Biology subjects

Hong, J.

Publications and source records attributed to Hong, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Distinct Repeat Architecture Landscapes in the Proteomes of Protozoan Parasites

Protozoan parasites cause major infectious diseases and pose persistent challenges to global health, particularly the emergence of drug-resistant strains. Tandem repeats (TRs) and other repetitive architectures are widespread in proteomes, especially in protozoan proteins, where they have been implicated in host-parasite interactions, immune evasion, and antigenicity. However, repeat-containing proteins (RPs) exhibit highly diverse architectures that often extend beyond the simple reiteration of a single motif, making comprehensive and quantitative characterization challenging. In this study, we performed bioinformatics analysis of repeat architectures in protozoan proteins. In addition to the established repeat-detection approaches, we developed a new algorithm, Drepper, which quantifies repeat-architecture complexity. By integrating diverse repeat-related features, we clustered RPs across species and identified distinct groups associated with parasite lineages. Notably, we detected a Plasmodium-specific RP cluster and a Trypanosoma/Leishmania-specific RP cluster; both were characterized by large repeat regions but exhibited contrasting repeat-structure complexity. The Plasmodium-specific RPs showed high complexity, whereas the Trypanosoma/Leishmania-specific RPs displayed significantly low complexity. Functional enrichment analyses indicated that these lineage-associated clusters were enriched in parasite-specific factors. Furthermore, evolutionary analyses suggested that low-complexity repeat architectures may be actively maintained through concerted evolution. Taken together, our results reveal lineage-specific strategies in protozoan repeat architectures and provide a quantitative framework for studying their biological and evolutionary roles.

bioinformatics↗

bioRxiv2026

The Caudate Nucleus Exhibits Distinct Pathology and Cell Type-Specific Responses Across Alzheimer's Disease

A{beta} presence in the caudate nucleus (Ca) partially defines Thal stage III in Alzheimers disease (AD), but little is known about ADs cellular impact on the region. Leveraging a public basal ganglia taxonomy of cellular populations, we generated a cellular resolution atlas of AD-associated pathological changes in Ca. Unlike cortex, we found that Ca AD pathology is dominated by two key features: phosphorylated tau (pTau)-containing neuropil threads enriched near oligodendrocytes in white matter tracts and amyloid-{beta} diffuse plaques enriched in gray matter. Although AD pathology in affected cortical regions results in neuronal loss, we find no AD-driven reductions in neuron proportions in Ca. However, there were observable changes in multiple cellular populations. Protoplasmic astrocytes and FLT1+/IL1B+ microglia increased in abundance with global pTau levels. We also observe gene expression changes in fast-spiking PTHLH-PVALB interneurons indicative of disrupted signaling pathways and altered intrinsic physiological properties. This work provides a cellular-resolution framework for understanding AD pathology in Ca.

neuroscience↗

bioRxiv2026

Mapping kinase-dependent tumor immune adaptation with multiplexed single-cell CRISPR screens

Immune dysfunction in cancer is enacted by multiple programs, including tumor cell-intrinsic responses to distinct immune subpopulations. A subset of these immune evasion programs can be systematically recapitulated through direct tumor-immune interactions in vitro. Here, we present an integrated, high-throughput single-cell CRISPR screening framework focused on the protein kinome for mapping the tumor-intrinsic regulation of T cell-driven immune pressure in glioblastoma (GBM). We combine pooled CRISPR interference and activation (CRISPRi/a) with immune-matched NY-ESO-1 antigen-specific allogeneic GBM-T cell co-culture and massively multiplexed single-cell transcriptomics to systematically quantify how genetic perturbation reshapes baseline tumor state and adaptive responses across graded effector-to-target ratios. We further leverage deep generative models for analyzing pooled CRISPR screens to decipher the effects of genetic perturbations on the mechanisms of tumor resistance. This framework resolves distinct modules of immune evasion and survival, including the regulation of the antigen-presentation machinery, interferon/NF-{kappa}B signaling, oxidative stress resilience, and checkpoint/cytokine programs, while identifying perturbations that reroute the continuous tumor transcriptional trajectory induced by T cell engagement. A secondary chemical screen in patient-derived GBM cultures identified putative kinase targets of immune evasion phenotypes (e.g., EPHA2 and PDGFRA), whose inhibition leads to the blockade of evasive programs and enhances T cell-mediated GBM killing. Together, this workflow provides a scalable blueprint for comprehensive charting of the genetic control of tumor-immune interactions.

genomics↗

bioRxiv2025

Foundational genomic resources for date palm: A gap-free, telomere-to-telomere phased assembly of Ajwa and 19 high-quality genome assemblies of Phoenix dactylifera.

Phoenix dactylifera L. is an economically, nutritionally, and culturally important fruit crop in the arid and semi-arid regions of the Middle East and North Africa. Here, we present a gap-free, telomere-to-telomere reference genome of the variety Ajwa, along with 19 additional high-quality assemblies (18 female and 1 male). These assemblies reveal novel chromosomal structures validated through cytogenetics, Hi-C, optical mapping, and synteny analyses with other palm genomes. Chromosome names were revised based on average lengths across all sequenced genomes. The Sex Determination Region (SDR) on chromosome 14 was confirmed through male-specific k-mer analysis, spanning approximately 14.7 Mb. Nucleolar organizing regions (NORs) were localized on chromosome 10, where a large 45S rDNA locus displayed unique repeat spacer motifs containing transposon-like sequences. In some accessions, a second NOR was identified on the female sex chromosome. This collection of date palm assemblies, anchored by the Ajwa reference genome, provides a critical resource for advancing breeding strategies aimed at enhancing the genetic resilience and productivity of date palm.

genomics↗

bioRxiv2025

Structural insights into the specific recognition of H2A.Z-H2B dimer by the catalytic subunit of SRCAP chromatin remodeling complex

Chromatin remodeling complexes (SRCAP-C) mediate the exchange of H2A.Z with H2A, leading to the incorporation of H2A.Z-H2B dimers into chromatin through a step-by-step manner. Here, we determined the crystal structure of the N-terminal region (531-560) of the catalytic subunit of SRCAP-C, termed SRCAP-Z domain, in complex with the engineered single-chain H2B-H2A.Z (scH2B-H2A.Z) at 2.53 [A] resolution. In the crystal structure, two molecules of scH2B-H2A.Z and one molecule of SRCAP-Z form a sandwich-like architecture. Histones form typical histone fold and SRCAP-Z domain adopts a short 310 helix. In details, Val551, Leu554, and Leu555 of the SRCAP-Z 310 helix specifically recognize the C helix of H2A.Z through hydrophobic interactions. The particular recognition has been validated in vitro by MBP-pulldown and isothermal titration calorimetry (ITC) experiments, and the MBP-pulldown assay results demonstrated that mutations in specific amino acids of SRCAP-Z attenuated its interaction with H2A.Z-H2B dimer. Finally, we demonstrated that SRCAP-Z recognizes H2A.Z-H2B dimer with a conservative structural basis and facilitates the nucleosome assembly by its chaperone activity. HighlightsFirstly, we determined the structure of the SRCAP-Z domain/scH2A.Z-H2B complex at 2.53 [A] resolution. Second, our results support a mechanism of H2A.Z-H2B/H2A-H2B exchange model in which YL1 enriches H2A.Z/H2B around nucleosomes, presenting them to SRCAP, which mediates the exchange of H2A.Z/H2B with H2A/H2B. Third, SRCAP-Z recognition of H2A.Z-H2B dimer is structurally conserved. Fourth, a distinct sandwich-like architecture: SRCAP-Z adopts a 310 helix that inserts precisely between H2A.Z and H2B, forming a stable, unexpected structural module.

biochemistry↗

bioRxiv2025

RPRM (Reprimo) triggers SCF(FBXW11)-mediated DNA-PKcs degradation to block non-homologous end joining and radiosensitize tumors

Targeting DNA damage repair pathways represents a promising strategy in cancer radiotherapy, however, the limited insight into the regulation of the non-homologous end joining (NHEJ) repair pathway in cancer cells severely restricts the development of precise radiosensitization approaches. Here, we identify Reprimo (RPRM), a p53-inducible tumor suppressor, is a potent inhibitor of NHEJ in which RPRM promotes proteasomal degradation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylated at the T2609 site. Upon irradiation, RPRM quickly translocates into the nucleus to enhance the transcription of FBXW11, a F-box protein gene that encodes the substrate recognition component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex. In the nucleus, RPRM is also able to scaffold the assembly of the SCFFBXW11 E3 ligase complex by binding to both CUL1 and FBXW11. This complex can specifically ubiquitylate phosphorylated DNA-PKcs (T2609), leading to reduced levels of both phosphorylated DNA-PKcs (T2609) and total DNA-PKcs, thereby suppressing NHEJ-driven repair and enhancing tumor radiosensitivity. In consistence, inhibiting the SCFFBXW11 E3 ligase complex eliminates RPRM-mediated radiosensitization by protecting DNA-PKcs. Clinically, high RPRM expression in non-small cell lung cancer (NSCLC) predicts better patient prognosis. Moreover, RPRM expression correlates positively with FBXW11 and PRKDC (DNA-PKcs) in the tumor tissues of the NSCLC patient cohort. Significantly, high expression of RPRM protein is linked to enhanced radiosensitivity of patient-derived organoids (PDOs) from these NSCLC patients. These results establish RPRM as a key negative regulator in NHEJ repair pathway and the RPRM-SCFFBXW11-DNA-PKcs axis as a potential target to enhance cancer radiosensitivity.

cancer biology↗

bioRxiv2025

Engineered Transdermal Peptide-Recombinant Type III Collagen Hydrogel with Biological Efficacy in Cell Proliferation and Wound Healing

Collagen, as a major structural protein in the extracellular matrix, plays a crucial role in tissue regeneration; however, traditional collagen sources often suffer from immunogenicity, poor stability, and batch-to-batch variability. In this study, a novel transdermal peptide-recombinant type III collagen hydrogel was developed, which has excellent biocompatibility, thermal stability, and skin repair effects. Transdermal peptide-recombinant type III collagen was expressed and purified by genetic engineering methods, and its potential for biomedical applications was further evaluated. The experimental results showed that the prepared collagen had a high purity (95%) and retained the unique secondary structure of collagen, showing good structural stability at different pH values. Through cell proliferation experiments and mouse wound healing experiments, we verified the superior effect of the collagen hydrogel in promoting wound healing and significantly accelerating wound healing at concentrations as low as 0.2 mg/mL. This achievement provides strong experimental support for its application in clinical skin wound repair.

biochemistry↗

bioRxiv2025

Development of Recombinant Anti-TLR2 Antibodies and PLGA Nanoparticle-based Gene Therapy for the Treatment of Neuropathic Pain

Neuroinflammation is a key contributor to neuropathic pain, with microglial Toll-like receptor 2 (TLR2) playing a central role in initiating and sustaining proinflammatory responses. However, existing TLR2-targeting antibody therapies are limited by poor delivery to the central nervous system and short-lived efficacy. We developed a non-viral gene therapy strategy using biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to deliver recombinant anti-TLR2 antibody genes. High-affinity nanobody and single-chain variable fragment candidates were selected through phage display screening and shown to suppress TLR2-dependent signaling both in vitro and in vivo. In mouse models of neuropathic pain, a single intrathecal administration of PLGA NP-encapsulated antibody genes produced robust and sustained analgesia, accompanied by reduced glial activation and proinflammatory cytokine expression. These findings demonstrate a modular NP-based platform for sustained antibody expression in the central nervous system and establish its potential for the treatment of chronic pain driven by innate immune activation.

neuroscience↗

bioRxiv2025

A novel allele of Sh1 underlines the conversion of waxy corn to wx-sweet corn

AbstractWaxy corn and sweet corn are two major types of fresh-eating corn. Each has significant market value due to its unique sensory and nutritional profiles. Developing a variety that combines both traits would meet growing consumer demand and create new market opportunities. From a fast neutron-mutagenized population of the waxy corn inbred line HB522, we identified a mutant, wx-sweet, whose kernels exhibit both waxy and sweet properties at the filling stage. Using bulked segregant analysis (BSA) and fine mapping, we localized the causal locus to SHRUNKEN1 (Sh1) on chromosome 9, which was confirmed by an allelic test with a known Mu-insertion mutant. PCR and sequencing indicated a putative large structural variation in the second and third exons of Sh1 in wx-sweet, likely disrupting the reading frame. This novel sh1 allele significantly reduced sucrose synthase activity. The sh1 and waxy1 mutant genes act synergistically to remodel carbohydrate metabolism in wx-sweet endosperms. This remodeling, revealed by integrated transcriptomic and metabolomic analyses, drives transcriptional reprogramming and restructures metabolic flux. These changes thereby enhance sucrose and raffinose accumulation, which underlies the unique waxy-sweet texture in fresh-eating maize endosperm. In summary, our work not provides valuable genetic resources for fresh-eating corn breeding, but also elucidates, for the first time, the molecular mechanism underlying the synergistic formation of waxy and sweet texture. Key messageThe novel allele of Sh1 acts synergistically with waxy1 to regulate carbohydrate metabolism, which elucidates the molecular mechanism for the unique waxy-sweet texture in fresh-eating corn endosperm. Our work provides new strategies for sweet-waxy maize breeding.

genetics↗

bioRxiv2025

Captamer: A Novel Quantitative Protein Detecting Method Depending on Aptamer-activated Molecular Switches and RPA Signal Amplification

There are various protein assays for specific and quantitative detection and widely used for laboratory and clinic purposes, but current methods still have limitations. Immunoassays based on antibodies, like ELISA, suffer from slow response and a long antibody-screening period, while physical or electrochemical methods are generally restricted by high cost or the stringent requirement of equipment or operating skills. In this study, we developed an in vitro sensitive protein quantification method: Captamer. The Captamer system comprises a molecular switch derived from aptamer sequence and an exponential fluorescence signal amplification pathway based on recombinase polymerase amplification (RPA). We demonstrated the Captamer for SARS-CoV-2 nucleocapsid protein detection and obtained results from samples within 30 min, displaying a wide detection window from 0.2 pg/mL to 200 pg/mL with high specificity. Furthermore, we tested the Captamer for Tau441 protein (a potential Alzheimers disease biomarker) and thrombin (a classic aptamer-protein interaction model), showing the limit of detection as low as 1 ng/mL and 0.02pg/mL respectively, which suggested the capacity of Captamer to be applied to various aptamer-protein pairs. Compared with the most commonly used and recently reported protein quantification methods, Captamer stands out for its high sensitivity, short response time, low cost, and simplicity, indicating its great potential to be widely used in protein quantification. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/686180v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e38ffaorg.highwire.dtl.DTLVardef@1019e72org.highwire.dtl.DTLVardef@1498b68org.highwire.dtl.DTLVardef@15f5ea2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Neural Inflammation in Thoracic Dorsal Root Ganglia Mediates Cardiopulmonary Spinal Afferent Sensitization in Chronic Heart Failure

The cardiac sympathetic afferent reflex (CSAR) and pulmonary spinal afferent reflex (PSAR) amplify sympathetic outflow, and their sensitization contributes to chronic heart failure (CHF). Using a myocardial infarction (MI) rat model, molecular profiling, imaging, and functional assays revealed that thoracic dorsal root ganglia (DRGs) undergo marked macrophage and glial activation and suppression of voltage-gated potassium (Kv) channels after MI. In vitro studies confirmed that pro-inflammatory cytokines and activated macrophages directly reduce Kv channel expression and activity in DRG neurons. Cardiac afferents mediated cytokine transport from the heart to DRGs, driving macrophage infiltration in a cytokine receptor-dependent manner. Anti-inflammatory strategies including systemic minocycline, liposomal clodronate-induced macrophage depletion, or local epidural dexamethasone prodrug delivery reduced neuroinflammation, restored Kv channel levels, attenuated the exaggerated CSAR and PSAR, and improved cardiac remodeling. These findings highlight a cytokine uptake-driven inflammatory pathway in cardiopulmonary spinal afferent sensitization and support targeted DRG anti-inflammatory therapy as a potential cardioprotective approach. AbstractThe cardiac sympathetic afferent reflex (CSAR) and pulmonary spinal afferent reflex (PSAR) amplify sympathetic activity and may contribute to chronic heart failure (CHF). We hypothesized that neural inflammation in thoracic dorsal root ganglia (DRGs) drives cardiopulmonary afferent sensitization through suppression of voltage-gated potassium (Kv) channels after myocardial infarction (MI). MI was induced in rats by coronary ligation. Molecular profiling, immunofluorescence, tissue clearing, and functional assays were used to assess neuroinflammation and reflex responses. Post-MI, thoracic DRGs showed macrophage infiltration, glial activation, cytokine upregulation, and reduced Kv channel expression. Bulk RNA-seq identified enrichment of macrophage activation-related genes, and in vitro studies confirmed that pro-inflammatory cytokines and activated macrophages suppressed Kv channels and increased DRG neuron excitability. Epicardial injection of biotinylated TNF- demonstrated cardiac afferent-mediated cytokine transport to DRGs, inducing macrophage infiltration via a cytokine receptor-dependent mechanism. Anti-inflammatory interventions including oral minocycline, systemic macrophage depletion, and local epidural delivery of thermo-responsive hydrogel-forming dexamethasone prodrug (ProGel-Dex) significantly reduced DRG neuroinflammation, restored Kv channel levels, and attenuated exaggerated CSAR and PSAR responses. ProGel-Dex also improved cardiac chamber dilation in the post-MI rats. These findings identify a cytokine uptake-glial activation- macrophage activation pathway as a driver of cardiopulmonary afferent sensitization after MI. Targeting DRG inflammation, particularly with sustained local dexamethasone delivery using ProGel-Dex, offers a precision medicine to dampen pathological sympathetic activation and improve cardiac outcomes in CHF. HighlightsO_LIBoth cardiac (CSAR) and pulmonary (PSAR) spinal afferent reflexes are sensitized after myocardial infarction, contributing to sympathetic overactivation. C_LIO_LIThoracic dorsal root ganglia (T1-T4) exhibit macrophage activation, glial responses, pro- inflammatory cytokine upregulation, and suppression of Kv channels following MI. C_LIO_LICardiac afferents mediate receptor-dependent uptake and transport of cytokines (e.g., TNF-) from the heart to DRGs, driving macrophage infiltration and inflammation. C_LIO_LIActivated macrophages and pro-inflammatory cytokines reduce Kv channel expression and Kv current density (Ito) in DRG neurons, enhancing excitability. C_LIO_LIAnti-inflammatory strategies including minocycline, liposomal clodronate-induced macrophage depletion, and local epidural dexamethasone prodrug attenuate neuroinflammation, restore Kv channel expression, and suppress exaggerated CSAR/PSAR. C_LIO_LITargeting DRG inflammation, particularly via sustained epidural dexamethasone delivery, represents a promising cardioprotective precise medicine. C_LI

neuroscience↗

bioRxiv2025

The Liver is an Inflammatory Mediator of Pulmonary Arterial Hypertension

The livers contribution to pulmonary arterial hypertension (PAH) pathogenesis remains unclear. We hypothesized that the liver promotes inflammatory injury to the pulmonary endothelium. PAH patients without liver disease with pulmonary artery endothelial cell (PAEC) biopsies were included. Liver serologies and imaging were analyzed by unsupervised classification and regression tree (CART) to identify subclinical liver dysfunction clusters. Two machine-learning models predicted cluster assignment and informed differential expression. PAEC transcriptomes were compared to liver and lung data from monocrotaline and Sugen-Hypoxia rats. Liver fibrosis was assessed in rat and human PAH livers. Among 25 PAH patients (76% female, median age 61 [30 - 84] years), CART identified clusters distinguished by Model for End-Stage Liver Disease Sodium (MELD-Na) [≥]12, predicting higher pulmonary vascular resistance ({beta}=0.5 Wood units per point increase in MELD-Na, 95% CI 0.2-0.8, p=0.005) after adjustment for right atrial pressure. Subjects with MELD-Na [≥]12 had decreased 6-minute walk distance (353 [120 - 576] m vs. 411[300 - 600] m, p=0.03), with upregulation of apelin, beta-catenin, and immune signaling. Rat lung ECs demonstrated survival and hepatic growth-factor signaling, while rat livers showed immune activation. Rat (20.8 vs 16.6 % area stained, p=0.09) and human PAH livers revealed fibrosis despite absent right ventricular failure, supporting a pathogenic lung-liver axis in PAH.

cell biology↗

bioRxiv2025

Domain-Invariant Feature Learning for Patient-Level Phenotype Prediction from Single-Cell Data

Accurate prediction of patient-level disease status from single-cell RNA sequencing (scRNA-seq) data is critical to enabling precision diagnostics. However, study-specific artifacts induce spurious correlations that limit generalization and interpretability. We studied this problem in the context of Multiple Instance Learning (MIL), a framework where each patient is modeled as a set of single-cell profiles. To improve robustness to domain shifts, we propose an adversarial and metric-based approach that learns domain-invariant representations while preserving task-relevant biological variation. We benchmarked our method on a systemic lupus erythematosus (SLE) dataset with synthetically added spurious features and evaluated its performance on two real-world scRNA-seq atlases: a cross-tissue immune dataset and a COVID-19 severity atlas. Across all settings, we observed consistent improvements in out-of-domain accuracy and more biologically faithful model attributions. Our findings establish a new standard for robust, interpretable patient-level prediction under domain shifts using scRNA-seq.

bioinformatics↗

bioRxiv2025

AMICI: Attention Mechanism Interpretation of Cell-cell Interactions

Spatial transcriptomic data enable study of cell-cell communication, yet current analysis tools often fail to provide dynamic, interpretable estimates of interactions and their spatial range across tissue. We present AMICI, an interpretable attention framework that jointly estimates interaction length scales, adaptively resolves sender-receiver subpopulations, and links communication to downstream gene programs. AMICI recovers ground-truth interactions in semi-synthetic data, uncovers gene programs linked to cell communication in the mouse cortex, and reveals length-scale-dependent tumor-immune signaling that reinforces estrogen receptor (ER) programs in breast cancer.

bioinformatics↗

bioRxiv2025

REV1 inhibition enhances trinucleotide repeat mutagenesis

Trinucleotide repeat (TNR) instability has been implicated in the pathogenesis of numerous neurodegenerative disorders. Because TNR instability causes mutagenesis of the underlying gene, we refer to the repeat instability phenomenon as TNR mutagenesis in this study. While germline expansions destabilize TNR to cause disease anticipation, somatic cell TNR instability drives earlier onset of symptoms and further disease progression. However, the drivers behind these repeat length changes remain unclear. Current models suggest that DNA replication slippage events and the action of genome instability pathways, such as DNA repair, cause TNR mutagenesis. Whether mutagenic polymerases from the translesion synthesis (TLS) pathway result in TNR instability is unclear. TLS polymerases are best at bypassing difficult-to-replicate DNA regions due to bulky lesions or gaps in DNA. While some effects of TLS polymerases on TNR instability have been explored in lower organisms, evidence in human cells is lacking. Using a quantitative GFP reporter with expanded CAG repeats, we show that inhibition of the TLS polymerase REV1 by its inhibitor, JH-RE-06, or siRNA knockdown increases TNR instability and the underlying mutability. These results suggest that REV1 protects Trinucleotide repeat length mutagenesis through potential continuous DNA synthesis when replicative polymerases stall ahead of repeat secondary structures. Collectively, we present evidence of the role of the TLS pathway in TNR instability, with potential implications for understanding mutability mechanisms, disease biology, and therapeutic targeting.

genetics↗

bioRxiv2025

High-fidelity CRISPR genome editing of single-nucleotide mutation with near-complementary guide RNA via enhanced target binding kinetics

The CRISPR-Cas9 system is a powerful genome editing tool capable of precisely recognizing and cleaving specific DNA sequences, and has been extensively investigated as a strategy for correcting mutations associated with genetic diseases and cancer. However, conventional CRISPR genome engineering often fail to discriminate single-nucleotide mutations from wild-type alleles when the mutation is located outside the protospacer adjacent motif (PAM) sequence. To address this limitation, we developed a RNA engineering approach for designing near-complementary single guide RNA (sgRNA) that contain intentional mismatches within the seed region of the sgRNA. Single molecule kinetic analyses showed that the near-complementary sgRNA selectively reduces the binding affinity of CRISPR ribonucleoprotein complex by via differentiated increment in the dissociation rates to the wild-type target DNA compared to the mutant allele. The engineered kinetic characteristics of near-complementary sgRNAs enable highly specific genome editing of single-base mutations without reliance on PAM proximity. We demonstrate the application of the strategy to the a cancer-specific single-nucleotide G228A (-124C > T) mutation in the TERT promoter, frequently found in glioblastomas and other tumors, that does not generate a canonical PAM sequence. Our near-complementary sgRNA successfully induced selective editing of the mutant allele while sparing the wild-type sequence. Furthermore, single-molecule fluorescence resonance energy transfer (smFRET) analyss revealed distinct differences in binding kinetics between mutant and wild-type DNA, providing kinetic insight into the discrimination process. We conclude that the near-complementary sgRNA CRISPR editing strategy facilitates precise PAM-independent targeting of single-nucleotide mutations without protein engineering and offers a molecular framework for expanding the specificity and applicability of CRISPR-based genome and epigenome editing technologies.

biochemistry↗

bioRxiv2025

Impaired Lung BCAA Metabolism Promotes Ferroptosis and Resultant Pulmonary Arterial Hypertension-Associated Hepatopathy

BackgroundDysregulated branched chain amino acid (BCAA) homeostasis occurs in pulmonary arterial hypertension (PAH) as BCAA metabolites accumulate and cause metabolic alterations in pulmonary artery smooth muscle cells (PASMC). In other cells, altered BCAA metabolism promotes ferroptosis, a PAH-inducing metabolic pathway. However, the interplay between BCAAs, lung ferroptosis, and PAH is unexplored, as is the impact of PAH severity on liver molecular regulation, a key unknown as recent clinical data highlight the importance of the lung-right heart-liver axis in PAH outcomes. MethodsHuman metabolomic and transcriptomic studies examined BCAA metabolism and ferroptosis pathways. The relationship between BCAAs and ferroptotic-phenotypes in PASMCs was evaluated. Multi-omics and physiological analyses evaluated how modulation of BCAA catabolism impacted preclinical PAH multi-organ physiology. Confocal microscopy and proteomic analyses assessed hepatic alterations in human PAH. ResultsMetabolomic analyses identified alterations in BCAA metabolites across multiple physiological gradients in patients with pulmonary vascular disease. RNA sequencing demonstrated deficits in the BCAA catabolic and ferroptosis pathways in PAH lungs and smooth muscle cells. In vitro, excess BCAAs induced mitochondrial fragmentation, reactive oxygen species generation, and lipid peroxidation in PASMC. Moreover, BCAAs promoted PASMC death, which ferrostatin-1, a ferroptosis antagonist, rescued. BT2, a small-molecule inducer of BCAA catabolism, reduced PAH severity, improved RV function, and enhanced maximal exercise capacity in monocrotaline rats. BT2 blunted pro-ferroptotic changes in lung metabolites and proteins, and combatted peri-vascular complement deposition. In the liver, BT2 blocked mechanical shear stress phenotypes including hepatocyte nuclear expansion and restructured mitochondrial protein regulation and the metabolomic signature. Additionally, a low BCAA diet modestly combatted preclinical PAH severity. Finally, human PAH livers exhibited increased hepatocyte nuclear size and derangements in liver metabolic regulation. ConclusionsImpaired BCAA metabolism promotes PAH via ferroptosis. PAH severity is associated with hepatic pathological shear stress phenotypes and metabolic alterations, which are combatted by a BCAA-targeted therapy. Graphical Abstract/Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/672819v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1649980org.highwire.dtl.DTLVardef@199c40aorg.highwire.dtl.DTLVardef@1586fdorg.highwire.dtl.DTLVardef@15166c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

The host protein cyclophilin A inhibits HIV-1 nuclear entry by decreasing capsid elasticity

Binding of the host protein cyclophilin A (CypA) to the HIV-1 capsid exerts a variety of effects on infection, including enhancement of reverse transcription, stabilization of the capsid, and promotion of nuclear entry. For several HIV-1 mutants, CypA binding inhibits nuclear entry by an unknown mechanism. We recently demonstrated that HIV-1 cores are elastic and that HIV-1 mutants with inelastic capsids are impaired for nuclear entry and infection of nondividing cells. Here we show that CypA prevents infection of nondividing cells by such mutants and inhibits their entry into the nucleus. CypA binding to mutant cores further reduced their elasticity in vitro, and this effect was reversed by suppressor mutations that restored nuclear entry. We suggest that HIV-1 nuclear entry involves temporal modulation of capsid elasticity by host proteins prior to and during traversal of the nuclear pore.

microbiology↗