Biology subjects

Natu, A.

Publications and source records attributed to Natu, A..

10 recordsLinked to original sources

bioRxiv2026

Delayed Tagging of ED-A Fibronectin-Mimetic Peptide in an RGD-Decorated Synthetic Matrix Induces Fibroblast-to-Myofibroblast Transition

Synthetic hydrogels with bioactive ligands have been utilized to develop 3D models to gain mechanistic insight into how discrete extracellular matrix (ECM) cues direct cell fate. While the RGD motif is ubiquitously present in healthy and diseased tissues, the EDGIHEL (EDG) sequence is present only in the extra domain A-containing fibronectin (ED-A FN), which is transiently deposited in the provisional matrix in the wound bed. Here, we explore the potential of covalently tethered EDG in conjunction with RGD to promote fibroblast-to-myofibroblast transition (FMT). Normal human lung fibroblasts (NHLFs) were maintained in bioorthogonally constructed, hyaluronan-based hydrogel (BOHAGel) with tethered RGD ligands. When EDG was introduced on day 0 during cell encapsulation, cellular expression of Toll-like receptor 4 (TLR4) was upregulated, and a pro-inflammatory matrix remodeling response was observed, but myofibroblast differentiation was not detected. To mimic the transition from a healthy to an injured state, we leveraged the temporal tunability of BOHAGel by supplementing cell culture media with trans-cyclooctene (TCO)-tagged EDG after cells were primed in the RGD environment for 8 days. As the TCO species diffused through the hydrogel, EDG was instantaneously coupled to the network through immobilized tetrazine functionalities. Delayed introduction of profibrotic EDG motifs increased mRNA levels of the myofibroblast marker (ACTA2), ECM proteins (COL1A1, COL3A1, FN1), and transforming growth factor beta1 (TGFbeta1) downstream targets (VEGFA, CTGF), as well as matrix remodeling enzymes (MMP2, TIMP1). These changes were accompanied by the formation of alpha-SMA stress fibers, confirming complete FMT. Delayed EDG conjugation also enhanced and reinforced alpha1 integrin expression. Importantly, removing the RGD signal from the gel failed to induce myofibroblast differentiation. Collectively, our results suggest that FMT depends on ligand identities and the timing of their emergence in engineered matrices.

bioengineering↗

bioRxiv2026

Bioorthogonal Tuning of Hydrogel Stiffness Promotes Zonal Redifferentiation of Passaged Chondrocytes

Generating bioengineered cartilage that recapitulates the depth-dependent phenotype, structure, and function of native articular cartilage remains a challenge. While cartilage is rich in aggrecan and type II collagen, proper function depends on depth-dependent protein expression. Superficial zone chondrocytes (SZCs) secrete proteoglycan-4 (PRG4) to lubricate the cartilage surface. Deep zone chondrocytes produce type X collagen (COLX) to support compressive loading and load transfer to subchondral bone. We previously demonstrated that passaged full-thickness chondrocytes (FTCs) and zonal chondrocytes can re-express cartilage and zone-specific markers following scaffold-free three-dimensional (3D) culture in redifferentiation media. However, in the absence of an instructive matrix, cells expressed low levels of zone-specific proteins and exhibited limited depth-dependent organization. We hypothesize that synthetic extracellular matrix with zone-specific microenvironmental cues will guide zonal differentiation. To this end, passaged primary bovine chondrocytes were encapsulated in a soft, hyaluronan (HA)-based, cell-adhesive, and protease-degradable hydrogel established via bioorthogonal tetrazine (Tz) ligation with norbornene (Nb). When supplemented with TGF{beta}3, FTCs deposited aggrecan and type II collagen with minimal type I collagen. Application of interfacial tetrazine ligation with trans-cyclooctene (TCO) during cell culture resulted in matrix stiffening, leading to upregulation of COLX expression. Conversely, SZCs cultured in soft hydrogels exhibited the greatest PRG4 expression. Establishment of a trilayered construct with region-specific stiffness via the diffusion-controlled reaction promoted PRG4 and COLX expression in defined zones. Together, these findings demonstrate that tunable HA-based hydrogels can enhance zone-specific chondrocyte phenotypes and promote the formation of zonally organized cartilage.

bioengineering↗

bioRxiv2025

A comprehensive view of somatic mosaicism by single-cell DNA analysis

Single-cell DNA sequencing offers a powerful means of studying somatic mosaicism but requires careful analysis to mitigate DNA amplification-related artifacts. We performed primary template-directed amplification (PTA) and sequencing of 102 nuclei from postmortem lung and colon tissues of a 74-year-old male. Single-cell mutation burdens and spectra were validated by duplex sequencing and revealed heterogeneity across organs and cells, including signatures of APOBEC activity and tobacco exposure. Cells from both tissues exhibited chromosomal aneuploidies, loss of chromosome Y, and chromosomal rearrangements including rearrangements of the T-cell receptor loci indicative of T-cells. Shared embryonic mutations between cells enabled reconstruction of cellular ancestries from the zygote, which were validated by bulk sequencing. Collectively, we demonstrate a comprehensive approach for single-cell genomics that yields an expansive view of diverse somatic mutation types from development through aging across diverse tissues--insights that are obscured in bulk sequencing and only partially captured by other single-cell methods.

genomics↗

bioRxiv2025

Single cell whole genome and transcriptome sequencing links somatic mutations to cell identity and ancestry

The role of somatic mutations in human development and disease is obscured by difficulties in characterizing mutations at the single cell level and identifying cell types carrying them. Here we analysed somatic genomes of clonal iPSC lines and of single-cells after whole-genome amplification (scWGA) by PTA and ResolveOme from skin fibroblasts, blood and urine of a live donor. Mutation burden and spectra converged across approaches, revealing heterogeneous mutational footprints across cells driven by environmental exposures (UV damage and chemotherapy) and lymphocyte differentiation. Aneuploidies in single cells were detected by all the approaches and were orthogonally validated by Strand-seq. Uniquely, ResolveOme enabled cell-type identification using single-cell transcriptomes. Using a newly developed method accounting for noise and allele drop-out in scWGA, we de novo reconstructed the cell phylogenetic tree for this donor. Together, scWGA establishes a powerful foundation for comprehensive, cell type-aware, lineage-aware profiling of somatic mutations at single cell level.

genomics↗

bioRxiv2024

Proximity labeling proteomics reveals Kv1.3 potassium channel immune interactors in microglia.

Microglia are the resident immune cells of the brain and regulate the brains inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to establish the potential proteins interacting with Kv1.3 during the TLR4-mendiated transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate the potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia during an immune response. Electrophysiology, western blots, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during an TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g. NUNDC, TIMM50). The C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (e.g. STAT1, TLR2, and C3). There are 70 proteins that rely on the c-terminal PDZ-binding domain to interact with Kv1.3 (i.e. ND3, Snx3, and Sun1). Overall, we highlight that the Kv1.3 potassium channel functions beyond outward flux of potassium in an inflammatory context and contributes to activity of key immune signaling proteins, such as STAT1 and C3. MAIN POINTSKv1.3 channels are highly abundant in pro-inflammatory microglia in neurological diseases. Kv1.3 channels may regulate microglial functions by interacting with other proteins via its N and C terminal domains. Using proximity-based proteomics, we identified several novel proteins that interact with the N and C terminus of Kv1.3 channels, some of which are domain-specific. Kv1.3 channels in microglia interact with many immune signaling proteins, including Tlr2, Stat1 and integrins. Under homeostatic conditions, the N-terminus of Kv1.3 interacts with proteins involved in protein trafficking, to the cell surface and mitochondria. The PDZ-binding region was an important determinant of the C terminal interactome. During an LPS-induced inflammatory response, the C-terminus of Kv1.3 uniquely interacts with immune and signaling proteins of disease relevance, including STAT1

neuroscience↗

bioRxiv2023

Aβ Amyloid Scaffolds the Accumulation of Matrisome and Additional Proteins in Alzheimer's Disease

We report a highly significant correlation between human Alzheimers disease (AD) brain proteome changes and those in CRND8 APP695NL/F transgenic mice. Comparing protein changes observed in the CRND8 mice with co-expression networks derived from human Alzheimers disease (AD), reveals both conserved and divergent module changes. Many proteins in the most highly conserved module (M42, matrisome) accumulate in plaques, cerebrovascular amyloid (CAA), dystrophic neuronal processes, or a combination thereof. Overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), in CRND8 mice brains leads to increased accumulation of A{beta} in plaques and in blood vessels; further, recombinant MDK and PTN enhance A{beta} aggregation into amyloid structures. Multiple M42 proteins bind to fibrillar A{beta}42 and a non-human amyloid fibril in vitro. Supporting this binding data, MDK and PTN co-accumulate with transthyretin (TTR) amyloid in the heart. Notably, our findings establish that proteomic changes in modules observed in human AD brains define an A{beta} amyloid "responsome" that is well conserved from mouse models to humans. Further, distinct amyloid structures appear to serve as scaffolds, facilitating the co-accumulation of proteins with signaling functions, and this co-accumulation may contribute to downstream pathological sequalae. Overall, this contextualized understanding of proteomic changes and their interplay with amyloid deposition provides valuable insights into the complexity of AD pathogenesis and potential biomarkers and therapeutic targets.

neuroscience↗

bioRxiv2023

Identification of state-specific proteomic and transcriptomic signatures of microglia-derived extracellular vesicles

Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation.

neuroscience↗

bioRxiv2023

Native-state proteomics of Parvalbumin interneurons identifies novel molecular signatures and metabolic vulnerabilities to early Alzheimer's disease pathology

One of the earliest pathophysiological perturbations in Alzheimers Disease (AD) may arise from dysfunction of fast-spiking parvalbumin (PV) interneurons (PV-INs). Defining early protein-level (proteomic) alterations in PV-INs can provide key biological and translationally relevant insights. Here, we use cell-type-specific in vivo biotinylation of proteins (CIBOP) coupled with mass spectrometry to obtain native-state proteomes of PV interneurons. PV-INs exhibited proteomic signatures of high metabolic, mitochondrial, and translational activity, with over-representation of causally linked AD genetic risk factors. Analyses of bulk brain proteomes indicated strong correlations between PV-IN proteins with cognitive decline in humans, and with progressive neuropathology in humans and mouse models of A{beta} pathology. Furthermore, PV-IN-specific proteomes revealed unique signatures of increased mitochondrial and metabolic proteins, but decreased synaptic and mTOR signaling proteins in response to early A{beta} pathology. PV-specific changes were not apparent in whole-brain proteomes. These findings showcase the first native state PV-IN proteomes in mammalian brain, revealing a molecular basis for their unique vulnerabilities in AD.

neuroscience↗

bioRxiv2022

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines.

1.0Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level, can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration in BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. These included endo-lysosome, translation, vesicle and signaling proteins in BV2 microglia, and synaptic, neuron projection and microtubule proteins in N2A neurons. The effect of LPS treatment on the microglial proteome was captured by MS analysis of biotinylated proteins (>500 differentially-abundant proteins) including increased canonical pro-inflammatory proteins (Il1a, Irg1, Oasl1) and decrease anti-inflammatory proteins (Arg1, Mgl2).

neuroscience↗

bioRxiv2021

Differential expression of H2A isoforms contribute to tissue and lineage specificity with HIST2H2AC as a potential cancer biomarker

Recent advancements in the field of histone biology imply non-redundancy in the function of histone H2A isoforms; however, the expression of H2A isoforms in various normal tissue types, the correlation among organs and tumor/tumor type-specific expression remain poorly investigated. The profiling of sixteen H2A isoforms in eleven different normal human tissue types strongly suggests their tissue-specific or predominant expression. Further, clustering analysis shows a lineage-specific correlation of H2A isoforms. In continuation, the expression analysis in twelve human tumor types shows overexpression of HIST2H2AC. Moreover, overexpression was observed exclusively in tumor samples but not with fetal samples; highlighting the cancer-specific association of HIST2H2AC. Further, in silico analysis of TCGA pan-cancer data also showed tumor-specific over-expression of the HIST2H2AC isoform. Our findings provide insights into tissue-type-specificity of histone H2A isoforms expression patterns and advance our understanding of their importance in lineage specification and cancer.

cancer biology↗