Biology subjects

Rao, Y.

Publications and source records attributed to Rao, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Lineage tracing and live-cell imaging reveal that NeuroD1 does not reprogram microglia into neurons

In situ glia-to-neuron conversion induced by a single transcription factor is a promising regenerative strategy for treating central nervous system injuries and neurodegenerative diseases. Previous studies reported that NeuroD1 can induce microglia-to-neuron cross-lineage conversion and improve recovery after brain injury. However, these findings remain controversial. To examine the ability of NeuroD1 in inducing microglia-to- neurons conversion, we employed virus-free genetic lineage tracing systems to specifically induce NeuroD1 expression in microglia, and track the cell fate of NeuroD1-expressing cells at multiple timepoints. Meanwhile, we longitudinally monitored NeuroD1-expressing microglia via two-photon imaging, and characterized their transcriptional profile by single-cell RNA sequencing. All observations across these methods revealed that NeuroD1-expressing cells retained their microglia identity, and cannot convert into neurons, no matter under physical and injury conditions. Instead, sustained expression of NeuroD1 facilitated microglia apoptosis in vivo. Collectively, our findings provide strong evidence that NeuroD1 alone is sufficient to induce microglia-to neuron conversion. This study further highlighted the importance of rigorous lineage-tracing and cell fate mapping strategies for validating in situ glia-to-neuron conversion.

neuroscience↗

bioRxiv2026

A multimodal perturbation atlas defines the phenotypic resolution of cellular morphology.

Modeling cellular behavior requires measurements that capture how cells evolve across time, environments, and interventions. Microscopy is uniquely suited to this goal: it is non-destructive and can be applied to living cells in their native context. Yet its phenotypic resolving power remains incompletely characterized relative to molecular assays. Here, we present a multimodal perturbation atlas of 1,000 pooled CRISPR knockouts in A549 cells, profiled by fluorescence microscopy (42 live, 13 fixed markers), label-free quantitative phase imaging of the same live cells (at single timepoints), and single-cell RNA sequencing (scRNA-seq). We develop deep learning frameworks to interpret the rich cell-biological signatures in these ~65M single-cell profiles. At matched reagent cost, phase imaging exceeds the phenotypic resolution of both fluorescence imaging and scRNA-seq, and more reliably recovers higher-order pathway organization. These results establish intrinsic morphology as a high-precision readout of cellular state, and lay a foundation for live-cell profiling of phenotypic trajectories.

systems biology↗

bioRxiv2026

An Essential Role of the AMPK-Related Kinase SNRK in Regulating Drosophila Sleep

Protein kinases are important regulators of sleep in animals, but their loss-of-function mutations usually show moderate phenotypes. We report here that sucrose non-fermenting related kinase (SNRK), a member of the AMP-activated protein kinase-related kinase (ARK) family, plays an essential role in sleep regulation: Snrk gene knockout resulted in a profound loss of night-time sleep phenotype, much stronger than that of the well-known ARK member Sik3. This phenotype was completely rescued by neuronal SNRK expression. SNRK in adulthood was required for sleep. Catalytic and regulatory sites in SNRK were important for sleep. A chemoconnectomic (CCT) screen revealed SNRK functioning in cholinergic neurons whose activation is known to inhibit sleep. Sleep loss in SNRK mutants was rescued by pharmacological blockade of nicotinic acetylcholine receptors. Our findings have uncovered a fundamental role for SNRK in cholinergic neurons to regulate sleep.

neuroscience↗

bioRxiv2025

On-target mutations confer resistance to WRN helicase inhibitors in Microsatellite Unstable Cancer Cells.

Werner helicase inhibitors (WRNi) are in clinical development for microsatellite-unstable (MSI) tumors with defective DNA mismatch repair. Here, we investigate how cancer cell evolution shapes response to WRN inhibition and informs potential resistance mechanisms. Genome-wide CRISPR screens combined with WRN knockout did not identify bypass mechanisms, underscoring WRNs essential, non-redundant function in MSI cells. Pharmacogenomic screens identified modulators of WRNi sensitivity, including SMARCAL1, which links it to WRN-MSI synthetic lethality. Semi-saturation mutagenesis of WRN and prolonged drug treatment identified on-target WRN mutations driving acquired resistance to multiple WRNi in vitro and in vivo, which was mitigated by combination with standard chemotherapies. Some resistance mutations conferred broad cross-resistance, whereas others preserved sensitivity to alternative clinical-grade WRNi with distinct mechanism of action. Our findings could inform clinical trial design by suggesting the feasibility of real-time tracking of emerging resistance and enabling early therapeutic adaptations. SignificanceWe present the first exploration of how MSI cancer cells evolve under the selective pressure of WRN helicase inhibition, providing a framework for understanding adaptive responses to this newly identified synthetic-lethal dependency. This study identifies on-target WRN mutations as key drivers of resistance in MSI cancers, supporting the use of combination strategies with other standard-of-care treatments to prevent resistance. It highlights how mutation tracking can guide therapeutic switching to clinically available WRN inhibitors with distinct mechanisms of action, thereby refining clinical development and potentially improving biomarker-informed patient outcomes.

cancer biology↗

bioRxiv2025

scE2TM improves single-cell embedding interpretability and reveals cellular perturbation signatures

Single-cell RNA sequencing technologies have revolutionized our understanding of cellular heterogeneity, yet computational methods often struggle to balance performance with biological interpretability. Embedded topic models have been widely used for interpretable single-cell embedding learning. However, these models suffer from the potential problem of interpretation collapse, where topics semantically collapse towards each other, resulting in redundant topics and incomplete capture of biological variation. Furthermore, the rise of single-cell foundation models creates opportunities to harness external biological knowledge for guiding model embeddings. Here, we present scE2TM, an external knowledge-guided embedded topic model that provides a high-quality cell embedding and interpretation for scRNA-seq analysis. Through embedding clustering regularization method, each topic is constrained to be the center of a separately aggregated gene cluster, enabling it to capture unique biological information. Across 20 scRNA-seq datasets, scE2TM achieves superior clustering performance compared with seven state-of-the-art methods. A comprehensive interpretability benchmark further shows that scE2TM-learned topics exhibit higher diversity and stronger consistency with underlying biological pathways. Modeling interferon-stimulated PBMCs, scE2TM simulates topic perturbations that drive control cells toward stimulated-like transcriptional states, faithfully mirroring experimental interferon responses. In melanoma, scE2TM identifies malignant-specific topics and extrapolates them to unseen patient data, revealing gene programs associated with patient survival.

bioinformatics↗

bioRxiv2025

Panax notoginseng saponins protect the blood brain barrier against oxidative stress by activating the Akap12/PI3K/AKT/AQP1 signaling axis

Oxidative stress is a key pathological factor that contributes to blood-brain barrier (BBB) disruption during ischemic injury. Panax notoginseng saponins (PNS), the primary bioactive components of Panax notoginseng, possess potent antioxidative and vasoprotective activities. This study aimed to elucidate the molecular mechanisms by which PNS regulate endothelial integrity and BBB permeability under oxidative stress conditions. Mouse brain microvascular endothelial cells (bEnd.3) were treated with hydrogen peroxide to induce oxidative damage, followed by intervention with PNS. Transcriptomic and proteomic analyses revealed that Akap12 (A-kinase anchoring protein 12) and Aquaporin 1 (AQP1) were key molecules involved in PNS-mediated endothelial protection. H2O2 exposure markedly suppressed Akap12 expression and inhibited the PI3K/AKT signaling pathway, leading to elevated AQP1 expression and increased BBB permeability. PNS treatment reversed these effects by restoring Akap12 expression, reactivating PI3K/AKT signaling, and reducing AQP1 levels. Functional validation further showed that AQP1 dysregulation altered the expression balance of tight junction proteins, including Claudin-5 and ZO-1, thereby impairing barrier function. Collectively, these findings demonstrate that PNS protect endothelial cells from oxidative injury by activating Akap12-dependent PI3K/AKT signaling and modulating AQP1-associated tight junction remodeling. This study provides new mechanistic insights into the molecular basis of PNS-mediated BBB protection and suggests potential therapeutic targets for oxidative or ischemic cerebrovascular diseases.

pharmacology and toxicology↗

bioRxiv2025

Adaptive Multimodal Temporal Transformer for Bio Signal Driven Stroke Rehabilitation Prognosis

Stroke rehabilitation requires continuous, individualized assessment of recovery progress to optimize treatment planning. Existing prognosis models often rely on static, single-timepoint predictions and lack adaptive fusion of multi-modal bio-signals, limiting their clinical interpretability and utility. To address these challenges, we propose AMTT-Net, an adaptive multimodal temporal transformer for dynamic stroke rehabilitation prognosis. AMTT-Net integrates wearable biosensor data, video keypoints, and clinical records through an attention-based Adaptive Fusion Module and Trajectory-Aware Prediction Heads to jointly predict continuous recovery trajectories and responder likelihoods under different rehabilitation modalities. Evaluations on the StrokeBalance-Sim dataset demonstrate that AMTT-Net achieves superior performance in both trajectory regression and responder classification tasks, while providing interpretable, patient-specific insights to support personalized rehabilitation strategies.

bioengineering↗

bioRxiv2025

High-throughput Experimental Validation of Synonymous Mutation Neutrality in the Human Genome

The assumption that synonymous mutations are fitness-neutral is central to many foundational results in the fields of genetics, genomics, evolutionary biology, and medicine. However, recent results suggest synonymous mutations have pervasive and strong fitness effects. These vigorously debated studies in non-human model systems have even suggested that the proportion of synonymous mutations and their fitness effect sizes are similar to non-synonymous mutations. To systematically test synonymous neutrality across the human genome we utilize recent advances in base editing to probe the fitness effects of 8,558 potential synonymous mutations in 128 highly essential genes. Combined with novel base editing controls and extensive individual sgRNA guide-level validation experiments that quantify false positive rates, we find that synonymous mutations rarely have fitness effects on growth, occurring 165-fold (95% CI, 35-2929 fold) less frequently than non-synonymous fitness-altering mutations. Thus, despite a decade of high-profile controversies, the neutrality assumption for spontaneous synonymous mutations is valid.

genetics↗

bioRxiv2025

Activity-based probes and chemical proteomics reveal the potential of targeting HMGCS1 for cancer therapy

Mevalonate is a precursor for essential metabolites, such as isoprenoids and sterols. Its synthesis starts with HMGCS1 producing HMG-CoA, which is then converted to mevalonate by HMGCR, a target of statins. Cancer cells often upregulate enzymes in the mevalonate pathway (MVP) to meet their metabolic demands, leading to the development of inhibitors targeting several enzymes in this pathway. However, current inhibitors have not yet shown significant anti-cancer activity. While HMGCS1 has unique biochemical properties that distinguish it from other MVP enzymes, the effects of inhibiting HMGCS1 have not been thoroughly investigated. Here, we present a set of chemical probes that enable us to systematically assess the proteome-wide selectivity and potency of Hymeglusin, the primary inhibitor of HMGCS1 used in the field, confirming it as a useful tool for short-term HMGCS1 inhibition. Inhibiting HMGCS1 with Hymeglusin causes proteome changes that are nearly identical to those caused by inhibiting HMGCR or degrading HMGCS1. Accordingly, simultaneously targeting HMGCS1 and HMGCR effectively suppresses the growth of statin-resistant cells and xenograft models, without increasing the risk of side effects. Finally, we find that while Hymeglusin is a valuable tool for short-term mechanistic studies, its usefulness is limited for long-term efficacy studies due to its poor stability in serum. Together, this study highlights the biological implications of targeting HMGCS1 as monotherapy or in combination with statins, and caution is required when using Hymeglusin as a tool.

biochemistry↗

bioRxiv2025

Production of Fc-fused receptor agonists for glucagon-like peptide-1/glucose-dependent insulinotropic polypeptide (GLP-1/GIP) in the milk of transgenic mice

Incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play pivotal roles in glucose homeostasis and metabolic regulation. Therapeutic incretin receptor agonists (RAs), such as tirzepatide, are widely used to manage type 2 diabetes and obesity. However, incretin RAs are facing production challenges at present. Therefore, we engineered transgenic (tg) mice to secrete incretin RAs in milk, leveraging mammary gland bioreactors for cost-effective peptide production. The goat beta-casein promoter-driven constructs encoding tirzepatide-derived peptide linked to human IgG4 Fc via a (GGGGS)3 spacer were used to produce tg mice. Founders tg-1 and tg-5 exhibited mammary-specific expression, yielding 0.8-1.42 g/L recombinant protein exclusively in milk. Progeny nursed by founders showed sustained hypoglycemia (10-39% reduction; p < 0.05) and marked weight loss (14-49%; p < 0.01) compared to wild-type controls, validating the bioactivity of milk-derived GLP-1/GIP RAs. Moreover, tg-5-nursed offspring experienced high mortality post-Day 16, likely due to overdosing. This proof-of-concept demonstrates the mammary gland bioreactor as a viable platform for incretin RAs production, circumventing complex synthesis and enabling scalable biologics manufacturing.

bioengineering↗

bioRxiv2024

Limited involvement of Ca2+/calmodulin-dependent kinase II alpha and beta in mammalian sleep

While sleep is important, our understanding of its molecular mechanisms is limited. Over the last two decades, protein kinases have been implicated in sleep regulation, with a prominent role for Ca++/calmodulin-dependent kinase II (CaMKII) {beta}. Of all the known mouse genetic mutants, the biggest changes in sleep was reported to be observed in Camk2b gene knockout mice: sleep was reduced by approximately 120 minutes (mins) over 24 hours (hrs). We have reexamined the sleep phenotype in Camk2a and Camk2b knockout mice, and while we have observed sleep reduction in Camk2a knockout mice, we did not find sleep reduction in Camk2b mutants.We did find both Camk2a and Camk2b participated in homeostatic sleep rebound, though. Because CamKII and {beta} are widely known to be crucial kinases with interesting properties, it is worthwhile to keep our results in record for a general service to the field.

animal behavior and cognition↗

bioRxiv2024

Evaluate the efficiency of Mr BMT in macrophage replacement of peripheral organs

The successful replacement of endogenous tissue-resident macrophages with healthy bone marrow-derived cells through transplantation presents a promising therapeutic approach for treating disease associated with macrophage dysfunction. Our previous development of the novel microglia replacement by bone marrow transplantation (Mr BMT) method achieved high engraftment efficiency in the central nervous system (CNS), overcoming the limitation of traditional bone marrow transplantation (tBMT). In this study, we aimed to investigate the potential impacts of Mr BMT approach on peripheral organs. The Mr BMT approach involves depleting resident microglia through colony stimulating factor-1 receptor (CSF-1R) inhibition, so we first evaluated the survival of peripheral macrophages after CSF-1R inhibition. We observed that the CSF-1R antagonist, PLX5622, exerted significant elimination of peripheral tissue macrophages, albeit with lower efficiency than in the CNS. Furthermore, we discovered that Mr BMT achieved replacement efficiencies comparable to tBMT in peripheral tissues such as the liver, kidney, lung, and spleen. Our findings suggested Mr BMT is a highly efficient strategy for allogeneic macrophage replacement in both the CNS and peripheral organs, and it could have potential clinical applications.

immunology↗

bioRxiv2024

RSG-YOLO: Detection of rice seed germination rate based on enhanced YOLOv8 and multi-scale attention feature fusion

The lack of obvious difference between germinated seeds and non-germinated seeds will cause the low accuracy of detecting rice seed germination rate, remains a challenging issue in the field. In view of this, a new model named Rice Seed Germination-YOLO (RSG-YOLO) is proposed in this paper. This model initially incorporates CSPDenseNet to streamline computational processes while preserving accuracy. Furthermore, the BRA, a dynamic and sparse attention mechanism is integrated to highlight critical features while minimizing redundancy. The third advancement is the employment of a structured feature fusion network, based on GFPN, aiming to reconfigure the original Neck component of YOLOv8, thus enabling efficient feature fusion across varying levels. An additional detection head is introduced, improving detection performance through the integration of variable anchor box scales and the optimization of regression losses. This paper also explores the influence of various attention mechanisms, feature fusion techniques, and detection head architectures on the precision of rice seed germination rate detection. Experimental results indicate that RSG-YOLO achieves a mAP50 of 0.981, marking a 4% enhancement over the mAP50 of YOLOv8 and setting a new benchmark on the RiceSeedGermination dataset for the detection of rice seed germination rate.

plant biology↗

bioRxiv2024

Microglia replacement effectively attenuates the disease progress of ALSP in the mouse model and human patient

Microglia play critical roles in the brain physiology and pathology. CSF1R is primarily expressed in microglia. The mono-allelic CSF1R mutation causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a lethal neurological disease and no rational cure in clinical trials. There are no animal models mimicking human ALSP. In this study, we first developed mouse models based on human ALSP hotspot mutations. We then utilized microglia replacement by bone marrow transplantation (Mr BMT) to replace the Csf1r-deficient microglia in ALSP mice by Csf1r-normal donor cells. With pathogenic gene correction, Mr BMT efficiently attenuated the pathologies. Previously, an ALSP patient received traditional bone marrow transplantation (tBMT) due to a misdiagnosis of metachromatic leukodystrophy. The disease progress was halted for 15 years with unknown reasons. We demonstrated that tBMT in ALSP is equivalent to or close to Mr BMT, achieving efficient microglia replacement and therefore attenuating the ALSP progress in the mouse model. Next, we applied tBMT to replace CSF1R-deficient microglia in human patients. Our clinical results show that after microglia replacement, the ALSP course is effectively halted. Together, microglia replacement corrects the pathogenic gene and thus halts the disease progress in the mouse model and human patients. This study strongly demonstrates clinical potentials of microglia replacement in neurological disease treatments.

neuroscience↗

bioRxiv2024

Hydrophobic bulk conservation and genetic code determine the pattern of prokaryotic genome organization

Prokaryotic genomes in general exhibit marked organizational asymmetry, and differ substantially in GC-skew, AT-skew, and GS-bias (gene strand bias). Despite enigmatic origins, these organizational features and genomic GC-content have been described to be widely associated with each other recently, providing an opportunity to probe the evolutionary mechanisms. By analyzing sequence data of 4,012 leading strands from all available representative genomes, we found that the unusual nucleotide usage of coding genes under low GC-content contributed to a shift between two organizational patterns in the context of mutational biases. Analysis of artificially established neutral and natural models further suggests that in genes both GC-skew and AT-skew increase with decreasing GC-content, mostly contributed by synonymous substitutions because of genetic code, and by nonsynonymous substitutions because of hydrophobic bulk conservation in amino acid usage, respectively. This novel mechanistic framework in our study highlights the importance of evolutionary processes "operating at lower levels of organization" in the microbial world.

evolutionary biology↗

bioRxiv2023

4D Crystallography Captures Transient IF1-Ribosome Dynamics in Translation Initiation

Initiation factor 1 (IF1) is one of multiple key ligands involved in the initiation of mRNA translation, a highly dynamic and carefully-orchestrated process. However, details surrounding IF1 transient interactions with the small 30S ribosomal subunit remain incompletely understood despite characterization of unbound and fully-bound 30S states. Improvements in X-ray light sources and crystallographic techniques are now enabling time-resolved structural studies at near-physiological temperature and near-atomic resolution and thus the structural investigation of such dynamic processes. Here, we employed time-resolved serial femtosecond X-ray crystallography (TR-SFX) to probe the binding of IF1 to the small 30S ribosomal subunit in real time. Our time-resolved structural data demonstrates transient cryptic short-, mid-, and long-range allostery among different regions of the small 30 ribosomal subunit during IF1 binding, revealing small- and large-scale protein-target interactions and dynamics within intermediate macromolecular states at unprecedented temporal and spatial resolution. These data represent one of the first such 4D crystallographic studies assessing protein-protein and protein-RNA interactions and could serve as the basis for subsequent studies of the ribosome and of the multitudinous dynamic processes which underpin biology, and therefore, of life.

biophysics↗

bioRxiv2023

Ambient Temperature Bacterial Large Ribosomal Subunit Structure Enabled by Serial Femtosecond X-ray Crystallography

Ribosomes are the supramolecular complexes responsible for protein synthesis. The large 50S ribosomal subunit catalyzes the peptidyl transferase reaction and peptide bond formation between amino acids. The 50S is targeted by many known clinically effective antibiotics. Available structures, obtained at cryogenic temperatures (CT), are used for drug discovery despite that active or important target sites may display a structural configuration that is CT-induced. The introduction of ultrafast and ultrabright X-ray free electron laser (XFEL) pulses has enabled the structural observation of biological macro- and supramolecules at previously unattainable, near-physiological temperatures. In this study, we use ultrafast and ultrabright XFEL pulses to solve the apo form of 50S ribosomal subunit isolated from the extremely thermophilic bacterium Thermus thermophilus at ambient temperature (AT). The dimeric structure of the 50S subunit presented in this work is among the largest ([~]3 megadalton) structures determined using an XFEL source to date. This study demonstrates the ability to obtain new information about ribosome structural dynamics at AT through serial femtosecond X-ray crystallography (SFX). This allowed us to capture previously unobserved dynamics of ribosomal protein uL23 and coordination by hexahydrated magnesium cations at a hitherto unseen resolution at near-physiological temperature. Also, residue A2602, at the core of the peptidyl transferase center (PTC), shows a rather different orientation of the sugar moiety if compared to CT structures. In addition, our structure highlights the importance of flexible residues at both the PTC and in the binding sites for antibiotics erythromycin and chloramphenicol. The method implemented here may also serve as a starting point for future structural research involving the 50S subunit complexes by employing time-resolved mix-inject and probe kineto-crystallography experiments at XFELs. Unveiling ligand-dependent 50S dynamics at physiological temperatures shall guide further development of next-generation antibiotics that target the translation machinery.

biophysics↗

bioRxiv2023

The Interferon-inducible NAMPT acts as a protein phosphoribosylase to restrict viral infection

As obligate intracellular pathogens, viruses often activate host metabolic enzymes to supply intermediates that support progeny production. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the salvage NAD+ synthesis, is an interferon-inducible protein that inhibits the replication of several RNA and DNA viruses with unknown mechanism. Here we report that NAMPT restricts herpes simplex virus 1 (HSV-1) replication via phosphoribosyl-hydrolase activity toward key viral structural proteins, independent of NAD+ synthesis. Deep mining of enriched phosphopeptides of HSV-1-infected cells identified phosphoribosylated viral structural proteins, particularly glycoproteins and tegument proteins. Indeed, NAMPT de-phosphoribosylates viral proteins in vitro and in cells. Chimeric and recombinant HSV-1 carrying phosphoribosylation-resistant mutations show that phosphoribosylation promotes the incorporation of structural proteins into HSV-1 virions and subsequent virus entry. Moreover, loss of NAMPT renders mice highly susceptible to HSV-1 infection. The work describes a hidden enzyme activity of a metabolic enzyme in viral infection and host defense, offering a system to interrogate roles of phosphoribosylation in metazoans.

molecular biology↗