Biology subjects

Lim, C. K.

Publications and source records attributed to Lim, C. K..

10 recordsLinked to original sources

bioRxiv2026

Impaired lipoprotein secretion by APOE4 leads to lysosomal and mitochondrial dysfunction in human microglia

While Apolipoprotein E4 (APOE4) is the greatest known genetic risk factor for late-onset Alzheimers disease, its mechanistic role in the brain-resident macrophage, microglia, remains elusive. Microglia are important in the clearance of pathology in disease, heavily relying on lysosome functionality; therefore, we sought to understand the impact of APOE4 on microglial function. APOE44 microglia have been shown to have lipid accumulation, yet the mechanisms leading to this accumulation are unknown. Using induced pluripotent stem cell-derived microglia, we found that the APOE4 haplotype resulted in transcriptional state shifts in microglia, suppressing activated-response microglia (ARMs) and promoting a G2 senescent-like state. We found that APOE44 microglia accumulate cholesterol esters and provide less lipid support to fibroblast-induced neurons, decreasing their synaptic connections. APOE44 microglia secrete significantly less lipoproteins, leading to the accumulation of lipoproteins within the cells including the lysosomes. APOE44 microglia exhibit impaired lysosomal acidification and degradation capacity. Further, our results elucidated that APOE44 microglia are proinflammatory and shift away from fatty acid oxidation towards glycolysis, due to dysfunctional mitochondria. Taken together, our findings indicate that a loss-of-function in lipoprotein secretion drives intracellular lipid accumulation, including within lysosomes, ultimately disrupting the lysosome-endoplasmic reticulum-mitochondrial axis. This drives a proinflammatory and metabolically compromised microglial phenotype with impaired neuro-supportive functions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/724612v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@18d6a2org.highwire.dtl.DTLVardef@b3644dorg.highwire.dtl.DTLVardef@17e3716org.highwire.dtl.DTLVardef@1529caf_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

AGO1 in neural progenitor cells orchestrates brain development and sociability via LIN28A-REELIN axis

AGO1, an essential RNA binding protein (RBP) in RNA interference, is associated with autism spectrum disorder (ASD). However, the precise functions of AGO1 in brain development and related disorders remain largely unexplored. Here, we report the critical roles of AGO1 in neural progenitor cells (NPCs) in promoting sociability and shaping the structure of the developing brain. Ago1 knockout (KO) in the mouse brain leads to hyposociability, a characteristic symptom of ASD. In human forebrain organoids, AGO1 KO disrupts the formation of ventricle-like structures and delays cortical layer development. We discovered that AGO1 KO results in a loss of polarity in NPCs, which subsequently reduces neuronal development. These AGO1 KO phenotypes originate from the failure to suppress LIN28A in NPCs. AGO1 is primarily localized in the nucleus within NPCs and binds to the LIN28A promoter region, thereby inhibiting LIN28A transcription. We found that increased LIN28A in AGO1 KO NPCs reduces REELIN expression, a key regulator of brain development, by binding directly to the REELIN mRNA. The aberrant polarity phenotype in AGO1 KO NPCs was successfully ameliorated by either LIN28A knockdown or recombinant REELIN treatment. Collectively, our findings elucidate the intricate molecular mechanisms orchestrated by nuclear AGO1 in NPCs and underscores its pivotal roles in brain development, providing significant insights into ASD.

neuroscience↗

bioRxiv2025

CK2 inhibition suppresses glial inflammation in the brain

Neuroinflammation plays a key role in Alzheimers disease (AD) and related neurodegenerative disorders. Chronic activation of astrocytes and microglia fuels neuronal damage via cytokine secretion, oxidative stress, and proteolysis. However, glial inflammatory regulation remains poorly understood. Using chemoproteomics, we identified CK2, particularly the brain-enriched catalytic subunit CK22, as a key driver of astrocytic inflammation. CK2 enhances NF-{kappa}B activity by phosphorylating NF-{kappa}B S529 and I{kappa}B S32, promoting pro-inflammatory gene expression. CK2 inhibition via genetic or chemical approaches dampens inflammation, including IL-6 and IL-8 expression in an acute neuroinflammation mouse model. CK22 is upregulated in AD postmortem tissues and patient-derived astrocytes. AD astrocytes exhibit a hyperinflammatory state that can be attenuated by CK2 inhibition. Overexpression of CK22 in cortical organoids mimics AD pathology, whereas CK2 inhibition using the potent, selective, and brain-penetrant probe TAL606 rescues inflammatory markers in transgenic AD mice. These findings position CK2 as a central regulator of neuroinflammation and a promising therapeutic target for AD and related disorders.

neuroscience↗

bioRxiv2023

Automated Microbial Classification System based on Deep Convolutional Neural Networks using Images from Colony Picker

Colony screening in single and multi-species environments is an essential step for microbiome studies. However, it possesses a high possibility of inaccurately classifying the species of interest and demands a high degree of manpower and time. Thus, automating the classification of microbes is beneficial to minimize the time and inaccuracy in the colony screening/picking step. Here, we developed an automated microbial classification system for five target species, based on deep Convolutional Neural Networks (CNN) using images captured by an automated robotic colony picker. Multiple possible scenarios of colony culture and diverse morphologies of colonies were examined in building the training and test datasets to train and validate the model and performance on real-life implementations. The final model trained using 60,000 training images, with 12,000 images per species and 3-fold cross-validation, achieved a test accuracy of 94.2% and a test loss of 0.154. Upon testing using a deployment dataset of 4,500 images (900 images per species) with different methods of applying cells onto the agar plate, high accuracy of up to 96.6% was obtained. Five evaluation metrics were implemented to evaluate diverse scenarios of the test data to justify the validity of the model in real-life applications. This model forms a foundation for the classification of more species through transfer learning in the future.

synthetic biology↗

bioRxiv2022

Human Liver Organoids as a Patient-derived Model for HBV Infection and Cellular Response

Background & AimsCurrent HBV in vitro model systems suffer from many physiological limitations that restrict understanding of complex viral-host interactions and thus prohibit prediction of disease in vivo. We developed and assessed adult stem cell (AdSC) derived liver organoids as a novel model system for characterisation of the HBV lifecycle, the cellular response to infection and demonstrate their utility in assessing antiviral and immunomodulator response. This model system has the potential to be used in predicting individual HBV responses to antivirals and viral reactivation in the setting of immunosuppressive agents. MethodsDuctal stem cells were isolated from healthy tissue acquired from liver resections or biopsy (n=12). Wnt3a & RSPO-1 containing medium was used to stimulate ductal stem cell expansion into organoids which were subsequently differentiated into hepatocyte-like cells. Mature hepatocyte metabolic markers (albumin, CYP3A4) and HBV entry receptor (Na-taurocholate co-transporting polypeptide, NTCP) expression were evaluated throughout differentiation using qRT-PCR and confocal microscopy. We assessed the organoids culture conditions required for HBV infection and HBV life cycle using HepAD38 (genotype D) and plasma derived HBV (genotype B & C). HBV infection was confirmed using immunofluorescence staining (HBcAg), qRT-PCR (RNA, cccDNA, extracellular DNA) and ELISA (HBsAg and HBeAg). We also assessed drug responsiveness using antivirals and an immunosuppressive agent, and cellular responses (interferon-stimulated genes) using interferon- and viral mimic (PolyI:C). ResultsFollowing differentiation, organoids underwent structural remodelling and changes in cellular polarity, accompanied with an increase in albumin, CYP3A4 and NTCP mRNA expression. Optimal HBV infection was achieved in well-differentiated organoids using spinoculation of at least 200 copies/cell of AD38 derived HBV. Infected organoids demonstrate time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBe and HBsAg consistent with viral replication and antigen secretion. Using these markers we assessed drug-responsiveness to the HBV entry inhibitor, Myrcludex B and the JAK inhibitor, Baricitinib. Despite having a very robust interferon stimulated gene response to interferon- and PolyI:C stimulation, HBV infection in liver organoids did not reveal innate immune activation. ConclusionsAdSC derived liver organoids support the full life cycle of HBV with significant donor dependent variation in viral replication and cellular responses. These features can be utilised for development of personalised drug testing platform for antivirals. Lay SummaryHuman liver organoid culture provides a personalised assessment of HBV infection, replication and responsiveness to antiviral therapy. This model system has a robust innate immune response and could be used to assess novel immune-modulating curative therapy.

microbiology↗

bioRxiv2022

Neuronal activity-related transcription is blunted in immature compared to mature dentate granule cells

Immature dentate granule cells (DGCs) generated in the hippocampus during adulthood are believed to play a unique role in dentate gyrus function. Although immature DGCs have hyperexcitable membrane properties in vitro, the consequences of this hyperexcitability in vivo remain unclear. In particular, the relationship between experiences that activate the dentate gyrus, such as exploration of a novel environment (NE), and downstream molecular processes that modify dentate gyrus circuitry in response to cellular activation are unknown in this cell population. We first performed quantification of immediate early gene proteins in immature (5-week-old) and mature (13-week-old) DGCs from mice exposed to a NE. Paradoxically, we observed lower immediate early gene protein expression in hyperexcitable immature DGCs. We then isolated nuclei from active and inactive immature DGCs and performed single nuclei RNA-Sequencing. Compared to mature nuclei collected from the same animal, immature DGC nuclei showed less activity-induced transcriptional change, even though they were classified as active based on expression of ARC protein. These results demonstrate that the coupling of spatial exploration, cellular activation, and transcriptional change differs between immature and mature DGCs, with blunted activity-induced changes in immature cells.

neuroscience↗

bioRxiv2021

The novel conserved NAD+ binding micropeptide SGHRT regulates mitochondrial function and metabolism in human cardiomyocytes

AbstractsNicotinamide adenine dinucleotide (NAD) is a critical metabolite and coenzyme for multiple metabolic pathways and cellular processes (1-4). In this study, we identified Singheart, SGHRT as a nuclear genome-encoded NAD+-binding mitochondrial micropeptide. SGHRT, present in both monomeric and dimeric forms, binds directly to NAD, but not NADH or flavin adenine dinucleotide (FAD). Localized to the inner mitochondrial membrane and mitochondrial matrix, SGHRT interacts with the mitochondrial enzymes Succinate-CoA Ligase and Succinate Dehydrogenase. SGHRT deletion in human embryonic stem cell derived cardiomyocytes disrupted mitochondria morphology, decreased total NAD and ATP abundance, and resulted in defective TCA cycle metabolism, the electron transport chain and in Ox-Phos processes. These results comprise the first report of an NAD+-binding micropeptide, SGHRT, required for mitochondrial function and metabolism.

cell biology↗

bioRxiv2021

Integrative genome-scale metabolic modeling reveals versatile metabolic strategies for methane utilization in Methylomicrobium album BG8

Methylomicrobium album BG8 is an aerobic methanotrophic bacterium that can mitigate environmental methane emission, and is a promising microbial cell factory for the conversion of methane to value-added chemicals. However, the lack of a genome-scale metabolic model (GEM) of M. album BG8 has hindered the development of systems biology and metabolic engineering of this methanotroph. To fill this gap, a high-quality GEM was constructed to facilitate a system-level understanding on the biochemistry of M. album BG8. Next, experimental time-series growth and exometabolomics data were integrated into the model to generate context-specific GEMs. Flux balance analysis (FBA) constrained with experimental data derived from varying levels of methane, oxygen, and biomass were used to model the metabolism of M. album BG8 and investigate the metabolic states that promote the production of biomass and the excretion of carbon dioxide, formate, and acetate. The experimental and modeling results indicated that the system-level metabolic functions of M. album BG8 require a ratio > 1:1 between the oxygen and methane specific uptake rates for optimal growth. Integrative modeling revealed that at a high ratio of oxygen-to-methane uptake flux, carbon dioxide and formate were the preferred excreted compounds; at lower ratios, however, acetate accounted for a larger fraction of the total excreted flux. The results of this study reveal a trade-off between biomass production and organic compound excretion and provide evidence that this trade-off is linked to the ratio between the oxygen and methane specific uptake rates.

microbiology↗

bioRxiv2020

Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

Viruses pose a constant threat to human health. As a result our innate immune system has evolved multiple strategies to detect the presence of intracellular viral pathogen-associated molecular patterns (PAMPs) (1). The full repertoire of human immune sensors and their PAMP ligands are not completely understood. Here we report that human NLRP1 senses and is activated by 3C proteases (3Cpros) of enteroviruses. Mechanistically, 3Cpros cleave human NLRP1 at a single site immediately after its primate-specific PYRIN domain, leading to oligomerization of its C-terminal fragment. Expression of 3Cpros in primary human cells cause NLRP1-dependent ASC oligomerization, pyroptotic cell death and IL-1 secretion. Consistent with our observation that NLRP1 is the predominant endogenous inflammasome sensor in human airway epithelium, we find that its genetic deletion, or that of ASC, abrogates IL-18 secretion from rhinovirus (HRV)-infected primary human bronchial epithelial cells. Our findings identify the first cognate PAMP ligand for human NLRP1 and assign a new function for the NLRP1 inflammasome in human antiviral immunity and airway inflammation. These results challenge the widely held notion that viral proteases largely serve to disable host immune sensing, and suggest that the human NLRP1 inflammasome may be a therapeutic target to treat inflammatory airway diseases including asthma. One Sentence SummaryHuman NLRP1 is activated by enteroviral 3C proteases

immunology↗

bioRxiv2017

Evidence-based gene models for structural and functional annotations of the oil palm genome

The advent of rapid and inexpensive DNA sequencing has led to an explosion of data that must be transformed into knowledge about genome organization and function. Gene prediction is customarily the starting point for genome analysis. This paper presents a bioinformatics study of the oil palm genome, including a comparative genomics analysis, database and tools development, and mining of biological data for genes of interest. We annotated 26,087 oil palm genes integrated from two gene-prediction pipelines, Fgenesh++ and Seqping. As case studies, we conducted comprehensive investigations on intronless, resistance and fatty acid biosynthesis genes, and demonstrated that the current gene prediction set is of high quality. 3,672 intronless genes were identified in the oil palm genome, an important resource for evolutionary study. Further scrutiny of the oil palm genes revealed 210 candidate resistance genes involved in pathogen defense. Fatty acids have diverse applications ranging from food to industrial feedstock, and we identified 42 key genes involved in fatty-acid biosynthesis in oil palm mesocarp and kernel. These results provide an important resource for studies on plant genomes and a theoretical foundation for marker-assisted breeding of oil palm and related crops.

bioinformatics↗