration vary at both the broad and focal scales, and offers a framework for investigating how local tissue properties and interactions among neighboring cells may contribute to disease progression.

neuroscience↗

bioRxiv2026

Myeloid STING restrains cardiac remodeling by suppressing macrophage amyloid precursor protein

Mitochondrial DNA (mtDNA) released into the cytosol activates innate immune signaling and promotes inflammation, yet its role in macrophages following sterile tissue injury remains poorly understood. Here, we show that cardiac macrophages from both patients and mice with myocardial infarction (MI) exhibit increased mitochondrial biogenesis, mitochondrial content, membrane potential, and expression of mitochondrial nucleases that facilitate mtDNA release. Consistently, macrophage-specific silencing of genes regulating mitochondrial biogenesis or mtDNA processing attenuated adverse cardiac remodeling after MI. Unexpectedly, despite the role of mtDNA in activating the cGAS-STING pathway, myeloid deletion or macrophage-specific silencing of Sting or cGas exacerbated ventricular dilation, fibrosis, and contractile dysfunction following MI. Single-cell transcriptomic and cell communication analyses identified amyloid precursor protein (APP) as a key downstream effector of STING in cardiac macrophages. Macrophage-specific in vivo App silencing rescued the detrimental effects of myeloid Sting deficiency, establishing APP as a critical mediator of adverse remodeling. Mechanistically, STING interacted with the transcriptional repressor MZF1, promoted its nuclear localization, facilitated its binding to the App promoter, and suppressed App transcription to restrain adverse cardiac remodeling. Together, our findings uncover an unexpected cardioprotective function of myeloid STING and identify the STING-MZF1-APP axis as a previously unrecognized mechanism governing cardiac repair after myocardial infarction.

cell biology↗

bioRxiv2026

Predicting pain location from resting-state brain fMRI

Low back pain is a prevalent issue with few reliable treatments. Although there is great variation in clinical presentation within the low back pain population, little is known about the neurobiological mechanisms underlying these differences. In this study, we sought to stratify chronic low back pain patients (N = 275) into phenotypes characterized by correlated patterns of resting-state brain activity and sensory abnormalities (pain, numbness, and pins and needles) indicated on hand-drawn body maps. Our cross-decomposition analysis yielded phenotypes that resemble previously documented mechanistic pain types, revealing distinct brain connectivity patterns associated with different clinical presentations. Our model was then used to predict pain body maps from fMRI data in a small novel dataset of chronic pain subjects, suggesting that these relationships may generalize to other chronic pain conditions. Our results support the utility of resting-state fMRI in understanding the heterogeneity of chronic pain, which may be leveraged to develop more targeted pain treatments.

neuroscience↗

bioRxiv2026

Gene Gradients Reveal Directed Structural Connectivity Across Species

Studying the brain's inter-regional structural connectivity (SC) has transformed neuroscience, yet noninvasive neuroimaging remains blind to the directionality of neural wiring. Structural directionality must influence observed brain dynamics, but estimating edge-level directionality from brain function is ill-posed and underdetermined. Here, we show that gene co-expression gradients define a low-dimensional biological manifold that locally biases brain regions to act as network sources or sinks. Embedding gene gradients into a linear, higher-order network diffusion model defines a gene-structure-function relationship that recovers ground-truth directionality in C. elegans (r=0.70), mouse (r=0.57), and macaque (r=0.46). Applying this framework to 770 healthy young adults from the Human Connectome Project yields subject-specific directed connectomes that reproduce established corticothalamic asymmetries and reveal distinct gene ontologies associated with network sources and sinks. Furthermore, integrating directed structure with observed functional covariance enables us to derive a principled measure of directed functional connectivity, termed "angular flow" (AF), related to probability angular momentum in non-equilibrium statistical mechanics. Regional AF recapitulates the principal unimodal--multimodal gradient of functional connectivity, suggesting that this macroscale functional hierarchy emerges from a source--sink organization of signal flow through the brain. This framework resolves a long-standing limitation in connectomics and opens new opportunities to study structural directionality and non-equilibrium brain dynamics in health and disease.

neuroscience↗

bioRxiv2026

Single-cell lineage tracing maps clonal and transcriptional dynamics in melanoma metastasis

Melanoma metastasis is driven by extensive intratumoral heterogeneity and phenotypic plasticity, yet how clonal identity relates to transcriptional programs during metastasis remains unclear. Here, we applied MeRLin, a single-cell lineage tracing platform, to dissect the clonal and transcriptional heterogeneity of metastatic melanoma in a patient-derived spontaneous metastasis model. Clonal analyses revealed hierarchical structures during tumor progression, with a subset of lineages from primary tumors consistently enriched across metastatic sites, supporting a model of polyclonal seeding followed by selective expansion of pre-existing highly metastatic subpopulations. Single-cell transcriptomic profiling identified two major metastatic subpopulations of distinct transcriptional programs, characterized by neural crest stem cell-like and lipid metabolism signatures. Both programs were enriched for invasion-associated genes and maintained across organs through distinct regulatory networks. Spatial mapping by barcode RNA-FISH linked these transcriptional states to their tissue context and showed that OLFML3 expression partially co-localized with a dominant subpopulation at the tumor-liver interface, marking the invasive fronts of metastatic growth. Together, these findings establish a framework in which clonal identity, transcriptional state, and spatial organization jointly shape metastatic melanoma progression.

cancer biology↗

bioRxiv2026

Global Signal Removal (GSR) as graph spatial filtering

Global Signal Removal (GSR) is a widely applied step in functional magnetic resonance imaging (fMRI) preprocessing. Although GSR conventionally denotes Global Signal Regression, we use Global Signal Removal to encompass a broader family of spatial filtering operations. GSR in general remains controversial due to concerns about introducing spurious anticorrelations and removing neurally meaningful signals. In this paper, we provide a precise geometric characterization by formalizing GSR as graph spatial filtering. We demonstrate that the most common form of GSR, Regression-GSR, equates to a rank-1 deflation of the covariance matrix (i.e. functional connectivity) by the degree vector. Empirically, the degree vector is dominated by the first principal component of the functional connectivity matrix (correlation = 0.88 {+/-} 0.12 in resting-state HCP data), making Regression-GSR an approximation to first eigenmode removal. This view of GSR as a spatial projection framework allows us to develop a family of GSR variants, each expressible in a unified spatial filter: Naive-GSR removes the uniform vector, PCA-GSR precisely removes the first eigenvector, and SC-GSR, a new variant we introduce that removes the first harmonic of the structural connectivity matrix. A key distinction emerges: while Naive, PCA, and SC-GSR are orthogonal projections, Regression-GSR is an oblique projection that computes regional weights proportional to the degree vector but removes a spatially uniform signal. All GSR variants induce numerical singularity in the covariance matrix, but they differ in their effects on task-state separability, which we examine empirically. In summary, we reframe GSR as a family of graph spatial filters that enable interpretability of its effects, with systematically varying effects on network connectivity across variants.

neuroscience↗

bioRxiv2026

MAMBAxBrain: A Multi-task Neural Framework Linking Brain Functional Dynamics to Individual Fingerprints, Cognitive and Disease States

Functional magnetic resonance imaging (fMRI) contains rich individual, cognitive, and pathological information, yet no universal model exists for multi-task modeling of these dimensions. Here, we introduce MAMBAxBrain, a multi-task neural framework that integrates Mamba architecture with functional connectivity analysis to jointly model the temporal dynamics and spatial coordination of neural activity. MAMBAxBrain achieves high accuracy across four distinct fMRI objectives--brain fingerprinting, cognitive task decoding, reaction time prediction, and schizophrenia classification--consistently outperforming state-of-the-art methods with robust crosssession generalization. Interpretability analyses show that each task engages distinct, biologically plausible circuitry--from higher-order association cortex for identity to subcortical-motor loops for reaction time and disrupted control-sensory connectivity for schizophrenia. These findings inform a longstanding debate: rather than operating through wholly separate or entirely shared systems, the brain preferentially recruits task-specific circuits while retaining common representational structure across functions.

neuroscience↗

bioRxiv2026

Biophysically realistic network-level transport model of tau progression with exosome-mediated release and uptake processes

The spatiotemporal progression of tau in neurodegenerative diseases like Alzheimers follows the brains structural connectome, yet a gap exists between the macroscopic spread observed over years and the protein kinetics occurring over hours. Current models fail to reconcile this disparity or incorporate the cellular mechanisms driving transmission. Here, we advance the Network Transport Model (NTM) to bridge these scales by integrating active transport along microtubules, continuous toxic tau production, and exosome-mediated release and uptake. This framework constitutes one of the most biologically detailed models of tau spread on a whole brain to date, representing a significant innovation in how multiscale proteinopathies are simulated. Simulations on the mouse connectome demonstrate that this framework replicates empirical tau propagation patterns. Our results identify trans-neuronal release and uptake rates as the primary "bottleneck" on macroscopic spread, providing a biologically grounded explanation for the diseases slow progression. Furthermore, we find that high aggregation sequesters tau within regions, limiting global transmission, while increasing the abundance of toxic polymeric tau fibrils. Meanwhile, tau transport polarity bias (anterograde vs. retrograde) dictates spatial patterning. By linking molecular mechanics to system-wide pathology, this model provides an "in-silico" framework to evaluate how cellular-targeted interventions might alter the trajectory of tauopathic dementias. Author SummaryIn Alzheimers disease and related dementias, a protein called tau forms misfolded aggregates and gradually spreads through the brain along its neuronal wiring. A long-standing puzzle is linking the microscopic protein dynamics taking minutes to hours to the multiyear macroscopic spread of the disease. We developed a new mathematical model that is capable to explain macroscopic spreading behavior of tau as an emerging process from the underlying biophysical mechanics of tau pathology. Our simulations reproduce real patterns of tau spread, and our framework will pave the way for testing how treatments targeting specific cellular mechanisms might reshape disease progression.

neuroscience↗

bioRxiv2026

Environmental Amino Acid Sensing Regulates the Rate of ASC Translation and NLRP3 Inflammasome Assembly

The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a multiprotein signaling complex that triggers pyroptotic cell death and interleukin (IL)-1 family cytokine release during infection and cell injury. Its assembly is driven by the adaptor protein, apoptosis-associated speck-like protein containing a CARD (ASC), whose filamentation forms a supramolecular speck upon NLRP3 activation to amplify inflammasome signaling. While the NLRP3 inflammasome is well appreciated as a sensor of environmental danger and damage, little is known about how homeostatic environmental factors like dietary metabolites regulate its activity. Here, we find that environmental availability of the branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, controls NLRP3 inflammasome assembly. While ASC is typically viewed as a constitutively expressed, unregulated inflammasome component, we find that Toll-like receptor 4 (TLR4) activation triggers localization of ASC mRNA to the perinuclear space. Moreover, our data demonstrate that ASC undergoes TLR4-driven translational bursting from polyribosomes during inflammasome priming. This translational engagement is dependent on BCAA availability and mechanistic target of rapamycin (mTOR) activity, which regulate the kinetics of inflammasome assembly. In contrast, the translation of NLRP3 and caspase-1 is largely insensitive to these inputs. Furthermore, we find that BCAAs regulate NLRP3 inflammasome activation in both mouse and human macrophages, in the context of bacterial infection, and during lipopolysaccharide (LPS)-induced sepsis in vivo. Altogether, this work unveils a novel inflammasome priming event governed by the amino acid environment. These findings further highlight how the activity of proteins maintained in equilibrium like ASC can be dynamically regulated through rapid changes in mRNA translation.

immunology↗

bioRxiv2026

A Joint Tau Propagation and Neuroinflammation Model Reinforces Inflammatory Modulation of Network-Driven Spread in Alzheimer's Disease

Alzheimers disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia. Despite over a century of research, AD remains untreatable due to an incomplete understanding of its underlying mechanisms. While amyloid beta has dominated therapeutic efforts, tau pathology and neuroinflammation represent critical disease drivers and intriguing therapeutic targets. We developed an extended computational model building upon the open-source Aggregation Network Diffusion (AND) framework by coupling spatial tau propagation and aggregation with ordinary differential equations representing key inflammatory cascades. The model incorporates M1/M2 microglia and astrocytic activation, cytokine-mediated feedback loops, and neuronal loss, all modulated via region-specific genetic expression matrices of ApoE and TREM2 with variant-specific weighting. The model maintains biologically plausible tau dynamics while generating robust inflammatory marker trends, serving as a computational testbed for hypothesis generation and mechanistic exploration. The inflammatory components independently capture experimental observations and display a marked M1/M2 microglia divergence. The model demonstrates enhanced similarity to regional tau propagation patterns due to inflammatory and genetic components, reinforcing neuroinflammations role in tau spread and highlighting the need to incorporate these processes in modeling efforts. Finally, through parsimony analysis, we identify microglia and pro-inflammatory rates (including microglia-facilitated tau spread) as key contributors to improved model accuracy, informing future modeling approaches.

neuroscience↗

bioRxiv2026

HONeD-in on Brain Activity: Deconvolving Passive Diffusion on the Structural Network from Functional Brain Signals

Brain regions perform distinct computations, and their signals propagate through the whole-brain white matter network. Yet, mathematical models that describe this signal propagation via purely passive diffusion can predict a considerable amount of the observed functional connectivity between regions. This raises a critical question: if so much functional connectivity can be explained by a passive process, how can we isolate the active process? Here, we calculate in closed-form an estimate for such an active signal in functional MRI by spatially deconvolving the effect of passive signal spread over the brains structural connectivity using a higher-order network diffusion (HONeD) model. Across 770 Human Connectome Project subjects, we show that the resulting HONeD-innovation (HONeD-in) signal 1) sparsifies functional connectivity while retaining a well-connected network, 2) remodels resting-state networks (RSNs), 3) mixes the unimodal- multimodal hierarchical organization of RSNs into a circle with no clear hierarchy, and 4) deblurs task-activation maps. Together, our results highlight HONeD deconvolution as a generalizable new way to study resting-state and task fMRI brain signals.

neuroscience↗

bioRxiv2025

Self-Organization Through Local Cell-Cell Communication Drives Intestinal Epithelial Zonation

The intestinal epithelium exhibits zonated gene expression along the crypt-villus axis, with distinct transcriptional programs in enterocytes at the villus top versus bottom. However, the mechanisms establishing these spatial patterns remain unclear. Three models could explain zonation: external gradients, cell-intrinsic temporal programs, or local self-organization. Using spatial transcriptomics and perturbations of two-dimensional intestinal organoids, we show that zonation emerges via spontaneous self-organization without mesenchymal, neural, or vascular inputs. Cell-intrinsic models were eliminated by transplanting cells into established monolayers; transplanted cells progressively adopted zonation profiles matching their new location, with strongly zonated genes showing the greatest adaptive responses. Pharmacological inhibition of EphA2 receptors disrupted zonation, revealing a previously unknown role for epithelial EphA-ephrin-A signaling in regulating enterocyte zonation. These findings demonstrate that self-organization through local epithelial cell-cell communication generates spatial patterns independently of external positional cues or cell-autonomous programs.

systems biology↗

bioRxiv2025

Clonal dynamics shaped by diverse drug-tolerant persister states in melanoma resistance

Most advanced melanomas initially respond to targeted therapy but eventually relapse. Rather than acquiring new mutations, resistance is driven by drug-tolerant persister cells that enter a reversible drug-refractory state. We developed MeRLin, a high-resolution lineage tracing platform integrating cellular barcoding, single-cell transcriptomics, RNA fluorescence in situ hybridization (FISH), and computational analyses to track clonal and transcriptional dynamics in patient-derived melanoma models during prolonged therapy. Clonal dynamics revealed that persister subpopulations first responded to treatment but persisted and expanded during minimal residual disease, ultimately leading to tumor recurrence. Pre-treatment melanoma populations diversified into four conserved persister states characterized by stress-like, lipid metabolism, PI3K signaling, and extracellular matrix remodeling programs associated with adaptive resistance. Spatial transcriptomics showed the organization of these adaptive programs and a complex signaling network of autocrine and paracrine interactions among persister subpopulations. Barcoded RNA-FISH enabled spatial mapping of clonal identity and gene expression, revealing in situ co-localization of a dominant resistant clone with SLC2A1 expression. MeRLin provides a robust framework for dissecting cancer heterogeneity and identifying vulnerabilities in persister populations.

cancer biology↗

bioRxiv2025

Dynamics of Ribosomal RNA Transcription and Abundance in Normal and Leukemic Hematopoiesis

Transcription of ribosomal RNAs (rRNAs) from rDNA repeats is a critical first step of ribosome biogenesis. Often deemed a housekeeping process, its cell-type-specific regulation is largely unexplored. We profiled nascent rRNA and mature rRNAs (ribosome subunits) across mouse hematopoiesis, and observed 10-fold, cell-type-specific variation in rRNA abundance that is largely uncoupled from proliferation and translation rates. We also tested and confirmed the longstanding assumption that acute myeloid leukemia progenitors show notably higher rRNA levels than matched normal counterparts. These trends in nascent rRNA across hematopoietic contexts parallel changes in the proportion of active rDNA repeats. Absolute quantification of rRNAs unexpectedly revealed an excess of large versus small ribosomal subunits in all cells, particularly in the myeloid lineage. Finally, using an RNA Polymerase I degron system, we found that reduced rRNA transcription promotes myeloid progenitor differentiation. Collectively, our work uncovers complex dynamics and functional significance of rRNA regulation in hematopoiesis.

molecular biology↗

bioRxiv2025

Gastruloid patterning reflects division of labor among biased stem cell clones

Division of labor, whereby individuals specialize in complementary roles to collectively achieve beneficial outcomes, is a recurring phenomenon in economics, ecology, and microbiology. In development, individual cells specialize, but this specialization is thought to arise from instruction by external signaling cues upon otherwise interchangeable progenitors. It is unclear if progenitors exhibit some degree of specialization, and if so, whether that specialization is optimized for developmental outcomes. Using fluorescence-based lineage tracing in combination with spatial transcriptomics, we show that, in the gastruloid model of early development, individual stem cell clones harbored reproducible spatial propensities for anterior or posterior fates that result in a spontaneous division of labor during axial organization. Gastruloids derived from pure clones generated elongated structures less frequently than a polyclonal population, but mixing clones allowed clones to follow their inherent propensity, restoring proper axial elongation. Spatial transcriptomics reveals that pure clones show disrupted gene expression with inappropriate coexpression of anterior and posterior markers, while clone combinations restore proper spatial organization. We further show that propensities are globally utilized: a clone with a particular propensity can adopt different fates depending on what is more optimal for development as a whole. Perturbations to key developmental signaling pathways disrupted this sorting and profiling revealed molecular characteristics of propensity. Proper developmental outcomes may thus emerge from the coordinated action of intrinsically biased clonal populations.

developmental biology↗

bioRxiv2025

Single-cell spatial mapping reveals reproducible cell type organization and spatially-dependent gene expression in gastruloids

Gastruloids are stem-cell-based models that recapitulate key aspects of mammalian gastrulation, including formation of an anterior-posterior (AP) axis. However, we do not have detailed spatial information about gene expression and cell type organization, particularly at the level of individual gastruloids. Here, we report a spatially resolved, single-cell molecular catalog of the transcriptomes of 26 individual gastruloids. We found that cell type composition and tissue-scale spatial organization were largely consistent across gastruloids, but meso-scale patterning of specific cell types varied between samples. Posterior cell types formed distinct, organized clusters, while anterior cell types were more disorganized. To distinguish progressive differentiation from cell type differences, we developed the L-score, a parameter-free quantification of mutually exclusive gene expression. This analysis revealed spatial organization without explicit encoding, recapitulated known cell type relationships, and identified novel gene expression states and spatial subclusters within cell types. We confirmed that in gastruloids, NMP differentiation occurred through a continuous, spatially-coordinated process. We also showed that endothelial precursors exhibited unique spatial organization and had distinct gene expression profiles dependent on their association with anterior somitic or posterior endodermal tissues. This work enables the rigorous use of gastruloids as models for studying the molecular mechanisms underlying mammalian development and tissue organization, and introduces new computational tools for analyzing spatially-resolved single-cell datasets.

developmental biology↗

bioRxiv2025

Lineage memory shapes viral resistance barriers in human skin

Individual cells within a given population exhibit striking variability in viral susceptibility, but it remains unknown whether this heterogeneity reflects memories encoded into the cellular lineage or true probabilistic variability. We used multi-color lineage tracing in a human primary organotypic skin model to reveal that viral resistance is encoded within specific cellular lineages. These lineages create distinct boundaries that block viral spread. Our lineage analyses in vitro confirmed that viral susceptibility exhibits strong heritability across cell generations, with siblings and cousins displaying remarkably similar infection outcomes. ATAC and proteomics profiling of resistant and susceptible clones revealed distinct epigenomic and proteomic states, with the transcription factor AP-1 emerging as a potential central regulator of lineage-encoded viral resistance. Inducing AP-1 activity with PMA rendered cells resistant to viral infection, suggesting a causative role in mediating resistance memory. Our findings demonstrate that antiviral resistance in human skin cells is encoded within cellular lineages and preserved through cell divisions, revealing how cell memory may shape infection dynamics and viral containment in tissues.

bioengineering↗

bioRxiv2025

Single-Allele Chromatin Tracing Reveals Genomic Clustering of Paralogous Transcription Factors as a Mechanism for Developmental Robustness in T Cells

In metazoans, gene duplication has given rise to paralogous transcription factors, which have functionally diversified to control cellular differentiation. While the majority of paralogous TFs are dispersed across different chromosomes, some remain clustered raising the question of whether genomic proximity confers any evolutionary advantage for TF clusters. To address this, we investigated a [~]1 Mbp locus containing two ETS family paralogs, Ets1 and Fli1. Using a sub-diffraction sequential imaging technique called Optical Reconstruction of Chromatin Architecture (ORCA), we traced the 3D organization of this region in single alleles of T cells from genetically engineered mice with targeted deletions of key regulatory elements. In wild-type T cells, the predominant chromatin conformation spatially links Ets1 to its proximal super-enhancer, segregating Ets1 from Fli1. This topology correlates with high Ets1 and low Fli1 expression. Deletion of the Ets1 super-enhancer abolishes this configuration, triggering locus-wide architectural rewiring that increases Ets1-Fli1 promoter-promoter interactions and subsequently the co-expression of two genes within individual cells. Remarkably, this compensatory interaction bypasses insulated chromatin domains, sustaining Ets1 levels necessary for T cell development despite enhancer loss. Our results reveal that genomic clustering of TF paralogs enables dynamic architectural plasticity: while a super-enhancer fine-tunes paralog expression balance in wild-type contexts, its deletion unmasks latent promoter-driven coordination, suggesting that proximity safeguards functional redundancy and transcriptional resilience critical for cellular fitness.

genomics↗