Biology subjects

Zhuo, S.

Publications and source records attributed to Zhuo, S..

5 recordsLinked to original sources

bioRxiv2025

Ultrahigh-Throughput Forster Resonance Energy Transfer-Activated Droplet Sorting for Terminator Polymerase Engineering

Droplet microfluidic screening systems enable high-throughput, labor-saving enzyme directed evolution by employing fluorescence, absorbance, and Raman-activated sorting strategies for library screening. Forster resonance energy transfer (FRET) - a nanoscale technique for monitoring intramolecular/intermolecular conformational changes - is yet to be integrated into this process. We upgraded a single-channel sorter to a dual-channel one without redesigning the microscopy setup, which can monitor FRET signals during enzyme reactions in droplets at kilohertz rates. We applied this upgraded sorter to improve the incorporation efficiency of KOD DNA polymerase towards reversible terminator deoxyribonucleotides, a property crucial for its application in next-generation sequencing (NGS). Our data show that a single-round sorting can achieve 30-fold enrichment of active variants. Five KOD variants enabling 100-cycle single-end runs of DNA sequencing were identified using a novel cyclic reversible termination (CRT) substrate featuring a terminator group {approx} 5-fold bulkier than the classical azidomethyl moiety. We also engineered a metagenome-derived novel polymerase, and a variant achieving 90% terminator incorporation efficiency within 2 minutes was identified after two rounds of enrichment. In sum, we provide a practical setup for dual-channel FRET-based droplet sorting, and demonstrate its ability in terminator polymerases engineering, thereby broaden the scope of microfluidic applications.

biochemistry↗

bioRxiv2025

3-Phenylpropionic acid, a microbiota-derived polyphenol metabolite linked to hippuric acid, ameliorates colitis as a colon-enriched PPAR-γ agonist

Polyphenols are promising therapeutics for Crohns disease (CD), yet their bioavailability is largely dependent on the gut microbiome. The bioactive metabolites, metabolic pathways, and anti-inflammatory mechanisms underlying their effects remain poorly defined. We identified hippuric acid (HIPA) as a biomarker of polyphenol-microbiome interaction. HIPA is a meta-organismal metabolite produced through a cross-organ pathway that involves synthesis from gut microbial polyphenol metabolism via 3-phenylpropionic acid (3-PPA), followed by renal clearance. We found that CD is characterised by impaired polyphenol metabolism, resulting in deficient colonic 3-PPA and reduced serum hippuric acid relative to healthy individuals. Furthermore, we demonstrate that 3-PPA is a colon-enriched, mild PPAR-{gamma} agonist that ameliorates colitis in murine model. Systemic bioinformatic profiling of metagenomic data suggested the existence of undiscovered 3-PPA producers. Through targeted microbial incubation with polyphenols, we identified the probiotic Bifidobacterium breve and a lab isolate Escherichia coli FAH as novel 3-PPA producers. Collectively, our findings establish 3-PPA as a diet-dependent, colon-produced, mild PPAR-{gamma} agonist with the potential to minimise adverse effects associated with traditional PPAR-{gamma} agonists. This work establishes a foundation for developing microbiota-targeted, polyphenol-based therapeutic strategies for CD patients.

microbiology↗

bioRxiv2024

YAP inhibits NF-κB signaling and ccRCC growth by opposing p65-ZHX2 cooperativity

The prevailing view in the cancer field is that Hippo signaling pathway functions as a tumor suppressor pathway by blocking the oncogenic potential of the pathway effectors Yes1 associated transcriptional regulator (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ). However, YAP can also function as a context-dependent tumor suppressor in several types of cancer including clear cell renal cell carcinomas (ccRCC). We find that, in additional to inhibiting hypoxia-inducible factor 2 (HIF2), a major oncogenic driver in Von Hippel-Lindau (VHL)-/- ccRCC, YAP also blocks nuclear factor {kappa}B (NF-{kappa}B) signaling in ccRCC to inhibit cancer cell growth under conditions where HIF2 is dispensable. Mechanistically, YAP inhibits the expression of Zinc fingers and homeoboxes 2 (ZHX2), a VHL substrate and critical co-factor of NF-{kappa}B in ccRCC. Furthermore, YAP competes with ZHX2 for binding to the NF-{kappa}B subunit p65. Consequently, elevated nuclear YAP blocks the cooperativity between ZHX2 and the NF-{kappa}B subunit p65, leading to diminished NF-{kappa}B target gene expression. Pharmacological inhibition of Hippo kinase blocked NF-{kappa}B transcriptional program and suppressed ccRCC cancer cell growth, which can be rescued by overexpression of ZHX2 or p65. Our study uncovers a crosstalk between the Hippo and NF-{kappa}B/ZHX2 pathways and its involvement in ccRCC growth inhibition, suggesting that targeting the Hippo pathway may provide a therapeutical opportunity for ccRCC treatment.

cancer biology↗

bioRxiv2022

YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth

Hippo signaling restricts tumor growth by inhibiting the oncogenic potential of YAP/TAZ-TEAD transcriptional complex. Here we uncover a context-dependent tumor suppressor function of YAP in androgen receptor (AR) positive prostate cancer (PCa) and show that YAP impedes AR+ PCa growth by antagonizing TEAD-mediated AR signaling. TEAD forms a complex with AR to enhance its promoter/enhancer occupancy and transcriptional activity. YAP and AR compete for TEAD binding and consequently, elevated YAP in the nucleus disrupts AR-TEAD interaction and prevents TEAD from promoting AR signaling. Pharmacological inhibition of Hippo/MST1/2 kinase or transgenic activation of YAP suppressed the growth of PCa expressing therapy resistant AR splicing variants. Our study uncovers an unanticipated crosstalk between Hippo and AR signaling pathways, reveals an antagonistic relationship between YAP and TEAD in AR+ PCa, and suggests that targeting the Hippo signaling pathway may provide a therapeutical opportunity to treat PCa driven by therapy resistant AR variants. O_LIYAP acts as a context-dependent tumor suppressor in AR+ PCa C_LIO_LITEAD interacts with AR to enhance its promoter/enhancer occupancy C_LIO_LIYAP inhibits AR activity by competing for TEAD binding C_LIO_LISmall molecule Hippo pathway inhibitor impedes AR+ PCa growth C_LI

cancer biology↗

bioRxiv2020

The electrically conductive pili of Geobacter soli

Electrically conductive pili (e-pili) enable electron transport over multiple cell lengths to extracellular environments and play an important role in extracellular electron transfer (EET) of Geobacter species. To date, the studies of e-pili have mainly focused on Geobacter sulfurreducens and the closely related Geobacter metallireducens because of their developed genetic manipulation systems. We investigated the role of G. soli pili in EET by directly deleting the pilin gene, pilA, which is predicted to encode e-pili. Deletion of pilA, prevented the production of pili, resulting in poor Fe(III) oxide reduction and low current production, implying that G. soli pili is required for EET. To further evaluate the conductivity of G. soli pili compared with G. sulfurreducens pili, the pilA of G. soli was heterologously expressed in G. sulfurreducens, yielding the G. sulfurreducens strain GSP. This strain produced abundant pili with similar conductivity to the control strain that expressed native G. sulfurreducens pili, consistent with G. soli as determined by direct measurement, which suggested that G. soli pili is electrically conductive. Surprisingly, strain GSP was deficient in Fe(III) oxide reduction and current production due to the impaired content of outer-surface c-type cytochromes. These results demonstrated that heterologous pili of G. sulfurreducens severely reduces the content of outer-surface c-type cytochromes and consequently eliminates the capacity for EET, which strongly suggests an attention should be paid to the content of c-type cytochromes when employing G. sulfurreducens to heterologously express pili from other microorganisms. IMPORTANCEThe studies of electrically conductive pili (e-pili) of Geobacter species are of interest because of its application prospects in electronic materials. e-Pili are considered a substitution for electronic materials due to its renewability, biodegradability and robustness. Continued exploration of additional e-pili of Geobacter soli will improve the understanding of their biological role in extracellular electron transfer and expand the range of available electronic materials. Heterologously expressing the pilin genes from phylogenetically diverse microorganisms has been proposed as an emerging approach to screen potential e-pili according to high current densities. However, our results indicated that a Geobacter sulfurreducens strain heterologously expressing a pilin gene produced low current densities that resulted from a lack of content of c-type cytochromes, which were likely to possess e-pili. These results provide referential significance to yield e-pili from diverse microorganisms.

microbiology↗