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Han, J.

Publications and source records attributed to Han, J..

At least 91 records · Page 5Linked to original sources

Methionine regulates antitumor function of CD8⁺ T cells through polyamine synthesis

Methionine is an essential amino acid critical for T cell activation. While methionine restriction (MR) combined with immune checkpoint blockade has been shown to enhance T cell function, the impact of methionine on adoptive T cell therapies is largely unexplored. Here, we examined the functionality of T cells under MR and pharmaceutical inhibition of the methionine cycle (MAT2Ai), using primary T cells and a murine adoptive T cell therapy model. In vitro, transient MR or MAT2Ai treatment increased interferon gamma (IFN{gamma}) expression in CD8+ T cells, whereas sustained MR led to the upregulation of T cell exhaustion-associated markers. Mechanistically, transient MR suppressed the polyamine synthesis pathway, and supplementation with polyamines reversed MR-induced IFN{gamma} expression. Genetic ablation of s-adenosylmethionine decarboxylase, an enzyme in the polyamine synthesis pathway, recapitulated the effect of MR, indicating that transient MR enhances T cell function by inhibiting polyamine synthesis. Despite this, transient MR treatment of ovalbumin (OVA)-specific (OT-I) CD8+ T cells prior to adoptive transfer did not improve antitumor efficacy against EG7-OVA tumors in vivo. In contrast, sustained dietary MR accelerated EG7-OVA tumor growth in mice treated with OT-I T cells, demonstrating that methionine availability is essential for the activity of adoptively transferred T cells. These findings suggest that enhancing methionine availability in the tumor microenvironment may improve the efficacy of adoptive T cell therapies.

immunology↗

AGO1 in neural progenitor cells orchestrates brain development and sociability via LIN28A-REELIN axis

AGO1, an essential RNA binding protein (RBP) in RNA interference, is associated with autism spectrum disorder (ASD). However, the precise functions of AGO1 in brain development and related disorders remain largely unexplored. Here, we report the critical roles of AGO1 in neural progenitor cells (NPCs) in promoting sociability and shaping the structure of the developing brain. Ago1 knockout (KO) in the mouse brain leads to hyposociability, a characteristic symptom of ASD. In human forebrain organoids, AGO1 KO disrupts the formation of ventricle-like structures and delays cortical layer development. We discovered that AGO1 KO results in a loss of polarity in NPCs, which subsequently reduces neuronal development. These AGO1 KO phenotypes originate from the failure to suppress LIN28A in NPCs. AGO1 is primarily localized in the nucleus within NPCs and binds to the LIN28A promoter region, thereby inhibiting LIN28A transcription. We found that increased LIN28A in AGO1 KO NPCs reduces REELIN expression, a key regulator of brain development, by binding directly to the REELIN mRNA. The aberrant polarity phenotype in AGO1 KO NPCs was successfully ameliorated by either LIN28A knockdown or recombinant REELIN treatment. Collectively, our findings elucidate the intricate molecular mechanisms orchestrated by nuclear AGO1 in NPCs and underscores its pivotal roles in brain development, providing significant insights into ASD.

neuroscience↗

Lysosome-dependent nutrient scavenging underliesstress adaptation during epithelial-to-mesenchymaltransition

Metastatic cancer cells invade tissue, overcome nutrient stress, and survive transit to distant sites. Many of the mechanisms that support these processes are incompatible with proliferation. This study defines cellular transition states in breast epithelial cells undergoing epithelial-mesenchymal transition (EMT) driven by ERK2 and TGF-{beta} signaling. EMT triggers robust endolysosomal system upregulation and metabolic adaptations that balance proliferative and invasive states. Surprisingly, invasive cells rely on scavenging via lysosomes and macropinocytosis to acquire amino acids, rather than plasma membrane transport, even in nutrient-rich conditions. Macropinocytosis increases intracellular amino acid storage, promoting survival during amino acid deprivation. This metabolic shift depends on c-MYC downregulation, an early EMT event. Reintroducing c-MYC suppresses the metabolic switch, endolysosomal induction, macropinocytosis, and the proliferation-to-migration transition. These findings reveal how cells dynamically balance proliferation and invasion, offering insights into transition states difficult to capture in models of breast cancer metastasis.

cell biology↗

High-throughput cell-free profiling of SARS-CoV-2 RBD variants enables rapid and quantitative in vitro affinity landscape mapping

SARS-CoV-2 variants continue to threaten public health, necessitating the study of cumulative and epistatic effects of receptor-binding domain (RBD) mutations on antibody evasion. We present a high-throughput platform combining cell-free protein synthesis and microfluidics to quantify the affinity of a large number of RBD triplet mutants covering the evolutionary space between wild-type and Omicron against two therapeutic antibodies and one engineered binder. Using rapid in vitro gene assembly and cell-free synthesis, we expressed 518 RBD variants and obtained 31,740 quantitative affinity measurements to generate three comprehensive binding energy landscapes. This approach enables rapid and large-scale in vitro affinity profiling and machine learning-based predictions, providing a valuable tool for studying emerging variants.

systems biology↗

Mycobacterium tuberculosis, Mycobacterium kansasii and Rhodococcus equi induce macrophage necroptosis in the presence of a caspase inhibitor acting on a non-canonical target(s)

Macrophages are the predominant cell type infected by Mycobacterium tuberculosis (Mtb) in tuberculosis (TB). Death of Mtb-infected macrophages promotes tissue pathology and releases Mtb to infect other cells, suggesting that inhibiting the death of Mtb-infected macrophages could be an adjunctive treatment of TB. Prospects for such an intervention depend on identifying the molecular pathways leading to cell death. We previously reported that the death of Mtb-infected mouse macrophages in vitro depends on type I interferon (IFN) and that the ensuing upregulation of cis-aconitate decarboxylase (ACOD1; IRG1) contributes to cell death by exacerbating Mtb-induced lysosomal membrane permeabilization. Here we report that death of Mtb-infected primary mouse macrophages in vitro became necroptotic and die faster in the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD) acting on a target other than caspase-8. Macrophages infected with Mycobacterium kansasii and Rhodococcus equi likewise underwent z-VAD-dependent necroptosis. In C57BL/6 mice, which are relatively TB-resistant, we saw no impact of MLKL deficiency on bacterial burden or pulmonary pathology. In contrast, in Sp140-/- mice on the C57BL/6 background, which express high levels of type I IFN after Mtb infection and develop necrotic pulmonary lesions, MLKL-deficiency reduced bacterial burden and pathology after high-dose infection. This report illustrates that off-target action(s) of a caspase-8 inhibitor can switch the cell death pathway to necroptosis in macrophages infected with various Gram-positive pathogens. In turn, this opens the possibility that pathophysiologic circumstances may lead to inhibition of the same target(s) that z-VAD inhibited in our studies. That may be what allows MLKL to exacerbate tuberculosis in mice that are prone to formation of necrotic lesions. Author SummaryTuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), led to about 1.3 million deaths in 2023. The rapid emergence of drug resistance and slow pace of new drug development prompt attention to adjunctive host-directed therapies, an approach that relies on a thorough and detailed understanding of host-Mtb interactions. Mtb infection of macrophages can kill them, promoting tissue pathology and releasing of Mtb to infect other cells. Here we find that a pan-caspase inhibitor z-VAD hastened the death of Mtb-infected macrophages and converted the mode of cell death to RIPK1-RIPK3-MLKL-dependent necroptosis. However, the functional target(s) of z-VAD-FMK and the activation mechanism of RIPK3 differ from the canonical pathways. The pan-caspase inhibitor also promoted rapid, necroptotic death of macrophages infected with Mycobacterium kansasii and Rhodococcus equi. We further found that MLKL-deficiency in Sp140-/- mice on the C57BL/6 background resulted in less weight loss, lower Mtb burdens and mitigation of lung pathology after high-dose Mtb infection. Our findings suggest that necroptosis can contribute to TB pathogenesis in some circumstances. Further identification of the death pathway-switching mechanism could deepen our understanding of host-Mtb interactions and aid in the design of adjunctive host-directed therapies.

microbiology↗

A clade 2.3.4.4b H5N1 virus vaccine that elicits cross-protective antibodies against conserved domains of H5 and N1 glycoproteins

The continuous evolution and widespread dissemination of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose critical challenges to global pandemic preparedness. In this study, we assessed a low-dose inactivated split virus vaccine derived from clade 2.3.4.4b H5N1, formulated with an Alum/CpG adjuvant, using a preclinical mouse model. This vaccine induced potent humoral and cellular immune responses, generating high titers of cross-reactive antibodies targeting both hemagglutinin (HA) and neuraminidase (NA) glycoproteins across homologous and heterologous H5 clades. The Alum/CpG adjuvant enabled significant antigen dose-sparing while promoting a balanced Th1/Th2 immune profile. Functional analyses demonstrated strong virus neutralization, neuraminidase inhibition, and potent antibody-dependent cellular cytotoxicity activity. Additionally, the vaccine elicited robust antigen-specific CD4+ and CD8+ T cell responses and effectively controlled viral replication in the lungs, accompanied by reduced lung inflammation. Importantly, vaccinated mice were fully protected against lethal challenges with both the homologous clade 2.3.4.4b and heterologous clade 1 H5N1 viruses, despite low hemagglutination inhibition titers. Electron microscopy polyclonal epitope mapping revealed serum antibodies targeting multiple epitopes on homologous HA and NA, with some cross-reacting to conserved epitopes on heterologous proteins, underscoring broad immune recognition. Collectively, these results highlight the potential of this vaccine candidate to provide broad, multifunctional, and durable immunity against both current and emerging H5N1 threats, supporting its further development for pandemic preparedness.

microbiology↗

Distinguishing cortical indexes of arousal and awareness in sleeping patients with unresponsive wakefulness syndrome

Consciousness is assumed to be defined by two components: awareness and arousal. However, compared with awareness, the cortical index for arousal remains lacking in clues. Since patients with unresponsive wakefulness syndrome (UWS) are supposed to have no awareness but arousal, sleep-wake cycle in patients with UWS may reflect pure arousal fluctuations, and then could highlight the cortical indexes for arousal. This study recorded nighttime polysomnography in patients with UWS, patients with minimally conscious state and healthy controls. The EEG signal showed that spectral slope could index arousal, fluctuating among sleep stages in all three groups. Both spectral entropy and Lempel-Ziv complexity indexed awareness, showing a significant difference between conscious and unconscious states. These findings provide fundamental evidence for the two-component hypothesis of consciousness.

neuroscience↗

CSF1R regulates monocyte subset differentiation and intracellular metabolism.

Monocytes are key circulating effectors of vascular homeostasis, innate immunity and inflammation. Following their generation in mouse bone marrow, classical (Ly6Chigh) monocytes are mobilized into the blood circulation where they mature into non-classical (Ly6Clow) patrolling monocytes or are recruited into peripheral tissues where they differentiate into tissue resident or inflammatory macrophages. Monocytes and macrophages express CSF1R (CD115), the receptor for lineage-specific growth factors CSF1 and IL34. Here, we report that acute CSF1R blockade or genetic deletion negatively interferes with monocyte intracellular metabolism and reduces blood Ly6Clow monocytes in part by blunting differentiation of Ly6Chigh monocytes. Based upon lineage-specific deletion of GFPT1 (Glutamine-Fructose-6-Phosphate Transaminase 1), the hexosamine biosynthetic pathway (HBP) is identified as a novel regulator of CSF1R expression and monocyte subset diversity. Our findings provide new insights into the link between CSF1R signaling, metabolic regulation, and monocyte survival and differentiation.

immunology↗

MEG3 Enhances Survival of Developing Human Neurons with CLCN4-Linked Autophagy Impairment

Genetic variations in CLCN4, encoding the H+/Cl- exchanger CLC-4, are associated with human neurodevelopmental disorders with highly variable phenotypes. A lack of physiologically relevant models has hampered molecular understanding of pathogenic mechanisms. We now establish engineered brain organoid and neuronal cell systems to examine impacts of patient-relevant CLCN4 genetic variations. We find that CLCN4 variants reduced excitatory neuron numbers due to early-stage cell death, accompanied by altered endo-lysosomal dynamics and disrupted autophagic flux. Transcriptomic profiling showed significant downregulation of long non-coding RNA MEG3 in CLCN4-variant neurons. Restoring MEG3 expression is sufficient to rescue cellular defects and improve survival of CLCN4-variant neurons. These findings link CLCN4 dysfunction with impaired autophagy and neuronal cell death, highlighting MEG3 as a potential therapeutic target for neurodevelopmental disorders involving autophagic dysfunction.

neuroscience↗

A 3D iPSC retina model reveals non-cell-autonomous and non-neuronal mechanism of photoreceptor degeneration in a lysosomal storage disorder.

Disruption of photoreceptor-retinal pigment epithelium (RPE) interface with loss of photoreceptor outer segments (POSs) in the retina is a pathological hallmark of several neurodegenerative and retinal diseases including lysosomal storage disorders like CLN3 disease. However, the retina is a functional composite in vivo; and in vitro stem cell models of retina that enable investigation of the photoreceptor-RPE interface in healthy and diseased retina are lacking. Here, we developed a 3D human pluripotent stem cell (hPSC)-derived retina model to investigate the photoreceptor-RPE interface in healthy and disease tissue. Using this 3D hPSC retina model, we demonstrated that the most common disease causing CLN3 mutation (CLN3{Delta}ex7-8) leads to reduced levels of acid ceramidase (AC) and consequently altered sphingolipid metabolism and signaling and POS loss in CLN3 disease. Consistent with the 3D hPSC retina model, altered sphingolipid metabolism and signaling coincided with POS loss in a large animal model of CLN3 disease, CLN3 miniswine. Therapeutically, recombinant human acid ceramidase (rhAC) targeted both altered sphingolipid metabolism and retina degeneration in the CLN3 hPSC retina model and the CLN3 miniswine eye. These findings demonstrate a proof-of-concept that rhAC can rescue disease phenotype in a large animal model of CLN3 disease and suggest that rhAC could be a therapeutic approach for CLN3 disease. One Sentence SummaryAcid ceramidase deficiency and consequently altered sphingolipid signaling promotes disease phenotype(s) in a lysosomal storage disorder, CLN3 disease.

neuroscience↗

RNA structural heterogeneity in a eukaryotic cell influences its heat shock response

RNA structure-based gene regulation remains under-explored in eukaryotes. While RNA can form different conformations in solution, the extent to which it folds into structure ensembles and how the different conformations regulate gene expression still needs to be fully understood. We coupled the SHAPE compound NAI-N3 with direct RNA sequencing to identify structure modifications along a single RNA molecule (sm-PORE-cupine). Using a combination of base mapping and direct signal alignment, we boosted the percentage of mappable RNA molecules from direct RNA sequencing. Using Bernoulli Mixture Model (BMM) clustering, we show that we can separate RNA structure ensembles from ligand-bound and unbound riboswitches accurately, identify isoform-specific structure ensembles along the SARS-CoV-2 genome, and determine RNA structure ensembles in the transcriptome of a eukaryote, C. albicans, at yeast (30{degrees}C) and hyphae (37{degrees}C) states. We observed that RNAs are more structurally homogenous at 37{degrees}C compared to 30{degrees}C, are more variable in vivo than in vitro, and show higher homogeneity in 3UTRs than in the coding region. We also identified structure ensembles that are associated with changes in translation efficiency and decay in C. albicans at 30{degrees}C and 37{degrees}C and validated translational changes using reporter assays. Our work shows that single-molecule RNA structure probing using direct RNA sequencing can be applied to diverse transcriptomes to study the complexity and function of RNA structures.

molecular biology↗

A conserved long-range RNA interaction in SARS-CoV-2 recruits ADAR1 to enhance virus proliferation

Long-range RNA-RNA pairing impacts the genome structure and function of SARS-CoV-2 variants. To understand the structure and function relationships of different SARS-CoV-2 variants that have emerged during the COVID-19 pandemic, we performed high-throughput structure probing and modelling of the genomic structures of the wildtype (WT), Alpha, Beta, Delta and Omicron variants of SARS-CoV-2. We observed that genomes of SARS-CoV-2 variants are generally structurally conserved, and that single-nucleotide variations and interactions with RNA binding proteins can impact RNA structures across the viruses. Importantly, using proximity ligation sequencing, we identified many conserved ultra-long-range RNA-RNA interactions, including one that spans more than 17 kb in both the WT virus and the Omicron variant. We showed that mutations that disrupt this 17 kb long-range interaction reduce viral fitness at later stages of its infection cycle, while compensatory mutations partially restore virus fitness. Additionally, we showed that this ultra-long-range RNA-RNA interaction structure binds directly to ADAR1 to alter the RNA editing levels on the viral genome. These studies deepen our understanding of RNA structures in the SARS-CoV-2 genome and their ability to interact with host factors to facilitate virus infectivity.

genomics↗

Isolation of genetically diverse influenza antibodies highlights the role of IG germline gene variation and informs the design of population-comprehensive vaccine strategies

The regular emergence of influenza strains with pandemic potential creates a strong incentive to develop vaccines that stimulate protective responses across all human populations. A critical consideration is how variation in the human immunoglobulin (IG) loci influences B cell recognition of viral epitopes and elicitation of neutralizing antibodies. Here, we applied personalized IG germline genotyping and high-throughput sequencing of paired antibody chains from influenza A virus hemagglutinin (HA)-binding B cells to demonstrate that the response to HA is highly individual. We show that a germline-encoded polymorphism in IGHV2-70 alters the functionality of the LPAF-a class of neutralizing antibodies, and we describe HA stem-targeting broadly neutralizing antibodies (bNAbs) that use germline IGHV genes other than the population-restricted IGHV genes used by many previously known stem bNAbs. Our results demonstrate that the approach used here can be used to discover and avert population vulnerabilities arising from IG gene variation when designing HA-based influenza vaccines aimed for the global human population.

immunology↗

Copper-Containing ROS-Scavenging Nanozyme Paradoxically Drives Alpha-Synucleinopathy by Amplifying Nitrosative Stress

Reactive oxygen and nitrogen species (RONS) are implicated in neurodegeneration, but their specific pathogenic roles remain unclear. Here, we developed a pair of iridium-based nanozymes with opposing functionalities to dissect these pathways. We show that a copper-tuned iridium nanozyme (Ir{square}Cu), despite being a superior ROS scavenger, paradoxically and dramatically exacerbated -synuclein (Syn) pathology in vivo. This pathology was causally linked to its ability to amplify RNS, as pharmacological inhibition of nitric oxide synthase (NOS) with L-NAME completely abrogated the pathology and reversed a human Parkinsons disease (PD)-like transcriptomic signature. In contrast, a copper-free, broad-spectrum RONS-scavenging iridium (Ir) nanozyme demonstrated substantial therapeutic efficacy across diverse brain-first, body-first, and Alzheimers disease with Lewy body co-pathology models. Our findings uncover the importance of the RNS pathway in driving -synucleinopathies and establish a critical design principle for nanomedicine, mandating caution in the use of redox-active copper for neuroprotective applications.

pathology↗

Plagl1 and Lrrc58 control mammalian body size by triggering target-directed microRNA degradation of miR-322 and miR-503

Precise control of microRNA (miRNA) expression is critical during development. An important mechanism of miRNA regulation is target-directed microRNA degradation (TDMD), a pathway in which the binding of miRNAs to specialized trigger RNAs induces ubiquitylation and decay of associated Argonaute (AGO) proteins by the ZSWIM8 ubiquitin ligase. Concomitant release of miRNAs results in their rapid turnover. ZSWIM8-deficient mice exhibit reduced body size, cardiopulmonary and neurodevelopmental defects, and perinatal lethality. Despite widespread dysregulation of miRNAs in these animals, the vast majority of presumptive trigger RNAs that induce decay of ZSWIM8-regulated miRNAs remain undefined. Here, using AGO crosslinking and sequencing of hybrids (AGO-CLASH), a high-throughput method for identifying miRNA binding sites, we report the identification of Plagl1 as a TDMD trigger for miR-322-5p, and Lrrc58 and Malat1 as TDMD triggers for miR-503-5p in mouse embryonic fibroblasts (MEFs). In mice, deletion of the miR-322-5p and miR-503-5p trigger sites in the Plagl1 and Lrrc58 3' UTRs, respectively, abrogated TDMD of these miRNAs and resulted in miR-322/503-dependent embryonic growth restriction, recapitulating a key feature of the Zswim8--/-- phenotype. Thus, Plagl1 and Lrrc58 act as triggers for degradation of miR-322-5p and miR-503-5p, revealing a noncoding function for these mRNAs as regulators of mammalian body size.

molecular biology↗

Structural and functional characterization of the antigenicity of influenza A virus hemagglutinin subtype H15

Avian H15 influenza viruses are closely related to H7 viruses, but feature a unique 9-amino acid insertion in their hemagglutinin head domain, creating an additional site for antigenic variation. Here, we characterized a panel of mouse monoclonal antibodies (mAbs) raised against the A/wedge-tailed shearwater/Western Australia/2576/1979 ancestral strain, and a human mAb isolated from an H7N9 vaccinee. We found differences in binding and neutralization profiles against the ancestral strain and drifted strains of H15 isolated after 2008. MAbs that have hemagglutination inhibition activity against the ancestral strain do not show binding to drifted strains, hinting at antigenic differences in the receptor binding site. We show that the mAbs protect in vivo and elucidate mAb-antigen interactions using negative stain and cryo-electron microscopy. The characterization of H15 antigenicity and mechanisms of antibody-mediated neutralization expands our knowledge of this rare avian influenza virus and informs our understanding of immune pressures on viral surface glycoproteins.

microbiology↗

Multicellularity, Culture Duration, and Hydrogel Stiffness Guide Induced Pluripotent Stem Cell-Derived Endothelial Progenitor Cell Contractility

Human induced pluripotent stem cells (hiPSCs) offer immense potential for tissue engineering, yet poor vascularization remains a significant hurdle. Understanding how hiPSC-derived endothelial progenitor cells (hiPSC-EPs) form networks is essential for therapeutic progress. This study investigates extracellular matrix (ECM) remodeling and cellular contractility during the early self-assembly of hiPSC-EPs within 3D hyaluronic acid-based hydrogels. By tracking microsphere displacements before and after cytochalasin-D treatment, we quantified contractile forces in single cells and clusters at days 4 and 7. We then applied a novel inverse modeling approach using a compressible material model to determine spatially varying changes in hydrogel modulus caused by enzymatic degradation and ECM deposition. Our findings reveal that basal contractility and remodeling are nonlinearly influenced by multicellularity, culture duration, and initial stiffness. Increased hydrogel stiffness, paired with synergistic rises in strain energy, resulted in high traction forces in longer culture times. These results provide critical mechanical insights into hiPSC-EP self-assembly, advancing our ability to engineer functional vascular networks.

bioengineering↗

Locomotion-induced neural activity independent of auditory feedback in the mouse inferior colliculus

Accumulating evidence indicates that the auditory pathway integrates movement-related signals with auditory input, yet the precise sources and mechanisms of this integration across various processing levels are incompletely understood. The inferior colliculus (IC), a major midbrain hub in the auditory pathway, shows widespread modulation of neural activity during locomotion, indicating that auditory neurons at this level are sensitive to ongoing movement. However, in hearing animals, it has been challenging to dissociate auditory feedback from other motor-related signals. In this study, to isolate non-auditory contributions, we recorded IC neural activity during locomotion in deafened mice, thereby eliminating all auditory feedback through both air and bone conduction. Even in the absence of auditory input, IC neurons exhibited robust, bidirectional modulation during locomotion. Timing analysis using electromyography revealed both predictive and feedback components relative to locomotion onset. Furthermore, the timing and direction of modulation varied considerably across different locomotion bouts, suggesting convergence of multiple non-auditory inputs. These findings demonstrate that non-auditory, movement-related signals significantly shape auditory midbrain activity through both predictive and feedback mechanisms.

neuroscience↗