Biology subjects

Wu, M.-R.

Publications and source records attributed to Wu, M.-R..

10 recordsLinked to original sources

bioRxiv2026

MKMC enables reference-free transcriptomic analysis using k-mer representations

Traditional RNA-seq analysis depends heavily on genome alignment and gene annotation, limiting its utility in non-model organisms and introducing biases that can obscure regulatory complexity. We present MKMC (Multi-sample Kmer Counter), a scalable, reference-free toolkit for RNA-seq analysis that leverages k-mer-based statistics to detect biological variation without requiring alignment. MKMC integrates fast k-mer counting, abundance matrix generation, normalization, dimensionality reduction, and differential analysis into a unified workflow. Across diverse datasets, MKMC recapitulates key biological signals--including sex differences in killifish liver--and matches alignment-based pipelines in differential expression analysis and transcriptomic age prediction. Notably, MKMC detects isoform-specific events missed by traditional methods, one of which we validated using in situ hybridization. These results reveal previously hidden isoform-level regulatory events that contribute to sex-and age-associated transcriptional programs. MKMC offers a robust, extensible alternative to alignment-based approaches, enabling transcriptomic discovery across both model and non-model systems. While we focus here on RNA-seq as a primary application, MKMC is broadly applicable to any k-mer-based analysis of next-generation sequencing data.

bioinformatics↗

bioRxiv2026

Mitochondrial Transplantation in the Eye: A Review and Evaluation of Surgical Approaches

PurposeMitochondrial dysfunction contributes to major blinding diseases, including age-related macular degeneration and glaucoma. Although mitochondrial transplantation has shown therapeutic potential in multiple organ systems, translation to the eye remains limited, partly due to uncertainty regarding optimal delivery. We summarize the biologic rationale and preclinical evidence supporting ocular mitochondrial transplantation and present feasibility data evaluating clinically relevant delivery routes. MethodsWe conducted a focused narrative review of ocular mitochondrial transplantation. For feasibility experiments, mitochondria with an endogenous fluorescent dye were isolated from liver donor mice. Postnatal day 7 pups received subretinal injections, and adult CD1 mice received intravitreal injections, including optic nerve head directed delivery. Eyes were analyzed using fluorescence microscopy and immunohistochemistry. Mitochondrial uptake was assessed in cultured retinal pigmental epithelial (RPE) cells using co-incubation assays. Suprachoroidal delivery feasibility was evaluated in cadaveric human near-real surgical specimens using a novel dedicated suprachoroidal injector. ResultsThe literature on ocular mitochondrial transplantation remains limited and consists primarily of small preclinical studies using intravitreal delivery and imaging-based detection. In our experiments, intravitreal delivery produced donor signals predominantly within inner retinal layers, with enrichment along retinal nerve fiber bundles when directed toward the optic nerve head. Cultured RPE cells demonstrated dose-dependent uptake of exogenous mitochondria. Subretinal delivery localized donors signal to the RPE and adjacent outer retina. Suprachoroidal injections demonstrated procedural feasibility with reliable access to the suprachoroidal space and visible injectate distribution. ConclusionsOcular mitochondrial transplantation is in an early stage of investigation. Our feasibility data indicate that established posterior-segment delivery routes expose distinct retinal compartments and that route selection strongly influences anatomic distribution. Further studies are needed to verify intracellular uptake, define dosing and durability, and evaluate safety in disease-relevant models.

cell biology↗

bioRxiv2025

A transcription factor-responsive enhancer discovery platform for targeted immunotherapy

Synthetic enhancers with high specificity are crucial for therapeutic gene control. However, experimental screens and machine learning-guided design typically require context-specific datasets, limiting generalizability. Because transcription factor (TF) activity reflects cellular state, TF-responsive enhancer libraries offer a universal starting point. Here, we developed TREND (transcription factor-responsive enhancer discovery), a massively parallel reporter assay of [~]2.7 million enhancer-barcode constructs representing 57,715 designs. TREND covers 1,068 motif-annotated proteins, including 729 confirmed TFs across 49 DNA-binding domain families. Applied to ovarian cancer, TREND identified enhancers that discriminate cancer from normal epithelial cells. These enhancers enabled protein-interaction-based AND-gate circuits with reduced OFF-state leakage and amplified ON-state output, driving tumor-restricted expression of combinatorial immune effectors and robust antitumor responses in murine ovarian cancer models. TREND also identified T-cell activation-responsive enhancers with greater inducibility and lower basal activity than conventional NFAT-motif-based elements. Together, TREND provides a generalizable framework for context-specific enhancer discovery and therapeutic gene regulation.

synthetic biology↗

bioRxiv2025

Sequestration-Based Neural Networks That Operate Out of Equilibrium

Classification of high-dimensional information is a ubiquitous computing paradigm across diverse biological systems, including organs such as the brain, down to signaling between individual cells. Inspired by the success of artificial neural networks in machine learning, the idea of engineering genetic circuits that operate as neural networks emerges as a strategy to expand the classification capabilities of living systems. In this work, we design these biomolecular neural networks (BNNs) based on the molecular sequestration reaction, and experimentally characterize their behavior as linear classifiers for increasing levels of complexity. Initially, we demonstrate that a static, DNA-based system can effectively prototype a linear classifier, though we also identify its limitations to easily tune the slope of the decision boundary it generates. We then propose and experimentally validate a BNN at the protein level using a cell-free transcription-translation (TXTL) system, which overcomes the DNA-based systems limitation and behaves as a linear classifier even before it reaches its steady state (or, equivalently, out-of-equilibrium). Ultimately, we test a CRISPR-based design and its out-of-equilibrium behavior in a biological context by successfully constructing a linear classifier within mammalian cells. Overall, by leveraging mathematical modeling and experimental automation, we establish molecular sequestration as a universal scheme for implementing neural networks within living systems, paving the way for transformative advances in synthetic biology and programmable biocomputing systems.

synthetic biology↗

bioRxiv2025

Multi-tissue transcriptomic aging atlas reveals predictive aging biomarkers in the killifish

Aging is associated with progressive tissue dysfunction, leading to frailty and mortality. Characterizing aging features, such as changes in gene expression and dynamics, shared across tissues or specific to each tissue, is crucial for understanding systemic and local factors contributing to the aging process. We performed RNA-sequencing on 13 tissues at 6 different ages in the African turquoise killifish, the shortest-lived vertebrate that can be raised in captivity. This comprehensive, sex-balanced atlas dataset reveals the varying strength of sex-age interactions across killifish tissues and identifies age-altered biological pathways that are evolutionarily conserved. Demonstrating the utility of this resource, we discovered that the killifish head kidney exhibits a myeloid bias during aging, a phenomenon more pronounced in females than in males. In addition, we developed tissue-specific transcriptomic clocks and identified biomarkers predictive of chronological age. We show the importance of sex-specific clocks for selected tissues and use the tissue clocks to evaluate a dietary intervention in the killifish. Our work provides a comprehensive resource for studying aging dynamics across tissues in the killifish, a powerful vertebrate aging model.

genomics↗

bioRxiv2024

Induction of a Muller glial-specific protective pathway safeguards the retina from diabetes induced damage

Diabetes can lead to cell-type-specific responses in the retina, including vascular lesions, glial dysfunction and neurodegeneration, all of which contribute to retinopathy. However, the molecular mechanisms underlying these cell type-specific responses, and the cell types that are sensitive to diabetes have not been fully elucidated. Employing single cell transcriptomic analyses, we profiled the transcriptional changes induced by diabetes in different retinal cell types in diabetic rat models as the disease progressed. Rod photoreceptors, a subtype of amacrine interneurons, and Muller glial cells exhibited rapid responses to diabetes at the transcript levels. Genes associated with ion regulation were upregulated in all three cell types, suggesting a common response to diabetes. Furthermore, focused studies revealed that while Muller glial cells initially increased the expression of genes playing protective roles, they cannot sustain this beneficial effect as the disease progressed. We explored one of the candidate protective genes, Zinc finger protein 36 homolog (Zfp36), and observed that depleting Zfp36 in rat Muller glial cells in vivo using AAV-based tools exacerbated early diabetes-induced phenotypes, including gliosis, neurodegeneration, and vascular defects. Notably, the over-expression of Zfp36 slowed the development of phenotypes associated with diabetic retinopathy. In summary, this work unveiled retinal cell types that are sensitive to diabetes and demonstrated that Muller glial cells can mount protective responses through Zfp36. The failure to maintain Zfp36 levels contributes to the development of diabetic retinopathy.

neuroscience↗

bioRxiv2022

Mettl14-mediated m6A modification ensures the cell cycle progression of late-born retinal progenitor cells

Neural progenitor cells lengthen their cell cycle to prime themselves for differentiation as development proceeds. It is currently not clear how they counter this lengthening and avoid being halted in the cell cycle. Here, we show that m6A (N6-methyladenosine) methylation of cell cycle-related mRNAs ensures the proper cell cycle progression of late-born retinal progenitor cells (RPCs), which are born towards the end of retinogenesis and have long cell cycle durations. Conditional deletion of Mettl14, which is required for depositing m6A on mRNAs, significantly reduced the level of m6A modification in the developing mouse retina. This led to delayed cell cycle exit of late-born RPCs and neuronal cell death in mature retina, but appeared to have no effect on retinal development prior to birth. M6A-seq, which maps m6A modified mRNAs, and single cell transcriptomic analyses revealed that mRNAs involved in slowing down cell cycle progression were highly enriched for m6A modification, which could target them for degradation and guarantee proper cell cycle progression of late-born RPCs. In addition, we identified Zfp292 as a novel target of m6A and potent inhibitor of RPC cell cycle progression. Overall, our work establishes m6A modification as an important mechanism countering cell cycle lengthening in late-born retinal progenitor cells.

developmental biology↗

bioRxiv2020

A Synthetic Transcription Platform for Programmable Gene Expression in Mammalian Cells

Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. We created a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements expressed mammalian genes in a highly predictable and tunable manner. We demonstrated the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more activity than seen with the EF1 promoter, in multiple cell types. We used these circuits to program the secretion of human monoclonal antibodies and to control T-cell effector function marked by interferon-{gamma} production. Antibody titers and interferon-{gamma} concentrations significantly correlated with synthetic promoter strengths, providing a platform for programming gene expression and cellular function in diverse applications.

synthetic biology↗

bioRxiv2020

A novel oxidase from Alcaligenes sp. HO-1 oxidizes hydroxylamine to N2

Hydroxylamine is a key intermediate of microbial ammonia oxidation and plays an important role in the biogeochemical cycling of N-compounds. Hydroxylamine is oxidized to NO or N2O by hydroxylamine oxidases or cytochrome P460 from heterotrophic or autotrophic bacteria, but its enzymatic oxidation to N2 has not yet been observed. Here, we report on the discovery of a novel oxidase that converts hydroxylamine to N2 from the newly isolated heterotrophic nitrifier Alcaligenes strain HO-1. Strain HO-1 accumulated hydroxylamine and produced N2 from ammonia oxidation. Using transcriptome analysis and heterologous expression via fosmid library screening, we identified three genes (dnfABC) of strain HO-1 that enabled E. coli cells not only to produce hydroxylamine from 15N-labelled ammonium but also to further convert it to 15N2. The three genes were individually cloned and expressed, and their translational products DnfA, DnfB, and DnfC were purified. In vitro DnfA bound to hydroxylamine and catalyzed the conversion of hydroxylamine to N2 in the presence of FAD, NADH and O2. Thus, DnfA was identified as a novel hydroxylamine oxidase and catalyzed a previously unknown N-N bond forming reaction with a yet-to-be discovered mechanism. DnfA homologs were detected in different bacterial groups, suggesting that hydroxylamine oxidation to nitrogen might occur in additional microbial taxa.

microbiology↗

bioRxiv2019

Cell type- and stage-specific expression of Otx2 is coordinated by a cohort of transcription factors and multiple cis-regulatory modules in the retina

Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is an advantageous strategy. However, the mechanisms that control the activation of TFs in different cell types and at different stages of development remain unclear. The neural retina serves as a model of the development of a complex tissue. We used this system to analyze how expression of the homeobox TF, Orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during retinogenesis. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. These include mature bipolar interneurons, photoreceptors, and retinal progenitor/precursor cells. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages, as revealed by ATAC-seq and DNase-seq analyses. We conclude that retinal cells utilize a cohort of TFs with different expression patterns, and multiple CRMs with different chromatin configurations, to precisely regulate the expression of Otx2 in a cell type- and stage-specific manner in the retina.

developmental biology↗