Biology subjects

Singh, S.

Publications and source records attributed to Singh, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

Senescent CD8+ T Effector Memory Cells are Functionally Impaired, Enriched in Aging and Disease, and a Barrier to Immunotherapy

Senescent cells play important roles in various biological processes that promote fitness and health, however, their timely elimination by immune cells is critical to maintain tissue homeostasis and prevent disease. Despite this, senescent cells progressively accumulate systemically with age, suggesting that certain immune cells also become senescent and dysfunctional during aging. Supporting this, we previously demonstrated that CD8 T cells, immune cells capable of targeting senescent cells, increasingly develop characteristics of senescence with advancing age in humans. Here, we further characterized the senescence state of human SA-{beta}Gal-expressing CD8 T effector cells, their functional capabilities, and their involvement in aging and disease. Single-cell RNA sequencing revealed that SA-{beta}Gal-expressing CD8 T cells with unique transcriptional signatures develop in all stages of T cell differentiation, including in effector memory (EM) T cells. SA-{beta}Gal-expressing CD8 TEM cells expressed various classical markers of senescence and were significantly impaired in their ability to proliferate, produce cytokines, and eliminate senescent human stromal cells, compared to CD8 TEM cells with low SA-{beta}Gal activity. Gene signatures of senescent SA-{beta}Gal-expressing CD8 TEM cells were enriched in CD8 T cells from older human donors, patients with age-related disorders, cancer, and smokers. Furthermore, our results demonstrate that T cell senescence is distinct from and dominant over T cell exhaustion, limiting the response of CD8 TEM cells to immunotherapy. Collectively, our study demonstrates that the senescence state impairs the functions of CD8 TEM cells and reveals the involvement of senescent and dysfunctional CD8 TEM cells in aging, disease, exposure to toxins, and responses to immunotherapy.

cell biology↗

bioRxiv2025

Activation of cannabinoid receptor CB1 leads to aberrant myelination in development

The endocannabinoid system (ECS) has a widespread role in the development and function of the central nervous system (CNS). Cannabinoid receptors like CB1 and CB2 can be activated with exogenous cannabinoids most popularly known as tetrahydrocannabinol (THC) or cannabis and cannabidiol (CBD). The components of the ECS are expressed early in fetal development, and prenatal exposure to cannabis can lead to structural changes in white matter. White matter is composed of neuronal axons ensheathed in myelin, a lipid-rich insulation that facilitates saltatory conduction and maintains axon integrity. In the CNS, myelin is made by specialized glial cells called oligodendrocytes (OLs), which in addition to neurons also express components of the ECS. However, while several studies have focused on how the ECS regulates neuronal development, there is a limited understanding of its impact on OL development or myelin formation. Therefore, our current study set out to understand how pharmacological activation of the ECS alters OL differentiation and myelin formation in vivo. We administered WIN 55,212-2 (WIN 55), a CB1 and CB2 agonist, to larval zebrafish and longitudinally analyzed OL development and myelination in vivo. Interestingly, we observed an increase in non-axonal ensheathments in the spinal cord, which appeared to be surrounding neuronal cell bodies. These non-axonal ensheathments were dependent on CB1, as the addition of WIN 55 in a global CB1 mutant prevented this phenotype. Furthermore, this ectopic cell body ensheathment occurred independently from normal myelination processes, as individual OLs did not exhibit changes in the number of myelin sheaths, sheath length, or total myelin output. This study shows that activation of CB receptors in vivo leads to increased non-axonal ensheathment without significantly changing OL differentiation or normal myelin formation. Future studies can further investigate the pathways that drive this phenotype to better understand how exogenous cannabinoid activation can regulate the precision of oligodendrocyte ensheathment.

neuroscience↗

bioRxiv2025

Phase separation as a concentrating mechanism of protein/peptide hormones for the secretory granule storage

Protein/peptide hormones in the regulated secretory pathway are stored within secretory granules (SGs) as densely packed aggregates for extended periods of time until their stimulated release. These aggregated dense cores are reported as functional amyloids for several pituitary hormones. However, the molecular events initiating hormone aggregation and SG biogenesis remain poorly understood. Using a diverse set of protein/peptide hormones with different sizes and structures, we demonstrated that all these hormones undergo phase separation in trans-Golgi network (TGN)-relevant conditions (including low pH, the presence of glycosaminoglycans, etc.), which rapidly transitioned into solid-like structures and eventually form amyloid fibrils. Further, the cellular model expressing two protein hormones (GH-EGFP and PRL-EGFP) showed the highly dynamic, liquid-like condensate formation at the TGN volume, which was transported via microtubule to the cellular periphery/tips and formed a ready pool of mature SGs containing amyloid-rich hormone aggregates. Importantly, both in vitro and cellular studies confirmed that solid-like amyloid aggregates are reversible and can release bioactive monomeric hormones, recapitulating regulated exocytosis. Together, our findings suggest hormone phase separation at the TGN as a common molecular principle for concentrating cargo, driving rapid functional aggregation, and ensuring efficient storage within SGs.

biochemistry↗

bioRxiv2025

Progranulin deficiency aggravates aging-induced vascular injury

SignificanceVascular aging is a major contributor to cardiovascular disease, yet the molecular mechanisms of age-associated vascular dysfunction remain incompletely defined. This study reveals a critical role for progranulin (PGRN) in regulating vascular senescence, function, and remodeling during aging. MethodsWe assessed PGRN expression in human and mouse arteries and senescent vascular smooth muscle cells (VSMCs). Functional vascular studies were performed in PGRN-deficient (PGRN-/-) mice. Senescence was modulated pharmacologically using the senolytic agent navitoclax (ABT-263), and vascular phenotype was evaluated in adult (6-month-old) and aged mice (18-month-old). ResultsPGRN expression increased with age in human and mouse arteries, correlating with elevated p21 expression. PGRN deficiency in adult mice induced endothelial dysfunction, increased vasoconstriction, and induced vascular inflammation and remodeling. Transcriptomic analysis of PGRN-/- VSMCs revealed a senescence-associated signature, including perturbed oxidative phosphorylation, altered epigenetic regulation, and collagen pathways. Pharmacological clearance of senescent cells improved endothelial function but increased vascular contractility in PGRN-/- mice. In aged mice, PGRN deficiency aggravated vascular dysfunction, remodeling, and renal injury without further increasing senescence markers--suggesting premature, rather than progressive, senescence in the PGRN-/- mice. ConclusionPGRN is a novel regulator of vascular aging, coordinating senescence, inflammation, and remodeling. While endothelial senescence contributes to dysfunction, VSMCs senescence may serve an adaptive role in modulating vascular tone. Targeting PGRN or senescence pathways may offer therapeutic opportunities for age-related vascular diseases, especially in patients with PGRN mutations associated with frontotemporal dementia.

cell biology↗

bioRxiv2025

Synaptopodin KO rat for assessing the dendritic spine apparatus and axonal cisternal organelle in synaptic plasticity, development, and behavior

The actin-binding protein synaptopodin (Synpo) regulates the cytoskeleton and organization of endoplasmic reticulum, thereby amplifying intracellular Ca2+ signaling. Knockout (KO) mouse models have been used to study the role of Synpo in kidney and brain functions, where it supports stress fiber formation, as well as long-term potentiation (LTP) and learning, respectively. Here, we generated Synpo KO rats using CRISPR-Cas9, and show they are viable but have reduced body weight after postnatal days 35-45, along with shorter limb bone length. Their basal kidney function is normal into early adulthood. Serial section electron microscopy from Synpo KO rat hippocampus reveals the absence of the spine apparatus in dendrites and cisternal organelle in the axon initial segment (AIS), two Synpo-dependent specializations of smooth endoplasmic reticulum. The AIS in KO was still innervated by inhibitory synapses despite the total loss of the cisternal organelle. Synpo KO rats also showed reduced LTP. Previously unknown KO effect of Synpo on body stature could have an inadvertent impact on behavioral outcomes. Furthermore, rats have a well-defined developmental onset of LTP, compared to the variable onset of LTP in mice. This, combined with known species differences in behavior, makes our KO rat model a valuable resource for assessing the role of Synpo in development, learning, synaptic plasticity, and a wide range of other biological functions.

neuroscience↗

bioRxiv2025

STcompare: comparative spatial transcriptomics data analysis of structurally matched tissues to characterize differentially spatially patterned genes

MotivationComparative analysis of spatial transcriptomics (ST) data is needed to identify genes that spatially change in their expression patterns between conditions, such as in diseased versus healthy tissues. Existing methods generally fail to distinguish changes in spatial patterning by focusing only on changes in gene expression magnitude for methods adapted from non-spatial data or on changes in significance of spatial variability for methods focusing on spatially-resolved data. ResultsTo address these limitations, we develop STcompare, a statistical framework for comparative analysis of ST data by testing for differences in spatial correlation and spatial fold-change across structurally matched locations. Using simulated data, we demonstrate how STcompare provides distinct insights from bulk differential gene expression analysis and spatially variable gene expression analysis as well as other spatial comparison methods. STcompare further robustly controls for false positives even in the presence of spatial autocorrelation common in ST data. We apply STcompare to real ST data of biological replicates of mouse brains to confirm high spatial correspondence of gene expression patterns across samples. We apply STcompare to identify genes that spatially change in mouse kidneys with acute kidney injury compared to a healthy control, revealing tissue compartment-specific molecular dysregulation. Overall, the application of this spatially-aware comparative analysis will enable the discovery of differential spatially patterned genes across various physiological and technological axes of interest. Availability and ImplementationSTcompare is implemented as an open-source R package at https://github.com/JEFworks-Lab/STcompare with additional documentation and tutorials available at https://jef.works/STcompare/.

bioinformatics↗

bioRxiv2025

CpG-induced regions associated compositional stratification of human essential proteins through mathematical genomics

CpG islands are genomic regions enriched in cytosine-phosphate-guanine dinucleotides, typically associated with gene promoters and regulatory elements. While their role in transcriptional regulation is well established, their influence on protein sequence composition remains underexplored. In this study, 3,222 essential proteins from Homo sapiens were analyzed to investigate the impact of CpG island architecture on amino acid usage, sequence complexity, and chromosomal distribution. Codons containing both cytosine and guanine were mapped to five CpG-induced amino acids, and their relative abundance was quantified across proteins. CpG-induced regions were identified using a sliding window approach, and metrics such as average CpG density, longest CpG-induced region length, and sequence coverage were computed. Motif composition within these regions was characterized using polarity and charge-based indices. Clustering revealed consistent bipartite subgroup structures, indicating compositional stratification among essential proteins. Shannon entropy quantified compositional complexity, revealing a significant shift between clusters (p = 5. x 50 10-145). Cluster 1 proteins showed lower entropy and greater heterogeneity, whereas Cluster 2 showed higher entropy and tighter distributions, indicating distinct regimes of variability. Additionally, we examined CpG island overlap within coding exons of essential genes and found that CpG-depleted proteins are predominantly encoded by genes with minimal CGI coverage, whereas CpG-induced proteins span both low- and high-overlap classes. CGI-associated CpGs were consistently enriched toward the 5' end of essential genes, indicating a positional bias in CpG distribution. These findings establish a quantitative framework linking CpG island distribution to proteomic architecture and offer a scalable strategy for motif annotation, epigenetic modeling, and functional stratification. Future applications include predictive modeling of protein function, disease association, and regulatory dynamics.

genomics↗

bioRxiv2025

A Modular Toolkit For Theophylline-Dependent Synthetic Auxotrophs Via Riboswitch-Gated Essential Genes

Synthetic auxotrophs are a useful means for developing genetically encoded biocontainment systems. Current methods for developing synthetic auxotrophs are complicated or expensive, limiting access and adoption of biocontainment technologies. To address this gap, we developed a simple and modular platform for creating synthetic auxotrophs based on ligand-dependent translational gating of essential genes. By inserting a theophylline-responsive riboswitch upstream of essential genes in Escherichia coli, we created strains whose viability depends on the presence of theophylline. We systematically applied this approach to 29 essential genes, obtained ligand-dependent growth phenotypes for 19 targets, and found that 18 of these essential genes yielded stringent live-die synthetic auxotrophs. These strains exhibited robust theophylline dependence with escape frequencies ranging from 1 x 10-5 to 1 x 10-6 (most below limit of detection). Our modular design allows for the rapid (<1 week), low-cost, and reliable generation of synthetic auxotrophs. This work introduces ligand-dependent translational control as a new mechanism for engineering synthetic auxotrophy and provides an accessible platform that expands the biocontainment toolkit.

synthetic biology↗

bioRxiv2025

Inhibiting Glycan Degradation Prevents HIV-Induced Inflammaging and Cognitive Impairment

Cognitive impairment is a frequent outcome of chronic viral infections linked to premature aging, including HIV. The mechanisms underlying this decline remain poorly understood. Here, we identify pro-inflammatory glycan degradation, characterized by loss of sialic acid and galactose, alterations that are hallmarks of premature aging, as key contributors to HIV-associated cognitive impairment (HIV-CI). In two independent cohorts of people living with HIV, these degradative changes were enriched in individuals with cognitive impairment, particularly females, and correlated with worse cognitive performance. In both a humanized mouse model of HIV and Eco-HIV, a complementary model that allows behavioral testing, pharmacological inhibition of glycan degradation with sialidase inhibitors prevented virally induced inflammation, immune activation, accelerated aging, and memory deficits. These findings implicate glycan degradation as a contributor to inflammation and cognitive impairment in HIV and highlight glycan-preserving therapies as a promising strategy to mitigate inflammation, premature aging, and cognitive decline during viral infections.

microbiology↗

bioRxiv2025

Cryo-EM structures of Mycobacterium tuberculosis pyruvate carboxylase reveal allosteric activation and domain dynamics during catalysis

Pyruvate carboxylase (PC) is a biotin-dependent enzyme essential for anaplerotic flux into the tricarboxylic acid cycle and gluconeogenesis. Despite longstanding biochemical knowledge, the structural mechanisms governing its allosteric regulation and domain coordination remain poorly defined. Here, we present the cryo-EM structures of Mycobacterium tuberculosis PC (MtPC) captured in both apo and substrate-bound forms. In the absence of acetyl-CoA, MtPC forms a flexible and unstable tetramer that readily dissociates into subcomplexes. Upon binding of acetyl-CoA and the substrates, the enzyme transitions into a compact, ordered assembly, with acetyl-CoA acting as a molecular clamp that stabilizes inter-subunit interactions. Remarkably, we captured the mobile biotinylated domain simultaneously engaging each catalytic center within monomers, providing direct structural evidence for the two steps of the catalytic cycle. 3D variability analysis further revealed how this mobile domain translocates between the active sites during catalysis. Together, these findings demonstrate how acetyl-CoA stabilizes MtPC by reinforcing inter-subunit contacts and elucidate the conformational dynamics underlying domain coordination during catalysis. These findings provide new structural insights into the allosteric regulation of this essential metabolic enzyme.

biophysics↗

bioRxiv2025

Non-Enzymatic Structural Modifications Reshape Peptide Presentation and Antigen Recognition

Cytotoxic T lymphocytes recognize infected and transformed cells through peptide antigens presented by MHC-I. Peptide sequence is assumed to dictate recognition, yet non-enzymatic post-translational modifications (PTMs) installed by reactive electrophiles can render a peptide chemically distinct from the form against which tolerance was established. Here we show that non-enzymatic PTMs arising from oxidative, inflammatory, metabolic, and carbonyl stress alter peptide-MHC-I stability and frequently disrupt recognition by a cognate TCR despite preserved MHC-I binding. Using a thioester probe, we demonstrate proof-of-concept capture of acylation-susceptible MHC-I ligands from the cellular immunopeptidome. Environmental chemicals, pharmaceuticals, and dietary isothiocyanates likewise modify antigenic peptides and impair T cell activation, and chemically distinct adducts at a single residue span the range from complete loss to full retention. Finally, methylglyoxal glycation impairs antigen-specific activation, and scavenging by creatine prevents it. Antigenic identity therefore reflects not only sequence but a peptides cumulative chemical history.

immunology↗

bioRxiv2025

A chromosome-level assembly and functional genomic resources for the model annelid Capitella teleta

BackgroundThe polychaete Capitella teleta is a commonly used annelid for studies in evolutionary developmental biology, comparative genomics, conservation, and ecotoxicology. Over a decade ago, it was the first polychaete to have its genome sequenced and assembled, contributing to pioneering studies that transformed our understanding of animal genomes and their evolution. However, this early resource is now outdated compared to current genome sequencing standards, limiting the use of modern functional genomic tools that could further our understanding of numerous biological processes. ResultsWe combine long-read and short-read sequencing with Hi-C chromatin conformation capture data to assemble the chromosome-level nuclear and mitochondrial genomes of the laboratory strain of C. teleta. This reference assembly more accurately reflects the expected genome size for this polychaete ([~]243.6 Mb) and contains a highly complete, evolutionarily conserved gene repertoire. Notably, the nuclear and mitochondrial genomes are heavily rearranged, indicating a decoupling between gene family repertoire and chromosomal evolution. The analyses of multi-omic datasets available for C. teleta, including developmental time courses of bulk and single-cell RNA-seq, ATAC-seq, and EM-seq, using the new reference assembly, resulted in a significant quality improvement, allowing us to identify new cell-type-specific gene markers and gain additional insights of biological relevance. Finally, we generated a publicly available genome browser that ensures all these resources are easily findable, accessible, interoperable, and reusable. ConclusionsOur study provides state-of-the-art genomic resources for the polychaete model C. teleta, addressing a pressing community need that will open new research opportunities in animal and genome evolution.

genomics↗

bioRxiv2025

Acute activation of Gq-signaling in islet macrophages inhibits β-cell insulin secretion through AMPK-sphingolipid axis

Obesity-associated inflammation disrupts pancreatic {beta}-cell function, but the immune-derived signals that directly regulate insulin secretion remain incompletely defined. Here, we identify myeloid Gq signaling as a critical immunometabolic node that links macrophage activation to {beta}-cell dysfunction. For the first time, we employed a chemogenetic approach (DREADDs) to selectively and temporally activate Gq-coupled GPCR signaling in myeloid cells to examine its effect on islet function. Our findings reveal that acute Gq activation in islet-resident macrophages impaired glucose-stimulated insulin secretion, uncovering a previously unrecognized immune-endocrine axis. Conversely, myeloid-specific Gq deletion improves systemic glucose homeostasis, underscoring the physiological relevance of this pathway. Mechanistic analysis revealed that Gq activation in macrophages stimulates AMPK signaling and drives the secretion of sphingolipids. These lipids suppress insulin secretion and introduce a new mechanism for immune-islet communication, extending beyond traditional cytokine-based models. We further identify the lipid-sensing receptor GPR18 as an upstream activator of the Gq-AMPK pathway in macrophages. GPR18 stimulation recapitulated the Gq-dependent sphingolipid secretion and {beta}-cell inhibitory phenotype, which was abolished in myeloid Gq-deficient mice. Collectively, these findings establish a mechanistic framework whereby macrophage Gq signaling integrates lipid sensing and metabolic stress to modulate {beta}-cell function. This work reveals a previously unrecognized macrophage-{beta}-cell communication axis with therapeutic potential for restoring insulin secretion in metabolic diseases such as obesity and type 2 diabetes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/680858v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@790c4dorg.highwire.dtl.DTLVardef@116cceborg.highwire.dtl.DTLVardef@1e76631org.highwire.dtl.DTLVardef@e65770_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Engineered RVG29-Conjugated Chitosan for Enhanced Brain Delivery of Kisspeptin Agonists and Antagonists in Reproductive Health.

Efficient delivery of intact therapeutic peptides across the blood-brain barrier (BBB) remains a central obstacle in translating neuroendocrine modulators to clinical practice. Kisspeptin receptor (KISS1R) agonists and antagonists precisely regulate gonadotropin-releasing hormone (GnRH) pulsatility, thereby controlling luteinizing hormone (LH) release, steroidogenesis, and fertility. While the KISS1R agonist KP10 accelerates GnRH-LH pulses to promote ovulation, the antagonist KP234 suppresses pulse generation, inducing reversible infertility. However, their poor BBB permeability limits clinical application. Here, we engineered a non-invasive brain delivery platform by covalently coupling the rabies virus glycoprotein-derived peptide RVG29 to oxidized chitosan nanoparticles via Schiff base chemistry. Sodium periodate oxidation introduced aldehyde groups, FT-IR confirmed aldehyde formation, and UV-Vis spectroscopy validated RVG29 conjugation through characteristic absorbance shifts. The resulting RVG29 - chitosan carriers demonstrated biocompatibility, enhanced neuronal uptake [~]2.1-fold in vitro, and achieved [~]2.5-fold higher brain accumulation in vivo compared to unconjugated controls. Systemic delivery of RVG29-chitosan-KP10 restored and amplified LH pulsatility in ovariectomized mice, accelerated estrous cycling, increased preovulatory follicle numbers, and enhanced litter size. In contrast, RVG29-chitosan-KP234 abolished LH pulses, maintained animals in persistent diestrus, depleted corpora lutea, and produced complete yet reversible infertility. Longitudinal dosing demonstrated stable neuroendocrine modulation without systemic toxicity, and ovarian histology confirmed ovulatory arrest in antagonist-treated animals. By enabling programmable, bidirectional control of a central hypothalamic circuit through systemic delivery of unmodified peptides, this platform addresses a long-standing barrier in neuroendocrine therapeutics. Beyond fertility regulation, this approach can be adapted to other hypothalamic-driven processes, including metabolic, stress-axis, and circadian control.

bioengineering↗

bioRxiv2025

A bioluminescence-based chemical screen identifies a bactericidal naphthalene scaffold targeting MmpL3 in Mycobacterium abscessus

Mycobacterium abscessus pulmonary disease (Mabs-PD) presents a significant and growing global health threat, particularly in individuals with underlying lung conditions like cystic fibrosis and chronic obstructive pulmonary disease. A key challenge in treating Mabs-PD is the lack of bactericidal antibiotics effective at therapeutically relevant concentrations, underscoring an urgent need for drug discovery. Targeting cell-wall synthesis is a promising approach, as evidenced by the success of broad-spectrum {beta}-lactam antibiotics and the frontline antituberculosis drug isoniazid. However, these agents exhibit limited efficacy against Mabs, often requiring concentrations unachievable in lung tissues. Here, we used a bioluminescence-based whole-cell assay optimized to identify drugs targeting both cell-wall synthesis and the oxidative phosphorylation pathway. Screening a small drug library against Mabs revealed multiple hits, including {beta}-lactam antibiotics, validating the effectiveness of this approach to identify cell wall-targeting agents. Among these, we identified a chemically tractable naphthalene scaffold with potent bactericidal activity. The optimized derivative GM47-1 targets MmpL3, disrupting cell wall integrity, inducing ATP leakage into the extracellular milieu, and uncoupling respiration, predominantly through the cytochrome bcc:aa3 branch. Further chemical optimization resulted in a new derivative exhibiting a nanomolar minimum inhibitory concentration, with potent activity against intracellular Mabs and in a zebrafish model of infection. This study offers a promising scaffold for future therapeutic development and highlights the utility of this approach as a rapid assay platform for identifying bactericidal compounds against Mabs.

microbiology↗

bioRxiv2025

Synthesis and Characterization of ICG-based Near-infrared Photoacoustic Contrast Agents

Near-infrared photoacoustic imaging (NIR-PAI) integrates optical excitation with ultrasound detection to enable high-resolution, deep-tissue imaging by taking advantage of reduced light scattering and absorption in this spectral window. Despite its potential, clinical translation of contrast-enhanced NIR-PAI is limited by the scarcity of effective contrast agents. Indocyanine green (ICG), an FDA-approved NIR dye, is a strong candidate due to its biocompatibility and photoacoustic efficiency. However, its concentration-dependent aggregation, lack of facile targeting strategies, instability in aqueous environments, and low photostability result in variable signal, high background noise, and reduced reliability in vivo. To address these challenges, we developed three biocompatible ICG-based nanoprobe platforms amenable to facile, scalable synthesis: 5-arm DNA-ICG nanostructures (5-arm DNA-ICG), lipid-shelled ICG nanobubbles (ICG-NBs), and Azide-modified ICG J-aggregates (JAAZ). These platforms are designed to preserve ICG monomers or control aggregation, enabling enhanced NIR-PAI performance. Spectroscopic and photoacoustic analyses revealed consistent absorbance and photoacoustic profiles, showing enhanced signals compared to free ICG. The greatest improvement was observed for JAAZ, followed by ICG-NBs and 5-arm DNA-ICG. Photostability studies showed that JAAZ aggregation protects ICG from light-induced photodegradation, whereas monomer preservation in 5-arm DNA-ICG and ICG-NBs provides less protection and moderate signal stability. All three probes demonstrated stable performance under physiological conditions, achieved strong signal-to-noise ratios at depth and under tissue-mimicking conditions, and required markedly reduced probe concentrations to generate robust signals. Their modular architectures allow incorporation of targeting ligands, offering molecular specificity and multimodal functionality. Collectively, these contrast agent platforms provide noninvasive, deep-tissue molecular imaging and biosensing, with strong potential for future preclinical and clinical translation, and represent a promising alternative to free ICG for biomedical applications.

bioengineering↗

bioRxiv2025

An unconventional HxD motif orchestrates coatomer-dependent coronavirus morphogenesis

Assembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to assembly sites with the viral M-protein. Disruption of HxD-coatomer interactions impairs S incorporation and provokes compensatory viral adaptations, including emergence of a canonical dibasic motif or mutations in M-protein. Electron microscopy further reveals profound alterations in virion surface architecture. These findings uncover HxD as a previously unrecognized coatomer-targeting motif, highlighting an unexpected flexibility in coronavirus assembly pathways and broadening understanding of the cellular machinery that shapes coronavirus morphogenesis.

biochemistry↗

bioRxiv2025

Identifying and targeting abnormal mitochondrial localization associated with psychoses

Therapeutics working by novel mechanisms are needed for patients with psychiatric conditions. Cell-based assays to identify candidates that reverse observed abnormalities could accelerate the process. Here, we imaged peripheral cells (skin fibroblasts) of 168 patients, stained for DNA, actin, and mitochondria. We found mitochondria tend to be farther from the cell border for patients who experience psychosis (including subsets of individuals with bipolar disorder, schizophrenia, and schizoaffective disorder). We observed a reverse trend, albeit not statistically significant, for patients diagnosed with major depression. Because the phenotype could be identified by a single metric, we could query existing databases of cells stained for their mitochondria and treated with various chemical or genetic perturbations. We identified compounds and genes both negatively and positively affecting the psychosis-associated phenotype, including some known to impact psychiatric conditions. Developing therapeutics with novel mechanisms is a complex multi-step challenge. This cell-based assay holds promise for virtual and physical screening to identify candidates for treating psychiatric conditions.

systems biology↗