Biology subjects

Lee, J.

Publications and source records attributed to Lee, J..

At least 343 records · Page 19Linked to original sources

bioRxiv2024

Volumetric imaging and computation to explore contractile function in zebrafish hearts

Despite advancements in cardiovascular engineering, heart diseases remain a leading cause of mortality. The limited understanding of the underlying mechanisms of cardiac dysfunction at the cellular level restricts the development of effective screening and therapeutic methods. To address this, we have developed a framework that incorporates light field detection and individual cell tracking to capture real-time volumetric data in zebrafish hearts, which share structural and electrical similarities with the human heart and generate 120 to 180 beats per minute. Our results indicate that the in-house system achieves an acquisition speed of 200 volumes per second, with resolutions of up to 5.02 {+/-} 0.54 {micro}m laterally and 9.02 {+/-} 1.11 {micro}m axially across the entire depth, using the estimated-maximized-smoothed deconvolution method. The subsequent deep learning-based cell trackers enable further investigation of contractile dynamics, including cellular displacement and velocity, followed by volumetric tracking of specific cells of interest from end-systole to end-diastole in an interactive environment. Collectively, our strategy facilitates real-time volumetric imaging and assessment of contractile dynamics across the entire ventricle at the cellular resolution over multiple cycles, providing significant potential for exploring intercellular interactions in both health and disease.

bioengineering↗

bioRxiv2024

Schistosome esophageal gland factor MEG-8.2 drives host cell lysis and interacts with host immune proteins

Schistosomes are blood flukes that ingest large amounts of host blood during their intra-mammalian stage. The ingested blood contains leukocytes that can be harmful, yet the parasites survive inside the host for decades, reflecting superb immune evasion mechanisms that remain poorly understood. Our previous work discovered that FoxA, a forkhead transcription factor, drives the production of the esophageal gland, an anterior digestive organ essential for degrading the ingested leukocytes and for in vivo survival. However, a comprehensive molecular makeup of the esophageal gland remains unclear. Importantly, which of the esophageal gland factors are responsible for degrading the ingested leukocytes, their mechanism of action, and how such a function relates to parasite survival and immune evasion remains unknown. Here, we identify additional esophageal gland genes by taking a comparative transcriptomics approach to identify transcripts altered in foxA knockdown adult schistosomes. A targeted RNAi screen coupled with biochemistry reveals that specific domains of the micro-exon gene MEG-8.2, can drive host cell lysis in a concentration-dependent manner. Using pull-down assays coupled with mass spectrometry, we discover that MEG-8.2 interacts with several host membrane and extracellular proteins that play important roles in activating innate and/or adaptive immunity. Together, our findings suggest a dual role for MEG-8.2 in effectively lysing the ingested cells in the esophageal lumen and interacting with specific host proteins to neutralize or suppress the host immunity. These findings lay an important foundation for exploiting esophageal gland factors to treat schistosomiasis.

microbiology↗

bioRxiv2024

Back-to-the-future motion analysis using machine intelligence predicts the potential risk of mood disorders

Motion encodes emotional value. Decoding the hidden information within the complexity and randomness of motion may aid in the early diagnosis and prevention of mood disorders. In this study, we utilized machine intelligence to predict the risk of mood disorders by analyzing random motion patterns in mice. The support vector machine, trained on motion data collected before chronic social defeat stress (CSDS), predicted a significant correlation between these data and the degree of CSDS-induced social avoidance. Unsupervised clustering revealed key motion biomarkers of stress susceptibility, accounting for approximately 70% of the onset of social avoidance in a new group of mice before exposure to CSDS. These findings suggest that motion analysis using machine intelligence could provide a non-invasive approach for predicting the risk of mood disorders.

animal behavior and cognition↗

bioRxiv2024

Automated cell tracking using 3D nnUnet and Light Sheet Microscopy to quantify regional deformation in zebrafish

Light Sheet Microscopy (LSM) in conjunction with embryonic zebrafish, is rapidly advancing three-dimensional, in vivo characterization of myocardial contractility. Preclinical cardiac deformation imaging is predominantly restricted to a low-order dimensionality image space (2i) or suffers from poor reproducibility. In this regard, LSM has enabled high throughput, non-invasive 4i (3d+time) characterization of dynamic organogenesis within the transparent zebrafish model. More importantly, LSM offers cellular resolution across large imaging Field-of-Views at millisecond camera frame rates, enabling single cell localization for global cardiac deformation analysis. However, manual labeling of cells within multilayered tissue is a time-consuming task and requires substantial expertise. In this study, we applied the 3i nnU-Net with Linear Assignment Problem (LAP) framework for automated segmentation and tracking of myocardial cells. Using binarized labels from the neural network, we quantified myocardial deformation of the zebrafish ventricle across 4-6 days post fertilization (dpf). Our study offers tremendous promise for developing highly scalable and disease-specific biomechanical quantification of myocardial microstructures.

bioengineering↗

bioRxiv2024

The MRAP2 accessory protein directly interacts with melanocortin-3 receptor to enhance signaling

The central melanocortin system links nutrition to energy expenditure, with melanocortin-4 receptor (MC4R) controlling appetite and food intake, and MC3R regulating timing of sexual maturation, rate of linear growth and lean mass accumulation. Melanocortin-2 receptor accessory protein-2 (MRAP2) is a single transmembrane protein that interacts with MC4R to potentiate its signalling, and human mutations in MRAP2 cause obesity. Previous studies have been unable to consistently show whether MRAP2 affects MC3R activity. Here we used single-molecule pull-down (SiMPull) to confirm that MC3R and MRAP2 interact in HEK293 cells. Analysis of fluorescent photobleaching steps showed that MC3R and MRAP2 readily form heterodimers most commonly with a 1:1 stoichiometry. Human single-nucleus and spatial transcriptomics show MRAP2 is co-expressed with MC3R in hypothalamic neurons with important roles in energy homeostasis and appetite control. Functional analyses showed MRAP2 enhances MC3R cAMP signalling, impairs {beta}-arrestin recruitment, and reduces internalization in HEK293 cells. Structural homology models revealed putative interactions between the two proteins and alanine mutagenesis of five MRAP2 and three MC3R transmembrane residues significantly reduced MRAP2 effects on MC3R signalling. Finally, we showed genetic variants in MRAP2 that have been identified in individuals that are overweight or obese prevent MRAP2s enhancement of MC3R-driven signalling. Thus, these studies reveal MRAP2 as an important regulator of MC3R function and provide further evidence for the crucial role of MRAP2 in energy homeostasis.

molecular biology↗

bioRxiv2024

Designed miniproteins potently inhibit and protect against MERS-CoV

Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic pathogen with 36% case-fatality rate in humans. No vaccines or specific therapeutics are currently approved to use in humans or the camel host reservoir. Here, we computationally designed monomeric and homo-oligomeric miniproteins binding with high affinity to the MERS-CoV spike (S) glycoprotein, the main target of neutralizing antibodies and vaccine development. We show that these miniproteins broadly neutralize a panel of MERS-CoV S variants, spanning the known antigenic diversity of this pathogen, by targeting a conserved site in the receptor-binding domain (RBD). The miniproteins directly compete with binding of the DPP4 receptor to MERS-CoV S, thereby blocking viral attachment to the host entry receptor and subsequent membrane fusion. Intranasal administration of a lead miniprotein provides prophylactic protection against stringent MERS-CoV challenge in mice motivating future clinical development as a next-generation countermeasure against this virus with pandemic potential.

biochemistry↗

bioRxiv2024

Defective glycosylation and ELFN1 binding of mGluR6 congenital stationary night blindness mutants

Synaptic transmission from photoreceptors to ON-bipolar cells (BCs) requires the postsynaptic metabotropic glutamate receptor mGluR6, located at BC dendritic tips. Binding of the neurotransmitter glutamate initiates G-protein signaling that regulates the TRPM1 transduction channel. mGluR6 also interacts with presynaptic ELFN adhesion proteins, and these interactions are important for mGluR6 synaptic localization. The mechanisms of mGluR6 trafficking and synaptic targeting remain poorly understood. In this study, we investigated mGluR6 missense mutations from patients with congenital stationary night blindness (CSNB), which is associated with loss of synaptic transmission to ON-BCs. We found that multiple CSNB mutations in the extracellular ligand-binding domain of mGluR6 impart a trafficking defect leading to lack of complex N-glycosylation but efficient plasma membrane insertion, suggesting a Golgi bypass mechanism. These mutants fail to bind ELFN1, consistent with lack of a necessary modification normally acquired in the Golgi. The same mutants were mislocalized in bipolar cells, explaining the loss of function in CSNB. The results reveal a key role of Golgi trafficking in mGluR6 function, and suggest a role for the extracellular domain in Golgi sorting.

cell biology↗

bioRxiv2024

APOBEC3A, not APOBEC3B, drives deaminase mutagenesis in human gastric epithelium

Cancer genomes frequently carry APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like)-associated DNA mutations, suggesting APOBEC enzymes as innate mutagens during cancer initiation and evolution. However, the pure mutagenic impacts of the specific enzymes among this family that are responsible for APOBEC-associated mutagenesis remain unclear in human normal cell lineage, particularly the comparative mutagenic activities of APOBEC3A and APOBEC3B. Here, we investigated the mutagenic contributions of these enzymes through whole-genome sequencing of human normal gastric organoid lines carrying doxycycline-inducible APOBEC3A or APOBEC3B cassettes. Our findings demonstrated that transcriptional APOBEC3A upregulation led to the acquisition of a massive number of genomic mutations in a few cell cycles. By contrast, APOBEC3B upregulation did not generate a substantial number of mutations in gastric epithelium. APOBEC3B-associated mutagenesis remained insignificant even after a combined inactivation of TP53. Based on the spectrum of acquired mutations after APOBEC3A upregulation, we further analyzed APOBEC3A-associated mutational signatures, encompassing indels mainly composed of 1bp deletions, characteristics of clustered mutations, and selective pressures operative on cells carrying the mutations. Our observations provide a clear foundation for understanding the mutational impact of APOBEC enzymes in human cells.

genomics↗

bioRxiv2024

Role of Complexin 2 in the regulation of hormone secretion from the islet of Langerhans

Regulated secretion of insulin from {beta}-cells, glucagon from -cells, and somatostatin from {delta}-cells is necessary for the maintenance of glucose homeostasis. The release of these hormones from pancreatic islet cells requires the assembly and disassembly of the SNARE protein complex to control vesicle fusion and exocytosis. Complexin 2 (Cplx 2) is a small soluble synaptic protein that participates in the priming and release steps of vesicle fusion. It plays a dual role as a molecular switch that first clamps and prevents fusion pore opening, and subsequently undergoes a conformational change upon Ca2+ binding to synaptotagmin to facilitate exocytosis. Using a Cplx 2 knockout (KO) mouse model, we show a direct inhibitory role of Cplx 2 for glucagon and somatostatin secretion, along with an indirect role in the paracrine inhibition of insulin secretion by somatostatin. Deletion of Cplx 2 increases glucagon and somatostatin secretion from intact mouse islets, while there is no difference in insulin secretion between WT and Cplx 2 KO islets. The normal paracrine inhibition of insulin secretion by somatostatin is disrupted in Cplx 2 KO islets. On the contrary, deletion of Cplx 2 did not affect the known role of somatostatin in the paracrine inhibition of glucagon at elevated glucose levels, since the paracrine inhibition of glucagon secretion by somatostatin is similar for both WT and Cplx 2 KO islets. In both {beta}- and -cells, the secretion profiles are parallel to Ca2+ activity changes following somatostatin treatment of WT and Cplx 2 KO islets. The loss of paracrine inhibition of insulin secretion is substantiated by direct measurements of insulin vesicle fusion events in Cplx 2 KO islets. Together, these data show a differential role for Cplx 2 in regulating hormone secretion from pancreatic islets.

cell biology↗

bioRxiv2024

Mechanistic insights into structure-based design of a Lyme disease subunit vaccine

The quality of protective immunity plays a critical role in modulating vaccine efficacy, with native antigens often not able to trigger sufficiently strong immune responses for pathogen killing. This warrants creation of structure-based vaccine design, leveraging high-resolution antigen structures for mutagenesis to improve protein stability and efficient immunization strategies. Here, we investigated the mechanisms underlying structure-based vaccine design using CspZ-YA, a vaccine antigen from Borrelia burgdorferi, the bacteria causing Lyme disease (LD), the most common vector-borne disease in the Northern Hemisphere. Compared to wild-type CspZ-YA, we found CspZ-YAI183Y and CspZ-YAC187S required lower immunization frequency to protect mice from LD-associated manifestations and bacterial colonization. We observed indistinguishable human and mouse antigenicity between wild-type and mutant CspZ-YA proteins after native infection or active immunization. This supports our newly generated, high-resolution structures of CspZ-YAI183Y and CspZ-YAC187S, showing no altered surface epitopes after mutagenesis. However, CspZ-YAI183Y and CspZ-YAC187S favored the interactions between helices H and I, consistent with their elevated thermostability. Such findings are further strengthened by increasing ability of protective CspZ-YA monoclonal antibodies in binding to CspZ-YA at a physiological temperature (37{degrees}C). Overall, this study demonstrated enhanced intramolecular interactions improved long-term stability of antigens while maintaining protective epitopes, providing a mechanism for structure-based vaccine design. These findings can ultimately be extended to other vaccine antigens against newly emerging pathogens for the improvement of protective immunity.

immunology↗

bioRxiv2024

Restoring the Multiple Sclerosis Associated Imbalance of Gut Indole Metabolites Promotes Remyelination and Suppresses Neuroinflammation

In multiple sclerosis (MS) the circulating metabolome is dysregulated, with indole lactate (ILA) being one of the most significantly reduced metabolites. We demonstrate that oral supplementation of ILA impacts key MS disease processes in two preclinical models. ILA reduces neuroinflammation by dampening immune cell activation as well as infiltration; and promotes remyelination and in vitro oligodendrocyte differentiation through the aryl hydrocarbon receptor (AhR). Supplementation of ILA, a reductive indole metabolite, restores the gut microbiomes oxidative/reductive metabolic balance by lowering circulating indole acetate (IAA), an oxidative indole metabolite, that blocks remyelination and oligodendrocyte maturation. The ILA-induced reduction in circulating IAA is linked to changes in IAA-producing gut microbiota taxa and pathways that are also dysregulated in MS. Notably, a lower ILA:IAA ratio correlates with worse MS outcomes. Overall, these findings identify ILA as a potential anti-inflammatory remyelinating agent and provide insights into the role of gut dysbiosis-related metabolic alterations in MS progression. One Sentence SummaryIndole lactate, a postbiotic metabolite reduced in MS, corrects gut microbiome metabolic imbalances associated with remyelination and neuroinflammation.

neuroscience↗

bioRxiv2024

FtsK Initiates the Assembly of a Unique Divisome Complex in the FtsZ-less Chlamydia trachomatis

Chlamydia trachomatis serovar L2 (Ct), an obligate intracellular bacterium that does not encode FtsZ, divides by a polarized budding process. In the absence of FtsZ, we show that FtsK, a chromosomal translocase, is critical for divisome assembly in Ct. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome in Ct has a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly in Ct and inhibits cell division and septal peptidoglycan synthesis. We further show that MreB does not function like FtsZ and serve as a scaffold for the assembly of the Ct divisome. Rather, MreB is one of the last proteins recruited to the chlamydial divisome, and it is necessary for the formation of septal peptidoglycan rings. Our studies illustrate the critical role of chlamydial FtsK in coordinating divisome assembly and peptidoglycan synthesis in this obligate intracellular bacterial pathogen.

microbiology↗

bioRxiv2024

Divergent and Convergent TMEM106B Pathology in Murine Models of Neurodegeneration and Human Disease

TMEM106B is a lysosomal/late endosome protein that is a potent genetic modifier of multiple neurodegenerative diseases as well as general aging. Recently, TMEM106B was shown to form insoluble aggregates in postmortem human brain tissue, drawing attention to TMEM106B pathology and the potential role of TMEM106B aggregation in disease. In the context of neurodegenerative diseases, TMEM106B has been studied in vivo using animal models of neurodegeneration, but these studies rely on overexpression or knockdown approaches. To date, endogenous TMEM106B pathology and its relationship to known canonical pathology in animal models has not been reported. Here, we analyze histological patterns of TMEM106B in murine models of C9ORF72-related amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD), SOD1-related ALS, and tauopathy and compare these to postmortem human tissue from patients with C9-ALS/FTD, Alzheimers disease (AD), and AD with limbic-predominant age-related TDP-43 encephalopathy (AD/LATE). We show that there are significant differences between TMEM106B pathology in mouse models and human patient tissue. Importantly, we also identified convergent evidence from both murine models and human patients that links TMEM106B pathology to TDP-43 nuclear clearance specifically in C9-ALS. Similarly, we find a relationship at the cellular level between TMEM106B pathology and phosphorylated Tau burden in Alzheimers disease. By characterizing endogenous TMEM106B pathology in both mice and human postmortem tissue, our work reveals considerations that must be taken into account when analyzing data from in vivo mouse studies and elucidates new insights supporting the involvement of TMEM106B in the pathogenesis and progression of multiple neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Decoding Spatial Tissue Architecture: A Scalable Bayesian Topic Model for Multiplexed Imaging Analysis

Recent progress in multiplexed tissue imaging is advancing the study of tumor microenvironments to enhance our understanding of treatment response and disease progression. Cellular neighborhood analysis is a popular computational approach for these complex image data. Despite its popularity, there are significant challenges, including high computational demands that limit feasibility for largescale applications and the lack of a principled strategy for integrative analysis across images. This absence hampers the precise and consistent identification of spatial features and tracking of their dynamics over disease progression. To overcome these challenges, we introduce SpatialTopic, a spatial topic model designed to decode high-level spatial architecture across multiplexed tissue images. SpatialTopic integrates both cell type and spatial information within a topic modelling framework, originally developed for natural language processing and adapted for computer vision. Spatial information is incorporated into the flexible design of documents, representing densely overlapping regions in images. We employ an efficient collapsed Gibbs sampling algorithm for model inference. We benchmarked the performance against five state-of-the-art algorithms through various case studies using different single-cell spatial transcriptomic and proteomic imaging platforms across different tissue types. We show that SpatialTopic is highly scalable on large-scale image datasets with millions of cells, along with high precision and interpretability. Our findings demonstrate that SpatialTopic consistently identifies biologically and clinically significant spatial "topics" such as tertiary lymphoid structures (TLSs) and tracks dynamic changes in spatial features over disease progression. Its computational efficiency and broad applicability across various molecular imaging platforms will enhance the analysis of large-scale tissue imaging datasets.

bioinformatics↗

bioRxiv2024

mRNA-based influenza vaccine expands breadth of B cell response in humans

Summary paragraphEliciting broad and durable antibody responses against rapidly evolving pathogens like influenza viruses remains a formidable challenge1,2. The germinal center (GC) reaction enables the immune system to generate broad, high-affinity, and durable antibody responses to vaccination3-5. mRNA-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines induce persistent GC B cell responses in humans6-9. Whether an mRNA-based influenza vaccine could induce a superior GC response in humans compared to the conventional inactivated influenza virus vaccine remains unclear. We assessed B cell responses in peripheral blood and draining lymph nodes in cohorts receiving the inactivated or mRNA-based quadrivalent seasonal influenza vaccine. Participants receiving the mRNA-based vaccine produced more robust plasmablast responses and higher antibody titers to H1N1 and H3N2 influenza A viruses and comparable antibody titers against influenza B virus strains. Importantly, mRNA-based vaccination stimulated robust recall B cell responses characterized by sustained GC reactions that lasted at least 26 weeks post-vaccination in three of six participants analyzed. In addition to promoting the maturation of responding B cell clones, these sustained GC reactions resulted in enhanced engagement of low-frequency pre-existing memory B cells, expanding the landscape of vaccine-elicited B cell clones. This translated to expansion of the serological repertoire and increased breadth of serum antibody responses. These findings reveal an important role for the induction of persistent GC responses to influenza vaccination in humans to broaden the repertoire of vaccine-induced antibodies.

immunology↗

bioRxiv2024

SAMPL-seq reveals micron-scale spatial hubs in the human gut microbiome

The local arrangement of microbes can profoundly impact community assembly, function, and stability. To date, little is known about the spatial organization of the human gut microbiome. Here, we describe a high-throughput and streamlined method, dubbed SAMPL-seq, that samples microbial composition of micron-scale sub-communities with split-and-pool barcoding to capture spatial colocalization in a complex consortium. SAMPL-seq analysis of the gut microbiome of healthy humans identified bacterial taxa pairs that consistently co-occurred both over time and across multiple individuals. These colocalized microbes organize into spatially distinct groups or "spatial hubs" dominated by Bacteroideceae, Ruminococceae, and Lachnospiraceae families. From a dietary perturbation using inulin, we observed reversible spatial rearrangement of the gut microbiome, where specific taxa form new local partnerships. Spatial metagenomics using SAMPL-seq can unlock new insights to improve the study of microbial communities. One Sentence SummaryHigh throughput micron-scale subcommunity sampling and sequencing identifies distinct spatial associations of gut bacteria within and across individuals.

systems biology↗

bioRxiv2024

Early disruption of entorhinal dopamine in a knock-in model of Alzheimer's disease

The entorhinal cortex (EC) is a critical brain area for memory formation, while also the region exhibiting the earliest histological and functional alterations in Alzheimers disease (AD). The EC thus has been long hypothesized as one of the originating brain areas of AD pathophysiology, although circuit mechanisms causing its selective vulnerability remain poorly understood. We found that dopamine neurons projecting their axons to the lateral EC (LEC), critical for memory formation in healthy brains, become dysfunctional and cause memory impairments in early AD brains. In amyloid precursor protein knock-in mice with associative memory impairment, LEC dopamine activity and associative memory encoding of LEC layer 2/3 neurons were disrupted in parallel from the early pathological stage. Optogenetic reactivation of LEC dopamine fibers, as well as L- DOPA treatment, rescued associative learning behavior. These results suggest that dysfunction of LEC-projecting dopamine neurons underlies memory impairment in AD from early stages, pointing to a need for clinical investigation of LEC dopamine in AD patients.

neuroscience↗

bioRxiv2024

CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

Precise transcriptional regulation is critical for cellular function and development, yet the mechanism of this process remains poorly understood for many genes. To gain a deeper understanding of the regulation of neuropsychiatric disease risk genes, we identified a total of 39 functional enhancers for four dosage-sensitive genes, APP, FMR1, MECP2, and SIN3A, using CRISPR tiling deletion screening in human induced pluripotent stem cell (iPSC)-induced excitatory neurons. We found that enhancer annotation provides potential pathological insights into disease-associated copy number variants. More importantly, we discovered that allelic enhancer deletions at SIN3A could be compensated by increased transcriptional activities from the other intact allele. Such allelic compensation effects (ACE) on transcription is stably maintained during differentiation and, once established, cannot be reversed by ectopic SIN3A expression. Further, ACE at SIN3A occurs through dosage sensing by the promoter. Together, our findings unravel a regulatory compensation mechanism that ensures stable and precise transcriptional output for SIN3A, and potentially other dosage-sensitive genes.

genetics↗