Biology subjects

Lim, J.

Publications and source records attributed to Lim, J..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Immune-modulatory effects of low dose γ-radiation on wax moth (Galleria mellonella) larvae

Larvae of the greater wax moth Galleria mellonella are common pests of beehives and commercial apiaries, and in more applied settings, these insects act as alternative in vivo bioassays to rodents for studying microbial virulence, antibiotic development, and toxicology. In the current study, our overall aim was to assess the putative adverse effects of background gamma radiation levels on G. mellonella. To achieve this, we exposed larvae to low (0.014 mGy/h), medium (0.056 mGy/h), and high (1.33 mGy/h) doses of caesium-137 and measured larval pupation events, weight, faecal discharge, susceptibility to bacterial and fungal challenges, immune cell counts, activity, and viability (i.e., haemocyte encapsulation) and melanisation levels. The effects of low and medium levels of radiation were distinguishable from the highest dose rates used - the latter insects weighed the least and pupated earlier. In general, radiation exposure modulated cellular and humoral immunity over time, with larvae showing heighted encapsulation/melanisation levels at the higher dose rates but were more susceptible to bacterial (Photorhabdus luminescens) infection. There were few signs of radiation impacts after 7 days exposure, whereas marked changes were recorded between 14 and 28 days. Our data suggest that G. mellonella demonstrates plasticity at the whole organism and cellular levels when irradiated and offers insight into how such animals may cope in radiologically contaminated environments.

immunology↗

bioRxiv2022

The Network Zoo: a multilingual package for the inference and analysis of biological networks

Inference and analysis of cellular biological networks requires software tools that integrate multi-omic data from various sources. The Network Zoo (netZoo; netzoo.github.io) is an open-source software suite to model biological networks, including context-specific gene regulatory networks and multi-omics partial correlation networks, to conduct differential analyses, estimate community structure, and model the transitions between biological states. The netZoo builds on our ongoing development of network methods, harmonizing the implementations in various computing languages (R, Python, MATLAB, and C) and between methods to allow a better integration of these tools into analytical pipelines. To demonstrate the value of this integrated toolkit, we analyzed the multi-omic data from the Cancer Cell Line Encyclopedia (CCLE) by inferring gene regulatory networks for each cancer cell line and associating network features with other phenotypic attributes such as drug sensitivity. This allowed us to identify transcription factors that play a critical role in both drug resistance and cancer development in melanoma. We also used netZoo to build a pan-cancer, multi-tiered CCLE map and used it to identify known metabolic hallmarks of cancer and to estimate novel context-specific elements that mediate post-transcriptional regulation. Because the netZoo tools are open-source and there is a growing community of both users and developers, we built an ecosystem to support community contributions, share use cases, and visualize networks online. As additional data types become available and our suite of methods grows, we will expand "the zoo" to incorporate an increasingly sophisticated collection of tools for network inference and analysis.

genomics↗

bioRxiv2022

Extensive mosaicism by somatic L1 retrotransposition in normal human cells

Over the course of an individuals lifetime, genomic alterations accumulate in somatic cells. However, the mutational landscape by retrotranspositions of long interspersed nuclear element-1 (L1), a widespread mobile element in the human genome, is poorly understood in normal cells. Here, we explored the whole-genome sequences of 892 single-cell clones established from various tissues collected from 28 individuals. Remarkably, 88% of colorectal epithelial cells acquired somatic L1 retrotranspositions (soL1Rs), carrying [~]3 events per cell on average with substantial intra- and inter-individual variances, which was accelerated at least 10-fold during tumourigenesis. Breakpoints of soL1Rs suggested that a few variant mechanisms can be involved in the L1 retrotransposition processes. Fingerprinting of donor L1s using source-specific unique sequences revealed 34 hot L1s, 44% of which were newly discovered in this study, and many ultra-rare hot L1s in the human population showed higher retrotransposition potential in somatic lineages than common sources. Multi-dimensional analysis of soL1Rs with early embryonic developmental relationships, genome-wide methylation, and gene expression profiles of the clones demonstrated that (1) soL1Rs occur from early embryogenesis at a substantial rate, (2) epigenetic activation of hot L1s is stochastically acquired during the wave of early global epigenomic reprogramming, rather than by the sporadic loss-of-methylation at the late stage, and (3) most L1 transcripts in the cytoplasm do not generate soL1Rs in somatic lineages. In summary, this study provides insights into the retrotransposition dynamics of L1s in the human genome and the resultant somatic mosaicism in normal human cells.

genomics↗

bioRxiv2022

A low-power communication scheme for wireless, 1000 channel brain-machine interfaces

ObjectiveBrain-machine interfaces (BMIs) have the potential to restore motor function but are currently limited by electrode count and long-term recording stability. These challenges may be solved through the use of free-floating "motes" which wirelessly transmit recorded neural signals, if power consumption can be kept within safe levels when scaling to thousands of motes. Here, we evaluated a pulse-interval modulation (PIM) communication scheme for infrared (IR)-based motes that aims to reduce the wireless data rate and system power consumption. ApproachTo test PIMs ability to efficiently communicate neural information, we simulated the communication scheme in a real-time closed-loop BMI with non-human primates. Additionally, we performed circuit simulations of an IR-based 1000-mote system to calculate communication accuracy and total power consumption. Main ResultsWe found that PIM at 1kb/s per channel maintained strong correlations with true firing rate and matched online BMI performance of a traditional wired system. Closed-loop BMI tests suggest that lags as small as 30 ms can have significant performance effects. Finally, unlike other IR communication schemes, PIM is feasible in terms of power, and neural data can accurately be recovered on a receiver using 3mW for 1000 channels. SignificanceThese results suggest that PIM-based communication could significantly reduce power usage of wireless motes to enable higher channel-counts for high-performance BMIs.

bioengineering↗

bioRxiv2021

Stain-free nucleus identification in holographic learning flow cyto-tomography

Quantitative Phase Imaging (QPI) has gained popularity because it can avoid the staining step, which in some cases is difficult or impossible. However, QPI does not provide the well-known specificity to various parts of the cell (e.g., organelles, membrane). Here we show a novel computational segmentation method based on statistical inference that bridges the gap between the specificity of Fluorescence Microscopy (FM) and the label-free property of QPI techniques to identify the cell nucleus. We demonstrate application to stain-free cells reconstructed through the holographic learning and in flow cyto-tomography modality. In particular, by means of numerical simulations and two cancer cell lines, we demonstrate that the nucleus-like regions can be accurately distinguished within the stain-free tomograms. We show that our experimental results are consistent with confocal FM data and microfluidic cytofluorimeter outputs. This is a significant step towards extracting the three-dimensional (3D) intracellular specificity directly from the phase-contrast data in a typical flow cytometry configuration.

bioengineering↗

bioRxiv2021

Gabra5LHA Mediate Astrocytic GABA-induced Obesity via Decreasing Energy Expenditure

The lateral hypothalamic area (LHA) regulates food intake and energy expenditure. Although LHA neurons innervate adipose tissues, the identity of neurons that regulate fat is undefined. Here we identify that Gabra5-positive neurons in LHA (GABRA5LHA) polysynaptically project to brown and white adipose tissues in the periphery. GABRA5LHA are a distinct subpopulation of GABAergic neurons and show decreased pacemaker firing in diet-induced obesity (DIO) mouse model. Chemogenetic inhibition of GABRA5LHA suppresses energy expenditure and increases weight gain, whereas gene-silencing of Gabra5 in LHA decreases weight gain. In DIO mouse model, GABRA5LHA are tonically inhibited by nearby reactive astrocytes releasing GABA, which is synthesized by MAOB. Gene-silencing of astrocytic MAOB in LHA reduces weight gain significantly without affecting food intake, which is recapitulated by administration of a MAOB inhibitor, KDS2010. We propose that firing of GABRA5LHA facilitates energy expenditure and selective inhibition of astrocytic GABA is a molecular target for treating obesity.

neuroscience↗

bioRxiv2021

Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as [~]36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy. One Sentence SummaryA new technology combining CRISPR-mediated genome editing and site-specific integrases enables efficient programmable gene integration at any targeted genomic locus without double-strand DNA breaks, leading to broad applications in basic science research, cell engineering, and gene therapy.

bioengineering↗

bioRxiv2021

Longitudinal single-cell transcriptional dynamics throughout neurodegeneration in SCA1

Neurodegeneration is a protracted process involving progressive changes in myriad cell types that ultimately result in neuronal death. Changes in vulnerable neuronal populations are highly influenced by concomitant changes in surrounding cells, complicating experimental approaches to interrogate the simultaneous events that underlie neurodegeneration. To dissect how individual cell types within a heterogeneous tissue contribute to the pathogenesis and progression of a neurodegenerative disorder, we performed longitudinal single-nucleus RNA sequencing of the mouse and human spinocerebellar ataxia type 1 (SCA1) cerebellum, establishing continuous dynamic trajectories of each population. Furthermore, we defined the precise transcriptional changes that precede loss of Purkinje cells and identified early oligodendroglial impairments that can profoundly impact cerebellar function. Finally, we applied a deep learning method to accurately predict disease state and identify drivers of disease. Together, this work uncovers new roles for diverse cerebellar cell types in SCA1 and provides a generalizable analysis framework for studying neurodegeneration.

neuroscience↗

bioRxiv2021

Differential effects of Wnt-β-catenin signaling in Purkinje cells and Bergmann glia in SCA1

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease characterized by progressive ataxia and degeneration of specific neuronal populations, including Purkinje cells (PCs) in the cerebellum. Previous studies have demonstrated a critical role for various evolutionarily conserved signaling pathways in cerebellar patterning, such as the Wnt-{beta}-catenin pathway; however, the roles of these pathways in adult cerebellar function and cerebellar neurodegeneration are largely unknown. In this study, we found that Wnt-{beta}-catenin activity was progressively enhanced in multiple cell types in the adult SCA1 mouse cerebellum, and that activation of this signaling occurs in an ataxin-1 polyglutamine (polyQ) expansion-dependent manner. Genetic manipulation of the Wnt-{beta}-catenin signaling pathway in specific cerebellar cell populations revealed that activation of Wnt-{beta}-catenin signaling in PCs alone was not sufficient to induce SCA1-like phenotypes, while its activation in astrocytes including Bergmann glia (BG) resulted in gliosis and disrupted BG localization, which was replicated in SCA1 mouse models. Our studies identify a novel mechanism in which polyQ-expanded ataxin-1 positively regulates Wnt-{beta}-catenin signaling, and demonstrate that different cell types have distinct responses to the enhanced Wnt-{beta}-catenin signaling in the SCA1 cerebellum, underscoring an important role of BG in SCA1 pathogenesis. Significance statementThe mechanisms underlying the degeneration of specific cellular populations in various neurodegenerative disorders remain unknown. Here, we show that the polyQ expansion of ataxin-1 activates the Wnt-{beta}-catenin signaling pathway in various cell types, including Purkinje cells and Bergmann glia, in the cerebellum of SCA1 mouse models. We used conditional mouse genetics to activate and silence this pathway in different cell types and found elevated activity of this signaling pathway impacted Bergmann glia and Purkinje cell populations differently. This study highlights the important role of Wnt-{beta}-catenin signaling pathway in glial cell types for SCA1 pathogenesis.

neuroscience↗

bioRxiv2021

Astrocytic urea cycle detoxifies Aβ-derived ammonia while impairing memory in Alzheimers Disease

Alzheimers disease (AD) is one of the foremost neurodegenerative diseases, characterized by beta-amyloid (A{beta}) plaques and significant progressive memory loss. In AD, astrocytes are known to take up and clear A{beta} plaques. However, how A{beta} induces pathogenesis and memory impairment in AD remains elusive. We report that normal astrocytes show non-cyclic urea metabolism, whereas A{beta}-treated astrocytes show switched-on urea cycle with upregulated enzymes and accumulated entering-metabolite aspartate, starting-substrate ammonia, end-product urea, and side-product putrescine. Gene-silencing of astrocytic ornithine decarboxylase-1 (ODC1), facilitating ornithine-to-putrescine conversion, boosts urea cycle and eliminates aberrant putrescine and its toxic by-products ammonia, H2O2, and GABA to recover from reactive astrogliosis and memory impairment in AD model. Our findings implicate that astrocytic urea cycle exerts opposing roles of beneficial A{beta} detoxification and detrimental memory impairment in AD. We propose ODC1-inhibition as a promising therapeutic strategy for AD to facilitate removal of toxic molecules and prevent memory loss.

neuroscience↗

bioRxiv2021

Molecular Drivers of Tumor Progression in Microsatellite Stable APC Mutation-Negative Colorectal Cancers

BackgroundThe tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC), underscoring the importance of aberrant regulation of intracellular WNT signaling in CRC development. Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify mechanisms underlying APC mutation-negative (APCmut-) CRCs. MethodsWe analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut- and APC mutation-positive (APCmut+) microsatellite stable CRCs. ResultsTranscriptionally, APCmut- CRCs clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut- CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3. ConclusionsAPCmut- CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways.

cancer biology↗

bioRxiv2021

Interactome and structural basis for targeting the human T-cell leukemia virus Tax oncoprotein

Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). Although ATL is a well-characterized T-cell neoplasm, linked to intermittent expression of the viral Tax-1 protein, there is currently no strategy to target Tax-1 functions using small molecules. Here, we report a comprehensive interaction map between Tax-1 and human PDZ domain-containing proteins (hPDZome). We show that Tax-1 interacts with more than one-third of hPDZome components, including proteins involved in cell cycle, cell-cell junctions, cytoskeleton organization, and membrane complex assembly. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the structural basis of the interaction between the C-terminal PDZ binding motif (PBM) of Tax-1, and the PDZ domains of syntenin-1, an evolutionary conserved hub that controls exosome trafficking. Finally, we have used confocal imaging, molecular modelling, NMR and mammalian cell-based assays to demonstrate that the Tax-1/syntenin-1 interaction is amenable to small-molecule inhibition. Altogether, our study highlights the biological significance of Tax-PDZ interactome and its interplay with exosome formation. It shows a direct link between extracellular vesicles and HTLV-1 transmission, providing a novel framework for the design of targeted therapies for HTLV-1-induced diseases.

biochemistry↗

bioRxiv2021

Natural variations in reproductive aging phenotypes reveal the importance of early reproductive period in Caenorhabditis elegans

Reproductive timing underlies self-sperm production and X-chromosome nondisjunction rate in Caenorhabditis elegans. These traits can be measured as brood size and male-production rate, and brood size decreases and male-production rate increases as worms age. These phenotypic changes occur simultaneously; however, whether these changes share common underlying genetic architectures still remains unclear. To enhance our understanding of reproductive timing in C. elegans, we measured and compared these two reproductive timing-related traits of 96 wild strains during early, late and total reproductive periods. We found that the two traits exhibited natural phenotypic variation with few outliers and a similar reproductive timing pattern as previous reports. The brood size and the male-production rate were not genetically correlated, implying that they might have different genetic architectures and that meiosis quality can be maintained despite increased progeny-production in the late reproductive period caused by more self-sperm. We also identified loci and candidate genetic variants significantly associated with male-production rate in the late and total reproductive periods. Our results provide an insight into life history traits in wild C. elegans strains.

genetics↗

bioRxiv2021

FEATURE DETECTION TO SEGMENT CARDIOMYOCYTE NUCLEI FOR INVESTIGATING CARDIAC CONTRACTILITY

In vivo quantitative assessment of structural and functional biomarkers is essential for understanding pathophysiology and identifying novel therapies for congenital heart disorders. Cardiac defect analysis through fixed tissue and histology has offered revolutionary insights into the tissue architecture, but section thickness limits the tissue penetration. This study demonstrated the potential of Light Sheet Fluorescence Microscopy (LSFM) for analyzing in vivo 4D (3d + time) cardiac contractility. Furthermore, we have described the utility of an improved feature detection framework for localizing cardiomyocyte nuclei in the zebrafish atrium and ventricle. Using the Hessian Difference of Gaussian (HDoG) scale space in conjunction with the watershed algorithm, we were able to quantify a statistically significant increase in cardiomyocyte nuclei count across different developmental stages. Furthermore, we assessed individual volumes and surface areas for the cardiomyocyte nuclei in the ventricles innermost and outermost curvature during cardiac systole and diastole. Using the segmented nuclei volumes from the feature detection, we successfully performed local area ratio analysis to quantify the degree of deformation suffered by the outermost ventricular region compared to the innermost ventricular region. This paper focuses on the merits of our segmentation and demonstrates its efficacy for cell counting and morphology analysis in the presence of anisotropic illumination across the field-of-view (FOV).

bioengineering↗

bioRxiv2021

Data-driven analysis of COVID-19 reveals specific severity patterns distinct from the temporal immune response

Key immune signatures of SARS-CoV-2 infection may associate with either adverse immune reactions (severity) or simply an ongoing anti-viral response (temporality); how immune signatures contribute to severe manifestations and/or temporal progression of disease and whether longer disease duration correlates with severity remain unknown. Patient blood was comprehensively immunophenotyped via mass cytometry and multiplex cytokine arrays, leading to the identification of 327 basic subsets that were further stratified into more than 5000 immunotypes and correlated with 28 plasma cytokines. Low-density neutrophil abundance was closely correlated with hepatocyte growth factor levels, which in turn correlated with disease severity. Deep analysis also revealed additional players, namely conventional type 2 dendritic cells, natural killer T cells, plasmablasts and CD16+ monocytes, that can influence COVID-19 severity independent of temporal progression. Herein, we provide interactive network analysis and data visualization tools to facilitate data mining and hypothesis generation for elucidating COVID-19 pathogenesis.

immunology↗

bioRxiv2021

Mutational impact and signature of ionizing radiation

Whole-genome sequencing (WGS) of human tumors and normal cells exposed to various carcinogens has revealed distinct mutational patterns that provide deep insights into the DNA damage and repair processes. Although ionizing radiation (IR) is conventionally known as a strong carcinogen, its genome-wide mutational impacts have not been comprehensively investigated at the single-nucleotide level. Here, we explored the mutational landscape of normal single-cells after exposure to the various levels of IR. On average, 1 Gy of IR exposure generated [~]16 mutational events with a spectrum consisting of predominantly small nucleotide deletions and a few characteristic structural variations. In [~]30% of the post-irradiated cells, complex genomic rearrangements, such as chromoplexy, chromothripsis, and breakage-fusion-bridge cycles, were resulted, indicating the stochastic and chaotic nature of DNA repair in the presence of the massive number of concurrent DNA double-strand breaks. These mutational signatures were confirmed in the genomes of 22 IR-induced secondary malignancies. With high-resolution genomic snapshots of irradiated cells, our findings provide deep insights into how IR-induced DNA damage and subsequent repair processes operate in mammalian cells.

genomics↗

bioRxiv2020

Master Transcription Factors Regulate the DNA Methylation Landscape During Hepatocyte Differentiation

BackgroundHepatocytes are the dominant cell type of the human liver, with functions in metabolism, detoxification, and in producing secreted proteins. During the process of hepatocyte differentiation, gene regulation and master transcription factors have been extensively investigated, whereas little is known about how the epigenome is regulated, particularly the dynamics of DNA methylation, and the upstream factors that have critical roles. ResultsBy examining changes in the transcriptome and the methylome during in vitro hepatocyte differentiation, we identified putative DNA methylation-regulating transcription factors, which are likely involved in DNA demethylation and maintenance of hypo-methylation in a differentiation stage-specific manner. Of these factors, we further reveal that GATA6 induces DNA demethylation together with chromatin activation at a binding-site-specific manner during endoderm differentiation. ConclusionsThese results provide an insight into the spatiotemporal regulatory mechanisms exerted on the DNA methylation landscape by transcription factors, and uncover a new role for transcription factors in early liver development.

genomics↗

bioRxiv2020

Clonal dynamics in early human embryogenesis inferred from somatic mutation

The trillions of cells that constitute the human body are developed from a fertilized egg through embryogenesis. However, cellular dynamics and developmental outcomes of embryonic cells in humans remain to be largely unknown due to the technical and ethical challenges. Here, we explored whole-genomes of 334 single-cell expanded clones and targeted deep-sequences of 379 bulk tissues obtained from various anatomical locations from seven individuals. Using the discovered 1,688,652 somatic mutations as an intrinsic barcode, we reconstructed cellular phylogenetic trees that provide novel insights into early human embryogenesis. Our findings suggest (1) endogenous mutational rate that is higher in the first cell division of life but decreases to ~1 per cell per cell division later in life, (2) universal unequal contribution of early cells into embryo proper resulting from early cellular bottlenecks that stochastically separate epiblasts from embryonic cells (3) uneven differential outcomes of early cells into three germ layers, left-right and cranio-caudal tissues, (4) emergence of a few ancestral cells that will contribute to the substantial fraction of adult blood cells, and (5) presence of mitochondrial DNA heteroplasmy in the fertilized egg. Our approach additionally provides insights into the age-related mutational processes including UV-mediated mutagenesis and loss of chromosome X or Y in normal somatic cells. Taken together, this study scrutinized somatic mosaicism, clonal architecture, and cellular dynamics in human embryogenesis at an unprecedented level and provides a foundation for future studies to complete cellular phylogenies in human embryogenesis.

genomics↗