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Allogeneic chimeric antigen receptor (CAR)-T cells hold great promise for expanding the accessibility of CAR-T therapy, whereas the risks of allograft rejection have hampered its application. Here, we genetically engineered healthy-donor-derived, CD19-targeting CAR-T cells using CRISPR-Cas9 to address the issue of immune rejection and treated one patient with refractory immune-mediated necrotizing myopathy and two patients with diffuse cutaneous systemic sclerosis with these cells. This study was registered at ClinicalTrials.gov (NCT05859997). The infused cells persisted for over 3 months, achieving complete B cell depletion within 2 weeks of treatment. During the 6-month follow-up, we observed deep remission without cytokine release syndrome or other serious adverse events in all three patients, primarily shown by the significant improvement in the clinical response index scores for the two diseases, respectively, and supported by the observations of reversal of inflammation and fibrosis. Our results demonstrate the high safety and promising immune modulatory effect of the off-the-shelf CAR-T cells in treating severe refractory autoimmune diseases.
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Intestinal stem cells (ISCs) are maintained by stemness signaling for precise modulation of self-renewal and differentiation under homeostasis. However, the way in which intestinal immune cells regulate the self-renewal of ISCs remains elusive. Here we found that mouse and human Lgr5+ ISCs showed high expression of the immune cell-associated circular RNA circPan3 (originating from the Pan3 gene transcript). Deletion of circPan3 in Lgr5+ ISCs impaired their self-renewal capacity and the regeneration of gut epithelium in a manner dependent on immune cells. circPan3 bound mRNA encoding the cytokine IL-13 receptor subunit IL-13Rα1 (Il13ra1) in ISCs to increase its stability, which led to the expression of IL-13Rα1 in ISCs. IL-13 produced by group 2 innate lymphoid cells in the crypt niche engaged IL-13Rα1 on crypt ISCs and activated signaling mediated by IL-13âIL-13R, which in turn initiated expression of the transcription factor Foxp1. Foxp1 is associated with ß-catenin in rendering its nuclear translocation, which caused activation of the ß-catenin pathway and the maintenance of Lgr5+ ISCs.
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Autorrenovación de las Células/inmunología , Interleucina-13/metabolismo , Mucosa Intestinal/inmunología , ARN/metabolismo , Células Madre/fisiología , Animales , Proteínas Portadoras/genética , Diferenciación Celular/inmunología , Autorrenovación de las Células/genética , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucina-13/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , ARN/genética , ARN/inmunología , ARN Circular , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regeneración/genética , Regeneración/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , beta Catenina/inmunología , beta Catenina/metabolismoRESUMEN
Chromatin organization undergoes drastic reconfiguration during gametogenesis. However, the molecular reprogramming of three-dimensional chromatin structure in this process remains poorly understood for mammals, including primates. Here, we examined three-dimensional chromatin architecture during spermatogenesis in rhesus monkey using low-input Hi-C. Interestingly, we found that topologically associating domains (TADs) undergo dissolution and reestablishment in spermatogenesis. Strikingly, pachytene spermatocytes, where synapsis occurs, are strongly depleted for TADs despite their active transcription state but uniquely show highly refined local compartments that alternate between transcribing and non-transcribing regions (refined-A/B). Importantly, such chromatin organization is conserved in mouse, where it remains largely intact upon transcription inhibition. Instead, it is attenuated in mutant spermatocytes, where the synaptonemal complex failed to be established. Intriguingly, this is accompanied by the restoration of TADs, suggesting that the synaptonemal complex may restrict TADs and promote local compartments. Thus, these data revealed extensive reprogramming of higher-order meiotic chromatin architecture during mammalian gametogenesis.
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Reprogramación Celular , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Meiosis , Espermatogénesis , Espermatozoides/metabolismo , Animales , Cromatina/química , Cromatina/genética , Regulación del Desarrollo de la Expresión Génica , Células HCT116 , Humanos , Macaca mulatta , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Conformación de Ácido Nucleico , Fase Paquiteno , Conformación Proteica , Relación Estructura-Actividad , Factores de Tiempo , Transcripción Genética , Inactivación del Cromosoma XRESUMEN
Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.
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Diabetes Mellitus Experimental , Insulina , Ratones , Animales , Insulina/metabolismo , Péptido Hidrolasas/metabolismo , Diabetes Mellitus Experimental/terapia , Endopeptidasas/metabolismo , Secreción de Insulina , Apoptosis/fisiologíaRESUMEN
BACKGROUND: Chronic overconsumption of lipids followed by their excessive accumulation in the heart leads to cardiomyopathy. The cause of lipid-induced cardiomyopathy involves a pivotal role for the proton-pump vacuolar-type H+-ATPase (v-ATPase), which acidifies endosomes, and for lipid-transporter CD36, which is stored in acidified endosomes. During lipid overexposure, an increased influx of lipids into cardiomyocytes is sensed by v-ATPase, which then disassembles, causing endosomal de-acidification and expulsion of stored CD36 from the endosomes toward the sarcolemma. Once at the sarcolemma, CD36 not only increases lipid uptake but also interacts with inflammatory receptor TLR4 (Toll-like receptor 4), together resulting in lipid-induced insulin resistance, inflammation, fibrosis, and cardiac dysfunction. Strategies inducing v-ATPase reassembly, that is, to achieve CD36 reinternalization, may correct these maladaptive alterations. For this, we used NAD+ (nicotinamide adenine dinucleotide)-precursor nicotinamide mononucleotide (NMN), inducing v-ATPase reassembly by stimulating glycolytic enzymes to bind to v-ATPase. METHODS: Rats/mice on cardiomyopathy-inducing high-fat diets were supplemented with NMN and for comparison with a cocktail of lysine/leucine/arginine (mTORC1 [mechanistic target of rapamycin complex 1]-mediated v-ATPase reassembly). We used the following methods: RNA sequencing, mRNA/protein expression analysis, immunofluorescence microscopy, (co)immunoprecipitation/proximity ligation assay (v-ATPase assembly), myocellular uptake of [3H]chloroquine (endosomal pH), and [14C]palmitate, targeted lipidomics, and echocardiography. To confirm the involvement of v-ATPase in the beneficial effects of both supplementations, mTORC1/v-ATPase inhibitors (rapamycin/bafilomycin A1) were administered. Additionally, 2 heart-specific v-ATPase-knockout mouse models (subunits V1G1/V0d2) were subjected to these measurements. Mechanisms were confirmed in pharmacologically/genetically manipulated cardiomyocyte models of lipid overload. RESULTS: NMN successfully preserved endosomal acidification during myocardial lipid overload by maintaining v-ATPase activity and subsequently prevented CD36-mediated lipid accumulation, CD36-TLR4 interaction toward inflammation, fibrosis, cardiac dysfunction, and whole-body insulin resistance. Lipidomics revealed C18:1-enriched diacylglycerols as lipid class prominently increased by high-fat diet and subsequently reversed/preserved by lysine/leucine/arginine/NMN treatment. Studies with mTORC1/v-ATPase inhibitors and heart-specific v-ATPase-knockout mice further confirmed the pivotal roles of v-ATPase in these beneficial actions. CONCLUSION: NMN preserves heart function during lipid overload by preventing v-ATPase disassembly.
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Cardiomiopatías , Resistencia a la Insulina , Animales , Ratones , Ratas , Adenosina Trifosfatasas , Arginina , Cardiomiopatías/inducido químicamente , Cardiomiopatías/prevención & control , Antígenos CD36/genética , Fibrosis , Inflamación , Leucina , Lípidos , Lisina , Diana Mecanicista del Complejo 1 de la Rapamicina , Miocitos Cardíacos , Mononucleótido de Nicotinamida , Receptor Toll-Like 4/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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In voltage-gated Na+ and K+ channels, the hydrophobicity of noncharged residues in the S4 helix has been shown to regulate the S4 movement underlying the process of voltage-sensing domain (VSD) activation. In voltage-gated proton channel Hv1, there is a bulky noncharged tryptophan residue located at the S4 transmembrane segment. This tryptophan remains entirely conserved across all Hv1 members but is not seen in other voltage-gated ion channels, indicating that the tryptophan contributes different roles in VSD activation. The conserved tryptophan of human voltage-gated proton channel Hv1 is Trp207 (W207). Here, we showed that W207 modifies human Hv1 voltage-dependent activation, and small residues replacement at position 207 strongly perturbs Hv1 channel opening and closing, and the size of the side chain instead of the hydrophobic group of W207 regulates the transition between closed and open states of the channel. We conclude that the large side chain of tryptophan controls the energy barrier during the Hv1 VSD transition.
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Activación del Canal Iónico , Canales Iónicos , Triptófano , Humanos , Activación del Canal Iónico/fisiología , Canales Iónicos/química , Canales Iónicos/genética , Canales Iónicos/metabolismo , Triptófano/genética , Triptófano/metabolismo , Dominios Proteicos/genética , MutaciónRESUMEN
Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.
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Glucosilceramidas , Virus ARN , Glucosilceramidas/metabolismo , Fusión de Membrana , Glicoproteínas/química , Lactosilceramidos , Virus ARN/metabolismoRESUMEN
Compared with young liver donors, aged liver donors are more susceptible to ischemia-reperfusion injury (IRI) following transplantation, which may be related to excessive inflammatory response and macrophage dysfunction, but the specific mechanism is unclear. Macrophage scavenger receptor 1 (MSR1) is a member of the scavenger receptor family, and plays an important regulatory role in inflammation response and macrophage function regulation. But its role in IRI following aged-donor liver transplantation is still unclear. This study demonstrates that MSR1 expression is decreased in macrophages from aged donor livers, inhibiting their efferocytosis and pro-resolving polarisation. Decreased MSR1 is responsible for the more severe IRI suffered by aged donor livers. Overexpression of MSR1 using F4/80-labeled AAV9 improved intrahepatic macrophage efferocytosis and promoted pro-resolving polarisation, ultimately ameliorating IRI following aged-donor liver transplantation. In vitro co-culture experiments further showed that overexpression of MSR1 promoted an increase in calcium concentration, which further activated the PI3K-AKT-GSK3ß pathway, and induced the upregulation of ß-catenin. Overall, MSR1-dependent efferocytosis promoted the pro-resolving polarisation of macrophages through the PI3K-AKT-GSK3ß pathway-induced up-regulating of ß-catenin leading to improved IRI following aged-donor liver transplantation.
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Closed head injury is a prevalent form of traumatic brain injury with poorly understood effects on cortical neural circuits. Given the emotional and behavioral impairments linked to closed head injury, it is vital to uncover brain functional deficits and their driving mechanisms. In this study, we employed a robust viral tracing technique to identify the alteration of the neural pathway connecting the medial prefrontal cortex to the basolateral amygdala, and we observed the disruptions in neuronal projections between the medial prefrontal cortex and the basolateral amygdala following closed head injury. Remarkably, our results highlight that ZL006, an inhibitor targeting PSD-95/nNOS interaction, stands out for its ability to selectively reverse these aberrations. Specifically, ZL006 effectively mitigates the disruptions in neuronal projections from the medial prefrontal cortex to basolateral amygdala induced by closed head injury. Furthermore, using chemogenetic approaches, we elucidate that activating the medial prefrontal cortex projections to the basolateral amygdala circuit produces anxiolytic effects, aligning with the therapeutic potential of ZL006. Additionally, ZL006 administration effectively mitigates astrocyte activation, leading to the restoration of medial prefrontal cortex glutamatergic neuron activity. Moreover, in the context of attenuating anxiety-like behaviors through ZL006 treatment, we observe a reduction in closed head injury-induced astrocyte engulfment, which may correlate with the observed decrease in dendritic spine density of medial prefrontal cortex glutamatergic neurons.
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Amígdala del Cerebelo , Ansiedad , Traumatismos Cerrados de la Cabeza , Corteza Prefrontal , Animales , Corteza Prefrontal/efectos de los fármacos , Masculino , Traumatismos Cerrados de la Cabeza/complicaciones , Ansiedad/tratamiento farmacológico , Amígdala del Cerebelo/efectos de los fármacos , Ratones , Vías Nerviosas/efectos de los fármacos , Ratones Endogámicos C57BL , Homólogo 4 de la Proteína Discs Large/metabolismoRESUMEN
Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC-MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.
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Cinesinas , Oocitos , Animales , Ratones , Transporte Biológico , Cinesinas/genética , Meiosis , MetafaseRESUMEN
Gene body methylation (GBM) is an ancestral mode of DNA methylation whose role in development has been obscured by the more prominent roles of promoter and CpG island methylation. The wasp Nasonia vitripennis has little promoter and CpG island methylation, yet retains strong GBM, making it an excellent model for elucidating the roles of GBM. Here we show that N. vitripennis DNA methyltransferase 1a (Nv-Dnmt1a) knockdown leads to failures in cellularization and gastrulation of the embryo. Both of these disrupted events are hallmarks of the maternal-zygotic transition (MZT) in insects. Analysis of the embryonic transcriptome and methylome revealed strong reduction of GBM and widespread disruption of gene expression during embryogenesis after Nv-Dnmt1a knockdown. Strikingly, there was a strong correlation between loss of GBM and reduced gene expression in thousands of methylated loci, consistent with the hypothesis that GBM directly facilitates high levels of transcription. We propose that lower expression levels of methylated genes due to reduced GBM is the crucial direct effect of Nv-Dnmt1 knockdown. Subsequently, the disruption of methylated genes leads to downstream dysregulation of the MZT, culminating in developmental failure at gastrulation.
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Avispas , Animales , Islas de CpG/genética , Metilación de ADN/genética , Genoma , Avispas/genética , Cigoto/metabolismoRESUMEN
Photocatalytic synthesis based on the oxygen reduction reaction (ORR) has shown great promise for H2O2 production. However, the low activity and selectivity of 2e- ORR result in a fairly low efficiency of H2O2 production. Herein, we propose a strategy to enhance the proton-coupled electron transfer (PCET) process in covalent organic frameworks (COFs), thereby significantly boosting H2O2 photosynthesis. We demonstrated that the construction of a hydrogen-bonding network, achieved by anchoring the H3PO4 molecular network on COF nanochannels, can greatly improve both proton conductivity and photogenerated charge separation efficiency of COFs. Thus, COF@H3PO4 exhibited superior photocatalytic performance in generating H2O2 without sacrificial agents, with a solar-to-chemical conversion efficiency as high as 0.69%. Results indicated that a much more localized spatial distribution of energy band charge density on COF@H3PO4 led to efficient charge separation, and the small energy barrier of the rate-limiting step from *OOH to H2O2 endowed COF@H3PO4 with higher 2e- ORR selectivity.
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We report, for the first time, a new synthetic strategy for the preparation of crystalline two-dimensional olefin-linked covalent organic frameworks (COFs) based on aldol condensation between benzodifurandione and aromatic aldehydes. Olefin-linked COFs can be facilely crystallized through either a pyridine-promoted solvothermal process or a benzoic anhydride-mediated organic flux synthesis. The resultant COF leaf with high in-plane π-conjugation exhibits efficient visible-light-driven photoreduction of carbon dioxide (CO2) with water (H2O) in the absence of any photosensitizer, sacrificial agents, or cocatalysts. The production rate of carbon monoxide (CO) reaches as high as 158.1 µmol g-1 h-1 with near 100% CO selectivity, which is accompanied by the oxidation of H2O to oxygen. Both theoretical and experimental results confirm that the key lies in achieving exceptional photoinduced charge separation and low exciton binding. We anticipate that our findings will facilitate new possibilities for the development of semiconducting COFs with structural diversity and functional variability.
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Extranodal extension of tumour on histopathology is known to be a negative prognostic factor in head and neck cancer. Compelling evidence suggests that extranodal extension detected on radiological imaging is also a negative prognostic factor. Furthermore, if imaging detected extranodal extension could be identified reliably before the start of treatment, it could be used to guide treatment selection, as patients might be better managed with non-surgical approaches to avoid the toxicity and cost of trimodality therapy (surgery, chemotherapy, and radiotherapy together). There are many aspects of imaging detected extranodal extension that remain unresolved or are without consensus, such as the criteria to best diagnose them and the associated terminology. The Head and Neck Cancer International Group conducted a five-round modified Delphi process with a group of 18 international radiology experts, representing 14 national clinical research groups. We generated consensus recommendations on the terminology and diagnostic criteria for imaging detected extranodal extension to harmonise clinical practice and research. These recommendations have been endorsed by 19 national and international organisations, representing 34 countries. We propose a new classification system to aid diagnosis, which was supported by most of the participating experts over existing systems, and which will require validation in the future. Additionally, we have created an online educational resource for grading imaging detected extranodal extensions.
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Consenso , Extensión Extranodal , Neoplasias de Cabeza y Cuello , Humanos , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/terapia , Extensión Extranodal/diagnóstico por imagen , Extensión Extranodal/patología , Técnica Delphi , Terminología como Asunto , PronósticoRESUMEN
Ultrasmall metallic nanoclusters (NCs) protected by surface ligands represent the most promising catalytic materials; yet understanding the structure and catalytic activity of these NCs remains a challenge due to dynamic evolution of their active sites under reaction conditions. Herein, we employed a single-nanoparticle collision electrochemistry method for real-time monitoring of the dynamic electrocatalytic activity of a single fully ligand-protected Au25(PPh3)10(SC2H4Ph)5Cl22+ nanocluster (Au252+ NC) at a cavity carbon nanoelectrode toward the oxygen reduction reaction (ORR). Our experimental results and computational simulations indicated that the reversible depassivation and passivation of ligands on the surface of the Au252+ NC, combined with the dynamic conformation evolution of the Au259+ core, led to a characteristic current signal that involves "ON-OFF" switches and "ON" fluctuations during the ORR process of a single Au252+ NC. Our findings reinvent the new perception and comprehension of the structure-activity correlation of NCs at the atomic level.
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Inverted p-i-n perovskite solar cells (PSCs) are easy to process but need improved interface characteristics with reduced energy loss to prevent efficiency drops when increasing the active photovoltaic area. Here, we report a series of poly ferrocenyl molecules that can modulate the perovskite surface enabling the construction of small- and large-area PSCs. We found that the perovskite-ferrocenyl interaction forms a hybrid complex with enhanced surface coordination strength and activated electronic states, leading to lower interfacial nonradiative recombination and charge transport resistance losses. The resulting PSCs achieve an enhanced efficiency of up to 26.08% for small-area devices and 24.51% for large-area devices (1.0208 cm2). Moreover, the large-area PSCs maintain >92% of the initial efficiency after 2000 h of continuous operation at the maximum power point under 1-sun illumination and 65 °C.
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BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) represents a prevalent malignant tumor, with approximately 40% of patients encountering treatment challenges or relapse attributed to rituximab resistance, primarily due to diminished or absent CD20 expression. Our prior research identified PDK4 as a key driver of rituximab resistance through its negative regulation of CD20 expression. Further investigation into PDK4's resistance mechanism and the development of advanced exosome nanoparticle complexes may unveil novel resistance targets and pave the way for innovative, effective treatment modalities for DLBCL. METHODS: We utilized a DLBCL-resistant cell line with high PDK4 expression (SU-DHL-2/R). We infected it with short hairpin RNA (shRNA) lentivirus for RNA sequencing, aiming to identify significantly downregulated mRNA in resistant cells. Techniques including immunofluorescence, immunohistochemistry, and Western blotting were employed to determine PDK4's localization and expression in resistant cells and its regulatory role in phosphorylation of Histone deacetylase 8 (HDAC8). Furthermore, we engineered advanced exosome nanoparticle complexes, aCD20@ExoCTX/siPDK4, through cellular, genetic, and chemical engineering methods. These nanoparticles underwent characterization via Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM), and their cellular uptake was assessed through flow cytometry. We evaluated the nanoparticles' effects on apoptosis in DLBCL-resistant cells and immune cells using CCK-8 assays and flow cytometry. Additionally, their capacity to counteract resistance and exert anti-tumor effects was tested in a resistant DLBCL mouse model. RESULTS: We found that PDK4 initiates HDAC8 activation by phosphorylating the Ser-39 site, suppressing CD20 protein expression through deacetylation. The aCD20@ExoCTX/siPDK4 nanoparticles served as effective intracellular delivery mechanisms for gene therapy and monoclonal antibodies, simultaneously inducing apoptosis in resistant DLBCL cells and triggering immunogenic cell death in tumor cells. This dual action effectively reversed the immunosuppressive tumor microenvironment, showcasing a synergistic therapeutic effect in a subcutaneous mouse tumor resistance model. CONCLUSIONS: This study demonstrates that PDK4 contributes to rituximab resistance in DLBCL by modulating CD20 expression via HDAC8 phosphorylation. The designed exosome nanoparticles effectively overcome this resistance by targeting the PDK4/HDAC8/CD20 pathway, representing a promising approach for drug delivery and treating patients with Rituximab-resistant DLBCL.
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Resistencia a Antineoplásicos , Exosomas , Linfoma de Células B Grandes Difuso , Nanopartículas , Rituximab , Humanos , Exosomas/metabolismo , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/terapia , Rituximab/farmacología , Rituximab/uso terapéutico , Animales , Ratones , Nanopartículas/química , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Apoptosis/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, but the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion as the heterozygous males (Tcfl5+/-) were infertile. However, we did observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally and post-transcriptionally regulated a set of genes participating in male germ cell development via TCFL5 ChIP-DNA and eCLIP-RNA high-throughput sequencing. We also identified a known RNA-binding protein, FXR1, as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and acts as a dual-function protein that mediates DNA and RNA to regulate spermatogenesis. This article has an associated First Person interview with the first author of the paper.
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Espermatogénesis , Testículo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , ADN/metabolismo , Fertilidad/genética , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Espermatogénesis/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Neuroblastoma (NB) is the most common and deadliest extracranial solid tumor in children. Targeting tumor-associated macrophages (TAMs) is a strategy for attenuating tumor-promoting states. The crosstalk between cancer cells and TAMs plays a pivotal role in mediating tumor progression in NB. The overexpression of Hexokinase-3 (HK3), a pivotal enzyme in glucose metabolism, has been associated with poor prognosis in NB patients. Furthermore, it correlates with the infiltration of M2-like macrophages within NB tumors, indicating its significant involvement in tumor progression. Therefore, HK3 not only directly regulates the malignant biological behaviors of tumor cells, such as proliferation, migration, and invasion, but also recruits and polarizes M2-like macrophages through the PI3K/AKT-CXCL14 axis in neuroblastoma. The secretion of lactate and histone lactylation alterations within tumor cells accompanies this interaction. Additionally, elevated expression of HK3 in M2-TAMs was found at the same time. Modulating HK3 within M2-TAMs alters the biological behavior of tumor cells, as demonstrated by our in vitro studies. This study highlights the pivotal role of HK3 in the progression of NB malignancy and its intricate regulatory network with M2-TAMs. It establishes HK3 as a promising dual-functional biomarker and therapeutic target in combating neuroblastoma.