Biomedical subjects
Publications and source records attributed to Ning Sun.
We report the draft genomes of two Enterococcus faecium blood isolates: one vancomycin-susceptible and one vancomycin-resistant. The assemblies comprised 2,991,512 bp (7 contigs) for the susceptible strain and 3,305,414 bp (7 contigs) for the resistant strain.
Deciphering the functionality of the noncoding genome, which includes important cis-regulatory elements (CREs) and transcribed noncoding RNA genes, remains technically challenging. Here, using massively parallel genetic screening, we systematically benchmark the performance of five representative loss-of-function perturbation tools, including single-guide RNA (gRNA) mediated SpCas9 cleavage or CRISPR interference, and paired gRNA (pgRNA) involved dual-SpCas9, Big Papi (paired SpCas9 and SaCas9) or dual-enAsCas12a fragment deletion methods, in decoding the roles of the noncoding genome. For targeting CREs such as enhancers, dual-SpCas9 outperforms other methods with superior efficiency in destroying functional genomic regions. For perturbing noncoding RNA genes, in addition to dual-SpCas9, other RNA-targeting methods such as RNA interference are recommended to discriminate transcript-dependent or -independent roles. A deep learning model, DeepDC, with an associated web server, is built to facilitate optimal dual-SpCas9 pgRNA design for efficiently deleting a genomic fragment. Together, our work provides practical guidance on selecting appropriate loss-of-function tools to resolve the functional complexity of the noncoding genome.
Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12 cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B) ≈ 9, coefficient of variance (CV) < 10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.
Enterococci are common commensals of the human gut and important opportunistic pathogens, with Enterococcus faecium and Enterococcus faecalis being the most clinically prevalent species. A significant epidemiological shift has emerged with an increasing clinical burden of E. faecium. To compare genomic evolution of E. faecium and E. faecalis, we performed whole-genome sequencing on 93 E. faecium and 32 E. faecalis isolates causing bloodstream infections at a single hospital (2022-2024). Analysis of patient demographics revealed that E. faecium infections originated from fewer sources than E. faecalis, with a higher proportion deriving from intra-abdominal infections. Multilocus sequence typing identified ST78 and ST789 as the predominant sequence types for E. faecium, whereas ST16 and ST179 were most common for E. faecalis. E. faecium carried more antimicrobial resistance genes and putative virulence marker (PVM)-type virulence genes than E. faecalis, with vancomycin resistance predominantly mediated by vanHAX (33/93, 35.5%) and a single E. faecalis isolate also carrying vanHAX (1/32, 3.1%); the structurally incomplete vanHMX gene cluster was detected in 11 E. faecium isolates. Pan-genome analysis indicated a larger core genome in E. faecalis compared to E. faecium, consistent with greater plasmid replicon diversity in the latter. Intra-host comparisons showed that two E. faecalis pairs from the same patient were clonally related, with one isolate acquiring a vanHAX plasmid conferring vancomycin resistance. In contrast, E. faecium isolates exhibited marked genomic diversity even among clonally related pairs. These findings suggest that E. faecium possesses greater genomic plasticity and adaptive potential to the clinical environment.IMPORTANCEThis study provides a detailed comparison of clinical and genomic features between Enterococcus faecium and Enterococcus faecalis from the same hospital setting. We show that E. faecium isolates, mainly ST78/ST789, carry more antimicrobial resistance genes and a higher number of putative virulence marker (PVM) genes than E. faecalis, reflecting their hospital-adapted nature. E. faecium also exhibits a smaller core genome and greater diversity of plasmid replicon types, indicating higher genomic plasticity and capacity for horizontal gene transfer. By contrast, E. faecalis retains a larger core genome and a set of classical virulence factors, and its within-host isolates are clonally related. These distinct genomic profiles help to understand how the two species adapt to clinical environments and may inform more targeted infection control strategies and resistance surveillance.