Biology subjects

Kuo, L.

Publications and source records attributed to Kuo, L..

8 recordsLinked to original sources

bioRxiv2026

In vivo reconstruction of the cell lineage history of a developing mouse with DNA Typewriter, from zygote to late organogenesis

The complete cell lineage of C. elegans, mapped over four decades ago, was tractable because the animal is small, transparent, and lineage invariant1. Most animals are none of these, having orders of magnitude more cells, being opaque, and developing with substantial stochasticity2. Since 2016, genome editing-based lineage tracing has opened the door to dense cell lineage reconstruction in such organisms3-8, but delivering on that promise has proven technically challenging. Here we apply DNA Typewriter9-11, a prime editing-based recorder that writes stochastic symbolic insertions to an engineered genomic TAPE in strictly sequential order, to trace mouse development from zygote (E0) to late organogenesis (E13.5). We introduce constructs encoding a prime editor, engineered prime editing guide RNAs (epegRNAs), and Pol-III-driven circularized TAPE RNA (circTAPE) into wildtype zygotes by pronuclear injection (PNI), then assay embryos by single-nucleus transcriptional profiling (sci-RNA-seq3)12 with paired circTAPE recovery. From one E13.5 embryo bearing [~]7 integrations of a constitutively expressed prime editor and 11 integrations of 6-unit circTAPE, we recover [~]1.75M single-nucleus transcriptomes and reconstruct a time-calibrated phylogeny of 1,340,794 annotated cells with parsimony-based node support. The first cell division is marked unequivocally, and although the resulting blastomeres contribute asymmetrically to the embryo proper, they are fate-neutral and serve as internal replicates that reproduce every finding. A modest cohort of pre-gastrulation founders dominates the embryo, with inequality exceeding neutral expectation within one to two cell cycles of founder allocation, yet these founders remain broadly multipotent; a second phase of clonal dominance arises in specific lineages during organogenesis. At the finest scale, sibling cells share cell type 9-fold in excess of chance, reaching 68- to 107-fold for cell types arising from spatially restricted founder pools, while the recent differentiations of organogenesis are legible in the heterotypic structure of terminal clades. From clade co-occurrence alone, we recover germ-layer organization and a dated hierarchy of cell-type couplings with branch points from E8.5 onwards. Finally, by integrating these data with our single-cell time-series of mouse development13, we impute transcriptional states and annotations for the majority of internal nodes and recover established state histories for diverse cell types. All data are made freely available, together with NextCell, an interactive browser for this annotated cellular phylogeny of mouse development from zygote to late organogenesis.

developmental biology↗

bioRxiv2023

An arginine-rich nuclear localization signal (ArgiNLS) strategy for streamlined image segmentation of single-cells

High-throughput volumetric fluorescent microscopy pipelines can spatially integrate whole-brain structure and function at the foundational level of single-cells. However, conventional fluorescent protein (FP) modifications used to discriminate single-cells possess limited efficacy or are detrimental to cellular health. Here, we introduce a synthetic and non-deleterious nuclear localization signal (NLS) tag strategy, called Arginine-rich NLS (ArgiNLS), that optimizes genetic labeling and downstream image segmentation of single-cells by restricting FP localization near-exclusively in the nucleus through a poly-arginine mechanism. A single N-terminal ArgiNLS tag provides modular nuclear restriction consistently across spectrally separate FP variants. ArgiNLS performance in vivo displays functional conservation across major cortical cell classes, and in response to both local and systemic brain wide AAV administration. Crucially, the high signal-to-noise ratio afforded by ArgiNLS enhances ML-automated segmentation of single-cells due to rapid classifier training and enrichment of labeled cell detection within 2D brain sections or 3D volumetric whole-brain image datasets, derived from both staining-amplified and native signal. This genetic strategy provides a simple and flexible basis for precise image segmentation of genetically labeled single-cells at scale and paired with behavioral procedures. Significance StatementQuantifying labeled cells in fluorescent microscopy is a fundamental aspect of modern biology. Critically, the use of short nuclear localization sequences (NLS) is a key genetic modification for discriminating single-cells labeled with fluorescent proteins (FPs). However, mainstay NLS approaches typically localize proteins to the nucleus with limited efficacy, while alternative non-NLS tag strategies can enhance efficacy at the cost of cellular health. Thus, quantitative cell counting using FP labels remains suboptimal or not compatible with health and behavior. Here, we present a novel genetic tagging strategy - named ArgiNLS - that flexibly and safely achieves FP nuclear restriction across the brain to facilitate machine learning-based segmentation of single-cells at scale, delivering a timely update to the behavioral neuroscientists toolkit.

neuroscience↗

bioRxiv2022

Combinatorial microRNA loading into extracellular vesicles for anti-inflammatory therapy

Extracellular vesicles (EVs) have emerged as promising therapeutic entities in part due to their potential to regulate multiple signaling pathways in target cells. This potential is derived from the broad array of constituent and/or cargo molecules associated with EVs. Among these, microRNAs (miRNAs) are commonly implicated as important and have been associated with a wide variety of EV-induced biological phenomena. While controlled loading of single miRNAs is a well-documented approach for enhancing EV bioactivity, loading of multiple miRNAs has not been fully leveraged to maximize the potential of EV-based therapies. Here, an established approach to extrinsic nucleic acid loading of EVs, sonication, was utilized to enable controlled loading of multiple miRNAs in HEK293T EVs. Combinations of carefully chosen miRNAs were compared to single miRNAs with respect to anti-inflammatory outcomes in assays of increasing stringency, with the combination of miR-146a, miR-155, and miR-223 found to have the most potential amongst tested groups.

bioengineering↗

bioRxiv2020

A Novel SARS-CoV-2 Multitope Protein/Peptide Vaccine Candidate is Highly Immunogenic and Prevents Lung Infection in an Adeno Associated Virus Human Angiotensin-Converting Enzyme 2 (AAV hACE2) Mouse Model

A novel multitope protein-peptide vaccine against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and disease is described in this report. The initial development and characterization experiments are presented along with proof-of-concept studies for the vaccine candidate UB-612. UB-612 consists of eight components rationally designed for induction of potently neutralizing antibodies and broad T cell responses against SARS-CoV-2: the S1-RBD-sFc fusion protein, six synthetic peptides (one universal peptide and five SARS-CoV-2-derived peptides), a proprietary CpG TLR-9 agonist at low concentration as an excipient, and aluminum phosphate adjuvant. Through immunogenicity studies in Guinea pigs and rats, we optimized the design of protein/peptide immunogens and selected an adjuvant system, yielding a vaccine that provides excellent S1-RBD binding and high neutralizing antibody responses, robust cellular responses, and a Th1-oriented response at low doses. In challenge studies, UB- 612 vaccination reduced viral load and prevented development of disease in mouse and non-human primate challenge models. With a Phase 1 trial completed, a Phase 2 trial ongoing in Taiwan, and additional trials planned to support global authorizations, UB-612 is a highly promising and differentiated vaccine candidate for prevention of SARS-CoV-2 infection and COVID-19 disease. Author SummarySARS-CoV-2 virus, the causative agent of Coronavirus Disease 2019 (COVID-19), has spread globally since its origin in 2019, causing an unprecedented public health crisis that has resulted in greater than 4.7 million deaths worldwide. Many vaccines are under development to limit disease spread and reduce the number of cases, but additional candidates that promote a robust immune response are needed. Here, we describe a multitope protein-peptide vaccine platform that is unique among COVID-19 vaccines. The advantages of our approach are induction of both high levels of neutralizing antibodies as well as a Th/CTL response in the vaccinated host, which mimics the immune response that occurs after natural infection with SARS-CoV-2. We demonstrate that our vaccine is immunogenic and effective in preventing disease in several animal models, including AAV- hACE-2 transduced mice, and both rhesus and cynomolgus macaques. Importantly, no immunopathology was observed in the lungs of immunized animals, therefore showing that antibody-dependent enhancement (ADE) does not occur. Our study provides an additional, novel vaccine candidate for advancement in clinical trials to treat and prevent SARS-CoV-2 infection and COVID-19 disease.

immunology↗

bioRxiv2019

Increased temperatures reduce the vectorial capacity of Aedes mosquitoes for Zika virus

Rapid and significant range expansion of both ZIKV and its Aedes host species has resulted in ZIKV being declared a global health threat. Mean temperatures are projected to increase globally, likely resulting in alterations of the transmission potential of mosquito-borne pathogens. The relationship between temperature and ZIKV transmission has not been well characterised for Aedes aegypti and Aedes albopictus.\n\nTo understand the effect of diurnal temperature range on the vectorial capacity of Aedes aegypti and Aedes albopictus for ZIKV, factors contributing to transmission potential were measured at different temperature regimens. Their longevity and blood feeding rates were assessed, and vector competence was determined following feeding on blood meals with 8.3 log10 PFU/ml ZIKV.\n\nHigher temperature resulted in decreased longevity of Ae. aegypti [Log-rank (Mantel-Cox) Test, Chi-square, df 35.66, 5 P (<0.0001)] and a significant decrease in blood feeding rates across groups [Z score (-5.8478) P (0.0444)]. Temperature had a population and species-specific impact on ZIKV infection rates. Overall, Ae. albopictus reared at the lowest temperature regimen demonstrated the highest vectorial capacity (1.63) and the highest transmission efficiency (57%). Temperature increases decreased vectorial capacity across groups, yet the largest decreases were measured for Ae. aegypti.\n\nThe results of this study suggest that future climate change could significantly impact vector competence, blood feeding behavior and longevity, and therefore decrease the overall vectorial capacity of Aedes mosquitoes. It is also clear that this impact is likely to be both species and population-specific.

microbiology↗

bioRxiv2019

Zika virus infection and temperature significantly impact Aedes albopictus gut microbiota

Elizabethkingia anophelis has been the cause of four outbreaks with significant morbidity and mortality. Its transmission routes remain unknown and no point source of infection has been identified. Here we show that E. anophelis can be found in the saliva of Aedes mosquitoes, suggesting the novel possibility of vector-borne transmission of this bacterium. We additionally characterized diverse microbial communities in Aedes midguts, salivary glands and saliva. To the best of our knowledge, this represents the first description of the microbiome of Aedes saliva. Further, we demonstrate that increased abundance of E. anophelis is associated with decreased susceptibility and replication of Zika virus (ZIKV) in the midgut of Aedes mosquitoes, suggesting a novel transmission barrier for arboviruses transmitted by Aedes mosquitoes. Together, these results demonstrate the complex relationships between the mosquito, the midgut microbial community and arboviruses and offer insights into the epidemiology and control of emerging bacterial and viral pathogens. Author SummaryElizabethkingia anophelis has in the recent past caused outbreaks different parts of the world resulting both in morbidity and mortality. Until now, to the best of our knowledge, no study has been able to demonstrate that this bacterium can be transmitted by mosquitoes. We have demonstrated for the first time that Elizabethkingia anophelis is present in the saliva of both infected and non-infected Aedes mosquitoes. Further, we have shown that it confers an inhibitory effect on Zika virus establishment in the midguts of Aedes mosquitoes. Together, these results potentially display the potential for vector borne transmission of E. anophelis as well as a novel transmission barrier of ZIKV. Lastly, we have for the first time characterized salivary microbes of Aedes mosquitoes necessitating the investigation of the impact of salivary microbes in severity of disease in vertebrate hosts.

microbiology↗

bioRxiv2019

Designer DNA architecture offers precise and multivalent spatial pattern-recognition for viral sensing and inhibition

DNA, when folded into nanostructures of customizable shapes, is capable of spacing and arranging external ligands in a desired geometric pattern with nanometer-precision. This allows DNA to serve as an excellent, biocompatible scaffold for complex spatial pattern-recognizing displays. In this report, we demonstrate that a templated designer DNA nanostructure achieves multi-ligand display with precise spatial pattern-recognition, representing a unique strategy in synthesizing potent viral sensors and inhibitors. Specifically, a star-shaped DNA architecture, carrying five molecular beacon-like motifs, was constructed to display ten dengue virus envelope protein domain-III (ED3)-binding aptamers into a 2D pattern precisely matching the pentagonal arrangement of ED3 clusters on the dengue viral surface. The resulting spatial pattern recognition and multivalent interactions achieve high dengue-binding avidity, conferring direct, highly-sensitive, facile, low-cost, and rapid sensing as well as potent viral inhibition capability. Our molecular-platform design strategy could be adapted to detect and combat other disease-causing pathogens, including bacteria and microbial-toxins, by generating the requisite ligand patterns on customized DNA nanoarchitectures.

bioengineering↗

bioRxiv2018

Assessing Combinability of Phylogenomic Data using Bayes Factors

With the rapid reduction in sequencing costs of high-throughput genomic data, it has become commonplace to use hundreds of genes/sites to infer phylogeny of any study system. While sampling large number of genes has given us a tremendous opportunity to uncover previously unknown relationships and improve phylogenetic resolution, it also presents us with new challenges when the phylogenetic signal is confused by differences in the evolutionary histories of sampled genes. Given the addition of accurate marginal likelihood estimation methods into popular Bayesian software programs, it is natural to consider using the Bayes Factor (BF) to compare different partition models in which genes within any given partition subset share both tree topology and edge lengths. We explore using marginal likelihood to assess data subset combinability when data subsets have varying levels of phylogenetic discordance due to deep coalescence events among genes (simulated within a species tree), and compare the results with our recently-described phylogenetic informational dissonance index (D) estimated for each data set. BF effectively detects phylogenetic incongruence, and provides a way to assess the statistical significance of D values. We discuss methods for calibrating BFs, and use calibrated BFs to assess data combinability using an empirical data set comprising 56 plastid genes from green algae order Volvocales.

evolutionary biology↗