Biology subjects

Ko, J.-H.

Publications and source records attributed to Ko, J.-H..

8 recordsLinked to original sources

bioRxiv2026

Quantitative Optimization of Sensitivity and Specificity in Targeted and Whole-Exome Sequencing Using Reference-Standard DNA Mixtures

BackgroundWe previously developed a benchmarking strategy using mixtures of homozygote and heterozygote DNAs as reference standards to simultaneously assess sensitivity and false positive (FP) error rates in targeted next-generation sequencing (T-NGS) and whole-exome sequencing (WES), revealing substantial variability across commercial platforms. However, optimal analytic conditions for clinical application remain undefined. MethodsWe systematically evaluated multiple sequencing kits and bioinformatics pipelines across various variant allele fraction (VAF) thresholds to identify conditions that maximize both sensitivity and specificity. Recurrent error-prone alleles were defined and filtered to enhance specificity. ResultsOptimal performance was achieved using the DRAGEN pipeline with recurrent FP allele filtering. For T-NGS, a 1% VAF cutoff yielded a 95% detection threshold of 2.99% and 1.21 FPs per megabase (FP/Mb); for WES, a 2% cutoff yielded a 95% threshold of 5.02% and 1.15 FP/Mb. These settings improved sensitivity >3-fold and reduced FP rates >96% versus suboptimal pipelines. Notably, VAF thresholds flattened sensitivity differences across platforms, obscuring key performance disparities--challenging assumptions that T-NGS is inherently more sensitive than WES. In-house and conventional pipelines undercalled up to 10% of true variants. Restricting reporting of 1-4% VAF variants to [~]1,000 predefined actionable sites enabled recovery of clinically relevant mutations while reducing FP risk >99%. ConclusionsThis study provides a quantitative framework for optimizing NGS performance. Our findings support actionable strategies to improve diagnostic accuracy in clinical genomics through tailored pipeline selection, VAF thresholding, and artifact filtering.

bioinformatics↗

bioRxiv2024

Targeted Degradation of MYC2 through MPK6 Phosphorylation Reveals Mechanisms and Implications for JA Signaling

MYC2 is a key regulator in the Jasmonic acid (JA) signaling pathway, yet the mechanisms governing its stability remain unclear. In this study, we demonstrate that MYC2 is degraded by MPK6 under JA signaling. Through yeast two-hybrid and co-immunoprecipitation assays, and in vitro kinase assay, we show that MPK6 directly interacts with and phosphorylates MYC2. Additionally, MYC2 transcriptional activity is enhanced in the mpk6 mutant. Further, MPK6 phosphorylates MYC2 at threonine 328, leading to its degradation. These findings suggest that MPK6 drives MYC2 degradation by specifically phosphorylating threonine 328 within the JA signaling pathway.

plant biology↗

bioRxiv2023

BCAT1 inhibition affects CD8+ T cell activation, exhaustion, and tumoral immunity by altering iron homeostasis

The present study explores the role of the cytosolic branched chain amino acid aminotransferase (BCAT1) in CD8+ T cell activation, in general, and tumor immunity, in particular, and identifies a non-canonical function of the protein in iron homeostasis. Pharmacologic inhibition of BCAT1 using the novel drug ERG245 abrogates the effector functions of CD8+ T cells in vitro and metabolically reprograms the cells towards increased OXPHOS. In vivo, it suppresses activation of CD8+ T cells in DSS colitis leading to improved disease outcomes. Remarkably, withdrawal of BCAT1 inhibition further amplifies OXPHOS and gives rise to CD8+ T cells with increased cytotoxicity in vitro and in vivo. When combined with an anti-PD-1 treatment, temporal BCAT1 inhibition dramatically increases anti-PD-1 efficacy inducing complete and durable tumor regressions in the moderately immunogenic CT26 tumor model. Single cell RNA-seq data link expression of Bcat genes to exhausted T cells within the tumor microenvironment of human cancer patients, whereas in vitro assays indicate that BCAT1 inhibition partially prevents the adoption of a terminally exhausted phenotype by CD8+ T cells. We propose BCAT1 as a target for cancer combinatory therapies. SIGNIFICANCEThe study explores for the first time the role of BCAT1 in CD8+ T cell activation and proposes novel strategies for using BCAT1 inhibitors in cancer and beyond. It demonstrates that BCAT1 exerts its function without significantly altering branched chain amino acid (BCAA) levels through a mechanism that controls iron homeostasis, a novel non-canonical mechanism of action, and implicates BCAT1 in the adoption of an exhausted phenotype by T cells found in human cancers. While the majority of metabolic drugs temper OXPHOS, it demonstrates that an agent that increases OXPHOS in CD8+ T cells can be used successfully as an immune-oncology drug.

immunology↗

bioRxiv2022

Single-cell Mendelian randomisation identifies cell-type specific genetic effects on human brain disease and behaviour

Translating genome-wide association loci to therapies requires knowledge of the causal genes, their directionality of effect and the cell-types in which they act. To infer these relationships in the human brain, we implemented Mendelian randomisation using single cell-type expression quantitative trait loci (eQTLs) as genetic anchors. Expression QTLs were mapped across 8 major cell-types in brain tissue exclusively ascertained from donors with no history of brain disease. We report evidence for a causal association between the change in expression of 118 genes and one or more of 16 brain phenotypes, revealing candidate targets for risk mitigation and opportunities for shared preventative therapeutic strategies. We highlight key causal genes for neurodegenerative and neuropsychiatric disease and for each, we report its cellular context and the therapeutic directionality required for risk mitigation. Our use of control samples establishes a new resource for the causal interpretation of GWAS risk alleles for human brain phenotypes.

neuroscience↗

bioRxiv2022

Multinucleation resets human macrophages for specialized functions at the expense of mononuclear phagocyte identity

Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign-body giant cells (FBGCs) in reaction to exogenous material. While osteoclast multinucleation is a prerequisite for vertebrate bone homeostasis, the effector function resulting from LGC and FBGC multinucleation is less well-defined. More generally, how multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrated comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced down-regulation of mononuclear phagocyte identity. We show enhanced lysosome-mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, only B7-H3 (CD276)-expressing human LGCs can form granuloma-like clusters in vitro, suggesting that LGC multinucleation potentiates T cell activation. These findings demonstrate how cell-cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC-specific functionality.

cell biology↗

bioRxiv2020

Cardiac glycosides cause selective cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis

Cardiac glycosides (CGs) inhibit the Na+,K+-ATPase and are widely prescribed medicines for chronic heart failure and cardiac arrhythmias. Recently, CGs have been described to induce inflammasome activation in human macrophages, suggesting a cytotoxicity that remains to be elucidated in tissues. Here we show that human monocyte-derived macrophages (hMDMs) undergo cell death following incubation with nanomolar concentrations of CGs, and in particular with ouabain (IC50=50 nM). The ouabain-induced cell death is more efficient in hMDMs compared to non-adherent PBMC populations and is through on-target inhibition of Na,K-ATPAse activity, as it causes an intracellular depletion of K+, while inducing accumulation of Na+ and Ca2+ levels. Consistently, the cell-death caused by these ion imbalances can be rescued by addition of potassium chloride in hMDMs. Strikingly, white adipose tissue (WAT) explants from morbidly obese patients cultured with nanomolar concentrations of ouabain causes depletion of macrophages, decreases type VI collagen levels, and ameliorates insulin-sensitivity ex vivo. These results suggest that the usage of nanomolar concentration of CGs can be an attractive therapeutic avenue in metabolic syndrome characterised by pathogenic infiltration and activation of macrophages.

immunology↗

bioRxiv2020

A trans-eQTL network regulates osteoclast multinucleation and bone mass

Functional characterisation of cell-type specific regulatory networks is key to establish a causal link between genetic variation and phenotype. The osteoclast offers a unique model for interrogating the contribution of co-regulated genes to in vivo phenotype as its multinucleation and resorption activities determine quantifiable skeletal traits. Here we took advantage of a trans-regulated gene network (MMnet, macrophage multinucleation network) which we found to be significantly enriched for GWAS variants associated with bone-related phenotypes. We found that the network hub gene Bcat1 and seven other co-regulated MMnet genes out of 13, regulate bone function. Specifically, global (Pik3cb-/-, Atp8b2+/-, Igsf8-/-, Eml1-/-, Appl2-/-, Deptor-/-) and myeloid-specific Slc40a1{Delta}LysMCre knockout mice displayed abnormal bone phenotypes. We report antagonizing effects of MMnet genes on bone mass in mice and osteoclast multinucleation/resorption in humans with strong correlation between the two. These results identify MMnet as a functionally conserved network that regulates osteoclast fusion and bone mass. Impact statementWe took advantage of the osteoclast whose multinucleation properties correlate with bone mass. We show that a trans-regulated gene network (MMnet) controls skeletal homeostasis through osteoclast multinucleation and function.

cell biology↗

bioRxiv2017

Deferiprone, an iron chelator, is preventive and therapeutic in experimental crescentic glomerulonephritis

Crescentic glomerulonephritis represents the most severe form of antibody-mediated glomerulonephritis. It is an important cause of renal dysfunction worldwide and there is a need for more effective treatment. Deferiprone, an orally active iron chelator, is widely used in patients with thalassemia. Here we present the preventive and therapeutic effects of deferiprone in experimental crescentic glomerulonephritis. Nephrotoxic nephritis was induced in Wistar Kyoto rats, and preventive treatment with deferiprone substantially lowered glomerular crescent formation by 84%, with 70% reduction in proteinuria. In established glomerulonephritis, deferiprone treatment effectively halted glomerular inflammation, reversed progression of proteinuria, and prevented deterioration of renal function. Deferiprone reduced glomerular inflammatory cell proliferation in vivo. It was internalised by monocyte/macrophages and inhibited their proliferation in vitro, without showing cellular toxicity. Interestingly, deferiprone showed a neutralizing effect on superoxide anions, and prevented the expression of monocyte chemoattractant protein-1 and matrix metalloproteinase 9, 12 and 14, by primary macrophages. These results suggest that deferiprone partly exerts its renal protective effect through inhibition of monocyte/macrophage proliferation and function by iron-chelating and anti-oxidant properties, respectively. We conclude that deferiprone is an effective treatment in a severe and reproducible model of antibody-mediated glomerular inflammation that resembles human crescentic glomerulonephritis, indicating its therapeutic potential.

immunology↗