Biology subjects

Kim, E.

Publications and source records attributed to Kim, E..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Activation mapping in multi-center rat sensory-evoked functional MRI datasets using a unified pipeline.

Functional Magnetic Resonance Imaging (fMRI) in rodents is pivotal for understanding the mechanisms underlying Blood Oxygen Level-Dependent (BOLD) signals and phenotyping animal models of disorders, amongst other applications. Despite its growing use, comparing rodent fMRI results across different research sites remains challenging due to variations in experimental protocols. Here, we aggregated and analyzed 22 sensory-evoked rat fMRI datasets from 12 imaging centers, totaling scans from 220 rats, to assess the consistency of results across diverse protocols. We applied a standardized preprocessing pipeline and evaluated the impact of different hemodynamic response function models on group and individual level activity patterns. Our analysis revealed inter-dataset variability attributed to differences in experimental design, anesthesia protocols, and imaging parameters. We identified robust activation clusters in all (22/22) datasets. The comparison between stock human models implemented in software and rat-specific models showed significant variations in the resulting statistical maps. Our findings emphasize the necessity for standardized protocols and collaborative efforts to improve the reproducibility and reliability of rodent fMRI studies. We provide open access to all datasets and analysis code to foster transparency and further research in the field.

neuroscience↗

bioRxiv2024

Therapeutic modulation of ROCK overcomes metabolic adaptation of cancer cells to OXPHOS inhibition and drives synergistic anti-tumor activity

Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer. Previously, we and others have identified that SMARCA4-mutant lung cancers are highly dependent on oxidative phosphorylation (OXPHOS). Despite initial excitements, therapeutics targeting metabolic pathways such as OXPHOS have largely been disappointing due to rapid adaptation of cancer cells to inhibition of single metabolic enzymes or pathways, suggesting novel combination strategies to overcome adaptive responses are urgently needed. Here, we performed a functional genomics screen using CRISPR-Cas9 library targeting genes with available FDA approved therapeutics and identified ROCK1/2 as a top hit that sensitizes cancer cells to OXPHOS inhibition. We validate these results by orthogonal genetic and pharmacologic approaches by demonstrating that KD025 (Belumosudil), an FDA approved ROCK inhibitor, has highly synergistic anti-cancer activity in vitro and in vivo in combination with OXPHOS inhibition. Mechanistically, we showed that this combination induced a rapid, profound energetic stress and cell cycle arrest that was in part due to ROCK inhibition-mediated suppression of the adaptive increase in glycolysis normally seen by OXPHOS inhibition. Furthermore, we applied global phosphoproteomics and kinase-motif enrichment analysis to uncover a dynamic regulatory kinome upon combination of OXPHOS and ROCK inhibition. Importantly, we found converging phosphorylation-dependent regulatory cross-talk by AMPK and ROCK kinases on key RHO GTPase signaling/ROCK-dependent substrates such as PPP1R12A, NUMA1 and PKMYT1 that are known regulators of cell cycle progression. Taken together, our study identified ROCK kinases as critical mediators of metabolic adaptation of cancer cells to OXPHOS inhibition and provides a strong rationale for pursuing ROCK inhibitors as novel combination partners to OXPHOS inhibitors in cancer treatment.

cancer biology↗

bioRxiv2024

Symmetry of loop extrusion by dimeric SMC complexes is DNA-tension-dependent

Structural maintenance of chromosome (SMC) complexes organize and regulate genomes by extruding DNA loops. During loop extrusion, DNA can be reeled into the growing loop from one or both sides, generating distinct extrusion directionality states whose physical basis remains unclear. Here, we combine single-molecule analysis and molecular dynamics simulations to investigate loop extrusion directionality across SMC complexes. We show that dimeric Smc5/6 and Wadjet predominantly perform two-sided loop extrusion during initial loop growth, whereas monomeric condensin exhibits one-sided extrusion, consistent with a relationship between stoichiometry and extrusion directionality. Surprisingly, however, cohesin predominantly exhibits one-sided extrusion despite functioning as a dimeric complex. Notably, dimeric Smc5/6 and Wadjet progressively transition from two-sided to one-sided extrusion as loop growth matures. Simulations and force-dependent analysis reveal that loop extrusion directionality is governed by a tension-dependent transition in dimeric motors. Increasing DNA tension erodes two-sided extrusion near the motor stalling force, driving a tug-of-war regime in which competing motors transiently dominate one another. This transition promotes one-sided extrusion during mature Smc5/6 and Wadjet-mediated loop extrusion and places cohesin, which has a comparatively low stalling force ([~]0.1 pN), constitutively near this regime. Together, our findings establish loop extrusion directionality as a dynamic emergent property governed by SMC stoichiometry, DNA tension, and stochastic motor competition.

biophysics↗

bioRxiv2024

FZD2 inhibits YAP and prevents cell cycle reentry in adult murine cardiomyocytes

RationaleFully differentiated cardiomyocytes (CMs) are post-mitotic and cannot repopulate damaged tissue after myocardial infarction (MI). Understanding the mechanisms preventing CM proliferation or promoting their survival after injury may lead to treatment strategies for MI. While the effects of canonical WNT/{beta}-catenin signaling in adult CMs have been examined, roles for non-canonical WNT signaling in cardiac homeostasis and repair remain unexplored. ObjectiveTo determine the function of the non-canonical WNT receptor frizzled 2 (FZD2) in adult cardiac homeostasis and injury. Methods and ResultsFZD2 was deleted from the CMs of adult mice to investigate its role in myocardial homeostasis. Fzd2 conditional knockout (CKO) mice had cardiomegaly but not hypertrophy. FZD2-deficient CMs expressed proliferation and cytokinesis markers, suggesting that they have increased proliferation potential. FZD2-deletion caused the accumulation of {beta}-catenin. However, {beta}-catenin localized to the membranes of FZD2-deficient CMs and did not activate target gene expression. Instead, the YES-associated protein (YAP) regulated genes v-myc avian myelocytomatosis viral oncogene 1 (Mycl), and B cell leukemia/lymphoma 2 (Bcl2l1) were upregulated in Fzd2 CKO CMs relative to controls. Knockdown of FZD2 increased YAP activity in neonatal ventricular CMs (NVCMs), while overexpressing FZD2 inhibited YAP. Neither {beta}-catenin knockdown nor mutating the large tumor suppressor 1 and 2 (LATS1/2) target site on YAP blocked the effects of FZD2 on YAP in NVCMs, suggesting that FZD2 utilizes different effectors than canonical WNT and Hippo signaling. Fzd2 CKO and control mice were subjected to MI to determine if FZD2-deletion affects cardiac repair. While ischemia and necrosis were similar 24 hours post MI, Fzd2 CKO mice had better cardiac function and less scarring than controls. ConclusionsFZD2 reduces YAP activity and prevents adult murine CMs from reentering the cell cycle. FZD2-deletion improves heart function and reduces scarring in mice after MI, implicating FZD2 as a target for pharmacological intervention.

cell biology↗

bioRxiv2024

Cortical inhibitory parvalbumin interneurons exhibit metabolic specializations coordinated by PGC-1α that are lost in rodents and humans after traumatic brain injury

Parvalbumin-positive interneurons (PV-INs) regulate neuronal and circuit activity, and their dysfunction is observed across neurological conditions, including traumatic brain injury (TBI), epilepsy, Alzheimers disease, and schizophrenia. PV-INs are particularly vulnerable to cell loss, potentially due to their increased metabolic demands arising from their uniquely high level of electrical activity, which render them susceptible to metabolic pressure. Here, we use single-nucleus RNA-sequencing (snRNAseq) data from a rodent model of TBI, as well as human TBI data, and demonstrate PV-INs have unique metabolic specializations that are lost after injury and can be rescued by in vivo treatment with the glycolytic inhibitor, 2-deoxyglucose. We generated a novel PV-IN transcriptional identity module comprised primarily of genes encoding specialized ion channels, metabolic enzymes, and synaptic machinery, that identifies heterogenous subsets of injury-associated PV-INs with loss of PV-IN transcriptional identity. We show that changes in metabolic specialization are coupled to changes in transcriptional identity in PV-INs and implicate the PV-IN-enriched transcriptional co-activator, Ppargc1a, as a key driver of PV-IN transcriptional metabolic dysfunction. We also identify a family of long non-coding RNAs enriched in this subset of transcriptionally dysfunctional PV-INs that negatively correlates with PV-IN metabolic specialization. Lastly, we utilize these tools to interrogate a published human TBI snRNAseq data set and find nearly identical changes, underscoring the importance of PV-IN metabolic dysfunction in the pathology of TBI.

neuroscience↗

bioRxiv2024

Lipid loss and compositional change during preparation of liposomes by common biophysical methods

Liposomes are widely used as model lipid membrane platforms in many fields, ranging from basic biophysical studies to drug delivery and biotechnology applications. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures have the potential to produce liposomes at specific concentrations and membrane compositions, and researchers often assume that the concentration and composition of their liposomes are similar to, if not identical, to what would be expected if no lipid loss occurred during preparation. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward method using HPLC-ELSD to quantify the lipid concentration and membrane composition of liposomes, and apply that method to study the preparation of simple POPC/cholesterol liposomes. We examine many common steps in liposome formation, including vortexing during re-suspension, hydration of the lipid film, extrusion, freeze-thaw, sonication, and the percentage of cholesterol in the starting mixture. We found that the resuspension step can play an outsized role in determining the overall lipid loss (up to [~]50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses ([~]10-20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 minutes and increasing cholesterol concentrations up to 50 mole% had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition more than [~]5% under the conditions we tested. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation protocols. We expect our results can be leveraged for improved preparation of model membranes by researchers in many fields. Statement of SignificanceLiposomes are spherical lipid membranes that can be prepared by a variety of biophysical techniques. Researchers use liposomes in a variety of ways, including fundamental biophysical studies of lipid membranes, in drug delivery, drug formulation, and other biotechnology applications. In this report, we study the process to prepare liposomes by several common techniques and validate how reliable each technique is at producing consistent liposome concentrations and lipid compositions. We identify best practices for researchers to produce reliable liposome preparations.

biophysics↗

bioRxiv2024

MSA: scalable DNA methylation screening BeadChip for high-throughput trait association studies

Epigenome-wide association studies (EWAS) are transforming our understanding of the interplay between epigenetics and complex human traits and phenotypes. We introduce the Methylation Screening Array (MSA), a new iteration of the Infinium technology for scalable and quantitative screening of trait associations of nuanced ternary-code cytosine modifications in larger, more inclusive, and stratified human populations. MSA integrates EWAS, single-cell, and cell-type-resolved methylome profiles, covering diverse human traits and diseases. Our first MSA applications yield multiple biological insights: we revealed a previously unappreciated role of 5-hydroxymethylcytosine (5hmC) in trait associations and epigenetic clocks. We demonstrated that 5hmCs complement 5-methylcytosines (5mCs) in defining tissues and cells epigenetic identities. In-depth analyses highlighted the cell type context of EWAS and GWAS hits. Using this platform, we conducted a comprehensive human 5hmC aging EWAS, discovering tissue-invariant and tissue-specific aging dynamics, including distinct tissue-specific rates of mitotic hyper- and hypomethylation rates. These findings chart a landscape of the complex interplay of the two forms of cytosine modifications in diverse human tissues and their roles in health and disease.

genomics↗

bioRxiv2024

Courtship song differs between African and European populations of Drosophila melanogaster and involves a strong effect locus

The courtship song of Drosophila melanogaster has long served as an excellent model system for studies of animal communication and differences in courtship song have been demonstrated among populations and between species. Here, we report that flies of African and European origin, which diverged approximately 13,000 years ago, show significant genetic differentiation in the use of slow versus fast pulse song. Using a combination of quantitative trait mapping and population genetic analysis we detected a single strong QTL underlying this trait and we identified candidate genes that may contribute to the evolution of this trait. Song trait variation between parental strains of our recombinant inbred panel enabled detection of genomic intervals associated with six additional song traits, some of which include known courtship-related genes. These findings improve the prospects for further genetic insights into the evolution of reproductive behavior and the biology underlying courtship song.

evolutionary biology↗

bioRxiv2024

Cannabinoid receptor 1 positive allosteric modulator ZCZ011 shows differential effects on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice

The cannabinoid receptor type 1 (CB1R) is a promising therapeutic target for various neurodegenerative diseases, including HIV-1-associated neurocognitive disorder (HAND). However, the therapeutic potential of CB1R by direct activation is limited due to its psychoactive side effects. Therefore, research has focused on indirectly activating the CB1R by utilizing positive allosteric modulators (PAMs). Studies have shown that CB1R PAMs (ZCZ011 and GAT211) are effective in mouse models of Huntingtons disease and neuropathic pain, and hence, we assess the therapeutic potential of ZCZ011 in a well-established mouse model of neuroHIV. The current study investigates the effect of chronic ZCZ011 treatment (14 days) on various behavioral paradigms and the endocannabinoid system in HIV-1 Tat transgenic female and male mice. Chronic ZCZ011 treatment (10 mg/kg) did not alter body mass, locomotor activity, or anxiety-like behavior regardless of sex or genotype. However, differential effects were noted in hot plate latency, motor coordination, and recognition memory in female mice only, with ZCZ011 treatment increasing hot plate latency and improving motor coordination and recognition memory. Only minor effects or no alterations were seen in the endocannabinoid system and related lipids except in the cerebellum, where the effect of ZCZ011 was more pronounced in female mice. Moreover, AEA and PEA levels in the cerebellum were positively correlated with improved motor coordination in female mice. In summary, these findings indicate that chronic ZCZ011 treatment has differential effects on antinociception, motor coordination, and memory, based on sex and HIV-1 Tat expression, making CB1R PAMs potential treatment options for HAND without the psychoactive side effects.

neuroscience↗

bioRxiv2024

Desmosomal Cadherin Tension Loss in Pemphigus Vulgaris Mediated by the Inhibition of Active RhoA at Cell-Cell Adhesions

Binding of autoantibodies to keratinocyte surface antigens, primarily desmoglein 3 (Dsg3) of the desmosomal complex, leads to the dissociation of cell-cell adhesion in the blistering disorder pemphigus vulgaris (PV). After the initial disassembly of desmosomes, cell-cell adhesions actively remodel in association with the cytoskeleton and focal adhesions. Growing evidence highlights the role of adhesion mechanics and mechanotransduction at cell-cell adhesions in this remodeling process, as their active participation may direct autoimmune pathogenicity. However, a large part of the biophysical transformations after antibody binding remains underexplored. Specifically, it is unclear how tension in desmosomes and cell-cell adhesions changes in response to antibodies, and how the altered tensional states translate to cellular responses. Here, we showed a tension loss at Dsg3 using fluorescence resonance energy transfer (FRET)-based tension sensors, a tension loss at the entire cell-cell adhesion, and a potentially compensatory increase in junctional traction force at cell-extracellular matrix adhesions after PV antibody binding. Further, our data indicate that this tension loss is mediated by the inhibition of RhoA at cell-cell contacts, and the extent of RhoA inhibition may be crucial in determining the severity of pathogenicity among different PV antibodies. More importantly, this tension loss can be partially restored by altering actomyosin based cell contractility. Collectively, these findings provide previously unattainable details in our understanding of the mechanisms that govern cell-cell interactions under physiological and autoimmune conditions, which may open the window to entirely new therapeutics aimed at restoring physiological balance to tension dynamics that regulates the maintenance of cell-cell adhesion.

biophysics↗

bioRxiv2024

An integrative single-cell atlas to explore the cellular and temporal specificity of neurological disorder genes during human brain development

Single-cell technologies have enhanced comprehensive knowledge regarding the human brain by facilitating an extensive transcriptomic census across diverse brain regions. Nevertheless, understanding the cellular and temporal specificity of neurological disorders remains ambiguous due to the developmental variations. To address this gap, we illustrated the dynamics of disorder risk gene expressions under development by integrating multiple single-cell RNA sequencing datasets. We constructed a comprehensive single-cell atlas of developing human brains, encompassing 393,060 single cells across diverse developmental stages. Temporal analysis revealed the distinct expression patterns of disorder risk genes, including autism, highlighting their temporal regulation in different neuronal and glial lineages. We identified distinct neuronal lineages diverged across developmental stages, each exhibiting temporal-specific expression patterns of disorder genes. Lineages of non-neuronal cells determined by molecular profiles also showed temporal-specific expressions, indicating a link between cellular maturation and the risk of disorder. Furthermore, we explored the regulatory mechanisms involved in early brain development, revealing enriched patterns of fetal cell types for neuronal disorders, indicative of the prenatal stages influence on disease determination. Our findings facilitate unbiased comparisons of cell type-disorder associations and provide insight into dynamic alterations in risk genes during development, paving the way for a deeper understanding of neurological disorders.

neuroscience↗

bioRxiv2024

Loss of Paneth cells dysregulates gut ILC subsets and enhances weight gain response to high fat diet in a mouse model.

Obesity has been associated with dysbiosis, but innate mechanisms linking intestinal epithelial cell subsets and obesity remain poorly understood. Using mice lacking Paneth cells (Sox9{Delta}IEC mice), small intestinal epithelial cells specialized in the production of antimicrobial products and cytokines, we show that dysbiosis alone does not induce obesity or metabolic disorders. Loss of Paneth cells reduced ILC3 and increased ILC2 numbers in the intestinal lamina propria. High-fat diet (HFD) induced higher weight gain and more severe metabolic disorders in Sox9{Delta}IEC mice. Further, HFD enhances the number of ILC1 in the intestinal lamina propria of Sox9{Delta}IEC mice and increases intestinal permeability and the accumulation of immune cells (inflammatory macrophages and T cells, and B cells) in abdominal fat tissues of obese Sox9{Delta}IEC. Transplantation of fecal materials from Sox9{Delta}IEC mice in germ-free mice before HFD further confirmed the regulatory role of Paneth cells for gut ILC subsets and the development of obesity.

immunology↗

bioRxiv2024

Molecular Mechanism of Condensin I Activation by KIF4A

During mitosis, the condensin I and II complexes compact chromatin into chromosomes. Loss of the chromokinesin, KIF4A, results in reduced condensin I chromosome association. However, the molecular mechanism behind this phenotype is unknown. Here, we show that KIF4A binds directly to condensin I HAWK subunit, NCAPG, via a conserved disordered short linear motif (SLiM) found in its C-terminal tail. KIF4A binding directly competes with two auto-inhibitory interactions in condensin I, mediated by SLiMs found in the N-terminus of NCAPH and the C-terminus of NCAPD2, which bind sites that overlap with KIF4A binding on NCAPG. Addition of the KIF4A SLiM peptide alone is sufficient to stimulate condensins ATPase and DNA loop extrusion activity. We also identify SLiMs in known yeast condensin interactors, Sgo and Lrs4, that bind yeast Ycg1, the HAWK equivalent to NCAPG. Our findings, together with previous work on condensin II and cohesin, demonstrate that SLiMs binding to HAWK subunits is a conserved mechanism of regulation in SMC complexes.

biochemistry↗

bioRxiv2024

Analysis of meiotic recombination in Drosophila simulans shows heterozygous inversions do not cause an interchromosomal effect

Chromosome inversions are of unique importance in the evolution of genomes and species because when heterozygous with a standard arrangement chromosome, they suppress meiotic crossovers within the inversion. In Drosophila species, heterozygous inversions also cause the interchromosomal effect, whereby the presence of a heterozygous inversion induces a dramatic increase in crossover frequencies in the remainder of the genome within a single meiosis. To date, the interchromosomal effect has been studied exclusively in species that also have high frequencies of inversions in wild populations. We took advantage of a recently developed approach for generating inversions in Drosophila simulans, a species that does not have inversions in wild populations, to ask if there is an interchromosomal effect. We used the existing chromosome 3R balancer and generated a new chromosome 2L balancer to assay for the interchromosomal effect genetically and cytologically. We found no evidence of an interchromosomal effect in D. simulans. To gain insight into the underlying mechanistic reasons, we qualitatively analyzed the relationship between meiotic double-strand break formation and synaptonemal complex assembly. We find that the synaptonemal complex is assembled prior to double-strand break formation as in D. melanogaster; however, we show that the synaptonemal complex is assembled prior to localization of the oocyte determination factor Orb, whereas in D. melanogaster, synaptonemal complex formation does not begin until Orb is localized. Together, our data show heterozygous inversions in D. simulans do not induce an interchromosomal effect and that there are differences in the developmental programming of the early stages of meiosis.

genetics↗

bioRxiv2024

Multiple resistance factors collectively promote inoculum-dependent dynamic survival during antimicrobial peptide exposure in Enterobacter cloacae

Antimicrobial peptides (AMPs) are a promising tool with which to fight rising antibiotic resistance. However, pathogenic bacteria are equipped with several AMP defense mechanisms, whose contributions to AMP resistance are often poorly defined. Here, we evaluate the genetic determinants of resistance to an insect AMP, cecropin B, in the opportunistic pathogen Enterobacter cloacae. Single-cell analysis of E. cloacaes response to cecropin revealed marked heterogeneity in cell survival, phenotypically reminiscent of heteroresistance (the ability of a subpopulation to grow in the presence of supra-MIC concentration of antimicrobial). The magnitude of this response was highly dependent on initial E. cloacae inoculum. We identified 3 genetic factors which collectively contribute to E. cloacae resistance in response to the AMP cecropin: The PhoPQ-two-component system, OmpT-mediated proteolytic cleavage of cecropin, and Rcs-mediated membrane stress response. Altogether, this evidence suggests that multiple, independent mechanisms contribute to AMP resistance in E. cloacae.

microbiology↗

bioRxiv2024

Thymidine Phosphorylase Mediates SARS-CoV-2 Spike Protein Enhanced Thrombosis in K18-hACE2TG Mice

COVID-19, caused by SARS-CoV-2, is associated with arterial and venous thrombosis, thereby increasing mortality. SARS-CoV-2 spike protein (SP), a viral envelope structural protein, is implicated in COVID-19-associated thrombosis. However, the underlying mechanisms remain unknown. Thymidine phosphorylase (TYMP), a newly identified prothrombotic protein, is upregulated in the plasma, platelets, and lungs of patients with COVID-19 but its role in COVID-19-associated thrombosis is not defined. In this study, we found that wild-type SARS-CoV-2 SP significantly promoted arterial thrombosis in K18-hACE2TG mice. SP-accelerated thrombosis was attenuated by inhibition or genetic ablation of TYMP. SP increased the expression of TYMP, resulting in the activation of signal transducer and activator of transcription 3 (STAT3) in BEAS-2B cells, a human bronchial epithelial cell line. A siRNA-mediated knockdown of TYMP inhibited SP-enhanced activation of STAT3. Platelets derived from SP-treated K18-hACE2TG mice also showed increased STAT3 activation, which was reduced by TYMP deficiency. Activated STAT3 is known to potentiate glycoprotein VI signaling in platelets. While SP did not influence ADP- or collagen-induced platelet aggregation, it significantly shortened activated partial thromboplastin time and this change was reversed by TYMP knockout. Additionally, platelet factor 4 (PF4) interacts with SP, which also complexes with TYMP. TYMP enhanced the formation of the SP/PF4 complex, which may potentially augment the prothrombotic and procoagulant effects of PF4. We conclude that SP upregulates TYMP expression, and TYMP inhibition or knockout mitigates SP-enhanced thrombosis. These findings indicate that inhibition of TYMP may be a novel therapeutic strategy for COVID-19-associated thrombosis. Key PointsO_LISARS-CoV-2 spike protein, thymidine phosphorylase, and platelet factor 4 form a complex that may promote clot formation. C_LIO_LIInhibiting thymidine phosphorylase attenuates SARS-CoV-2 spike protein-enhanced thrombosis, platelet activation, and coagulation. C_LI

cell biology↗

bioRxiv2024

Single-cell multimodal profiling of monocytes reveals diverse phenotypes and alterations linked to cardiovascular disease risks

Monocytes are a critical innate immune system cell type that serves homeostatic and immunoregulatory functions. The Cell surface expression of CD14 and CD16 has historically identified them, however, recent single-cell studies have uncovered that they are much more heterogeneous than previously realized. We utilized cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing (scRNA-seq) to describe the comprehensive transcriptional and phenotypic landscape of 437,126 monocytes. This high-dimensional multimodal approach identified vast phenotypic diversity and functionally distinct subsets, including IFN-responsive, MHCIIhi, monocyte-platelet aggregates, and non-classical, as well as several subpopulations of classical monocytes. Using flow cytometry, we validated the existence of MHCII+CD275+ MHCIIhi, CD42b+ monocyte-platelet aggregates, CD16+CD99- non-classical monocytes, and CD99+ classical monocytes. Each subpopulation exhibited unique functions, developmental trajectories, transcriptional regulation, and tissue distribution. Moreover, we revealed alterations associated with cardiovascular disease (CVD) risk factors, including race, smoking, and hyperlipidemia, and the effect of hyperlipidemia was recapitulated in mouse models of elevated cholesterol. This integrative and cross-species comparative analysis provides a unique resource to compare alterations in monocytes in pathological conditions and offers insights into monocyte-driven mechanisms in CVD and the potential for targeted therapies. SummaryMultimodal profiling provides a comprehensive phenotypic and transcriptional understanding of monocytes in health and cardiovascular disease risk states.

genomics↗

bioRxiv2024

Loop extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex

Structural maintenance of chromosomes (SMC) protein complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC family Wadjet complexes structurally resemble the widespread Muk-BEF genome-organizing complexes but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves circular DNA; however, the molecular mechanism underlying DNA substrate recognition remains unclear. Here, we use in vitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric DNA loop extruder that simultaneously reels in DNA from both sides of a growing loop and that this activity requires a dimeric JetABC supercomplex containing two dimers of the JetC motor subunit. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44 kilobase pair plasmid, stalls, and then cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet, and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across kingdoms of life.

molecular biology↗