Biology subjects

Kuo, J.

Publications and source records attributed to Kuo, J..

9 recordsLinked to original sources

bioRxiv2026

Five decades of human H1N1 evolution reveal conserved glycan receptor specificity

H1N1 influenza viruses have circulated continuously among human populations for five decades. During evolution, the hemagglutinin has accumulated amino acid substitutions in and around the receptor-binding site (RBS), mainly due to antigenic pressure, potentially altering receptor recognition and binding profiles. Here, we investigated the glycan-binding properties of H1N1 strains collected from 1977 through 2024 using ELISA, flow cytometry, and tissue histochemistry to characterize changes in receptor-binding specificity over time. Across the strains examined, we observe a recurring preference for binding 2,6-sialylated di-LacNAc N-glycans. Binding to 2,3-sialylated glycans was generally undetectable, although A/Hongkong/117/1977 and A/New Caledonia/20/1999 bind chicken tracheal tissues. Interestingly, post-pandemic H1N1 viruses fluctuate in their relative binding avidities. The responsible amino acids, in turn, have different effects in pre-pandemic HA proteins. We also report a classical egg-adapted H1N1 mutant that shows null binding across platforms. These findings highlight the contribution of RBS mutations during initial adaptation and evolution of human H1N1 influenza viruses to glycan-binding properties, which, in contrast to H3N2 viruses, never fail to bind glycans that correlate with avian erythrocyte hemagglutination.

microbiology↗

bioRxiv2025

The Secreted Metabolite Isopentenyladenine from Faecalibacterium prausnitzii Mitigates Gut Inflammation

Colonic microbiome dysbiosis is correlated with inflammatory bowel disease (IBD), and depletion of the commensal bacterium Faecalibacterium prausnitzii (F. prausnitzii) is routinely observed in the metagenomic analyses of IBD patient microbiome samples. F. prausnitzii is likely beneficial to hosts, as oral administration of F. prausnitzii strain A2-165 has anti-inflammatory properties in murine models of colitis. Previous studies attribute the anti-inflammatory effects of F. prausnitzii A2-165 to production of the short-chain fatty acid butyrate, as well as a 15 kDa protein known as microbial anti-inflammatory molecule (MAM). Here, we verified that oral dosing of strain A2-165 protects against DSS-induced murine colitis and further show the aqueous-soluble secreted fraction of overnight cultures from a collection of F. prausnitzii strain inhibit inflammatory signatures, including the activation of the hosts NF-{kappa}B pathway, production of IL-8, and differentiation of naive T cells into the TH17 lineage. We find that MAM is secreted in extracellular vesicles; however, MAM-containing vesicles do not have anti-inflammatory properties in our collection of assays and suggests that MAM is likely not a direct contributor. Untargeted and targeted mass spectrometry metabolomics analyses on the soluble anti-inflammatory secretome yielded several unique F. prausnitzii metabolites, including isopentenyladenine. We demonstrated isopentenyladenine independently modulates host cellular signaling and immune responses and suggests this newly identified metabolite with human immunomodulatory properties may be useful towards the discovery of IBD-focused therapeutics.

microbiology↗

bioRxiv2025

Polyserine-tau interactions modulate tau fibrilization

Tau aggregates are the defining feature of multiple neurodegenerative diseases and contribute to the pathology of disease. However, the molecules affecting tau aggregation in cells are unclear. We previously determined that polyserine-rich domain containing proteins enrich in tau aggregates, assemble into puncta that can serve as sites of tau aggregation, and exacerbate tau aggregation in cells and mice. Herein, we show that polyserine domains are sufficient to define assemblies as sites of tau aggregation, in part, through localization of tau seeds. Purified polyserine self-assembles and directly interacts with monomeric and fibrillar tau. Moreover, polyserine-tau assemblies recruit RNA, leading to faster rates of tau fibrillization in vitro. Using polyserine variants, we found that enrichment in tau aggregates and stimulation of tau aggregation are separable functions of polyserine domains, with polyserine self-assembly stimulating tau aggregation. Together, our results show that polyserine self-assembles and directly interacts with tau to form preferred sites of tau aggregation.

biochemistry↗

bioRxiv2022

Cytosolic condensates enriched in polyserine repeats are preferred sites of tau fiber propagation

Tau aggregates are a hallmark of multiple neurodegenerative diseases and can contain RNAs and RNA binding proteins, including SRRM2 and PNN. How these resident nuclear proteins mislocalize and their influence on the prion-like propagation of tau fibers remains unknown. We demonstrate that polyserine repeats in SRRM2 and PNN are necessary and sufficient for recruitment to tau aggregates. Moreover, we demonstrate tau fibers preferentially grow in association with endogenous cytoplasmic assemblies - mitotic interchromatin granules and cytoplasmic speckles - which contain SRRM2 and PNN. Polyserine undergoes self-assembly in vitro and in cells, where polyserine-assemblies are sites of tau fiber propagation. Modulating the levels of polyserine containing proteins results in a corresponding change in tau aggregation. These findings define a specific protein motif, and cellular condensates, that promote tau fiber propagation. As cytoplasmic speckles form in iPSC neurons under inflammatory or hyperosmolar stress, they may promote tau fiber propagation in various neurodegenerative diseases.

neuroscience↗

bioRxiv2022

Phylogeny and Metadata Network Database for Epidemiologic Surveillance

AO_SCPLOWBSTRACTC_SCPLOWThe ongoing SARS-CoV-2 pandemic has highlighted the difficulty in integrating disparate data sources for epidemiologic surveillance. To address this challenge, we have created a graph database to integrate phylogenetic trees, associated metadata, and community surveillance data for phylodynamic inference. As an example use case, we divided 22,713 SARS-CoV-2 samples into 5 groups, generated maximum likelihood trees, and inferred a potential transmission network from a forest of minimum spanning trees built on patristic distances between samples. We then used Cytoscape to visualize the resultant graphs.

evolutionary biology↗

bioRxiv2020

Towards a translationally-independent RNA-based synthetic oscillator using deactivated CRISPR-Cas

In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nucleotide guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging ∼9 generations, a dCas12a oscillator design with 40-nucleotide CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ hours at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally-independent behavior, as the RNAs control each others’ transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Complete Reconstitution and Deorphanization of the 3 MDa NOCAP (NOCardiosis-Associated Polyketide) Synthase

Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly-line polyketide synthase (PKS) that presumably synthesizes an unknown natural product. Here, we report the discovery and structure elucidation of the NOCAP (NOCardiosis-Associated Polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly-line PKS either in vitro or in E. coli, and of using these approaches to "deorphanize" a complete assembly-line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.

biochemistry↗

bioRxiv2017

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

Harnessing CRISPR-Cas9 technology has provided an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We have analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in the budding yeast Saccharomyces cerevisiae and find that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a one base pair (+1) insertion that appears to result from Cas9 creating a 1-bp 5 overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which were biased toward homonucleotide insertions. Using inverted PAM (iPAM) sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9::gRNA complex influences subsequent NHEJ processing. As with well-studied events induced by the site-specific HO endonuclease, CRISPR-Cas9 mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events, however, are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells (van Overbeek et al., 2016) reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

molecular biology↗

bioRxiv2017

Rapid genome recoding by iterative recombineering of synthetic DNA

Genome recoding will provide a deeper understanding of genetics and transform biotechnology. We bypass the reliance of previous genome recoding methods on site-specific enzymes and demonstrate a rapid recombineering based strategy for writing genomes by Stepwise Integration of Rolling Circle Amplified Segments (SIRCAS). We installed the largest number of codon substitutions in a single organism yet published, creating a strain of Salmonella typhimurium with 1557 leucine codon changes across 200 kb of the genome.

synthetic biology↗