Biology subjects

Lei, K.

Publications and source records attributed to Lei, K..

12 recordsLinked to original sources

bioRxiv2025

TaoChongBao: A Large-Scale Caenorhabditis elegans Mutagenesis and Missense Variant Database Bridging Worm and Human Genomes

We generated and sequenced 12,069 viable Caenorhabditis elegans strains produced by ethyl methanesulfonate mutagenesis, identifying 20,315,536 variants, including 541,102 unique missense mutations across 20,914 genes. To organize and visualize this resource, we developed TaoChongBao, an open-access database and strain repository that integrates C. elegans mutation data with AlphaMissense-predicted pathogenicity scores and ClinVar clinical annotations. TaoChongBao enables users to explore worm missense variants, identify conserved residues corresponding to human pathogenic sites, and access viable strains for experimental validation. Compared with the previous Million Mutation Project in C. elegans, TaoChongBao expands mutation coverage over twenty-fold and emphasizes amino acid-altering variants. This resource provides a scalable platform for functional residuomics, variant interpretation, and comparative analyses between C. elegans and human genomes.

bioinformatics↗

bioRxiv2025

Inherited retinal degenerations: PARP regulates calpain activation via TRPM2 channels in rd1 mouse photoreceptors

Inherited retinal degeneration (IRD) refers to untreatable blinding diseases characterized by progressive photoreceptor loss. Photoreceptor degeneration is often associated with an excessive activation of poly (ADP-ribose) polymerase (PARP) and Ca2+-dependent calpain-type proteases. To explore the interplay between PARP and calpain activity, we employed organotypic retinal explant cultures derived from wild-type mice and from the rd1 mouse model for IRD. Retinae were treated with the PARP inhibitors INO1001 or Olaparib, the poly (ADP-ribose) glycohydrolase (PARG) inhibitor JA2131, or the transient receptor potential channel M2 (TRPM2) blocker 8-Br-ADPR. Readouts included the TUNEL assay to detect cell death, in situ activity assays for histone-deacetylases (HDAC), PARP, and calpain, as well as immunostaining for activated calpain-2, and poly (ADP-ribose) (PAR). PARP, PARG, and TRPM2 inhibition reduced calpain activity and calpain-2 activation. PARP activity was decreased by PARP and TRPM2 inhibitors but not by PARG inhibition. Remarkably, the PARP inhibitor INO1001 increased HDAC activity unlike any of the other compounds. When combined with the PARG inhibitor JA2131, INO1001 reduced photoreceptor cell death in a synergistic fashion, although such synergy was not observed for calpain or PARP activity. Moreover, synergistic photoreceptor preservation was not observed when JA2131 was combined with the PARP inhibitor Olaparib. Overall, these results indicate that in rd1 photoreceptors, PARP controls calpain activity via PARG and TRPM2-induced Ca2+ influx. We also characterize INO1001 as potentially more beneficial for IRD treatment than Olaparib. Our study details the complexity of PARP-signalling in photoreceptors and identifies PARG and TRPM2 as new targets for IRD therapy development.

neuroscience↗

bioRxiv2025

Adeno-associated viruses escort nanomaterials to specific cells and tissues

The delivery of nanotherapeutics to specific tissues relies on bespoke targeting strategies or invasive surgeries. Conversely, adeno-associated viruses (AAVs) can target specific tissues following intravenous injections. Here we show that cell-targeting properties of AAVs could be broadly conferred to nanomaterials. We develop a strategy to couple AAV capsids to nanoparticles that is invariant of viral serotype or nanomaterial chemistry and permits control over stoichiometry of the AAV-nanoparticle chimeras. The chimeras selectively escort nanoparticles into cell classes governed by AAV serotypes. When applied to magnetic nanoparticles, the AAV-nanoparticle chimeras enable magnetically localized gene delivery. In vivo, we show that leveraging the brain-targeting AAV serotype CAP-B10 achieves nanoparticle delivery to the parenchyma with [~]10% efficiency (% injected dose/g[brain]) while avoiding accumulation in the liver. The enhanced delivery efficiency and tissue specificity highlight the potential of AAV-chimeras as a versatile strategy to escort broad classes of nanotherapeutics to the brain and beyond.

bioengineering↗

bioRxiv2024

Loss of Neurofibromin Induces Inflammatory Macrophage Phenotypic Switch and Retinal Neovascularization via GLUT1 Activation

Persons with neurofibromatosis type 1 (NF1), a tumor predisposition syndrome, are largely protected from diabetes and exhibit evidence of enhanced glucose metabolism, which is replicated in mice harboring Nf1 mutations. A hallmark of NF1-associated neurofibromas and sarcomas is the high density of inflammatory macrophages and targeting macrophages appears efficacious in models of NF1. Inflammatory macrophages rely on glycolysis to rapidly generate ATP; thus, identifying whether neurofibromin, the protein encoded by the NF1 gene, controls glucose uptake and/or glycolysis in macrophages is therapeutically compelling. Using neurofibromin-deficient macrophages and macrophage-specific Nf1 knockout mice, we demonstrate that neurofibromin complexes with glucose transporter 1 (GLUT1) to restrain its activity and that loss of neurofibromin permits Akt2 to facilitate GLUT1 translocation to the membrane in macrophages. In turn, glucose internalization and glycolysis are highly up regulated and provoke putative reparative (M2) macrophages to undergo inflammatory phenotypic switch. Inflammatory M1 macrophages and inflammatory-like M2 macrophages invest the perivascular stroma of tumors and induce pathologic angiogenesis in mice harboring macrophage-specific Nf1 deletion. These studies identify a clear mechanism for the enhanced glycolysis and low risk for diabetes observed in persons with NF1 and provide a novel therapeutic target for manifestations of NF1.

cell biology↗

bioRxiv2024

3D Reconstruction of Neuronal Allometry and Neuromuscular Projections in Asexual Planarians Using Expansion Tiling Light Sheet Microscopy

The intricate coordination of the neural network in planarian growth and regeneration has remained largely unrevealed, partly due to the challenges of imaging the central nervous system (CNS) in three dimensions (3D) with high resolution and within a reasonable timeframe. To address this gap in systematic imaging of the CNS in planarians, we adopted high-resolution, nanoscale imaging by combining tissue expansion and tiling light-sheet microscopy, achieving up to 4-fold linear expansion. Using an automatic 3D cell segmentation pipeline, we quantitatively profiled neurons and muscle fibers at the single-cell level in over 400 wild-type planarians during homeostasis and regeneration. We validated previous observations of neuronal cell number changes and muscle fiber distribution. We found that the increase in neuron cell number tends to lag behind the rapid expansion of somatic cells during the later phase of homeostasis. By imaging the planarian with up to 120 nm resolution, we also observed distinct muscle distribution patterns at the anterior and posterior poles. Furthermore, we investigated the effects of {beta}-catenin RNAi on muscle fiber distribution at the posterior pole, consistent with changes in anterior-posterior polarity. The glial cells were observed to be close in contact with dorsal-ventral muscle fibers. Finally, we observed disruptions in neural-muscular networks in inr-1 RNAi planarians. These findings provide insights into the detailed structure and potential functions of the neural-muscular system in planarians and highlight the accessibility of our imaging tool in unraveling the biological functions underlying their diverse phenotypes and behaviors.

developmental biology↗

bioRxiv2024

Activation of two noncanonical R proteins by an insect effector confers plant immunity to aphid infestation

Molecular characterization of resistance genes is crucial for efficiently understanding and fortifying plant immunity against insect herbivores. Here we report that RMES1A and RMES1B proteins confer resistance to the sorghum aphid Melanaphis sorghi when activated by an insect effector MsEF1. Map-based cloning plus genetic analysis of knockout mutants confirm that RMES1A and RMES1B are both required for aphid resistance. Upon aphid attack, RMES1A and RMES1B expression is elevated in the sclerenchyma cells and vascular bundles of leaves; the two proteins interact with MsEF1 in the exocysts, thus upregulating key defense processes such as reactive oxygen species burst. Structural modeling predicts that RMES1A and RMES1B each carry an ATP binding site and two leucine-rich-repeat domains but lack coiled-coil or Toll/Interleukin-1 receptor/resistance domain, thus likely representing a new type of resistance controlling proteins in plants. Our work reveals new genes and mechanisms for further deciphering and improving plant immunity to insect pests.

genetics↗

bioRxiv2023

Transposase-Assisted Tagmentation: An Economical and Scalable Strategy for Single-Worm Whole-Genome Sequencing

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in C. elegans, E. coli, S. cervisiae, and C. reinhardtii. This method, demands merely 20 minutes of hands-on time for a single worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the oncogenic BRAF(V600E) mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species.

genomics↗

bioRxiv2023

Biosynthesis- and Metabolomics-guided discovery of antimicrobial cyclopeptides against drug-resistant clinical isolates

Antimicrobial resistance remains a significant global threat, contributing significantly to mortality rates worldwide. Ribosomally synthesized and post-translationally modified peptides (RiPPs) have emerged as a promising source of novel peptide antibiotics due to their diverse chemical structures. Here, we reported the discovery of new Avi(Me)Cys-containing cyclopeptide antibiotics through a synergistic approach that combines rule-based genome mining, automated metabolomic analysis, and heterologous expression. We first bioinformatically identified 1,172 RiPP biosynthetic gene clusters (BGCs) responsible for Avi(Me)Cys-containing cyclopeptides from a vast pool of over 50,000 bacterial genomes. Subsequently, we successfully established the connection between three newly identified BGCs and the synthesis of five new peptide antibiotics. Notably, massatide A displayed excellent activity against a spectrum of gram-positive pathogens, including drug-resistant clinical isolates like linezolid-resistant S. aureus and methicillin-resistant S. aureus, with a minimum inhibitory concentration (MIC) of 0.25 g/mL. The remarkable performance of massatide A in an animal infection model, coupled with a low risk of resistance and favorable safety profile, positions it as a promising candidate for antibiotic development. Our study highlights the potential of Avi(Me)Cys-containing cyclopeptides in expanding the arsenal of antibiotics against multi-drug-resistant bacteria, offering promising drug leads in the ongoing battle against infectious diseases.

biochemistry↗

bioRxiv2022

Fibrillarin-1 and Fibrillarin-2 are required for divergent cell lineage development in planarian homeostasis and regeneration

Ribosome heterogeneity has been revealed to exist in different cell types during development. However, the function and regulatory mechanisms of ribosome heterogeneity in missing tissue regeneration have yet to be reported. We used the planarian Schmidtea mediterranea with whole-body regenerative capability as a model and revealed the function of the rRNA modification protein fibrillarin in cell lineage development and tissue regeneration. We identified two fibrillarin homologs in planarian, Smed-fbl-1 (fbl-1) and Smed-fbl-2 (fbl-2), with distinct expression patterns. While fbl-2 regulates stem cell proliferation and multiple progenitor cell differentiation, fbl-1 participates in epidermal lineage late-stage specification and wound response. This study indicates that fibrillarin, a nucleolar protein, can respond to wounds and function in distinct cell types, suggesting the existence and critical roles of ribosome heterogeneity in stem cells and tissue regeneration.

developmental biology↗

bioRxiv2020

Mitochondrial state determines functionally divergent stem cell population in planaria

Mitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared to its progenitors. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PIWI-1High neoblasts. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration.

cell biology↗

bioRxiv2019

Cultured pluripotent planarian stem cells retain potency and express proteins from exogenously introduced mRNAs

Planarians possess naturally occurring pluripotent adult somatic stem cells (neoblasts) required for homeostasis and whole-body regeneration. However, no reliable neoblast culture methods are currently available, hindering mechanistic studies of pluripotency and the development of transgenic tools. We report the first robust methods for neoblast culture and delivery of exogenous mRNAs. We identify optimal culture media for maintaining neoblasts in vitro, and show via transplantation that cultured stem cells retain pluripotency for two days. We developed a new procedure that significantly improves neoblast yield and purity by modifying standard flow cytometry methods. These methods enable the introduction and expression of exogenous mRNAs in neoblasts, overcoming a key hurdle impeding the application of transgenics in planarians. The advances in cell culture reported here create new opportunities for mechanistic studies of planarian adult stem cell pluripotency, and provide a systematic framework to develop cell culture techniques in other emerging research organisms.

cell biology↗

bioRxiv2017

Comparative Analysis of Gene Expression Identifies Distinct Molecular Signatures of Bone Marrow- and Periosteal-Skeletal Stem/Progenitor Cells

Periosteum and bone marrow (BM) both contain skeletal stem/progenitor cells (SSCs) that participate in fracture repair. However, the functional difference and selective regulatory mechanisms of SSCs in different location are unknown due to the lack of specific markers. Here, we report a comprehensive gene expression analysis of bone marrow SSCs (BM-SSCs), periosteal SSCs (P-SSCs), and more differentiated osteoprogenitors by using reporter mice expressing Interferon-inducible Mx1 and NestinGFP, previously known SSC markers. We first defined that the BM-SSCs can be enriched by the combination of Mx1 and NestinGFP expression, while endogenous P-SSCs can be isolated by positive selection of Mx1, CD105 and CD140a (known SSC markers) combined with the negative selection of CD45, CD31, and osteocalcinGFP (a mature osteolineage marker). Comparative gene experession analysis with FACS-sorted BM-SSCs, P-SSCs, Osterix+ (OSX) preosteoblasts, CD51+ stroma cells and CD45+ hematopoietic cells as controls revealed that BM-SSCs and P-SSCs have high similarity with few potential differences without statistical significance. We also found that CD51+ cells are highly heterogeneous and little overlap with SSCs. This was further supported by the microarray cluster analysis, and the two populations clustered together. However, when comparing SSC population to controls, we found several genes that were uniquely upregulated in endogenous SSCs. Amongst these genes, we found KDR (aka Flk1 or VEGFR2) to be most interesting and discovered that it is highly and selectively expressed in P-SSCs. This finding suggests that endogenous P-SSCs are functionally very similar to BM-SSCs with undetectable significant differences in gene expression but there are distinct molecular signatures in P-SSCs, which can be useful to specify P-SSC subset in vivo.

cell biology↗