Kyung-A Kim

Fusobacterium nucleatum (Fn) is increasingly recognized as a cancer-associated bacterium, yet reliable quantification in human specimens is challenging due to low bacterial burden and abundant host DNA. We analyzed 145 Fusobacterium genomes to design primers targeting conserved regions of the fadA adhesin gene and developed a duplex quantitative real-time PCR (qPCR) assay for simultaneous detection of fadA and a human PGT as an internal control. Analytical sensitivity, specificity, precision, and reproducibility were evaluated using serially diluted Fn DNA, spike-in experiments with human DNA, and cross-platform/operator validation. Clinical performance was assessed in colorectal cancer patient tissues, including fresh tissue (n = 24) and formalin-fixed paraffin-embedded (FFPE) samples (n = 22), using 16S rRNA-based methods as references. The assay successfully detected all four major Fn subspecies (nucleatum, animalis, polymorphum, and vincentii). The limit of detection was ≤0.1 pg, with no interference between duplex targets. Spike-in experiments demonstrated consistent target detection in human-DNA-rich samples, with strong linearity (R² = 0.998) across dilutions. High precision (coefficient of variations < 5%) was observed across intra-day, inter-day, inter-instrument, and inter-operator evaluations. In fresh tissues, the assay yielded 86% sensitivity, 94% specificity, and 92% accuracy. Using the FFPE samples, the assay achieved 91% sensitivity and 100% specificity, confirming robust classification in both clinical samples. This duplex qPCR assay enables broad detection of Fn with high analytical performance in both fresh and FFPE tissues. Its simplicity, reproducibility, and compatibility with pathology workflows support deployment in multi-center studies and downstream applications in diagnostic studies and prognostic modeling.

Lipid nanoparticles (LNPs) are widely recognized as crucial carriers of mRNA in therapeutic and vaccine development. The typical lipid composition of mRNA-LNP systems includes an ionizable lipid, a helper lipid, a polyethylene glycol (PEG)-lipid, and cholesterol. Concerns arise regarding cholesterol's susceptibility to oxidation, potentially leading to undesired immunological responses and toxicity. In this study, we formulated novel LNPs by replacing cholesterol with phytochemical-derived compounds, specifically ginsenoside Rg2 and its derivative phytosterol protopanaxadiol (PPD), and validated their efficacy as mRNA delivery systems. The mRNA–LNP complexes were manually prepared through a simple mixing process. The biocompatibility of these Rg2-based LNPs (Rg2-LNP) and PPD-based LNPs (PPD-LNP) was assessed through cell viability assays, while the protective function of LNPs for mRNA was demonstrated by RNase treatment. Enhanced green fluorescent protein (EGFP) mRNA delivery and expression in A549 and HeLa cells were analyzed using optical microscopy and flow cytometry. The expression efficiency of Rg2-LNP and PPD-LNP was compared with that of commercially available LNPs, with both novel formulations demonstrating superior transfection and EGFP expression. Furthermore, in vivo tests following intramuscular (I.M.) injection in hairless mice demonstrated efficient luciferase (Luc) mRNA delivery and effective Luc expression using Rg2-LNP and PPD-LNP compared to commercial LNPs. Results indicated that the efficiency of EGFP and Luc expression in Rg2-LNP and PPD-LNP surpassed that of the cholesterol-based LNP formulation. These findings suggest that Rg2-LNP and PPD-LNP are promising candidates for future drug and gene delivery systems.

This study aimed to investigate the levels of aqueous humour cytokines in the eyes of patients with central serous chorioretinopathy (CSC), who received an intravitreal injection of bevacizumab, and to evaluate the differences in cytokine levels between bevacizumab responders and non-responders. Aqueous humour samples were collected from 39 eyes of 39 patients with CSC and 25 eyes of 25 control patients undergoing cataract surgery and were analysed using Multiplex bead assays. We compared the cytokine levels in patients with CSC (responders and non-responders) and control subjects. After intravitreal injections of bevacizumab in patients with CSC, we evaluated the changes in visual acuity and optical coherence tomographic parameters. Significantly increased levels of interferon gamma-induced protein (IP-10) were detected in the aqueous humour of eyes from patients with CSC compared to controls (p = 0.036). Vascular endothelial growth factor (VEGF) levels were significantly higher in bevacizumab responders than in non-responders (p = 0.021). The concentrations of the other cytokines investigated did not differ significantly between patients with CSC and control subjects or between responders and non-responders. The results of the present study suggest that IP-10 and VEGF may contribute to the pathogenesis of CSC. It is clear that intravitreal bevacizumab was helpful in some of the patients with CSC, who showed higher levels of VEGF in the aqueous humour. However, the true effect and the rationale of anti-VEGF injection treatment in patients with CSC remain to be elucidated. Our results suggest that patients with CSC may be more susceptible to VEGF and/or other cytokines than the normal population.

Park KS, et al. Korean J Physiol Pharmacol. 2013 Dec;17(6):511-516. http://dx.doi.org/10.4196/kjpp.2013.17.6.511