Biology subjects

Kim, S. Y.

Publications and source records attributed to Kim, S. Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

CD14 plays a critical role in pain and inflammation across multiple models of post-traumatic osteoarthritis

Inflammation is a primary driver of osteoarthritis (OA), and no therapies exist to halt or delay disease progression or substantially ameliorate the chronic pain, inflammation and disability that are characteristic of disease. Soluble CD14 (sCD14), a co-factor that enhances inflammatory toll-like receptor signaling, is present in synovial fluid in patients with OA and positively associates with joint space narrowing, synovial macrophage content, and pain. In this study, we show that increased sCD14 within human synovial fluid correlates with joint effusion volume and increased knee hyperalgesia. Next, we evaluated CD14 as a potential therapeutic target in three pre-clinical models of post traumatic OA (PTOA), using both genetic deficiency and pharmacologic blockade to modulate its activity. We demonstrate that deficiency or blockade of CD14 results in significant protection from increased evoked pain behaviors and from OA driven mobility impairments (i.e. decreased cage activity) across models that differ in severity, and across male and female cohorts. Using flow cytometry, single cell transcriptomics, and spatial proteomics, we further show that CD14-deficiency drastically influences the local synovial inflammatory landscape post-injury, reducing monocyte and macrophage populations and modulating local fibroblast populations. Targeting CD14 via genetic deficiency or therapeutic blockade revealed no substantial protection, but no worsening, of cartilage degeneration. Ultimately, our results provide strong support that targeting synovial inflammation through blockade of CD14 can safely ameliorate OA pain and disability after a pre-disposing injury. One Sentence SummaryThe study demonstrates the key role of CD14 in pain, mobility loss, and inflammation in PTOA, and demonstrates the therapeutic potential of CD14 blockade for OA pain relief.

immunology↗

bioRxiv2025

CREB5 promotes immunotherapy resistance via tumor-intrinsic collagenmatrix deposition

Treatment with immune checkpoint inhibitors induces remarkable clinical responses in several cancer types. However, most cancer patients fail to respond to immunotherapy, and patients who initially respond often exhibit acquired resistance. Understanding the universe of immune evasion strategies will enable design of more effective immunotherapies. Here, we identify genes that drive immune evasion using genome-scale in vivo CRISPR gain-of-function screens in tumors treated with anti-PD-1 antibodies and found that the transcription factor CREB5 drives immune checkpoint blockade resistance. Using transcriptional profiling and functional studies, we show that CREB5 promotes a mesenchymal-like phenotype in melanoma characterized by upregulation of extracellular matrix genes including collagen and collagen-stabilizing factors. Using engineered tumor models and knockout mice, we found that immunotherapy resistance is functionally mediated by tumor-intrinsic collagen deposition. Collagen is the major ligand for the inhibitory receptor LAIR1, broadly expressed on T cells, B cells, NK cells, and myeloid cells. Deletion of LAIR1 in mice or overexpression of the decoy receptor LAIR2 in tumors abrogated the resistance induced by CREB5 overexpression, demonstrating that collagen-LAIR1 inhibitory signaling drives resistance to immune checkpoint inhibitors. These observations define a transcriptional program that remodels the tumor microenvironment to promote immunotherapy resistance via extracellular matrix deposition and indicates that targeting this pathway may enhance immunotherapy efficacy. One-Sentence Summary: In vivo gain-of-function screening in immunotherapy-treated mice reveals a transcription factor, Creb5, that drives the mesenchymal state in melanoma and facilitates immune escape by promoting tumor-intrinsic collagen matrix deposition.

cancer biology↗

bioRxiv2025

Human giant GTPase GVIN1 forms an antimicrobial coatomer around the intracellular bacterial pathogen Burkholderia thailandensis

Several human pathogens exploit the kinetic forces generated by polymerizing actin to power their intracellular motility. Human cell-autonomous immune responses activated by the cytokine interferon-gamma (IFNg) interfere with such microbial actin-based motility, yet the underlying molecular mechanisms are poorly defined. Here, we identify the IFNg-inducible human giant GTPases GVIN1 as a novel host defense protein that blocks the bacterial pathogen Burkholderia thailandensis from high-jacking the hosts actin polymerization machinery. We found that GVIN1 proteins form a coatomer around cytosolic bacteria and prevent Burkholderia from establishing force-generating actin comet tails. Coatomers formed by a second IFNg-inducible GTPase, human guanylate binding protein 1 (GBP1), constitute a GVIN1-independent but mechanistically related anti-motility pathway. We show that coating with either GVIN1 or GBP1 displaces the Burkholderia outer membrane protein BimA, an actin nucleator that is essential for actin tail formation. Both GVIN1 and GBP1 coatomers require additional IFNg-inducible co-factors to disrupt the membrane localization of BimA, demonstrating the existence of two parallel-acting IFNg-inducible defense modules that evolved to target a virulence trait critical for the pathogenesis of numerous bacterial infectious agents.

immunology↗

bioRxiv2024

Morphological and Molecular Phylogenetic Characterization of Three New Marine Goniomonad Species

Goniomonads are commonly found heterotrophic biflagellates in both marine and freshwater environments. Despite the high genetic diversity inferred from 18S rDNA data, many goniomonad species remain undescribed. In this study, we established a total of 21 marine goniomonad culture strains, and from these, describe three new species by using light microscopy, electron microscopy, and 18S rDNA phylogeny. Molecular sequence analyses suggest the presence of several distinct sub-lineages within the marine goniomonad clade. Two of these are Goniomonas ulleungensis sp. nov. and G. lingua sp. nov., which are similar in size, flagellar length, appendage & orientation and by having a tongue-like protrusion at the anterior. The two species can be differentiated by the periplast plate pattern with G. ulleungensis displaying one additional plate on the right side. G. duplex sp. nov. differed from these two species by having two unequal flagella with the longer one trailing posteriorly and having the opposite cell orientation when skidding. Comparative analyses of five marine goniomonad species showed that genetically distinct goniomonad groups can be delineated by morphological data as well, and of several morphological features that are of taxonomic utility, the periplast plate pattern, observable by SEM, is particularly informative in goniomonad taxonomy.

evolutionary biology↗

bioRxiv2024

ATF4 regulates mitochondrial dysfunction, mitophagy, and autophagy, contributing to corneal endothelial apoptosis under chronic ER stress in Fuchs dystrophy

PURPOSEEndoplasmic reticulum (ER) stress, mitochondrial dysfunction, and mitophagy are known to contribute independently to corneal endothelial cell (CEnC) apoptosis in Fuchs endothelial corneal dystrophy (FECD). However, the role of a specific ER stress pathway (PERK-ATF4-CHOP) in regulating these events is unknown. This study aims to investigate the role of ATF4 in regulating mitochondrial dysfunction and mitophagy, which ultimately leads to CEnC apoptosis in FECD. METHODSHuman corneal endothelial cell line (21T), Fuchs corneal endothelial cell line (F35T), and primary human corneal endothelial cells were treated with ER stressor tunicamycin (Tun). ATF4 siRNA was used to knock down ATF4 in 21T cell line and primary corneal endothelial cells. Mitophagy and apoptotic proteins were analyzed using Western blotting. ATF4+/- and ATF4 +/+ mice were irradiated with UVA to assess ER stress and corneal endothelial apoptosis. RESULTSF35T cell line had significantly increased expression of the ER stress pathway as well as caspase-mediated apoptotic molecules compared to 21T at baseline, which further increased after tunicamycin treatment. F35T cells exhibited significantly decreased ATP and MMP, and increased mitochondrial fragmentation, which was further exacerbated after Tunicamycin. F35T cell line also demonstrated inhibition of mitophagy, similar to 21T, after treatment with Tunicamycin, despite the upregulation of mitophagy initiators. ATF4 knockdown attenuated ER and mitochondrial stress proteins, rescued mitochondrial membrane potential (MMP) loss, downregulated mitochondrial fragmentation, activated mitophagy, and prevented cell death under chronic ER stress. ATF4+/-mice had increased CE numbers, with improved cellular morphology and decreased ER stress CHOP expression, compared to ATF4+/+ mice post-UVA. CONCLUSIONSPro-apoptotic ATF4 induction following ER stress disrupts mitochondrial function, leading to mitophagy inhibition and CEnC apoptosis. This study highlights the importance of ATF4 in ER-mitochondrial crosstalk and its contribution to CEnC apoptosis in FECD.

cell biology↗

bioRxiv2024

The role of phosphorylation and dephosphorylation in the regulation of Rubisco activase

Rubisco activase is an ATP-dependent chaperone that facilitates dissociation of inhibitory sugar phosphates from the catalytic sites of ribulose-1,5-bisphosphate carboxylase/oxygenase during photosynthesis. In Arabidopsis, Rubisco activase is negatively regulated by dark-dependent phosphorylation of threonine 78. The prevalence of threonine 78 in Rubisco activase was investigated across sequences from 91 plant species, finding 29 ([~]32%) species shared a threonine in the same position. Analysis of seven C3 species with an antibody raised against a threonine 78 phospho-peptide demonstrated that this position is phosphorylated in multiple genera. However, light-dependent dephosphorylation of threonine 78 was observed only in Arabidopsis. Further, phosphorylation of threonine 78 could not be detected in any of the four C4 grass species examined. The results suggest that despite conservation of threonine 78 in Rubisco activase from a wide range of species, a regulatory role for phosphorylation at this site is more limited. This provides a case study for how variation in post-translational regulation can amplify functional divergence across the phylogeny of plants beyond what is explained by sequence variation in a metabolically important protein.

plant biology↗

bioRxiv2024

The molecular framework underlying the growth and defense tradeoff in Arabidopsis roots in response to danger signals

Elevated stress signaling often compromises plant growth by suppressing proliferative and formative divisions at the meristem. Plant Elicitor Peptide 1 (PEP1), an endogenous danger signal triggered by both biotic and abiotic stresses in Arabidopsis thaliana, suppresses proliferative divisions, alters xylem vessel organizations, and disrupts cell-to-cell symplastic connections in the root. To gain an insight into the dynamic molecular framework that modulates root development under elevated danger signals, we performed a time course RNA-sequencing analysis at the root meristem following PEP1 treatment. A series of data analyses revealed that STZ and its homologs are a potential nexus between the stress response and proliferative cell cycle regulation. We observed that STZ differentially controls the cell cycle, cell differentiation, and stress response genes at various tissue layers in the root meristem through various functional, phenotypic, and transcriptomic analyses. Our study further indicated that the expression level of STZ in response to stresses is critically important to enable the growth and defense tradeoff. The findings here provide valuable information about the dynamic gene expression changes upon perceiving danger signals at the root meristem and future engineering schemes to generate stress-resilient plants.

plant biology↗

bioRxiv2024

In-situ microscopy-assisted meniscus-guided coating for highly sensitive reduced graphene oxide-based nanocomposite biosensor

Meniscus-guided coating provides great potential for fabricating the nanomaterial-based thin film into high-performance biomedical devices due to the strong relationship between its experimental parameters and the resulting structural properties. However, the complex leverages of various fluid dynamics phenomena hamper optimization of structural properties and device performances. This is due to the absence of in-depth analytical techniques to observe, interpret, and control the solidification process. In this work, we propose an analytical strategy based on the rheological properties of a rGO-based solution using computational fluid dynamics modeling and in situ high-speed microscopy. Through this, we reveal the principles of the solidification mechanism that creates a rGO-based nanocomposite in the form of highly- and evenly-wrinkled thin film and the experimental condition at which this mechanism occurs. The optimized thin film presents high electroconductivity, low chip-to-chip signal variation, and multiplexed electrochemical biosensing performance for three classes of antibodies related to the excessive enrichment of endoplasmic reticulum stress, with detection limits of picomolar levels. This optimizing technique can be universally applied to understanding various solution-based coating systems, and can streamline the production of large-area and high-quality nanocomposite biosensors.

bioengineering↗

bioRxiv2023

Recommendations For a Standardized Approach to Histopathologic Evaluation of Synovial Membrane in Murine Models of Experimental Osteoarthritis

BackgroundSynovial pathology has been linked to osteoarthritis (OA) pain in patients. Microscopic grading systems for synovial changes in human OA have been described, but a standardized approach for murine models of OA is needed. We sought to develop a reproducible approach and set of minimum recommendations for synovial histopathology in mouse models of OA. MethodsCoronal and sagittal sections from male mouse knee joints subjected to destabilization of medial meniscus (DMM) or partial meniscectomy (PMX) were collected as part of other studies. Stains included Hematoxylin and Eosin (H&E), Toluidine Blue (T- Blue) and Safranin O/Fast Green (Saf-O). Four blinded readers graded pathological features (hyperplasia, cellularity, and fibrosis) at specific anatomic locations in the medial and lateral compartments. Inter-reader reliability of each feature was determined. ResultsThere was acceptable to very good agreement between raters. After DMM, increased hyperplasia and cellularity and a trend towards increased fibrosis were observed 6 weeks after DMM in the medial locations, and persisted up to 16 weeks. In the PMX model, cellularity and hyperplasia were evident in both medial and lateral compartments while fibrotic changes were largely seen on the medial side. Synovial changes were consistent from section to section in the mid-joint area mice. H&E, T-blue, and Saf-O stains resulted in comparable reliability. ConclusionsTo allow for a standard evaluation that can be implemented and compared across labs and studies, we recommend using 3 readers to evaluate a minimum set of 3 pathological features at standardized anatomic areas. Pre-defining areas to be scored, and reliability for each pathologic feature should be considered.

pathology↗

bioRxiv2023

Physiologic Doses of TGF-beta Improve the Composition of Engineered Articular Cartilage

For cartilage regeneration applications, transforming growth factor beta (TGF-{beta}) is conventionally administered at highly supraphysiologic doses (10-10,000 ng/mL) in an attempt to cue cells to fabricate neocartilage that matches the composition, structure, and functional properties of native hyaline cartilage. While supraphysiologic doses enhance ECM biosynthesis, they are also associated with inducing detrimental tissue features, such as fibrocartilage matrix deposition, pathologic-like chondrocyte clustering, and tissue swelling. Here we investigate the hypothesis that moderated TGF-{beta} doses (0.1-1 ng/mL), akin to those present during physiological cartilage development, can improve neocartilage composition. Variable doses of media-supplemented TGF-{beta} were administered to a model system of reduced-size cylindrical constructs (O2-O3 mm), which mitigate the TGF-{beta} spatial gradients observed in conventional-size constructs (O4-O6 mm), allowing for a novel assessment of the intrinsic effect of TGF-{beta} doses on macroscale neocartilage properties and composition. The administration of physiologic TGF-{beta} to reduced-size constructs yields neocartilage with native-matched sGAG content and mechanical properties while providing a more hyaline cartilage-like composition, marked by: 1) reduced fibrocartilage-associated type I collagen, 2) 77% reduction in the fraction of cells present in a clustered morphology, and 3) 45% reduction in the degree of tissue swelling. Physiologic TGF-{beta} appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating hyaline cartilage compositional deficits. These results can guide the development of novel physiologic TGF-{beta}-delivering scaffolds to improve the regeneration clinical-sized neocartilage tissues.

bioengineering↗

bioRxiv2023

Widespread regulation of the maternal transcriptome by Nanos in Drosophila

The translational repressor Nanos (Nos) regulates a single target, maternal hunchback (hb) mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited specifically to sites in the 3-UTR of hb mRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via two approaches. In the first method, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition to hb, Upf1-Nos depletes [~]2600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical, hb-like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos from nos- females. Most (86%) of the 1185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Approximately 60% of mRNAs targeted by Upf1-Nos are not stabilized in the absence of Nos. However, Upf1-Nos mRNA targets are hypo-adenylated and inefficiently translated at the ovary-embryo transition, whether or not they suffer Nos-dependent degradation in the embryo. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors during the MZT in the embryo.

developmental biology↗

bioRxiv2023

Unilateral ephaptic program underlying sweetness dominance

In ephaptic coupling, physically adjacent neurons influence one anothers activity via the electric fields they generate. To date, the molecular mechanisms that mediate and modulate ephaptic couplings effects remain poorly understood. Here, we show that the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel lateralizes the potentially mutual ephaptic inhibition between Drosophila gustatory receptor neurons (GRNs). While sweet-sensing GRNs (sGRNs) engage in ephaptic suppression of the adjacent bitter-sensing GRNs (bGRNs), HCN expression in sGRNs enables them to resist ephaptic suppression from the bGRNs. Such one-sided ephaptic inhibition confers sweetness dominance, facilitating ingestion of bitter-laced sweets. Flies with HCN-deficient sGRNs exhibited dramatically decreased attraction to sucrose mixed with moderate levels of caffeine, highlighting the behavioral significance that gustatory ephaptic inhibition promotes ingestion of carbohydrates buried in bitterness. Our findings indicate a role for the gating of ephaptic coding to ensure the intake of the essential nutrient despite bitter contaminants present in the feeding niche of Drosophila, as the gating establishes a hierarchy of gustatory neuron excitation. Such refinement provides a previously unappreciated mechanism for controlling the activity of a neuronal network with potential implications in the mammalian brain, given the evolutionary conservation of the HCN genes.

neuroscience↗

bioRxiv2023

Angiogenic Steroids Induce Pathologic Scarring in a Preclinical Swine Model Via Dysfunctional Extracellular Matrix Deposition.

BackgroundHypertrophic scarring is a major source of morbidity for surgery patients. Sex hormones are not classically considered to be modulators of scarring. However, based on clinical observations of increased frequency of hypertrophic scarring in patients on testosterone, we hypothesized that androgenic steroids induce abnormal scarring and developed a preclinical swine model to explore these effects. MethodsA total of six male (XY) and female (XX) mini-swine underwent castration and were randomly assigned to no testosterone (noT) or biweekly testosterone therapy (+T). Ten dorsal excisional wounds were created on each pig. To mimic a chronic wound, a subset of wounds were re-excised at two weeks. Scars (POD42) and chronic wounds (POD28) were harvested six weeks after initial wounding for analysis via histology, RNA-seq, and mechanical testing. ResultsHistologic analysis of POD42 scars from +T swine showed increased mean fibrosis area (16mm2 noT, 28mm2 +T; p=0.007) and thickness (0.246mm2 noT, 0.406mm2 +T; p<0.001) compared to noT swine. Scars in XX+T and XY+T pigs had greater tensile burst strength (p=0.024 and p=0.013 respectively) compared to scars in noT swine. Color deconvolution analysis showed greater deposition of type I and type III collagen as well as increased type I to type III collagen ratio in +T scars. Dermatopathologist scores of POD42 scars show +T exposure was associated with worse overall scarring scores compared to controls (p<0.05). On RNAseq, gene ontology analysis showed testosterone exposure was associated with significant upregulation of cellular metabolism and immune response gene sets. Pathway analysis showed testosterone upregulated Reactome pathways related to keratinization and formation of collagen and laminin. ConclusionWe developed a novel preclinical porcine model to study the effects of the sex hormone testosterone on scarring. Testosterone induces early proliferation of excessive granulation tissue, which eventually leads to increased scar tissue. T also appears to increase the physical strength of scars via supraphysiologic deposition of collagen and other ECM factors. The increase in burst strength observed for both XX and XY suggests that hormonal administration has a stronger influence on mechanical properties than chromosomal sex. Antiandrogen topical therapies may be a promising future area of research.

systems biology↗

bioRxiv2023

Methylglyoxal is the main culprit to impairing neuronal function: mediated through tryptophan depletion.

Depression is a common and prevalent illness and the exact cause of major depressive disorder is not known. Here, we investigated how methylglyoxal (MGO) stress induces depression and unveiled the potential molecular mechanism. Our in vivo results suggested that MGO caused depression in mice, confirmed by several behavioral tests. Interestingly, it halted the mices brains tryptophan levels and its related neurotransmitters. In addition, MGO induced a reduction in the number of cells in different hippocampal regions. Moreover, it decreased tryptophan hydroxylase 1 (TPH1) and tryptophan hydroxylase 1 (TPH2) levels in the brain and large intestine. Surprisingly, MGO showed the highest affinity and trapping ability toward tryptophan. Most importantly, combined treatment with MGO-tryptophan displayed similar effects as those exhibited by the tryptophan-null treatment in neuronal cells, which included neuronal apoptosis, decrease TPH1 and TPH2 levels, and inhibition of neuronal outgrowth. However, tryptophan treatment improved MGO induced depression-like behavior of mice and recovered the loss of neuronal and hippocampal cells. Subsequently, it also induced MGO detoxifying factors, tryptophan levels, and reduces inflammation in the intestine. Collectively, our data revealed that MGO induced depression facilitated by neuronal and synaptic dysfunction is mediated through the disturbance of tryptophan metabolism in the brain and intestine.

neuroscience↗

bioRxiv2022

A synthetic lethal screen for Snail-induced enzalutamide resistance identifies JAK/STAT signaling as a therapeutic vulnerability in prostate cancer

Despite substantial improvements in the treatment landscape of prostate cancer, the evolution of hormone therapy-resistant and metastatic prostate cancer remains a major cause of cancer-related death globally. The mainstay of treatment for advanced prostate cancer is targeting of androgen receptor signaling, including androgen deprivation therapy plus second-generation androgen receptor blockade (e.g., enzalutamide, apalutamide, darolutamide), and/or androgen synthesis inhibition (abiraterone). While these agents have significantly prolonged the lives of patients with advanced prostate cancer, the evolution of resistance to these treatments in nearly universal. This therapy resistance is mediated by diverse mechanisms, including both androgen receptor-dependent mechanisms, such as androgen receptor mutations, amplifications, alternatively spliced isoforms, and structural rearrangements, as well as non-androgen receptor-mediated mechanisms, such as lineage plasticity toward neuroendocrine-like or epithelial-mesenchymal transition (EMT)-like lineages. Our prior work identified the EMT transcriptional regulator Snail as critical to hormonal therapy resistance and commonly detected in human metastatic prostate cancer. In the current study, we sought to interrogate the actionable landscape of EMT-mediated hormone therapy-resistant prostate cancer to identify synthetic lethality and collateral sensitivity approaches to treating this aggressive disease state. Using a combination of high-throughput drug screens and multi-parameter phenotyping by confluence imaging, ATP production, and phenotypic plasticity reporters of EMT, we identified candidate synthetic lethalities to Snail-mediated EMT in prostate cancer. These analyses identified multiple actionable targets, such as XPO1, PI3K/mTOR, aurora kinases, c-MET, polo-like kinases, and JAK/STAT as synthetic lethalities in Snail+ prostate cancer. We validated these targets in a subsequent validation screen in an LNCaP-derived model of resistance to sequential androgen deprivation and enzalutamide. This follow-up screen provided validation of inhibitors of JAK/STAT and PI3K/mTOR as therapeutic vulnerabilities for Snail+ and enzalutamide-resistant prostate cancer.

cancer biology↗

bioRxiv2022

Murine macrophage choline metabolism underpins IL-4 polarization and RELMα up-regulation

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated upregulation of choline transport compared to naive macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELM (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naive or mice infected with intestinal helminths Heligmosomoides polygyrus or Nippostrongylus brasiliensis were treated with the choline kinase inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELM expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus with increased egg burden, but there were no differences in intestinal worm count nor differences in N. brasiliensis parasite burden. Together, these data demonstrate that choline metabolism is required for macrophage RELM induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/510305v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@2f83a3org.highwire.dtl.DTLVardef@16b74b1org.highwire.dtl.DTLVardef@1ba1f8dorg.highwire.dtl.DTLVardef@6da06a_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Neuronal mitochondrial morphology is significantly affected by both fixative and oxygen level during perfusion

Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs) dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating ATP and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stresses such as oxidative stress, or in various neurodegenerative diseases including Alzheimers disease and Parkinsons disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address the pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure induce dramatic fragmentation of mitochondria both in vitro and in vivo. This disruption was not observed following the use of glutaraldehyde addition or oxygen supplementation, respectively. Finally, using pre-formed fibril -synuclein treated neurons, we show a difference between mitochondrial morphology when samples were fixed with PFA/glutaraldehyde or PFA/sucrose containing solutions, but not PFA alone. Our study provides optimized methods for examining mitochondrial morphology in neurons, and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.

neuroscience↗

bioRxiv2022

Mouse genomic rewriting and tailoring: synthetic Trp53 and humanized ACE2

Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection.

genomics↗