Biology subjects

Jang, M.

Publications and source records attributed to Jang, M..

8 recordsLinked to original sources

bioRxiv2025

Rapid CAR Screening and circRNA-driven CAR-NK Cells for Persistent Shed-resistant Immunotherapy

Chimeric antigen receptor (CAR)-based immunotherapies against solid tumors face two major hurdles, the "decoy effect" of shedding antigens that sequester CARs, and the limited persistence of immune effectors within the immunosuppressive tumor microenvironment. Here, we present a mechanistic approach to overcome these barriers by integrating a physiologically relevant screening platform with circular RNA (circRNA) engineering. Unlike conventional screens using immortalized cell lines, we performed rapid functional screening directly in human primary natural killer (NK) cells to identify a novel scFv, CLMS10. Structural modeling revealed that CLMS10 targets a membrane-proximal epitope that overlaps the proteolytic cleavage site, thereby evading inhibition by soluble mesothelin (solMSLN). Furthermore, we demonstrated that circRNA-mediated CAR expression, when codelivered with interleukin-21 (IL-21), confers sufficient stability to withstand the continuous antigen shedding induced by cancer-associated fibroblasts (CAFs), resulting in reduced CAR downregulation. In an in vivo metastatic pancreatic cancer model, IL-21-augmented circCAR-MS10-NK cells exhibited potency comparable to that of lentivirally engineered CAR-NK cells while offering superior manufacturability. Overall, this study establishes a paradigm for generating shed-resistant CAR therapeutics through the strategic integration of epitope-specific functional selection and enhanced RNA stability.

bioengineering↗

bioRxiv2024

Elucidating DNA-binding protein dynamics in Salmonella Typhimurium within macrophages using a breakthrough low-input ChIP-exo approach

Genome-wide identification of binding profiles for DNA-binding proteins in Salmonella Typhimurium within macrophages is crucial for understanding virulence gene expression and cellular processes. However, this task remains challenging due to the limited amount of intracellular bacterial cells. Here, we present ChIP-mini, a low-input ChIP-exo utilizing 5,000-fold reduced number of initial bacterial cells and analysis pipeline (DiffExo), to identify genome-wide binding dynamics of DNA-binding proteins in host-infected pathogens. Applying ChIP-mini to intracellular S. Typhimurium, we identified 642 and 1,837 binding sites of H-NS and RpoD, respectively, with near-base pair resolution, elucidating their roles in transcriptional initiation. Post-infection, we observed 21 significant reductions in H-NS binding at intergenic regions, exposing the promoter region of virulence genes, such that those in Salmonella pathogenicity islands-2, 3 and effectors, facilitating RpoD binding for transcription initiation. Furthermore, we identified 24 novel RpoD binding sites and 19 significantly increased RpoD bindings at the transcription start sites of virulence genes. These findings substantially enhance our understanding of how H-NS and RpoD simultaneously coordinate the transcription initiation of virulence genes within macrophages. Collectively, this optimized method demonstrates a tool that can be broadly adapted to elucidate transcriptional regulatory networks of host-infected pathogens, revealing critical interactions between host and microbe.

systems biology↗

bioRxiv2023

Variant annotation across homologous proteins (Paralogue Annotation) identifies disease-causing missense variants with high precision, and is widely applicable across protein families

BackgroundDistinguishing pathogenic variants from those that are rare but benign remains a key challenge in clinical genetics, especially for variants not previously observed and characterised in humans. In vitro and in vivo functional characterisation are typically resource intensive, and model systems may not accurately predict influence on human disease. Many in silico tools have been developed to predict which variants are disease-causing, but typically lack precision. Here we demonstrate the applicability of a framework, called Paralogue Annotation, that draws on information from previously-characterised variants in homologous proteins to predict whether variants in a gene of interest are likely disease causing. MethodsWe assessed the performance of Paralogue Annotation through three orthogonal approaches: (1) comparison to established in silico variant prediction tools using 47,016 missense variants from ClinVar across 3,524 genes representing a broad range of diverse protein classes, by calculating precision and sensitivity; (2) evaluation against large-scale functional assays of variant effect; and (3) comparing odd ratios calculated from case-control association tests for inherited cardiac arrhythmia syndromes, and neurodevelopmental disorders with epilepsy, stratifying variants by Paralogue Annotation. ResultsParalogue Annotation correctly annotates 4,328 ClinVar pathogenic variants, with 245 false positives, yielding a precision of 0.95. This increases to 0.99 with more stringent annotation parameters (requiring greater conservation of amino acids between annotated orthologues) at the expense of sensitivity. Compared to established tools, Paralogue Annotation has higher precision for the identification of pathogenic variants, albeit with lower sensitivity across diverse test sets. Extending the technique by transferring annotations between homologous protein domains, rather than full-length protein paralogues, increases sensitivity. Rare variants predicted pathogenic by Paralogue Annotation were more strongly disease-associated (increased odds ratio) than unstratified rare variants for six out of eight genes tested with case-control cohort approaches. ConclusionsWe demonstrate that Paralogue Annotation has high precision for predicting pathogenic missense variants, providing a useful line of evidence for clinical variant interpretation that is complementary to other approaches in use. As the number of characterised variants increases in reference datasets such as ClinVar, Paralogue Annotation will further increase in sensitivity and applicability.

bioinformatics↗

bioRxiv2022

Glia-mediated cerebellar pain modulation

Clinical studies have revealed that the cerebellum is activated by noxious stimuli or pathological pain, and its removal results in somatosensory dysfunction. However, the neural circuits and molecular mechanisms underlying the processing of noxious information in the cerebellum remain unknown. Using two-photon microscopy and optogenetics in mice, we found that the locus coeruleus (LC) terminals in the cerebellar cortex release noradrenaline (NA) in response to cutaneous noxious electrical stimuli. Most Bergmann glia (BG) accumulated this LC-NA noxious information by increasing intracellular calcium in an integrative manner. This global calcium activation of BG, referred to as "flare," was also elicited in response to an intraplantar capsaicin injection. Chemogenetic inactivation of LC terminals or BG in the cerebellar cortex suppressed BG flares and reduced licking, a nocifensive behavior associated with capsaicin-induced pain. BG-specific knockdown of -1 adrenergic receptors also suppressed capsaicin-induced BG flares and licking. Chemogenetic activation of BG or an intraplantar capsaicin injection reduced Purkinje cell firings, which disinhibited the output activity of the deep cerebellar nuclei. These results suggest that BG in the cerebellar cortex play an essential role in computing noxious information ascending from the LC and modulate pain-related behaviors by controlling the activity of the cerebellar neural circuits. One Sentence SummaryBergmann glia mediate noxious information processing in the cerebellum

neuroscience↗

bioRxiv2021

In vitro validation of neoantigen prediction algorithm for developing personalized cancer vaccine therapy

BackgroundThe development of personalized neoantigen-based therapeutic cancer vaccines relies on computational algorithm-based pipelines. One of the critical issues in the pipeline is obtaining higher positive predictive value (PPV) performance, i.e., how many are immunogenic when selecting the top 5 to 20 candidate neoepitopes for the vaccination. We attempted to test the PPV of a neoepitope prediction algorithm Neopepsee. MethodsSix breast cancer patients and patient-derived xenografts from three lung cancer patients and their paired peripheral blood samples were subjected to whole-exome and RNA sequencing. Neoantigen was predicted using two different algorithms (Neopepsee and pVACseq). Response of induced memory T cells to neopeptide candidates was evaluated by IFN-{gamma} Enzyme-linked immune absorbent spot (ELISpot) assays of peripheral blood mononuclear cell (PBMC) from three HLA-matched donors. Positive ELISpot response to a candidate peptide in at least 2 of 3 donor PBMC was regarded as an immunogenic response. ResultsNeopepsee predicted 159 HLA-A matched neoepitope candidates out of 898 somatic mutations in nine patients (six breast and three lung cancer patients), whereas pVACseq predicted 84 HLA-A matched candidates. A total of 26 neopeptide candidates overlapped between the two predicted candidate pools. Among the candidates, 28 (20%, 28/ 137) and 15 (20%, 15/ 75) were positive by ELISpot assay, respectively. Among 26 overlapped candidates, 20 could be tested, and 7 of them (35%) were validated by ELISpot. Neopepsee identified at least one neoepitope in 7 of 9 patients (range 0-6), compared to 6 by pVACseq (range 0-5). ConclusionAs suggested by Tumor Neoantigen Selection Alliance (TESLA), our results demonstrate low PPV of individual prediction models as well as the complementary nature of the Neopepsee and pVACseq and may help design neoepitope targeted cancer vaccines. Our data contribute a significant addition to the database of tested neoepitope candidates that can be utilized to further train and improve the prediction algorithms.

immunology↗

bioRxiv2020

LH LepR neurons directly drive sustained food seeking behavior after AgRP neuronal deactivation via neuromodulation of NPY

The physiological need for energy evokes motivated feeding behaviours that help to ensure survival. However, the neural mechanisms underlying the generation of food motivation remain poorly understood. We investigated these mechanisms by subdividing feeding-related motivated behaviours into food-seeking, evaluating, and swallowing. Micro-endoscopic results indicated that neurons containing leptin receptors (LepRs) in the lateral hypothalamus (LH) are the major food-specific subpopulation of LHGABA neurons. Optogenetic manipulation of LHLepR neurons bidirectionally regulated both food-seeking and evaluating. Furthermore, micro-endoscope data revealed that distinct LHLepR neurons encode seeking and evaluating. Computational modelling analysis demonstrated that LHLepR neurons encode motivation, whereas neurons containing agouti-related peptide and neuropeptide Y (AgRP/NPY) encode the need for food. Additionally, slice studies revealed that NPY decreases inhibitory input to LHLepR neurons via LHGABA interneurons. This mechanism explains the permissive gate role of hunger (food need) in seeking/evaluating motivation. Together, the present study provides a comprehensive neural mechanism of how physiological needs drive distinct motivated behaviours.

neuroscience↗

bioRxiv2020

The Histone Chaperone CAF-1 Sustains Myeloid Lineage Identity

During hematopoiesis, stem and progenitor cells become progressively restricted in their differentiation potential. This process is driven by lineage-specific transcription factors and is accompanied by dynamic changes in chromatin structure. The chromatin assembly factor complex CAF-1 is a key regulator of cellular plasticity in various cell lineages in different organisms. However, whether CAF-1 sustains lineage identity during normal homeostasis is unclear. To address this question, we investigated the role of CAF-1 in myeloid progenitor cells. CAF-1 suppression in myeloid progenitors triggered their rapid commitment but incomplete differentiation toward granulocyte, megakaryocyte, and erythrocyte lineages, resulting in a mixed cellular state. Through comparison with a canonical paradigm of directed terminal myeloid differentiation, we define changes in chromatin accessibility that underlie a unique transcriptome of the aberrantly matured CAF-1 deficient cells. We further identify C/EBP and ELF1 as key transcription factors whose control of myeloid lineage commitment is kept in check by CAF-1. These findings shed new light on molecular underpinnings of hematopoiesis and suggest that manipulation of chromatin accessibility through modulating CAF-1 levels may provide a powerful strategy for controlled differentiation of blood cells.

molecular biology↗

bioRxiv2020

Lopinavir-ritonavir is not an effective inhibitor of the main protease activity of SARS-CoV-2 in vitro

COVID-19 has caused over 900,000 deaths worldwide as of September 2020, and effective medicines are urgently needed. Lopinavir was identified as an inhibitor of the HIV protease, and a lopinavir-ritonavir combination therapy was reported to be beneficial for the treatment of SARS and MERS. However, recent clinical tests could not prove that lopinavir-ritonavir therapy was an effective treatment for COVID-19. In this report, we examined the effect of lopinavir and ritonavir to the activity of the purified main protease (Mpro) protein of SARS- CoV-2, the causative virus of COVID-19. Unexpectedly, lopinavir and ritonavir did not inhibit Mpro activity. These results will aid the drug candidate selection for ongoing and future COVID-19 clinical trials.

microbiology↗