RESUMO
Radiotherapy as a mainstay of in-depth cervical cancer (CC) treatment suffers from its radioresistance. Radiodynamic therapy (RDT) effectively reverses radio-resistance by generating reactive oxygen species (ROS) with deep tissue penetration. However, the photosensitizers stimulated by X-ray have high toxicity and energy attenuation. Therefore, X-ray responsive diselenide-bridged mesoporous silica nanoparticles (DMSNs) are designed, loading X-ray-activated photosensitizer acridine orange (AO) for spot blasting RDT like Trojan-horse against radio-resistance cervical cancer (R-CC). DMSNs can encapsulate a large amount of AO, in the tumor microenvironment (TME), which has a high concentration of hydrogen peroxide, X-ray radiation triggers the cleavage of diselenide bonds, leading to the degradation of DMSNs and the consequent release of AO directly at the tumor site. On the one hand, it solves the problems of rapid drug clearance, adverse distribution, and side effects caused by simple AO treatment. On the other hand, it fully utilizes the advantages of highly penetrating X-ray responsive RDT to enhance radiotherapy sensitivity. This approach results in ROS-induced mitochondria damage, inhibition of DNA damage repair, cell cycle arrest and promotion of cancer cell apoptosis in R-CC. The X-ray responsive DMSNs@AO hold considerable potential in overcoming obstacles for advanced RDT in the treatment of R-CC.
Assuntos
Nanopartículas , Dióxido de Silício , Humanos , Animais , Raios X , Nanopartículas/química , Feminino , Dióxido de Silício/química , Camundongos , Neoplasias do Colo do Útero/terapia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Espécies Reativas de Oxigênio/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Tolerância a Radiação/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Camundongos Nus , Células HeLa , Camundongos Endogâmicos BALB C , Apoptose/efeitos dos fármacos , Linhagem Celular TumoralRESUMO
As a reactive sulfur species, sulfur dioxide (SO2) and its derivatives play crucial role in various physiological processes, which can maintain redox homeostasis at normal levels and lead to the occurrence of many diseases at abnormal levels. So, the development of a suitable fluorescent probe is a crucial step in advancing our understanding of the role of SO2 derivatives in living organisms. Herein, we developed a near-infrared fluorescent probe (SP) based on the ICT mechanism to monitor SO2 derivatives in living organisms in a ratiometric manner. The probe SP exhibited excellent selectivity, good sensitivity, fast response rate (within 50 s), and low detection limit (1.79 µM). In addition, the cell experiment results suggested that the SP has been successfully employed for the real-time monitoring of endogenous and exogenous SO2 derivatives with negligible cytotoxicity. Moreover, SP was effective in detecting SO2 derivatives in mice.
Assuntos
Corantes Fluorescentes , Dióxido de Enxofre , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Dióxido de Enxofre/análise , Animais , Camundongos , Humanos , Limite de Detecção , Espectrometria de Fluorescência , Imagem Óptica , Células HeLaRESUMO
Tyramine signaling amplification (TSA) technology is generally applied in immunofluorescence, enzyme-linked immunoassays, in situ hybridization techniques, etc. Successful amplification of fluoresence signals cannot be achieved without excellent fluorescent dyes. BODIPY fluorophore is an ideal probe for cell fluorescence imaging, but pristine BODIPY cannot be direct used in the TSA system. In the paper, the new red-shifted tyramide-conjugated BODIPY (BDP-B/C/D) was synthesized via the Knoevenagel condensation reaction, which based on the tyramide-conjugated BODIPY (BDP-A). The synthesized dyes were combined with tyramine to obtain which could be used as a fluorescent substrate for enzymatic reaction of TSA. By using the selected substrate (BDP-C) in TSA, we found it to be more sensitive than the commercial dye 594 styramide for the detection of low-abundance antigen proteins.
Assuntos
Compostos de Boro , Corantes Fluorescentes , Porfobilinogênio , Tiramina , Tiramina/química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Compostos de Boro/química , Compostos de Boro/síntese química , Porfobilinogênio/análogos & derivados , Porfobilinogênio/química , Células HeLa , Espectrometria de Fluorescência , Imagem ÓpticaRESUMO
Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana.
Assuntos
Cisteína , Corantes Fluorescentes , Análise de Alimentos , Glutationa , Lisossomos , Espectrometria de Fluorescência , Peixe-Zebra , Humanos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Lisossomos/química , Lisossomos/metabolismo , Células HeLa , Cisteína/análise , Animais , Análise de Alimentos/métodos , Glutationa/análise , Espectrometria de Fluorescência/métodos , Homocisteína/análise , Arabidopsis/química , Limite de Detecção , Microscopia ConfocalRESUMO
The role of ClO- in the physiological functioning of organisms is significant. In this paper, the four fluorescent probes HONx (HON1, HON2, HON3 and HON4) were prepared based on oxyanthracene through the introduction of different substituents, and their photophysical properties were investigated, among which the AIE effect of HON1 was the most significant, and therefore the fluorescent "turn-off" ClO- probe HON1-CN was chosen to be prepared by constructing the ClO- recognition site hydrazone bond at HON1. The ClO- recognises the hydrazone group in the probe HON1-CN, and when the hydrazone bond is broken, the aldehyde group is released, generating HON1 with yellow fluorescence. The probe HON1-CN is highly selective and stable for the detection of ClO- with a detection limit of 0.48 µM and a more than 10-fold increase in fluorescence intensity when the fluorescence is 'switched on', and to a lesser extent, the probe is also very good for the detection of hypochlorite ClO- in the pericarp. Finally, HON1-CN has also been used to detect the presence of ClO- in HeLa cells and zebrafish.
Assuntos
Corantes Fluorescentes , Ácido Hipocloroso , Espectrometria de Fluorescência , Xantonas , Peixe-Zebra , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Xantonas/química , Animais , Ácido Hipocloroso/análise , Humanos , Células HeLa , Frutas/química , Limite de DetecçãoRESUMO
Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.
Assuntos
Membrana Celular , Ferroptose , Corantes Fluorescentes , Microscopia de Fluorescência , Membrana Nuclear , Ferroptose/fisiologia , Humanos , Corantes Fluorescentes/química , Membrana Celular/metabolismo , Membrana Nuclear/metabolismo , Microscopia de Fluorescência/métodos , Exocitose/fisiologia , Células HeLaRESUMO
BACKGROUND The environmentally friendly production of silver nanoparticles (AgNPs) has gained significant attention as a sustainable alternative to traditional chemical methods. This study focused on synthesizing AgNPs using extract of Dracocephalum kotschyi (D. kotschyi), a medicinal plant. MATERIAL AND METHODS The biosynthesis of AgNPs was monitored using UV-visible spectrophotometry. The role of phytoconstituents from D. kotschyi in stabilizing AgNPs was analyzed using Fourier-transform infrared (FTIR) spectroscopy. Dynamic light scattering (DLS) spectroscopy was used to determine the size, charge, and polydispersity of the nanoparticles, while scanning electron microscopy (SEM) was employed to assess their morphology. We evaluated the antimicrobial efficacy of the synthesized AgNPs against various bacteria, their antioxidant properties via a 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, and their cytotoxic activity against the HeLa cervical cancer cell line. RESULTS The formation of AgNPs was indicated by a color change and the emergence of a surface plasmon resonance peak at 418 nm. The nanoparticles demonstrated significant antimicrobial, antioxidant, cytotoxic, and anticancer activities. Morphology, size, and shape analysis revealed nearly spherical particles with an average size of 43 nm. FTIR confirmed the presence of phenolic compounds in the extract, serving as reducing and capping agents. X-ray diffraction (XRD) analysis confirmed the crystalline structure of the nanoparticles. Antimicrobial assessments showed effectiveness against Escherichia coli and Staphylococcus aureus. The DPPH scavenging assay demonstrated efficient antioxidant activity, and potent apoptotic anticancer effects were observed on cervical cancer cells. CONCLUSIONS The extract of D. kotschyi was effective as a reducing agent in the environmentally friendly synthesis of AgNPs, which exhibited noteworthy antimicrobial, antioxidant, and anticancer properties. These findings suggest potential biomedical applications for the synthesized AgNPs.
Assuntos
Anti-Infecciosos , Antineoplásicos , Antioxidantes , Química Verde , Nanopartículas Metálicas , Extratos Vegetais , Prata , Nanopartículas Metálicas/química , Prata/farmacologia , Prata/química , Antioxidantes/farmacologia , Antioxidantes/síntese química , Antioxidantes/química , Humanos , Química Verde/métodos , Anti-Infecciosos/farmacologia , Anti-Infecciosos/síntese química , Células HeLa , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Testes de Sensibilidade Microbiana , Lamiaceae/química , Espectroscopia de Infravermelho com Transformada de Fourier , Antibacterianos/farmacologia , Antibacterianos/síntese química , Staphylococcus aureus/efeitos dos fármacosRESUMO
Ca2+ ion as a second messenger in signaling pathway plays many vital roles in many biological phenomena. Thus, it is of significance for developing effective probes to detect Ca2+ ion specifically. Herein, a new Schiff base fluorescent probe FPH, fluorescein monoaldehyde (2-aminomethylpyridine) hydrazone, was designed and synthesized to identify Ca2+ in DMSO aqueous solution. The probe FPH revealed significant responses to Ca2+ with a fluorescence enhancement at 540 nm, exhibiting an evident fluorescence change from ultraweak luminescence to bright green. Otherwise, the FPH displayed a good linear range of 0.67 × 10-6 to 3.33 × 10-6 mol/L with a lower detection limit at 7.02 × 10-8 mol/L. The probe FPH were further successfully utilized to detect Ca2+ in living cells by an increased bright green fluorescence.
Assuntos
Cálcio , Corantes Fluorescentes , Bases de Schiff , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Bases de Schiff/química , Humanos , Cálcio/análise , Imagem Óptica , Espectrometria de Fluorescência , Estrutura Molecular , Células HeLa , Limite de DetecçãoRESUMO
VPS13B/COH1 is the only known causative factor for Cohen syndrome, an early-onset autosomal recessive developmental disorder with intellectual inability, developmental delay, joint hypermobility, myopia, and facial dysmorphism as common features, but the molecular basis of VPS13B/COH1 in pathogenesis remains largely unclear. Here, we identify Sec23 interacting protein (Sec23IP) at the ER exit site (ERES) as a VPS13B adaptor that recruits VPS13B to ERES-Golgi interfaces. VPS13B interacts directly with Sec23IP via the VPS13 adaptor binding domain (VAB), and the interaction promotes the association between ERES and the Golgi. Disease-associated missense mutations of VPS13B-VAB impair the interaction with Sec23IP. Knockout of VPS13B or Sec23IP blocks the formation of tubular ERGIC, an unconventional cargo carrier that expedites ER-to-Golgi transport. In addition, depletion of VPS13B or Sec23IP delays ER export of procollagen, suggesting a link between procollagen secretion and joint laxity in patients with Cohen disease. Together, our study reveals a crucial role of VPS13B-Sec23IP interaction at the ERES-Golgi interface in the pathogenesis of Cohen syndrome.
Assuntos
Retículo Endoplasmático , Complexo de Golgi , Deficiência Intelectual , Microcefalia , Hipotonia Muscular , Miopia , Proteínas de Transporte Vesicular , Humanos , Complexo de Golgi/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Hipotonia Muscular/genética , Hipotonia Muscular/metabolismo , Hipotonia Muscular/patologia , Miopia/metabolismo , Miopia/genética , Miopia/patologia , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Dedos/anormalidades , Células HeLa , Transporte Proteico , Ligação Proteica , Células HEK293 , Mutação de Sentido Incorreto , Deficiências do Desenvolvimento , Obesidade , Degeneração RetinianaRESUMO
Phosphatidylinositol is a precursor of various phosphoinositides, which play crucial roles in intracellular signaling and membrane dynamics and have impact on diverse aspects of cell physiology. Phosphoinositide synthesis and turnover occur in the cytoplasmic leaflet of the organellar and plasma membranes. P4-ATPases (lipid flippases) are responsible for translocating membrane lipids from the exoplasmic (luminal) to the cytoplasmic leaflet, thereby regulating membrane asymmetry. However, the mechanism underlying phosphatidylinositol translocation across cellular membranes remains elusive. Here, we discovered that the phosphatidylcholine flippases ATP8B1, ATP8B2, and ATP10A can also translocate phosphatidylinositol at the plasma membrane. To explore the function of these phosphatidylinositol flippases, we used cells depleted of CDC50A, a protein necessary for P4-ATPase function and ATP8B1 and ATP8B2, which express in HeLa cells. Upon activation of the Gq-coupled receptor, depletion of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was accelerated in CDC50A knockout (KO) and ATP8B1/8B2 double KO cells compared with control cells, suggesting a decrease in PtdIns(4,5)P2 levels within the plasma membrane of the KO cells upon stimulation. These findings highlight the important role of P4-ATPases in maintaining phosphoinositide homeostasis and suggest a mechanism for asymmetry of phosphatidylinositol in the cytoplasmic leaflet of the plasma membrane.
Assuntos
Adenosina Trifosfatases , Membrana Celular , Homeostase , Fosfatidilinositóis , Humanos , Membrana Celular/metabolismo , Células HeLa , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Fosfatidilinositóis/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Hydrazine (N2H4) serves as a crucial industrial raw material and finds extensive applications in the fields of medicine, pesticides, ecological environment, and textile dyes. Excessive residue of hydrazine will cause significant toxicity risks to the ecosystem and human health. Traditional detection methods often require multi-step pretreatment of samples, and complex instrumentation, and are time-consuming, which is not conducive to rapid on-site detection. Therefore, it is imperative to develop a method suitable for rapid detection of N2H4 in multiple fields. RESULTS: In this study, we constructed a red emission fluorescent probe (BCM). BCM can recognize N2H4 by colorimetric and fluorescence dual-channel response with a good anti-interference ability and a low detection limitation. The fluorescence emission of BCM is attributed to the ICT effect by DFT calculations, and a new product 3H-benzo[f]chromene-2-carbaldehyde hydrazine is formed after BCM recognition of N2H4. A linear relationship was established between the ratio of red-blue (R/B) coming from the fluorescence color of BCM and the N2H4 level. Hence, a BCM-based smartphone sensing platform for detecting N2H4 was developed, and the N2H4 content can be rapidly detected with satisfactory accuracy in the lake water samples. In addition, the residues of N2H4 in soils, plants and food samples can be visualized, and BCM can image for N2H4 in living cells, as well as N2H4 vapor can be detected by using the electrospinning film loaded with BCM. SIGNIFICANCE: In particular, the fluorescent probe BCM can be combined with a smartphone for the detection and visual imaging of hydrazine in environmental samples. We believe the BCM and smartphone-based sensing platforms constructed in this paper will be a powerful tool for visual quantitative detection of N2H4 in the fields of food safety assessment, bioimaging, and environmental protection.
Assuntos
Corantes Fluorescentes , Hidrazinas , Smartphone , Hidrazinas/análise , Hidrazinas/química , Corantes Fluorescentes/química , Humanos , Espectrometria de Fluorescência , Solo/química , Células HeLa , Colorimetria , Plantas/química , Imagem Óptica , Água/química , Limite de DetecçãoRESUMO
Rationale: Theranostic nanoplatforms exert a vital role in facilitating concurrent real-time diagnosis and on-demand treatment of diseases, thereby making contributions to the improvement of therapeutic efficacy. Nevertheless, the structural intricacy and the absence of well-defined integration of dual functionality persist as challenges in the development of theranostic nanoplatforms. Methods: We develop an atomically precise theranostic nanoplatform based on metal-organic cage (MOC) to provide magnetic resonance imaging (MRI) guided chemodynamic therapy (CDT) for cancer therapy and assess the theranostic performance both in vitro and in vivo. Through UV-vis spectroscopy, electron paramagnetic resonance (EPR), confocal microscopy, flow cytometry, immunofluorescence staining, and western blotting, the ability of MOC-Mn to generate â¢OH and the subsequent inhibition of HeLa cells was confirmed. Results: The MOC-Mn composed of manganese and calixarene was successfully synthesized and comprehensively characterized. The catalytic activity of manganese within MOC-Mn facilitated the efficient generation of hydroxyl radicals (â¢OH) through a Fenton-like reaction, leveraging the high concentrations of hydrogen peroxide in the tumor microenvironment (TME). Additionally, its capacity to prolong the T1 relaxation time and augment the MR signal was observed. The theranostic efficacy was verified via rigorous in vitro and in vivo experiments, indicating that MOC-Mn offered clearer visualization of tumor particulars and substantial suppression of tumor growth. Conclusion: This study showcases a precise MRI-guided CDT theranostic nanoplatform for cancer therapy, thereby promoting the advancement of precise nanomedicine and structure-function research.
Assuntos
Imageamento por Ressonância Magnética , Nanomedicina Teranóstica , Nanomedicina Teranóstica/métodos , Humanos , Animais , Células HeLa , Imageamento por Ressonância Magnética/métodos , Camundongos , Manganês/química , Camundongos Nus , Feminino , Radical Hidroxila/metabolismo , Radical Hidroxila/química , Neoplasias/tratamento farmacológico , Neoplasias/diagnóstico por imagem , Camundongos Endogâmicos BALB C , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Nanopartículas/químicaRESUMO
Importin ß-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUSIBB), carrying the importin ß binding (IBB) domain, is solubilized by importin ß in vitro, translocated to the nucleus in cultured cells, and downregulates the expression of endogenous FUS. In this study, we posit that guiding the mutual recognition of NLSs and NIRs will aid the development of therapeutics, illustrated by the highly soluble FUSIBB replacing the aggregation-prone endogenous FUS.
Assuntos
Regulação para Baixo , Sinais de Localização Nuclear , Proteína FUS de Ligação a RNA , beta Carioferinas , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Humanos , beta Carioferinas/metabolismo , beta Carioferinas/genética , Núcleo Celular/metabolismo , Ligação Proteica , Células HEK293 , Agregados Proteicos , Células HeLa , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Transporte Ativo do Núcleo CelularRESUMO
Endoplasmic reticulum quality control is crucial for maintaining cellular homeostasis and adapting to stress conditions. Although several ER-phagy receptors have been identified, the collaboration between cytosolic and ER-resident factors in ER fragmentation and ER-phagy regulation remains unclear. Here, we perform a phenotype-based gain-of-function screen and identify a cytosolic protein, FKBPL, functioning as an ER-phagy regulator. Overexpression of FKBPL triggers ER fragmentation and ER-phagy. FKBPL has multiple protein binding domains, can self-associate and might act as a scaffold connecting CKAP4 and LC3/GABARAPs. CKAP4 serves as a bridge between FKBPL and ER-phagy cargo. ER-phagy-inducing conditions increase FKBPL-CKAP4 interaction followed by FKBPL oligomerization at the ER, leading to ER-phagy. In addition, FKBPL-CKAP4 deficiency leads to Golgi disassembly and lysosome impairment, and an increase in ER-derived secretory vesicles and enhances cytosolic protein secretion via microvesicle shedding. Taken together, FKBPL with the aid of CKAP4 induces ER fragmentation and ER-phagy, and FKBPL-CKAP4 deficiency facilitates protein secretion.
Assuntos
Citosol , Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Humanos , Citosol/metabolismo , Animais , Células HEK293 , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Camundongos , Células HeLa , Ligação Proteica , Estresse do Retículo EndoplasmáticoRESUMO
Two closely related connexins, Cx26 and Cx30, share widespread expression in the cochlear cellular networks. Gap junction channels formed by these connexins have been shown to have different permeability profiles, with Cx30 showing a strongly reduced preference for anionic tracers. The pore-forming segment of the first extracellular loop, E1, identified by computational studies of the Cx26 crystal structure to form a parahelix and a narrowed region of the pore, differs at a single residue at position 49. Cx26 contains an Ala and Cx30, a charged Glu at this position, and cysteine scanning in hemichannels identified this position to be pore-lining. To assess whether the Ala/Glu difference affects permeability, we modeled and quantified Lucifer Yellow transfer between HeLa cell pairs expressing WT Cx26 and Cx30 and variants that reciprocally substituted Glu and Ala at position 49. Cx26(A49E) and Cx30(E49A) substitutions essentially reversed the Lucifer Yellow permeability profile when accounting for junctional conductance. Moreover, by using a calcein efflux assay in single cells, we observed a similar reduced anionic preference in undocked Cx30 hemichannels and a reversal with reciprocal Ala/Glu substitutions. Thus, our data indicate that Cx26 and Cx30 gap junction channels and undocked hemichannels retain similar permeability characteristics and that a single residue difference in their E1 domains can largely account for their differential permeabilities to anionic tracers. The higher anionic permeability of Cx26 compared with Cx30 suggests that these connexins may serve distinct signaling functions in the cochlea, perhaps reflected in the vastly higher prevalence of Cx26 mutations in human deafness.
Assuntos
Conexina 26 , Conexina 30 , Junções Comunicantes , Humanos , Conexina 26/metabolismo , Conexina 26/genética , Células HeLa , Conexina 30/metabolismo , Conexina 30/genética , Junções Comunicantes/metabolismo , Conexinas/metabolismo , Conexinas/genética , Ânions/metabolismo , Permeabilidade , Ácido Glutâmico/metabolismo , Alanina/metabolismo , Alanina/genética , Isoquinolinas/metabolismo , Permeabilidade da Membrana Celular/fisiologiaRESUMO
Ionic liquids (ILs) have found diverse applications in research and industry. Biocompatible ILs, a subset considered less toxic than traditional ILs, have expanded their applications into biomedical fields. However, there is limited understanding of the toxicity profiles, safe concentrations, and underlying factors driving their toxicity. In this study, we investigated the cytotoxicity of 13 choline-based ILs using four different cell lines: Human dermal fibroblasts (HDF), epidermoid carcinoma cells (A431), cervical cancer cells (HeLa), and gastric cancer cells (AGS). Additionally, we explored the haemolytic activity of these ILs. Our findings showed that the cytotoxic and haemolytic activities of ILs can be attributed to the hydrophobicity of the anions and the pH of the IL solutions. Furthermore, utilising quartz crystal microbalance with dissipation (QCM-D), we delved into the interaction of selected ILs, including choline acetate [Cho][Ac] and choline geranate [Cho][Ge], with model cell membranes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The QCM-D data showed that ILs with higher toxicities exhibited more pronounced interactions with membranes. Increased variations in frequency and dissipation reflected substantial changes in membrane fluidity and mass following the addition of the more toxic ILs. Furthermore, total internal reflection fluorescence microscopy study revealed that [Cho][Ac] could cause lipid rearrangements and pore formation in the membrane, while [Cho][Ge] disrupted the bilayer packing. This study advances our understanding of the cellular toxicities associated with choline-based ILs and provides valuable insights into their mechanisms of action concerning IL-membrane interactions. These findings have significant implications for the safe and informed utilisation of biocompatible ILs in the realm of drug delivery and biotechnology.
Assuntos
Acetatos , Aminoácidos , Ânions , Membrana Celular , Colina , Líquidos Iônicos , Líquidos Iônicos/química , Líquidos Iônicos/toxicidade , Humanos , Colina/química , Ânions/química , Membrana Celular/efeitos dos fármacos , Acetatos/química , Acetatos/toxicidade , Aminoácidos/química , Interações Hidrofóbicas e Hidrofílicas , Células HeLa , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacosRESUMO
Resistance to cisplatin presents a major obstacle in managing advanced-stage cervical cancer. Cuproptosis, a newly identified form of cell death induced by copper ions, has potential in overcoming chemoresistance. But the application of cuproptosis in cervical cancer resistant to cisplatin has not yet been reported. In this study, treatment with Elsm-Cu in cervical cancer cells induced cuproptosis, affecting cell proliferation and apoptosis was found. Moreover, cuproptosis in cervical cancer cells was significantly induced by baicalein. The combination of baicalein and cisplatin exhibited a synergistic effect on cervical cancer cells by promoting apoptosis and inhibiting cell viability via the induction of cuproptosis. Animal experiments demonstrated that this combination significantly suppressed tumor growth. Upon treating cells with SC79 (Akt agonist), a significant inhibition of the expression of cuproptosis-related proteins SDHB and FDX1 were observed, indicating that baicalein induced cuproptosis through the Akt pathway. These results indicated that baicalein, mediated through the Akt pathway to induce cuproptosis, had the potential to improve the sensitivity of cervical cancer cells to cisplatin.
Assuntos
Apoptose , Cisplatino , Sinergismo Farmacológico , Flavanonas , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Neoplasias do Colo do Útero , Cisplatino/farmacologia , Flavanonas/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/metabolismo , Humanos , Feminino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos Nus , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cobre/farmacologia , Camundongos Endogâmicos BALB C , Antineoplásicos/farmacologia , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Sobrevivência Celular/efeitos dos fármacos , Células HeLaRESUMO
Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.
Assuntos
DNA Helicases , Replicação do DNA , RecQ Helicases , Helicase da Síndrome de Werner , Humanos , Cromatina/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , Endonucleases Flap/metabolismo , Endonucleases Flap/genética , Células HEK293 , Células HeLa , RecQ Helicases/metabolismo , RecQ Helicases/genética , Telômero/metabolismo , Telômero/genética , Homeostase do Telômero/efeitos dos fármacos , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genéticaRESUMO
Spontaneous forward-reverse mutations were reported by us earlier in clinical samples from various types of cancers and in HeLa cells under normal culture conditions. To investigate the effects of chemical stimulations on such mutation cycles, the present study examined single nucleotide variations (SNVs) and copy number variations (CNVs) in HeLa and A549 cells exposed to wogonin-containing or acidic medium. In wogonin, both cell lines showed a mutation cycle during days 16-18. In acidic medium, both cell lines displayed multiple mutation cycles of different magnitudes. Genomic feature colocalization analysis suggests that CNVs tend to occur in expanded and unstable regions, and near promoters, histones, and non-coding transcription sites. Moreover, phenotypic variations in cell morphology occurred during the forward-reverse mutation cycles under both types of chemical treatments. In conclusion, chemical stresses imposed by wogonin or acidity promoted cyclic forward-reverse mutations in both HeLa and A549 cells to different extents.
Assuntos
Variações do Número de Cópias de DNA , Flavanonas , Mutação , Humanos , Células HeLa , Flavanonas/farmacologia , Variações do Número de Cópias de DNA/genética , Mutação/genética , Células A549 , Polimorfismo de Nucleotídeo Único/genética , Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Linhagem Celular TumoralRESUMO
Enteroviruses such as coxsackievirus B3 are identified as a common cause of viral myocarditis, but the potential mechanism of its replication and pathogenesis are largely unknown. The genomes of a variety of viruses contain N6-methyladenosine (m6A), which plays important roles in virus replication. Here, by using the online bioinformatics tools SRAMP and indirect immunofluorescence assay (IFA), we predict that the CVB3 genome contains m6A sites and found that CVB3 infection could alter the expression and cellular localization of m6A-related proteins. Moreover, we found that 3-deazaadenosine (3-DAA), an m6A modification inhibitor, significantly decreased CVB3 replication. We also observed that the m6A methyltransferases methyltransferase-like protein 3 (METTL3) and METTL14 play positive roles in CVB3 replication, whereas m6A demethylases fat mass and obesity-associated protein (FTO) or AlkB homolog 5 (ALKBH5) have opposite effects. Knockdown of the m6A binding proteins YTH domain family protein 1 (YTHDF1), YTHDF2 and YTHDF3 strikingly decreased CVB3 replication. Finally, the m6A site mutation in the CVB3 genome decreased the replication of CVB3 compared with that in the CVB3 wild-type (WT) strain. Taken together, our results demonstrated that CVB3 could exploit m6A modification to promote viral replication, which provides new insights into the mechanism of the interaction between CVB3 and the host.