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Park, Y.

Publications and source records attributed to Park, Y..

At least 37 records · Page 2Linked to original sources

Levosimendan inhibits HIV-1 infection in myeloid cells in the RIOK1-dependent manner

Despite of the highly potent antiretroviral therapies, HIV-1 establishes persistent infection and causes chronic inflammation in AIDS patients. Beyond CD4+ T cells, HIV-1 infects myeloid cells, including circulating monocytes and tissue-resident macrophages, and integrates with host genomes to form stable viral reservoirs. To achieve a functional HIV cure, latency-promoting agents (LPAs) have been developed for the "block-and-lock" strategy to reinforce deep HIV-1 latency and permanently silence proviruses. However, most LPAs have been tested mainly in CD4+ T cells, and their efficacy in myeloid cells remains unclear. In this study, we reported that levosimendan (LSM), a drug approved for clinic use to treat heart failures, is able to inhibit HIV lytic infection and reactivation in myeloid cells. LSM blocked viral lytic reactivation in HIV-1 latently infected monocytic cells (TH89GFP, U1) and microglial cells (HC69). LSM also inhibited HIV infection in human induced pluripotent stem cell (iPSC) derived microglia (iMG), primary human resident liver macrophages (Kupffer cells) as well as human monocyte-derived macrophages (MDMs). Furthermore, we demonstrated that overexpression of a predicted drug target of LSM, the conserved serine/threonine kinase RIOK1 (RIO kinase 1), overcomes LSMs anti-HIV effect. Overall, our studies concluded that LSM is a promising LPA to inhibit HIV-1 infection in myeloid cells in the RIOK1-dependent manner.

microbiology↗

Label-Free 4D Holotomography with Depth-Adaptive Segmentation for Quantitative Analysis of Lipid Droplet Dynamics in Hepatic Organoids

SignificanceQuantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. AimWe aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. ApproachTime-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index-derived concentration and dry mass at single-droplet resolution. ResultsOleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets. ConclusionsLabel-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid- dependent temporal modes of lipid storage. Statement of DiscoveryWe developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies--oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number--while palmitic acid rapidly compromises organoid integrity.

biophysics↗

Janus kinase 2 regulates Nurr1 protein stability in dopaminergic neurons of the aging midbrain

The nuclear receptor Nurr1 (NR4A2) is an essential transcription factor that governs the differentiation, maturation, and long-term maintenance of midbrain dopaminergic (mDA) neurons in the substantia nigra. Reduced Nurr1 expression has been closely linked to age-related dopaminergic neuronal loss and the pathogenesis of Parkinsons disease. However, the molecular mechanisms regulating Nurr1 expression and protein stability in the aging midbrain remain poorly understood. Here, we identify Janus kinase 2 (JAK2) as a previously unrecognized regulator of Nurr1 in mDA neurons. In the substantia nigra of aged mice (12-and 18-month-old), JAK2 was robustly expressed in Nurr1-positive mDA neurons, whereas its expression was minimal in young adult mice. In SK-N-BE(2)C neuroblastoma cells, overexpression of JAK2 modestly enhanced Nurr1 transcriptional activity, while the constitutively active mutant JAK2V617F markedly increased it. Notably, this effect was not blocked by pharmacological inhibition of STAT, PI3K, or Akt signaling pathways, indicating that JAK2 regulates Nurr1 independently of canonical JAK/STAT or PI3K/Akt signaling. Mechanistically, JAK2 did not promote tyrosine phosphorylation of Nurr1 but instead physically interacted with Nurr1, leading to enhanced nuclear stability of the Nurr1 protein. Consistent with this mechanism, expression of JAK2V617F increased Nurr1 protein levels without altering its mRNA expression. Functionally, co-expression of JAK2V617F and Nurr1 attenuated oxidative stress-induced cytotoxicity and reduced reactive oxygen species accumulation. Together, these findings reveal a phosphorylation-independent mechanism by which JAK2 stabilizes Nurr1 protein and enhances its transcriptional activity. Our results further suggest that age-associated induction of JAK2 in dopaminergic neurons may promote neuronal resilience by maintaining Nurr1 protein stability during aging.

neuroscience↗

Modulation of the RNAse P/MRP complex and mitochondrial ribosome enhances cytosolic ribosome coordination and sustains longevity

Aging is accompanied by a progressive decline in protein synthesis and ribosome abundance, yet paradoxically, genetic or pharmacological attenuation of translation extends lifespan across species. Whether the age-associated decline in translation is adaptive or reflects a pathological failure of ribosome homeostasis remains unclear. Here, we show that shortened lifespan is driven by dysregulated ribosome biogenesis (RiBi) and impaired ribosome assembly. Using Caenorhabditis elegans ncl-1 loss-of-function mutants as a model of accelerated aging, we find that nucleolar enlargement coincides with decoupling of precursor and mature rRNA, ribosomal protein (RP) transcripts, and protein abundance, loss of RP stoichiometry, defective ribosomal subunit joining, and compromised proteostasis. Strikingly, lifespan can be restored downstream of the nucleolus by targeting either the RNAse P/MRP complex or the mitochondrial ribosome. These interventions rebalance mature rRNA and RP abundance, improve ribosomal assembly, and reduce protein aggregation despite persistent nucleolar enlargement and elevated pre-rRNA levels. Our findings identify accelerated age-associated ribosome dysfunction as a qualitative failure of ribosomal biogenesis, and demonstrate that restoring ribosomal homeostasis is sufficient to improve proteostasis and extend lifespan.

cell biology↗

Co-repression of Yap1 and Sox9 Abrogates Established Cholangiocarcinoma by Eliminating Transcriptional Compensation

Background/AimsIntrahepatic cholangiocarcinoma (iCCA) represents an unmet clinical need due to its increasing incidence, aggressive biology, and limited treatment options. The extremely low-response rates to current systemic regimens and the emergence of adaptive resistance to targeted therapies underscore the urgent need for alternative therapeutic strategies. Given that the lineage-defining transcription factors SOX9 and YAP1 are central regulators of cholangiocyte and iCCA identity, we investigated their functional roles as potential therapeutic vulnerabilities across multiple preclinical models. MethodsPatient tissue-microarray (TMA) analysis, Sleeping-Beauty hydrodynamic tail vein injection-based iCCA models, and Cre-mediated inducible gene deletion systems were used to investigate the roles of Sox9 and Yap1. Deep-learning-based prediction, RNA-seq, ChIP-seq and immunohistochemistry analyses were performed to delineate transcriptional networks and downstream effectors associated with SOX9/YAP1 signaling. ResultsDual deletion of Sox9 and Yap1 effectively eradicated advanced iCCA while preserving intrahepatic bile ducts, regardless of oncogenic drivers. Mechanistically, SOX9 and YAP1 transcriptionally compensated for each other when one was absent, and ILF2, MGAT5, and WWTR1 were identified as key downstream effectors mediating this compensatory mechanism. Loss of Ilf2, Mgat5, or Taz suppressed iCCA, whereas overexpression of Ilf2 or Taz following Sox9/Yap1 co-deletion restored tumor development, indicating that ILF2 or TAZ can functionally substitute for YAP1 and SOX9 in sustaining iCCA. ConclusionsCo-targeting SOX9 and YAP1 offers a promising and safe broad-spectrum preventive/therapeutic approach for iCCA, potentially overcoming resistance to YAP1 inhibition. The adaptive resistance mechanism identified may extend to other malignancies, providing insights for addressing the advanced resistant to YAP1-TEAD-directed therapies.

cancer biology↗

Shared Maturation Pathways of HIV-1 Envelope-reactive V3-glycan bnAb Lineages in Human and Rhesus Macaque

ABSTRACTUnderstanding broadly neutralizing antibody (bnAb) lineage development in rhesus macaques (RMs) infected with Simian-HIV (SHIV) may inform HIV-1 vaccine designs. We analyzed HIV-1 envelope-antibody coevolution in 18 RMs infected by SHIV.BG505 (subtype A) and found highly conserved patterns of antibody recognition and Env escape, including in three animals that developed V3-glycan bnAbs. From one RM with V3-glycan targeted plasma breadth, we isolated 203 members of a single clonal Ab lineage designated DH1030. DH1030 Abs demonstrated striking genetic, functional and structural similarities with the human V3-glycan bnAb lineage DH270, which was isolated from an individual with subtype C HIV-1 CH848 infection. Notably, human-DH270 and macaque-DH1030 bnAbs shared early improbable mutations in HCDR2 that were critical for bnAb development. Thus, key improbable mutations and convergent patterns of antibody evolution and epitope recognition were shared across primate species and distinct HIV-1 subtypes, findings that may be leveraged in new HIV-1 vaccine designs. ONE SENTENCE SUMMARYBroadly neutralizing antibodies targeting HIV-1 envelope surface protein develop via a common maturation pathway across primate phylogeny.

immunology↗

Inhibition of KDM5A/B promotes antitumor innate immune responses in HHV-8/KSHV2 positive B-cell lymphomas

Summary/AbstractHistone methylation is a dynamic and reversible epigenetic modification that critically controls the progression of human diseases, including infections and cancers. Here we reported that histone lysine demethylases (KDMs) in the KDM5 family KDM5A/B play profound roles in suppressing lytic reactivation of oncogenic human herpesvirus 8 (HHV-8), i.e., Kaposis sarcoma-associated herpesvirus (KSHV), as well as antiviral/antitumor innate immune responses in KSHV-infected B-cell lymphomas. We showed that KSHV lytic replication decreases KDM5A/B protein stability by enhancing their K-48 linked polyubiquitination while KDM5A/B depletion facilitates KSHV lytic reactivation. Mechanistic studies illustrated that KDM5A/B associate with KSHV LANA protein and dampen its chromatin association at both KSHV viral lytic promoter and promoters of antitumor immune-responsive genes (IRGs). In comparisons to normal B cells, KDM5A/B expression significantly increased in B-cell lymphoma cells, including KSHV-positive primary effusion lymphoma (PEL). We demonstrated that KDM5A/B inhibition remarkably induces both KSHV lytic reactivation and innate immune responses in PEL cells, resulting in a strong viral oncolytic effect, both in vitro in cell cultures and in vivo using a PEL xenograft mouse model. Overall, our studies identified the novel functions of KDM5A/B to silence KSHV lytic replication and antiviral/antitumor innate immune responses, which can be blocked to benefit the treatment of KSHV-associated B-cell lymphomas that are usually aggressive and difficult to treat.

microbiology↗

Label-free quantitative phenotyping of hepatic stellate cell activation using holotomography with AI-enabled subcellular segmentation

Hepatic stellate cell (HSC) activation is a central driver of liver fibrosis, yet its quantitative characterization in living cells remains limited by endpoint assays that rely on fixation and labeling. Here we introduce a label-free framework that combines three-dimensional holotomography (HT) with automated, AI-assisted analysis to study HSC activation dynamics in live cells. Using refractive index tomography, we non-invasively visualize hallmark structural features of HSC activation, including lipid droplet depletion, cytoskeletal remodeling, and changes in cellular morphology. Correlative fluorescence imaging validates the biological relevance of HT-derived features, while longitudinal imaging reveals continuous activation trajectories at single-cell resolution. Automated segmentation of whole cells and subcellular organelles enables scalable extraction of multi-parametric biophysical descriptors, defining a quantitative phenotypic fingerprint that distinguishes quiescent and activated states. Together, this work establishes holotomography-based quantitative phenotyping as a powerful approach for studying HSC activation and, more broadly, dynamic cell-state transitions in living systems.

cell biology↗

NetCrafter: Ontology-derived gene network modeling and interpretation

Understanding the complex nature of multi-functional interactions among genes is crucial for interpreting omics data. We developed NetCrafter, an ontology-driven platform for constructing de novo gene networks that are specific to each input gene list and quantitatively defined by ontology-weighted similarity. By incorporating the probabilistic association of ontology or curated gene sets into a weighted Tanimoto similarity metric, NetCrafter transforms enrichment results into quantitative semantic similarity scores between genes, enabling the creation of context-specific statistical networks. These networks can be further decomposed into optimal sub-networks, facilitating multi-functional interpretation and the identification of gene interaction hotspots. NetCrafter also supports the integration of heterogeneous omics-derived gene lists through consensus ontology scoring. Importantly, this list-specific, quantitative framework reveals functional hotspots and target-biomarker relationships - even in cases where ontology terms alone are not predictive of node-level attributes such as CRISPR efficacy. NetCrafter provides an interactive platform for constructing and interpreting dynamic, context-specific gene networks, leveraging ontology-based functional associations to uncover underlying mechanisms and identify key nodes. It is freely available at https://netcrafter.sookmyung.ac.kr and integrated into Q-omics platform (https://qomics.ai) to enhance the utility of cancer omics data.

bioinformatics↗

Consumption of processed foods impairs memory function through dietary advanced glycation end-products

Consumption of processed foods is associated with dementia, obesity, and other negative health outcomes. Sustained heat treatment, a common food processing approach to enhance flavor, induces the chemical Maillard reaction that promotes the formation of dietary advanced glycation end-products (AGEs). The neurocognitive impacts of consuming dietary AGEs are poorly understood. Here we modeled an AGE-rich diet through heat treatment fed to rats during adolescence, a critical period of neural development, to mechanistically evaluate the long-term impact of early life dietary AGEs on behavioral and neural processes. Consuming the AGE-rich diet impaired hippocampal-dependent memory function and altered the gut microbiome without inducing obesity or nonspecific behavioral deficits. AGE-induced memory deficits were coupled with impaired hippocampal glutamatergic synaptic neurotransmission and altered expression in the synapse-pruning complement system. Hippocampal synaptic deficits likely result from direct AGE-complement interactions, as our extended studies reveal competitive antagonist action of AGEs on complement receptors. Memory impairments were prevented by administration of the AGE-inhibitor, alagebrium, and by supplementation with an AGE-inhibiting bacterial taxon, Lactococcus lactis, which was depleted in the heat-treated diet. These findings reveal a functional connection between early life dietary AGEs, the microbiome, and memory impairments, thus illuminating mechanisms through which food processing negatively impacts neurocognition.

neuroscience↗

Chronic alcohol exposure drives inflammaging and transposon derepression in hematopoietic stem and progenitor cells

Chronic alcohol use can cause pancytopenia and diminished immune responses against pathogens. However, its underlying molecular mechanisms remain unclear. Furthermore, whether chronic alcohol consumption directly induces inflammation in human hematopoietic stem progenitor cells (HSPCs) or whether it affects aging hematopoiesis differently is unknown. To examine how chronic alcohol use influences HSPCs, we performed single-cell RNA-seq in murine and human HSPCs and single-cell ATAC-seq in aged murine HSPCs following alcohol exposure. In the native murine bone marrow, chronic alcohol exposure primed HSPCs to differentiate into myeloid cells and to exhibit heightened inflammation, DNA damage, and epigenetic reactivation of transposable elements (TEs) in an age-dependent manner. Alcohol-exposed aged long-term hematopoietic stem cells (LT-HSCs) displayed increased chromatin accessibility at TE-containing loci correlated with aberrant TE transcription. This transposon derepression was associated with the accumulation of dsRNAs in aged bone marrow cells, and activation of innate immune pathways, perpetuating HSC inflammaging. Furthermore, we identified two epigenetically distinct LT-HSC clusters, LT-HSC1 and LT-HSC2, with the LT-HSC2 cluster expanding in response to chronic alcohol consumption, resembling activated HSCs. In xenotransplanted human HSPCs, chronic alcohol feeding resulted in a significant myeloid bias, heightened inflammation, upregulation of double-stranded RNA (dsRNA) sensors, activation of type I interferon responses, and increased expression of endogenous retroviruses. Despite these molecular alterations, we did not observe a decrease in long-term repopulation capacity in either human or murine HSCs. This suggests that HSC function may recover following alcohol cessation. However, previous chronic alcohol exposures imprint murine HSPCs to exhibit long-term myeloid bias and reduced cell cycle entry upon bacterial LPS challenge. Our data illuminate potential interactions between alcohol and aging that can reinforce inflammaging and epigenetic dysregulation in HSPCs. KeypointsO_LIAging perpetuates alcohol-induced myeloid bias, inflammation, DNA damage, and TE upregulation in murine HSPCs C_LIO_LIPrior chronic alcohol consumption does not affect long-term repopulation but causes persistent myeloid bias and inefficient stress responses after LPS challenge C_LIO_LIChronic alcohol consumption alters chromatin accessibility in TE-overlapping regions C_LIO_LIChronic alcohol consumption promotes myeloid bias, inflammation, and upregulation of endogenous retroviruses in xenotransplanted human HSPCs. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/696246v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@e0357aorg.highwire.dtl.DTLVardef@1d702fdorg.highwire.dtl.DTLVardef@10553ddorg.highwire.dtl.DTLVardef@c4e62e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

Agonism of mosquito and human transient receptor potential ankyrin 1 (TRPA1) channels by the natural drimane sesquiterpene cinnamodial

TRPA1 channels are multi-modal receptors in animals for sensing noxious physical and chemical factors. They are considered promising biochemical targets for developing repellents of arthropod disease vectors and drugs to treat a variety of medical conditions including pain, inflammation, and itch. Recently, we discovered that cinnamodial (CDIAL), a natural drimane sesquiterpene produced by the medicinal plant Cinnamosma fragrans, agonizes mosquito TRPA1 channels and is antifeedant and repellent to Aedes aegypti. However, the selectivity of CDIAL for mosquito vs. human TRPA1 and how CDIAL binds to TRPA1 channels are unknown. Here we characterize the agonism by CDIAL of Aedes aegypti (Aa) and Homo sapiens (Hs) TRPA1 channels and compare it to that induced by two other known TRPA1 agonists, nepetalactone and JT010. Heterologous expression of AaTRPA1 and HsTRPA1 in Xenopus laevis oocytes revealed that CDIAL agonized both channels with similar potency. Nepetalactone was less potent of an agonist than CDIAL for both AaTRPA1 and HsTRPA1, but was a more specific agonist for AaTRPA1 over HsTRPA1 compared to CDIAL. JT010 did not detectably activate AaTRPA1, but was a more potent agonist of HsTRPA1 than CDIAL. To generate insights into a putative binding site of CDIAL on AaTRPA1 we mutated six Lys residues (656, 678, 681, 728, 738, 744) and/or a Cys residue (684) in the coupling domain previously hypothesized to be involved. Simultaneous mutation of the six Lys residues to Ala dampened agonistic responses of AaTRPA1 to CDIAL, whereas changing Cys684 to Ser had no detectable impact. Protein modeling and molecular docking simulations for both AaTRPA1 and HsTRPA1 suggest species-specific CDIAL-binding mechanisms to the coupling domain, which involve Lys681 in AaTRPA1 and Cys621 in HsTRPA1.

pharmacology and toxicology↗

Allele-specific correction of ATAD3A pathogenic variants via template-free CRISPR-Cas9 editing and gene conversion

Gene conversion is a specific form of homologous recombination (HR), involving the unidirectional transfer of genetic information from one genomic locus to another. CRISPR-Cas9-directed double strand breaks (DSBs) induce both interallelic and interlocus gene conversion in early human embryos and somatic cells, suggesting its potential for correcting pathogenic mutations. However, the key features in mitotic gene conversion, including its efficiency, the length of conversion track, and its dependency on specific recombination proteins, remain largely undefined. Here, we show that allele-specific CRISPR-Cas9-induced DSBs, without exogenous donor templates, can efficiently correct a heterozygous pathogenic variant (c.1582C>T; p.Arg528Trp) in the ATAD3A gene of patient-derived induced pluripotent stem cells (iPSCs). Amplicon-based next-generation sequencing (NGS) revealed that approximately 38%[~]53% of edited iPSCs carried two wild-type ATAD3A alleles. Notably, over 99% of the corrected alleles derived from the homologous chromosome, indicating that the repair occurred mainly via interallelic gene conversion. Long-range amplicon nanopore sequencing coupled with haplotype analysis showed that the majority of gene conversion tracts was less than 2 kilobases in length. Whole-genome sequencing of three corrected iPSC clones showed the absence of large deletions or structural rearrangements at the ATAD3A target site. However, one clone carried a heterozygous deletion in ATAD3B locus, suggesting that CRISPR-Cas9 can introduce off-target genomic alterations. Knockdown of key HR proteins, including RAD51, CtIP, and BRCA1/2, significantly reduced the correction efficiency, indicating that the gene conversion relies on a RAD51-dependent HR pathway. Together, our findings provide compelling evidence that template-free CRISPR-Cas9-mediated interallelic gene conversion can be harnessed to correct disease causing variants in human iPSCs.

genomics↗

Targeted Intestinal Cooling Offers Superior Brain Protection in the Mouse Stroke Model

BackgroundIntestinal immune and inflammatory response plays a detrimental role following a stroke. This study aims to evaluate the brain protective efficacies of a novel intestinal cooling (CC) technique relative to the body surface cooling (SC) and the normothermic (NT) condition in a mouse stroke model. MethodsMice were randomly assigned to CC (n=13), SC (n=15), or NT (n=11) groups. They underwent 60 min of middle cerebral artery occlusion (MCAO) followed by 7-day reperfusion. Both head and intra-colon temperatures were maintained at 37{degrees}C for 30 min before, during, and 30 min after MCAO. At 30 min reperfusion, a cooling catheter was placed to maintain intra-colon at 37{degrees}C in NT or 12{degrees}C in CC. The head temperature was maintained at 37{degrees}C in NT and 30{degrees}C in CC. In SC, both intra-colon and head temperatures were maintained at 30{degrees}C. Cooling lasted 3 hours. Bodyweight, behavioral deficits (nesting and pole test), and survival rate were assessed post-MCAO. At day 7 post-MCAO, mice were perfusion-fixed for histopathological analysis. ResultsPost-stroke histopathological brain injury areas and volume were significantly reduced in CC, and appeared reduced though not statistically significant in SC, relative to NT. Compared with NT, body weight, nest building activity, and pole test were all significantly recovered in CC post-MCAO. In SC, only nest building improved significantly, while body weight and pole test showed marginal, nonsignificant trends. Consistent with functional recovery, survival was significantly improved in CC but not in SC, compared with NT. ConclusionIn a murine model, our novel CC technique successfully achieved targeted intestinal cooling while preserving safe upper-body temperatures necessary for normal cardiopulmonary function. Targeted intestinal cooling provides significant benefits superior to SC and NT, including smaller stroke volume, fewer functional deficits, and lower mortality rates, thus supporting the novel concept that the intestines are potential therapeutic targets for stroke management. Graphical Abstracts O_FIG O_LINKSMALLFIG WIDTH=131 HEIGHT=200 SRC="FIGDIR/small/681764v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1aa07cdorg.highwire.dtl.DTLVardef@1e64569org.highwire.dtl.DTLVardef@d6f178org.highwire.dtl.DTLVardef@1871862_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPost-stroke body surface cooling-induced hypothermia demonstrated marginally neuroprotective effects compared to normothermic conditions in the mouse model. C_LIO_LITargeted deep cooling of the intestine after a stroke resulted in a significantly greater reduction in stroke injury areas and volume, as well as lower mortality rates and fewer functional deficits, compared to body surface cooling-induced hypothermia and normothermic conditions. C_LI

neuroscience↗

Consistent Induction of Broadly Neutralizing HIV Antibodies by a Novel Two-Step Mechanism Informs Immunogen Design

A major obstacle confronting HIV-1 vaccine and cure research is the lack of an outbred animal model for rapid and consistent induction of broadly neutralizing antibodies (bNAbs). We designed an epitope-focused simian-human immunodeficiency virus (SHIV.5MUT) that elicited broad and potent V3-glycan-targeted antibodies within a year of infection in 14 of 22 macaques compared with 0 of 14 control animals. SHIV.5MUT elicited bNAbs by a novel two-step mechanism, inducing an initial wave of V1-directed antibodies that selected for Envs with shortened, hypoglycosylated V1 loops, which in turn primed V3-glycan bNAb precursors. Rhesus bNAbs were immunogenetically and structurally diverse, closely resembling human V3-glycan bNAbs. Env-bNAb coevolution revealed a diverse repertoire of bNAb precursors and the Env variants that matured them, yielding a molecular blueprint for vaccine design.

immunology↗

CytoScan: Automated detection of technical anomalies for cytometry quality control

Studies evaluating cellular phenotypes by cytometry techniques are increasingly facing analytical challenges due to the multitudes of samples and parameters that are evaluated concurrently. Spurious technical effects resulting from a lack of standardization can affect marker distributions and further complicate multi-sample analyses. User-friendly tools for exploratory data analysis to identify such technical effects in large datasets are lacking. To fill this gap, we present a novel R package, CytoScan, that evaluates inter-measurement variation in cytometry datasets and allows for detecting anomalous measurements after data acquisition. CytoScan can detect two types of anomalies: files with limited similarity to others within a dataset (outliers) and files with limited similarity to previously acquired high-quality reference data (novelties). Using simulations of skewed marker distributions and real-life technical effects we demonstrate that CytoScan can accurately detect such anomalies. CytoScan can be applied to large cytometry datasets on consumer-grade hardware with informative visualizations, providing accessible quality-control for more reliable analyses.

bioinformatics↗

A cross model spatial and single-cell atlas reveals the conserved involvement of osteopontin in polycystic kidney disease

Polycystic kidney disease (PKD) arises from mutations in cilia-associated genes, such as PKD1 and PKD2, expressed in renal epithelial cells, leading to progressive kidney dysfunction and end- stage kidney disease (ESKD). PKD patients exhibit significant heterogeneity in disease progression, largely due to genetic and environmental modifiers. Like patients, mouse models of PKD also exhibit significant heterogeneity with regards to the gene mutated, age of disease onset, and rate of disease progression. To elucidate the cellular and molecular consequences of these variables, we constructed an integrated single-cell and spatial transcriptomics atlas across mouse models of PKD, mapping changes in cell type composition, gene expression, and intercellular signaling networks across the whole atlas and within individual models. Consistently across models, single cell RNA sequencing (scRNAseq) data revealed increased Spp1 (osteopontin) expression and signaling from PKD-enriched clusters to Ly6clo monocytes. Global deletion of Spp1 in Pkd1RC/RC mice resulted in reduced cyst severity, improved kidney function, and reduced Ly6clo monocyte numbers, suggesting that SPP1 signaling to Ly6clo monocytes promotes PKD progression. We also created a freely available, searchable website (https://bmblx.bmi.osumc.edu/scPKD/) that can be used to identify cross- and intra-model specific changes in gene expression, guiding researchers to new therapeutic targets for treating PKD.

bioinformatics↗

Site-specific replacement of large-scale DNA fragments in human cells

Despite advances in genome editing1-4, precisely replacing large-scale fragments in human cells remains a significant challenge. Here, we present a site-specific gene replacement tool, named Prime Assembly (PA), which adapts prime editors to produce one or two pairs of 3-flaps on both the genome and donor plasmids. These 3-flaps anneal to each other precisely, similar to "Gibson Assembly" in DNA oligonucleotides5, allowing megabase-scale genomic excision and/or kilobase-scale donor insertion at the gene of interest. The PA system achieves an efficiency of up to 57.8% in replacing endogenous sequences with a 2.9 kbp donor DNA fragment in HEK293T cells, and presents an accuracy of more than 99% for integrated PA fragments. Ultimately, the site-specific replacement of large-scale coding sequences (CDSs) in disease-related genes can restore gene function across numerous patients with different mutations, providing a gene implantation technique for genome editing and a universal therapeutic approach.

molecular biology↗