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Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.
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Proliferación Celular , Melanoma , Metástasis de la Neoplasia , Animales , Comunicación Celular , Diferenciación Celular , Linaje de la Célula , Rastreo Celular , Reprogramación Celular , Células Endoteliales , Melanoma/genética , Melanoma/patología , Mesodermo/patología , Ratones , Metástasis de la Neoplasia/patología , Cresta Neural/embriología , Fenotipo , Análisis de la Célula Individual , Transcriptoma , Microambiente TumoralRESUMEN
Genetically encoded calcium indicators (GECIs) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of suboptimal peak signal-to-baseline ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and wide dynamic ranges (>100-fold). NEMO indicators report Ca2+ transients with peak SBRs around 20-fold larger than the top-of-the-range GCaMP6 series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared with GCaMP6s, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in vivo, thereby outperforming most existing state-of-the-art GECIs. Given their high sensitivity and resolution to report intracellular Ca2+ signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.
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Calcio , Neuronas , Animales , Calcio/metabolismo , Neuronas/fisiología , Señalización del Calcio/fisiología , Indicadores y Reactivos , Mamíferos/metabolismoRESUMEN
Mitochondria are in a constant balance of fusion and fission. Excessive fission or deficient fusion leads to mitochondrial fragmentation, causing mitochondrial dysfunction and physiological disorders. How the cell prevents excessive fission of mitochondria is not well understood. Here, we report that the fission yeast AAA-ATPase Yta4, which is the homolog of budding yeast Msp1 responsible for clearing mistargeted tail-anchored (TA) proteins on mitochondria, plays a critical role in preventing excessive mitochondrial fission. The absence of Yta4 leads to mild mitochondrial fragmentation in a Dnm1-dependent manner but severe mitochondrial fragmentation upon induction of mitochondrial depolarization. Overexpression of Yta4 delocalizes the receptor proteins of Dnm1, i.e., Fis1 (a TA protein) and Mdv1 (the bridging protein between Fis1 and Dnm1), from mitochondria and reduces the localization of Dnm1 to mitochondria. The effect of Yta4 overexpression on Fis1 and Mdv1, but not Dnm1, depends on the ATPase and translocase activities of Yta4. Moreover, Yta4 interacts with Dnm1, Mdv1, and Fis1. In addition, Yta4 competes with Dnm1 for binding Mdv1 and decreases the affinity of Dnm1 for GTP and inhibits Dnm1 assembly in vitro. These findings suggest a model, in which Yta4 inhibits mitochondrial fission by inhibiting the function of the mitochondrial divisome composed of Fis1, Mdv1, and Dnm1. Therefore, the present work reveals an uncharacterized molecular mechanism underlying the inhibition of mitochondrial fission.
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Demencia Frontotemporal , Schizosaccharomyces , Humanos , ATPasas Asociadas con Actividades Celulares Diversas/genética , Dinámicas Mitocondriales , Adenosina Trifosfatasas , Mitocondrias , Schizosaccharomyces/genéticaRESUMEN
Microglial phagocytosis is an energetically demanding process that plays a critical role in the removal of toxic protein aggregates in Alzheimer's disease (AD). Recent evidence indicates that a switch in energy production from mitochondrial respiration to glycolysis disrupts this important protective microglial function and may provide therapeutic targets for AD. Here, we demonstrate that the translocator protein (TSPO) and a member of its mitochondrial complex, hexokinase-2 (HK), play critical roles in microglial respiratory-glycolytic metabolism and phagocytosis. Pharmacological and genetic loss-of-function experiments showed that TSPO is critical for microglial respiratory metabolism and energy supply for phagocytosis, and its expression is enriched in phagocytic microglia of AD mice. Meanwhile, HK controlled glycolytic metabolism and phagocytosis via mitochondrial binding or displacement. In cultured microglia, TSPO deletion impaired mitochondrial respiration and increased mitochondrial recruitment of HK, inducing a switch to glycolysis and reducing phagocytosis. To determine the functional significance of mitochondrial HK recruitment, we developed an optogenetic tool for reversible control of HK localization. Displacement of mitochondrial HK inhibited glycolysis and improved phagocytosis in TSPO-knockout microglia. Mitochondrial HK recruitment also coordinated the inflammatory switch to glycolysis that occurs in response to lipopolysaccharide in normal microglia. Interestingly, cytosolic HK increased phagocytosis independent of its metabolic activity, indicating an immune signaling function. Alzheimer's beta amyloid drastically stimulated mitochondrial HK recruitment in cultured microglia, which may contribute to microglial dysfunction in AD. Thus, targeting mitochondrial HK may offer an immunotherapeutic approach to promote phagocytic microglial function in AD.
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Enfermedad de Alzheimer , Animales , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Hexoquinasa/genética , Hexoquinasa/metabolismo , Microglía/metabolismo , Fagocitosis , Mitocondrias/metabolismoRESUMEN
Stem cells can generate a diversity of cell types during development, regeneration and adult tissue homeostasis. Differentiation changes not only the cell fate in terms of gene expression but also the physical properties and functions of cells, e.g. the secretory activity, cell shape, or mechanics. Conversely, these activities and properties can also regulate differentiation itself. Membrane trafficking is known to modulate signal transduction and thus has the potential to control stem cell differentiation. On the other hand, membrane trafficking, particularly from and to the plasma membrane, depends on the mechanical properties of the cell surface such as tension within the plasma membrane or the cortex. Indeed, recent findings demonstrate that cell surface mechanics can also control cell fate. Here, we review the bidirectional relationships between these three fundamental cellular functions, i.e. membrane trafficking, cell surface mechanics, and stem cell differentiation. Furthermore, we discuss commonly used methods in each field and how combining them with new tools will enhance our understanding of their interplay. Understanding how membrane trafficking and cell surface mechanics can guide stem cell fate holds great potential as these concepts could be exploited for directed differentiation of stem cells for the fields of tissue engineering and regenerative medicine.
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Medicina Regenerativa , Células Madre , Adulto , Humanos , Membrana Celular , Diferenciación Celular , Forma de la CélulaRESUMEN
Drought stress is one of the dominating challenges to the growth and productivity in crop plants. Elucidating the molecular mechanisms of plants responses to drought stress is fundamental to improve fruit quality. However, such molecular mechanisms are poorly understood in apple (Malus domestica Borkh.). In this study, we explored that the BTB-BACK-TAZ protein, MdBT2, negatively modulates the drought tolerance of apple plantlets. Moreover, we identified a novel Homeodomain-leucine zipper (HD-Zip) transcription factor, MdHDZ27, using a yeast two-hybrid (Y2H) screen with MdBT2 as the bait. Overexpression of MdHDZ27 in apple plantlets, calli, and tomato plantlets enhanced their drought tolerance by promoting the expression of drought tolerance-related genes [responsive to dehydration 29A (MdRD29A) and MdRD29B]. Biochemical analyses demonstrated that MdHDZ27 directly binds to and activates the promoters of MdRD29A and MdRD29B. Furthermore, in vitro and in vivo assays indicate that MdBT2 interacts with and ubiquitinates MdHDZ27, via the ubiquitin/26S proteasome pathway. This ubiquitination results in the degradation of MdHDZ27 and weakens the transcriptional activation of MdHDZ27 on MdRD29A and MdRD29B. Finally, a series of transgenic analyses in apple plantlets further clarified the role of the relationship between MdBT2 and MdHDZ27, as well as the effect of their interaction on drought resistance in apple plantlets. Collectively, our findings reveal a novel mechanism by which the MdBT2-MdHDZ27 regulatory module controls drought tolerance, which is of great significance for enhancing the drought resistance of apple and other plants.
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Resistencia a la Sequía , Malus , Proteínas de Plantas , Factores de Transcripción , Ubiquitinación , Resistencia a la Sequía/genética , Regulación de la Expresión Génica de las Plantas , Malus/genética , Malus/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Itching is an aversive somatosensation that triggers the desire to scratch. Transient receptor potential (TRP) channel proteins are key players in acute and chronic itch. However, whether the modulatory effect of fibroblast growth factor 13 (FGF13) on acute and chronic itch is associated with TRP channel proteins is unclear. Here, we demonstrated that conditional knockout of Fgf13 in dorsal root ganglion neurons induced significant impairment in scratching behaviors in response to acute histamine-dependent and chronic dry skin itch models. Furthermore, FGF13 selectively regulated the function of the TRPV1, but not the TRPA1 channel on Ca2+ imaging and electrophysiological recordings, as demonstrated by a significant reduction in neuronal excitability and current density induced by TRPV1 channel activation, whereas TRPA1 channel activation had no effect. Changes in channel currents were also verified in HEK cell lines. Subsequently, we observed that selective modulation of TRPV1 by FGF13 required its microtubule-stabilizing effect. Furthermore, in FGF13 knockout mice, only the overexpression of FGF13 with a tubulin-binding domain could rescue TRP channel function and the impaired itch behavior. Our findings reveal a novel mechanism by which FGF13 is involved in TRPV1-dependent itch transduction and provide valuable clues for alleviating pathological itch syndrome.
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Factores de Crecimiento de Fibroblastos , Ratones Noqueados , Microtúbulos , Prurito , Canales Catiónicos TRPV , Animales , Humanos , Masculino , Ratones , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Ganglios Espinales/metabolismo , Células HEK293 , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , Prurito/metabolismo , Prurito/genética , Canal Catiónico TRPA1/metabolismo , Canal Catiónico TRPA1/genética , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genéticaRESUMEN
Previous studies have shown that excessive alcohol consumption is associated with poor sleep. However, the health risks of light-to-moderate alcohol consumption in relation to sleep traits (e.g., insomnia, snoring, sleep duration and chronotype) remain undefined, and their causality is still unclear in the general population. To identify the association between alcohol consumption and multiple sleep traits using an observational and Mendelian randomization (MR) design. Observational analyses and one-sample MR (linear and nonlinear) were performed using clinical and individual-level genetic data from the UK Biobank (UKB). Two-sample MR was assessed using summary data from genome-wide association studies from the UKB and other external consortia. Phenotype analyses were externally validated using data from the National Health and Nutrition Examination Survey (2017-2018). Data analysis was conducted from January 2022 to October 2022. The association between alcohol consumption and six self-reported sleep traits (short sleep duration, long sleep duration, chronotype, snoring, waking up in the morning, and insomnia) were analysed. This study included 383,357 UKB participants (mean [SD] age, 57.0 [8.0] years; 46% male) who consumed a mean (SD) of 9.0 (10.0) standard drinks (one standard drink equivalent to 14 g of alcohol) per week. In the observational analyses, alcohol consumption was significantly associated with all sleep traits. Light-moderate-heavy alcohol consumption was linearly linked to snoring and the evening chronotype but nonlinearly associated with insomnia, sleep duration, and napping. In linear MR analyses, a 1-SD (14 g) increase in genetically predicted alcohol consumption was associated with a 1.14-fold (95% CI, 1.07-1.22) higher risk of snoring (P < 0.001), a 1.28-fold (95% CI, 1.20-1.37) higher risk of evening chronotype (P < 0.001) and a 1.24-fold (95% CI, 1.13-1.36) higher risk of difficulty waking up in the morning (P < 0.001). Nonlinear MR analyses did not reveal significant results after Bonferroni adjustment. The results of the two-sample MR analyses were consistent with those of the one-sample MR analyses, but with a slightly attenuated overall estimate. Our findings suggest that even low levels of alcohol consumption may affect sleep health, particularly by increasing the risk of snoring and evening chronotypes. The negative effects of alcohol consumption on sleep should be made clear to the public in order to promote public health.
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Consumo de Bebidas Alcohólicas , Bancos de Muestras Biológicas , Estudio de Asociación del Genoma Completo , Análisis de la Aleatorización Mendeliana , Trastornos del Inicio y del Mantenimiento del Sueño , Sueño , Humanos , Análisis de la Aleatorización Mendeliana/métodos , Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/epidemiología , Masculino , Reino Unido/epidemiología , Femenino , Persona de Mediana Edad , Sueño/genética , Sueño/fisiología , Anciano , Trastornos del Inicio y del Mantenimiento del Sueño/genética , Trastornos del Inicio y del Mantenimiento del Sueño/epidemiología , Ronquido/genética , Ronquido/epidemiología , Adulto , Fenotipo , Trastornos del Sueño-Vigilia/genética , Trastornos del Sueño-Vigilia/epidemiología , Polimorfismo de Nucleótido Simple/genética , Biobanco del Reino UnidoRESUMEN
Dysregulated T cell activation underpins the immunopathology of rheumatoid arthritis (RA), yet the machineries that orchestrate T cell effector program remain incompletely understood. Herein, we leveraged bulk and single-cell RNA sequencing data from RA patients and validated protein disulfide isomerase family A member 3 (PDIA3) as a potential therapeutic target. PDIA3 is remarkably upregulated in pathogenic CD4 T cells derived from RA patients and positively correlates with C-reactive protein level and disease activity score 28. Pharmacological inhibition or genetic ablation of PDIA3 alleviates RA-associated articular pathology and autoimmune responses. Mechanistically, T cell receptor signaling triggers intracellular calcium flux to activate NFAT1, a process that is further potentiated by Wnt5a under RA settings. Activated NFAT1 then directly binds to the Pdia3 promoter to enhance the expression of PDIA3, which complexes with STAT1 or PKM2 to facilitate their nuclear import for transcribing T helper 1 (Th1) and Th17 lineage-related genes, respectively. This non-canonical regulatory mechanism likely occurs under pathological conditions, as PDIA3 could only be highly induced following aberrant external stimuli. Together, our data support that targeting PDIA3 is a vital strategy to mitigate autoimmune diseases, such as RA, in clinical settings.
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Artritis Reumatoide , Proteína Disulfuro Isomerasas , Factor de Transcripción STAT1 , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Humanos , Artritis Reumatoide/metabolismo , Ratones , Animales , Factor de Transcripción STAT1/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Transporte Activo de Núcleo Celular , Proteínas Portadoras/metabolismo , Transducción de Señal , Proteínas de Unión a Hormona Tiroide , Factores de Transcripción NFATC/metabolismo , Activación de Linfocitos , Hormonas Tiroideas/metabolismo , Regulación de la Expresión Génica , Células Th17/metabolismo , Células Th17/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Modelos Animales de Enfermedad , Piruvato QuinasaRESUMEN
Communication between interacting organisms via bioactive molecules is widespread in nature and plays key roles in diverse biological processes. Small RNAs (sRNAs) can travel between host plants and filamentous pathogens to trigger transkingdom RNA interference (RNAi) in recipient cells and modulate plant defense and pathogen virulence. However, how fungal pathogens counteract transkingdom antifungal RNAi has rarely been reported. Here we show that a secretory protein VdSSR1 (secretory silencing repressor 1) from Verticillium dahliae, a soil-borne phytopathogenic fungus that causes wilt diseases in a wide range of plant hosts, is required for fungal virulence in plants. VdSSR1 can translocate to plant nucleus and serve as a general suppressor of sRNA nucleocytoplasmic shuttling. We further reveal that VdSSR1 sequesters ALY family proteins, adaptors of the TREX complex, to interfere with nuclear export of the AGO1microRNA (AGO1miRNA) complex, leading to a great attenuation in cytoplasmic AGO1 protein and sRNA levels. With this mechanism, V. dahliae can suppress the accumulation of mobile plant miRNAs in fungal cells and succedent transkingdom silencing of virulence genes, thereby increasing its virulence in plants. Our findings reveal a mechanism by which phytopathogenic fungi antagonize antifungal RNAi-dependent plant immunity and expand the understanding on the complex interaction between host and filamentous pathogens.
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MicroARNs , Verticillium , Transporte Activo de Núcleo Celular , Antifúngicos , MicroARNs/genética , MicroARNs/metabolismo , Enfermedades de las Plantas/microbiología , Plantas/genética , ARN de Planta , Verticillium/metabolismoRESUMEN
BACKGROUND & AIMS: The precise pathomechanisms underlying the development of non-alcoholic steatohepatitis (NASH, also known as metabolic dysfunction-associated steatohepatitis [MASH]) remain incompletely understood. In this study, we investigated the potential role of EF-hand domain family member D2 (EFHD2), a novel molecule specific to immune cells, in the pathogenesis of NASH. METHODS: Hepatic EFHD2 expression was characterized in patients with NASH and two diet-induced NASH mouse models. Single-cell RNA sequencing (scRNA-seq) and double-immunohistochemistry were employed to explore EFHD2 expression patterns in NASH livers. The effects of global and myeloid-specific EFHD2 deletion on NASH and NASH-related hepatocellular carcinoma were assessed. Molecular mechanisms underlying EFHD2 function were investigated, while chemical and genetic investigations were performed to assess its potential as a therapeutic target. RESULTS: EFHD2 expression was significantly elevated in hepatic macrophages/monocytes in both patients with NASH and mice. Deletion of EFHD2, either globally or specifically in myeloid cells, improved hepatic steatosis, reduced immune cell infiltration, inhibited lipid peroxidation-induced ferroptosis, and attenuated fibrosis in NASH. Additionally, it hindered the development of NASH-related hepatocellular carcinoma. Specifically, deletion of myeloid EFHD2 prevented the replacement of TIM4+ resident Kupffer cells by infiltrated monocytes and reversed the decreases in patrolling monocytes and CD4+/CD8+ T cell ratio in NASH. Mechanistically, our investigation revealed that EFHD2 in myeloid cells interacts with cytosolic YWHAZ (14-3-3ζ), facilitating the translocation of IFNγR2 (interferon-γ receptor-2) onto the plasma membrane. This interaction mediates interferon-γ signaling, which triggers immune and inflammatory responses in macrophages during NASH. Finally, a novel stapled α-helical peptide targeting EFHD2 was shown to be effective in protecting against NASH pathology in mice. CONCLUSION: Our study reveals a pivotal immunomodulatory and inflammatory role of EFHD2 in NASH, underscoring EFHD2 as a promising druggable target for NASH treatment. IMPACT AND IMPLICATIONS: Non-alcoholic steatohepatitis (NASH) represents an advanced stage of non-alcoholic fatty liver disease (NAFLD); however, not all patients with NAFLD progress to NASH. A key challenge is identifying the factors that trigger inflammation, which propels the transition from simple fatty liver to NASH. Our research pinpointed EFHD2 as a pivotal driver of NASH, orchestrating the over-activation of interferon-γ signaling within the liver during NASH progression. A stapled peptide designed to target EFHD2 exhibited therapeutic promise in NASH mice. These findings support the potential of EFHD2 as a therapeutic target in NASH.
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Modelos Animales de Enfermedad , Interferón gamma , Enfermedad del Hígado Graso no Alcohólico , Transducción de Señal , Animales , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/inmunología , Ratones , Humanos , Interferón gamma/metabolismo , Masculino , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Hígado/metabolismo , Hígado/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Macrófagos/metabolismo , Macrófagos/inmunología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/etiología , Ferroptosis/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunologíaRESUMEN
Patients with caspase-associated recruitment domain-9 (CARD9) deficiency are more likely to develop invasive fungal disease that affect CNS. However, the understanding of how Candida invades and persists in CNS is still limited. We here reported a 24-year-old woman who were previously immunocompetent and diagnosed with CNS candidiasis. A novel autosomal recessive homozygous CARD9 mutation (c.184 + 5G > T) from this patient was identified using whole genomic sequencing. Furthermore, we extensively characterized the impact of this CARD9 mutation on the host immune response in monocytes, neutrophils and CD4 + T cells, using single cell sequencing and in vitro experiments. Decreased pro-inflammatory cytokine productions of CD14 + monocyte, impaired Th17 cell differentiation, and defective neutrophil accumulation in CNS were found in this patient. In conclusion, this study proposed a novel mechanism of CNS candidiasis development. Patients with CNS candidiasis in absence of known immunodeficiencies should be analyzed for CARD9 gene mutation as the cause of invasive fungal infection predisposition.
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Proteínas Adaptadoras de Señalización CARD , Humanos , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/deficiencia , Femenino , Adulto Joven , Mutación , Neutrófilos/inmunología , Células Th17/inmunología , Candidiasis Mucocutánea Crónica/genética , Candidiasis Mucocutánea Crónica/inmunología , Monocitos/inmunología , CitocinasRESUMEN
Diabetic nephropathy (DN) is one of the most common complications of diabetes. Our previous study showed that CD38 knockout (CD38KO) mice had protective effects on many diseases. However, the roles and mechanisms of CD38 in DN remain unknown. Here, DN mice were generated by HFD feeding plus streptozotocin (STZ) injection in male CD38KO and CD38flox mice. Mesangial cells (SV40 MES 13 cells) were used to mimic the injury of DN with palmitic acid (PA) treatment in vitro. Our results showed that CD38 expression was significantly increased in kidney of diabetic CD38flox mice and SV40 MES 13 cells treated with PA. CD38KO mice were significantly resistant to diabetes-induced renal injury. Moreover, CD38 deficiency markedly decreased HFD/STZ-induced lipid accumulation, fibrosis and oxidative stress in kidney tissue. In contrast, overexpression of CD38 aggravated PA-induced lipid accumulation and oxidative stress. CD38 deficiency increased expression of SIRT3, while overexpression of CD38 decreased its expression. More importantly, 3-TYP, an inhibitor of SIRT3, significantly enhanced PA-induced lipid accumulation and oxidative stress in CD38 overexpressing cell lines. In conclusion, our results demonstrated that CD38 deficiency prevented DN by inhibiting lipid accumulation and oxidative stress through activation of the SIRT3 pathway.
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Damage of reactive oxygen species to various molecules such as DNA has been related to many chronic and degenerative human diseases, aging, and even cancer. 8-Oxo-7,8-dihydroguanine (OG), the most significant oxidation product of guanine (G), has become a biomarker of oxidative stress as well as gene regulation. The positive effect of OG in activating transcription and the negative effect in inducing mutation are a double-edged sword; thus, site-specific quantification is helpful to quickly reveal the functional mechanism of OG at hotspots. Due to the possible biological effects of OG at extremely low abundance in the genome, the monitoring of OG is vulnerable to signal interference from a large amount of G. Herein, based on rolling circle amplification-induced G-triplex formation and Thioflavin T fluorescence enhancement, an ultrasensitive strategy for locus-specific OG quantification was constructed. Owing to the difference in the hydrogen-bonding pattern between OG and G, the nonspecific background signal of G sites was completely suppressed through enzymatic ligation of DNA probes and the triggered specificity of rolling circle amplification. After the signal amplification strategy was optimized, the high detection sensitivity of OG sites with an ultralow detection limit of 0.18 amol was achieved. Under the interference of G sites, as little as 0.05% of OG-containing DNA was first distinguished. This method was further used for qualitative and quantitative monitoring of locus-specific OG in genomic DNA under oxidative stress and identification of key OG sites with biological function.
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ADN , Guanina , Humanos , ADN/genética , Estrés Oxidativo , Especies Reactivas de Oxígeno , Técnicas de Amplificación de Ácido NucleicoRESUMEN
G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.
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Benzotiazoles , ADN , Fluorescencia , Uracil-ADN Glicosidasa , Humanos , Benzotiazoles/química , Benzotiazoles/metabolismo , Técnicas Biosensibles/métodos , Dicroismo Circular , ADN/química , ADN/metabolismo , Colorantes Fluorescentes/química , Espectrometría de Fluorescencia , Uracil-ADN Glicosidasa/metabolismo , Uracil-ADN Glicosidasa/químicaRESUMEN
A mechanistic understanding of the principles governing the hierarchical organization of supramolecular polymers offers a paradigm for tailoring synthetic molecular architectures at the nano to micrometric scales. Herein, the unconventional crystal growth mechanism of a supramolecular polymer of superbenzene(coronene)-diphenylalanine conjugate (Cr-FFOEt ) is demonstrated. 3D electron diffraction (3D ED), a technique underexplored in supramolecular chemistry, is effectively utilized to gain a molecular-level understanding of the gradual growth of the initially formed poorly crystalline hairy, fibril-like supramolecular polymers into the ribbon-like crystallites. The further evolution of these nanosized flat ribbons into microcrystals by oriented attachment and lateral fusion is probed by time-resolved microscopy and electron diffraction. The gradual morphological and structural changes reveal the nonclassical crystal growth pathway, where the balance of strong and weak intermolecular interactions led to a structure beyond the nanoscale. The role of distinct π-stacking and H-bonding interactions that drive the nonclassical crystallization process of Cr-FFOEt supramolecular polymers is analyzed in comparison to analogous molecules, Py-FFOEt and Cr-FF forming helical and twisted fibers, respectively. Furthermore, the Cr-FFOEt crystals formed through nonclassical crystallization are found to improve the functional properties.
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Salt scaling poses a significant obstacle to the practical implementation of solar-driven evaporation for desalination. Attempts to mitigate scaling by enhancing mass transfer often lead to a compromise in evaporation efficiency due to associated heat loss. In the present work, a novel seesaw evaporator with a Janus structure to harness scaling for periodic self-descaling is reported. The seesaw evaporators are facilely fabricated by delignifying balsa wood and subsequently single-sided spray-coating it with soot and polydimethylsiloxane (PDMS). This unique Janus structure enables the evaporator to float on the brine while ensuring an ample supply of solution for evaporation. During evaporation, salt ions are transported directionally toward the cocked end of the evaporator to form scaling, triggering the seesaw evaporator to flip once a threshold is reached. The accumulated salts re-dissolve back into the solution. By adjusting the tilt angle, the evaporator can achieve an impressive evaporation rate of up to 2.65 kg m-2 h-1 when evaporating an 8 wt.% NaCl solution. Remarkably, these evaporators maintain a stable evaporation rate during prolonged 120 h operation and produce ≈3.93-6.35 L mâ»2·day⻹ of freshwater from simulated brines when assembled into an evaporation device.
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Softening in fruit adversely impacts their edible quality and commercial value, leading to substantial economic losses during fruit ripening, long-term storage, long-distance transportation, and marketing. As the apple fruit demonstrates climacteric respiration, its firmness decreases with increasing ethylene release rate during fruit ripening and postharvest storage. However, the molecular mechanisms underlying ethylene-mediated regulation of fruit softening in apple remain poorly understood. In this study, we identified a WRKY transcription factor (TF) MdWRKY31, which is repressed by ethylene treatment. Using transgenic approaches, we found that overexpression of MdWRKY31 delays softening by negatively regulating xyloglucan endotransglucosylase/hydrolases 2 (MdXTH2) expression. Yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), and dual-luciferase assays further suggested that MdWRKY31 directly binds to the MdXTH2 promoter via a W-box element and represses its transcription. Transient overexpression of ethylene-induced MdNAC7, a NAC TF, in apple fruit promoted softening by decreasing cellulose content and increasing water-soluble pectin content in fruit. MdNAC7 interacted with MdWRKY31 to form a protein complex, and their interaction decreased the transcriptional repression of MdWRKY31 on MdXTH2. Furthermore, MdNAC7 does not directly regulate MdXTH2 expression, but the protein complex formed with MdWRKY31 hinders MdWRKY31 from binding to the promoter of MdXTH2. Our findings underscore the significance of the regulatory complex NAC7-WRKY31 in ethylene-responsive signalling, connecting the ethylene signal to XTH2 expression to promote fruit softening. This sheds light on the intricate mechanisms governing apple fruit firmness and opens avenues for enhancing fruit quality and reducing economic losses associated with softening.
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IMPORTANCE: Although Micropterus salmoides rhabdovirus (MSRV) causes serious fish epidemics worldwide, the detailed mechanism of MSRV entry into host cells remains unknown. Here, we comprehensively investigated the mechanism of MSRV entry into epithelioma papulosum cyprinid (EPC) cells. This study demonstrated that MSRV enters EPC cells via a low pH, dynamin-dependent, microtubule-dependent, and clathrin-mediated endocytosis. Subsequently, MSRV transports from early endosomes to late endosomes and further into lysosomes in a microtubule-dependent manner. The characterization of MSRV entry will further advance the understanding of rhabdovirus cellular entry pathways and provide novel targets for antiviral drug against MSRV infection.
Asunto(s)
Lubina , Rhabdoviridae , Animales , Rhabdoviridae/metabolismo , Lubina/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Endocitosis , Dinaminas/metabolismo , Microtúbulos/metabolismo , Clatrina/metabolismo , Concentración de Iones de Hidrógeno , Internalización del VirusRESUMEN
Uncovering the immune response to an inactivated SARS-CoV-2 vaccine (In-Vac) and natural infection is crucial for comprehending COVID-19 immunology. Here we conducted an integrated analysis of single-cell RNA sequencing (scRNA-seq) data from serial peripheral blood mononuclear cell (PBMC) samples derived from 12 individuals receiving In-Vac compared with those from COVID-19 patients. Our study reveals that In-Vac induces subtle immunological changes in PBMC, including cell proportions and transcriptomes, compared with profound changes for natural infection. In-Vac modestly upregulates IFN-α but downregulates NF-κB pathways, while natural infection triggers hyperactive IFN-α and NF-κB pathways. Both In-Vac and natural infection alter T/B cell receptor repertoires, but COVID-19 has more significant change in preferential VJ gene, indicating a vigorous immune response. Our study reveals distinct patterns of cellular communications, including a selective activation of IL-15RA/IL-15 receptor pathway after In-Vac boost, suggesting its potential role in enhancing In-Vac-induced immunity. Collectively, our study illuminates multifaceted immune responses to In-Vac and natural infection, providing insights for optimizing SARS-CoV-2 vaccine efficacy.