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Rationale: Microglia with a repertoire of functions are critical in pathological regulation of angiogenesis in the retina. However, retinal microglia with beneficial contributions and corresponding mechanisms during pathological neovascularization are poorly understood. Methods: We conducted a bioinformatic comparison of public single-cell RNA transcriptome data between retinal microglia from mice with oxygen-induced retinopathy (OIR) and an antiangiogenic microglial population named MG3 from the spine. The essential beneficial factor thrombospondin-1 (Tsp-1) from microglia was discovered and then validated in the retina of mice with OIR at P17. Exosomes were isolated from Tsp-1-knockout microglia (KO-Exos) and Tsp-1+ microglia (NT-Exos). Human umbilical vein endothelial cells (HUVEC) morphology studies, exosomes' miRNA sequencing, luciferase reporter assay, miRNA loss of function studies, and intravitreal injection were used to explore the mechanism of Tsp-1 and microglia-associated retinal angiogenesis. Results: The bioinformatic analyses of single-cell RNA-seq data indicated that a subtype of retinal microglia named RMG1 shares features with MG3 in regulating wound healing, cell adhesion, and angiogenesis. Remarkably, Tsp-1, an extracellular matrix protein with robust inhibition of angiogenesis, was especially expressed in both MG3 and RMG1. However, the scarcity of Tsp-1+ cells was observed in RMG1, which could be an obstacle to attenuating retinal neovascularization. Subsequently, we found that exosomes derived from Tsp-1+ microglia inhibit the migration and tube formation of HUVEC. Moreover, the knockout of Tsp-1 led to the enrichment of miR-27a-5p in exosomes from microglia and promoted angiogenesis compared to that of NT-Exos in vitro. Furthermore, in the luciferase reporter assay on the transcriptional activity of the promoter, we demonstrated that Tsp-1 negatively regulates miR-27a-5p expression. In addition, SMAD family member 3 (Smad3), a receptor-activated Smad protein that is conducive to vascular homeostasis, was defined as a functional target gene of miR-27a-5p. These data were consistently confirmed in vivo in the retina of mice with OIR. Conclusion: Collectively, the Tsp-1/miR-27a-5p/Smad3 axis is involved in microglia-related and exosome-mediated antiangiogenic regulation of the retina. Therefore, this study reveals a novel mechanism by which retinal microglia maintain vascular homeostasis, thereby providing a new therapeutic target for pathological neovascularization.
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Exosomas , MicroARNs , Neovascularización Retiniana , Humanos , Ratones , Animales , Neovascularización Retiniana/metabolismo , Microglía/metabolismo , Exosomas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Neovascularización Patológica/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismoRESUMEN
Activation of microglia plays a key role in the development of neovascular retinal diseases. Therefore, it is essential to reveal its pathophysiological and molecular mechanisms to interfere with disease progression. Here a publicly available single-cell RNA sequencing dataset is used to identify that intercellular communications from M1 microglia toward M0 microglia are increased in the retinal angiogenesis model via exosomes. Moreover, the results both in vitro and in vivo demonstrate that M1 microglia-derived exosomes promote the activation and enhance the proangiogenic ability of resting microglia. Based on miRNA sequencing of exosomes combined with gene interference, further results show that activated microglia-derived exosomes promoted microglial activation by transmitting polarized signals to M0 microglia via miR-155-5p. Subsequently, miR-155-5p suppresses Socs1 and activates the NFκB pathway, which ultimately causes the inflammatory cascade and amplifies the proangiogenic effect. In addition, upregulated Irf1 drives the expression of miR-155-5p in activated microglia, thus leading to an increase in the tendency of miR-155-5p to be encapsulated by exosomes. Thus, this study elucidates the critical role of intercellular communication among various types of microglia in the complex retinal microenvironment during angiogenesis, and contributes to the novel, targeted, and potential therapeutic strategies for clinical retinal neovascularization.
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Exosomas , MicroARNs , Exosomas/genética , Exosomas/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Retina , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Factor 1 Regulador del Interferón/metabolismoRESUMEN
Purpose: Chemokine receptor 4 (CXCR4) plays an essential role in the early stage of corneal neovascularization (CNV), but the underlying key molecular mechanism has yet to be addressed. This study aimed to explore the new molecular mechanism of CXCR4 in CNV and the related pathological events. Methods: CXCR4 was assayed by immunofluorescence or Western blotting. The function of the supernatant from hypoxia-treated human corneal epithelial cells (HCE-T) cells was examined by culturing with human umbilical vein endothelial cells. MicroRNA sequencing was used to detect the downstream microRNAs upon CXCR4 knockdown and analyzed by preliminary bioinformatics. The proangiogenic functions and downstream target genes of microRNA were investigated by gene interference and luciferase assay. An alkali-burned murine model was introduced to examine the function and mechanism of miR-1910-5p in vivo. Results: High CXCR4 expression was confirmed in corneal tissues of patients with CNV and hypoxic HCE-T cells. The supernatant from hypoxia-treated HCE-T cells is involved in the CXCR4-mediated angiogenesis of human umbilical vein endothelial cells. Notably, miR-1910-5p was demonstrated to be at a high level in wild-type HCE-T cells and its supernatant, and in CNV patient tears. The proangiogenic functions of miR-1910-5p were demonstrated with the assays of cell migration, tube formation, and aortic ring. Moreover, miR-1910-5p significantly inhibited multimerin-2 expression by targeting its 3' untranslated region and caused significant extracellular junctional defects in human umbilical vein endothelial cells. MiR-1910-5p antagomir could significantly increase multimerin-2 level and decrease vascular leakage, and ultimately inhibit CNV in a murine model. Conclusions: Our results revealed a novel CXCR4-mediated mechanism and proved that targeting the miR-1910-5p/multimerin-2 pathway could be a promising therapeutic target for CNV.
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Neovascularización de la Córnea , MicroARNs , Animales , Humanos , Ratones , Permeabilidad Capilar , Neovascularización de la Córnea/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Hipoxia/metabolismo , MicroARNs/genética , Receptores CXCR4/metabolismoRESUMEN
Soil bacteria participate in self-immobilization processes for survival, persistence, and production of virulence factors in some niches or hosts through their capacities for autoaggregation, cell surface hydrophobicity, biofilm formation, and antibiotic and heavy metal resistance. This study investigated potential virulence, antibiotic and heavy metal resistance, solvent adhesion, and biofilm-forming capabilities of six cellulolytic bacteria isolated from soil samples: Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Strains were subjected to phenotypic methods, including heavy metal and antibiotic susceptibility and virulence factors (protease, lipase, capsule production, autoaggregation, hydrophobicity, and biofilm formation). The effect of ciprofloxacin was also investigated on bacterial susceptibility over time, cell membrane, and biofilm formation. Strains MKAL2, MKAL5, and MKAL6 exhibited protease and lipase activities, while only MKAL6 produced capsules. All strains were capable of aggregating, forming biofilm, and adhering to solvents. Strains tolerated high amounts of chromium, lead, zinc, nickel, and manganese and were resistant to lincomycin. Ciprofloxacin exhibited bactericidal activity against these strains. Although the phenotypic evaluation of virulence factors of bacteria can indicate their pathogenic nature, an in-depth genetic study of virulence, antibiotic and heavy metal resistance genes is required.
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Antibacterianos , Metales Pesados , Virulencia , Antibacterianos/farmacología , Suelo , Metales Pesados/toxicidad , Metales Pesados/análisis , Metales Pesados/metabolismo , Bacterias/genética , Biopelículas , Factores de Virulencia/genética , Factores de Virulencia/farmacología , Ciprofloxacina/farmacología , Péptido Hidrolasas/farmacología , Lipasa/farmacologíaRESUMEN
Purpose: To explore the anti-inflammatory and neuroprotective effects of lithium chloride (LiCl) in LPS-induced retinal injury. Methods: In vitro, primary retinal microglia were pretreated with LiCl and stimulated with lipopolysaccharide (LPS). Pro-inflammatory cytokine production, microglial morphological changes, and inflammation-associated signaling pathways were measured by real-time PCR (RT-PCR), western blotting, and immunofluorescence. Primary retinal neurons were cultured with microglial-derived conditioned medium in the absence or presence of LiCl. Neurotoxicity was evaluated by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and γ-H2AX detection. In vivo, an endotoxin-induced uveitis mice model was established, and each animal was given intraperitoneal injection of LiCl or vehicle. The retinal inflammatory response was measured by hematoxylin and eosin and fluorescent staining, RT-PCR, western blotting, and TUNEL assay. Retinal thickness and function were evaluated by spectral-domain optical coherence tomography and electroretinography. Results: In vitro, LiCl exerted no obvious toxic effects on microglia and significantly decreased proinflammatory factor (inducible nitric oxide synthase, tumor necrosis factor α, interleukin 6) production, inhibited microglial activation in morphology, and suppressed nuclear factor kappa B (NF-κB), Akt, and phosphatidylinositol 3-kinase (PI3K) phosphorylation. Moreover, LiCl promoted retinal neuron survival and reduced cell apoptosis and the expression of γ-H2AX. In vivo, LiCl reduced inflammatory infiltrating cells in the vitreous cavity and decreased proinflammatory cytokine expression in retinas. LiCl suppressed LPS-induced microglial activation, proliferation, and migration. Additionally, LiCl reduced LPS-induced apoptosis of ganglion cells and retinal edema and rescued retinal functional damage. Conclusions: This study demonstrates that LiCl exerts anti-inflammatory and neuroprotective effects by inhibiting microglial activation via the PI3K/Akt/NF-κB pathway in LPS-induced retinal injury. LiCl provides a novel and promising option to treat retinal inflammatory diseases.
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Fármacos Neuroprotectores , Enfermedades de la Retina , Ratones , Animales , Lipopolisacáridos/toxicidad , FN-kappa B/metabolismo , Microglía/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Fármacos Neuroprotectores/metabolismo , Cloruro de Litio/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Línea Celular , Antiinflamatorios/farmacología , Enfermedades de la Retina/patología , Citocinas/genética , Citocinas/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
Previous studies have indicated that Brca1 (Breast cancer suppressor gene 1) plays an important role in neural development and degenerative diseases. However, the bioactivity and regulatory mechanism of Brca1 expression in retinal neurocytes remain unclear. In the present study, our data indicated that Brca1 maintains the state of neuronal precursor cells. Brca1 silencing induces differentiation in 661W cells. Nestin, a marker of precursor cells, was significantly decreased in parallel with Brca1 silencing in 661W cells, whereas Map2 (Microtubule associated protein 2), a marker of differentiated neurons, was significantly increased. Neurite outgrowth was increased by ~4.0-fold in Brca1-silenced cells. Moreover, DNA affinity purification assays and ChIP assays demonstrated that Gata3 (GATA binding protein 3) regulates Brca1 transcription in 661W cells. Silencing or overexpressing Gata3 could significantly regulate the expression of Brca1 and affect its promoter inducibility. Furthermore, the expression of Gata3 generally occurred in parallel with that of Brca1 in developing mouse retinas. Both Gata3 and Brca1 are expressed in the neonatal mouse retina but are developmentally silenced with age. Exogenous Gata3 significantly inhibited neural activity by decreasing synaptophysin and neurite outgrowth. Thus, this study demonstrated that Brca1 is transcriptionally regulated by Gata3. Brca1/Gata3 silencing is involved in neuronal differentiation and maturation.
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Factor de Transcripción GATA3 , Neuronas Retinianas , Animales , Ratones , Diferenciación Celular/genética , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Proyección Neuronal , Regiones Promotoras Genéticas , Neuronas Retinianas/metabolismoRESUMEN
Purpose: Retinoblastoma (RB) is the most common type of aggressive intraocular malignancy in children. The alteration of immunity during RB progression and invasion has not yet been well defined. This study investigated significantly altered immune-associated genes and cells related to RB invasion. Methods: The differentially expressed immune-related genes (IRGs) in noninvasive RB and invasive RB were identified by analysis of two microarray datasets (GSE97508 and GSE110811). Hub IRGs were further identified by real time PCR. The single-sample gene set enrichment analysis algorithm and Pearson correlation analysis were used to define immune cell infiltration and the relationships between hub IRGs and immune cells. Cell viability and migration were evaluated by CCK-8 and Transwell assays. A xenograft mouse model was used to verify the relationship between Src homology 3 (SH3) domain GRB2-like 2 (SH3GL2) expression and myeloid-derived suppressor cells (MDSCs). Results: Eight upregulated genes and six downregulated IRGs were identified in invasive RB. Seven IRGs were confirmed by real-time PCR. Moreover, the proportions of MDSCs were higher in invasive RB tissues than in noninvasive RB tissues. Furthermore, correlation analysis of altered immune genes and cells suggested that SH3GL2, Langerhans cell protein 1 (LCP1) and transmembrane immune signaling adaptor TYROBP have strong connections with MDSCs. Specifically, decreased SH3GL2 expression promoted the migration of RB cells in vitro, increased the tumor size and weight, and increased the numbers of MDSCs in the tumor and spleen in vivo. Conclusions: This study indicated that SH3GL2 and MDSCs play a critical role in RB progression and invasion and provide candidate targets for the treatment of RB.
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Neoplasias de la Retina , Retinoblastoma , Animales , Humanos , Ratones , Línea Celular Tumoral , Proliferación Celular/genética , Biología Computacional , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Retina/patología , Retinoblastoma/patología , Células Tumorales CultivadasRESUMEN
Maintaining DNA stability in induced pluripotent stem cells (iPSCs) and iPSCs-derived neurons is a challenge in their clinical application. In the present study, we compared DNA stability between primary retinal neurons and differentiated neurons. We found that the basal level of γ-H2AX phosphorylation, a specific marker of DNA breaks, was notably higher (~26-folds) in human iPSCs compared to iPSCs-derived neurons. However, iPSCs-derived neurons are more sensitive to UV treatment compared to primary rat retinal neurons (postnatal Day 1). UV treatment induced a significantly decreasing in the cell viability of iPSCs-derived neurons by ~76.1%, whereas ~20.8% in primary retinal neurons. After analyzing the expression levels of genes involved in DNA stability, such as Brca1, Ligase IV, Ku80, and Mre11, we found that Ku80 and its heterodimeric partner, Ku70 were positive in iPSCs-derived neurons. However, both Ku80 and Ku70 are not expressed in primary retinal neurons and cerebellar neurons. Similarly, both Ku80 and Ku70 are also expressed in 3D retinal organoids from human embryonic stem cells (ESCs), except for a few Map2-negative cells and the hyaloid vessels of mice E12.5 retinas. Hence, Ku80, and Ku70 are specifically expressed in stem cell-derived neurons. Moreover, using the Ku80 inhibitor Compound L, our data showed that Ku80 promotes the DNA stability and cell viability of iPSCs-derived neurons. Thus, our results demonstrated that iPSCs-, ESCs-derived neurons have specific characteristics of DNA stability. This study provides new insights into the neural differentiation of stem cells but might also warrant the future clinical application of stem cells in neurodegenerative diseases.
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Células Madre Pluripotentes Inducidas , Neuronas Retinianas , Animales , Diferenciación Celular , ADN , Células Madre Embrionarias , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , RatasRESUMEN
The characterization of bacteria with hydrolytic potential significantly contributes to the industries. Six cellulose-degrading bacteria were isolated from mixture soil samples collected at Kingfisher Lake and the University of Manitoba campus by Congo red method using carboxymethyl cellulose agar medium and identified as Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Their cellulase production was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, substrate concentration, nitrogen, and carbon sources using the dinitrosalicylic acid and response surface methods. Except for Paenarthrobacter sp. MKAL1, all strains are motile. Only Bacillus sp. MKAL6 was non-salt-tolerant and showed gelatinase activity. Sucrose enhanced higher cellulase activity of 78.87 ± 4.71 to 190.30 ± 6.42 U/mL in these strains at their optimum pH (5-6) and temperature (35-40 °C). The molecular weights of these cellulases were about 25 kDa. These bacterial strains could be promising biocatalysts for converting cellulose into glucose for industrial purposes.
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Bacillus , Celulasa , Celulasas , Celulasa/química , Celulosa , Suelo , Carboximetilcelulosa de Sodio , Agar , Rojo Congo , Nitrógeno , Temperatura , Carbono , Glucosa , Sacarosa , Gelatinasas , Concentración de Iones de HidrógenoRESUMEN
Thrombospondin-1 (Tsp-1), a matricellular protein, could protect retinal neurons from endogenous or exogenous insults; however, its underlying mechanism remains unclear. Thus, this study aimed to investigate Tsp-1-mediated neuron-protection effect in retinal cells. Our data showed that Tsp-1 downregulation would aggravate UV irradiation-induced DNA damage in 661 W cells and cone photoreceptor cells. The increasing levels of poly (ADP ribose) polymer (PAR) and γ-H2AX in Tsp-1-silenced 661 W cells indicate severe DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). By utilizing an error-prone substrate, Tsp-1 silencing significantly increased deleted DNA end joining in 661 W cells with spontaneous DNA damage (SDD). Moreover, Tsp-1 is indirectly involved in DNA stability in 661 W cells as UV treatment caused a significant Tsp-1 decreasing in cytoplasm, but no obvious Tsp-1 alteration in cell nuclear of 661 W cells. Furthermore, our data indicate that Tgf-ß1 activation domain in Tsp-1 plays a critical role in DNA stability in 661 W cells through expressing mutated exogenous Tsp-1 and Tgf-ß inhibitor, LSKL. Therefore, this study provides new insights into the mechanism of the neuroprotective action positively mediated by Tsp-1, which might be a therapeutic target for the treatment of retinal pathology.
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Células Fotorreceptoras Retinianas Conos , Factor de Crecimiento Transformador beta1 , Regulación hacia Abajo , Células Fotorreceptoras Retinianas Conos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
GATA binding protein 3 (Gata3), a zinc-finger transcription factor, plays an important role in neural development. However, its expression and bioactivity in the retina remain unclear. In the present study, our data indicated that Gata3 maintains the precursor state of 661W cells, and Gata3 silencing induces cell differentiation. The expression of Nestin, a marker of precursor cells, was significantly decreased in parallel, whereas the expression of Map2, a marker of differentiated neurons, was significantly increased following the decrease in Gata3. Neurite outgrowth was increased by 2.78-fold in Gata3-silenced cells. Moreover, Gata3 expression generally paralleled that of Nestin in developing mouse retinas. Both Gata3 and Nestin were expressed in the retina at postnatal day 1 and silenced in the adult mouse retina. Exogenous Gata3 significantly inhibited the neural activity of primary retinal neurocytes (postnatal day 1) by decreasing synaptophysin levels, neurite outgrowth, and cell viability. Furthermore, in vivo, exogenous Gata3 significantly induced apoptosis and the contraction of retinal outlay filaments and decreased the a- and b-waves in adult mouse intravitreal injected with AAV-Re-Gata3-T2A-GFP. Thus, Gata3 silencing promotes neuronal differentiation and neurite outgrowth. Its abnormal expression impedes neural activity in adult retinal neurocytes. This study provides new insights into Gata3 bioactivity in retinal neurocytes.
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Neuronas , Retina , Animales , Diferenciación Celular/genética , Supervivencia Celular , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Ratones , Nestina/genética , Nestina/metabolismo , Proyección Neuronal/fisiología , Retina/metabolismoRESUMEN
Krüppel-like factor 2 (KLF2) belongs to the KLF family of zinc-finger transcription factors and mediates the occurrence and progression of various cancers. However, little is known about its expression pattern and biological role in retinoblastoma (RB). In the present study, we showed that KLF2 was markedly downregulated in human RB tissue compared with retina. KLF2 overexpression significantly inhibited RB cell proliferation and decreased proliferating cell nuclear antigen (PCNA) expression. Subsequently, we confirmed that KLF2 arrested cells at the G1-S phase transition, accompanied by the upregulation of p21 and downregulation of CyclinD1, as well as the activation of mitochondria-mediated apoptosis in RB cells. In addition, KLF2 overexpression contributed to suppressing RB cell migration and invasion by downregulating matrix metallopeptidase 9 (MMP9). On the contrary, KLF2 downregulation promoted RB cells proliferation, migration and invasion. Notably, the KLF2 expression pattern was opposite to that of C-X-C chemokine receptor 4 (CXCR4) in the two RB cell lines, KLF2 overexpression significantly decreased CXCR4 expression, silencing KLF2 had the opposite effect. Furthermore, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays confirmed that KLF2 directly bound to the CXCR4 promoter and negatively regulated its expression in RB cells. Collectively, our results suggested that KLF2 function as a tumor suppressor in RB and may represent a potential therapeutic target for RB.
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Factores de Transcripción de Tipo Kruppel/fisiología , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Proteínas Supresoras de Tumor/fisiología , Apoptosis/fisiología , Western Blotting , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Ciclina D1/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Etiquetado Corte-Fin in Situ , Plásmidos , Antígeno Nuclear de Célula en Proliferación/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias de la Retina/patología , Retinoblastoma/patología , Transfección , Quinasas p21 Activadas/genéticaRESUMEN
The emergence of multidrug-resistant (MDR) pathogens and the gradual depletion of available antibiotics have exacerbated the need for novel antimicrobial agents with minimal toxicity. Herein, we report functionally substituted pyridine carbohydrazide with remarkable antimicrobial effect on multi-drug resistant strains. In the series, compound 6 had potent activity against four MDR strains of Candida spp., with minimum inhibitory concentration (MIC) values being in the range of 16-24 µg/mL and percentage inhibition up to 92.57%, which was exceptional when compared to broad-spectrum antifungal drug fluconazole (MIC = 20 µg/mL, 81.88% inhibition). Substitution of the octyl chain in 6 with a shorter butyl chain resulted in a significant anti-bacterial effect of 4 against Pseudomonas aeruginosa (ATCC 27853), the MIC value being 2-fold superior to the standard combination of ampicillin/cloxacillin. Time-kill kinetics assays were used to discern the efficacy and pharmacodynamics of the potent compounds. Further, hemolysis tests confirmed that both compounds had better safety profiles than the standard drugs. Besides, molecular docking simulations were used to further explore their mode of interaction with target proteins. Overall results suggest that these compounds have the potential to become promising antimicrobial drugs against MDR strains.
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Antiinfecciosos , Antifúngicos , Antifúngicos/farmacología , Simulación del Acoplamiento Molecular , Farmacorresistencia Bacteriana Múltiple , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antiinfecciosos/farmacología , Piridinas/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Purpose: Retinoblastoma (RB) is the most common primary malignant intraocular cancer. The etiology of RB is complex, and the mechanisms driving its progression remain unclear. Here, we used a series of bioinformatics approaches and experimental methods to investigate the potential regulatory mechanism involved in RB progression. Methods: The common differentially expressed genes were obtained from the public dataset GSE97508. Protein-protein interaction (PPI) network, correlation, and functional enrichment analyses were carried out. The candidate genes were verified in different RB cell lines, and ARPE19 cells served as control. miRNA-mRNA interaction analysis was performed and confirmed by real-time PCR. The CCK-8 assay was conducted to detect cell viability, and the transwell assay was utilized for evaluating the abilities of cell migration and invasion. Results: Overall, a total of 258 common differentially expressed genes associated with RB progression were screened out. The PPI network analysis further identified eight downregulated genes mainly enriched in the protein ubiquitination pathway. Moreover, we confirmed UBE2E1, SKP1, FBXO9, FBXO15, and RNF14 from among eight genes through experimental validation in vitro. Furthermore, miRNA-mRNA interaction and real-time PCR analysis of five hub genes revealed that ubiquitination-related miR-548k was involved in RB progression. Loss- and gain-of-function experiments demonstrated that miR-548k and its targets were essential for cell viability, migration, and invasion in the RB cells. Conclusions: Our data indicate that the dysregulation of protein ubiquitination may play an important role in RB progression, and ubiquitination-related miR-548k may be a promising therapeutic target for RB.
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Proteínas del Ojo/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , ARN Mensajero/genética , Neoplasias de la Retina/genética , Retinoblastoma/genética , Ubiquitinación , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Biología Computacional/métodos , Proteínas del Ojo/metabolismo , Humanos , ARN Neoplásico/genética , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/metabolismo , Retinoblastoma/patologíaRESUMEN
Exosomes derived from tumor cells play a key role in tumor development. In the present study, we identified the bioactivity of exosomes released from WERI-Rb1 retinoblastoma cells in tumor angiogenesis, as well as the underlying mechanism, through biochemical methods and animal experiments. Our in vitro data showed that exosomes could be engulfed by human vesicle endothelial cells (HUVECs), significantly promote cell viability and induce an inflammatory response in HUVECs by increasing the expression of a series of related genes, such as IL-1, IL-6, IL-8, MCP-1, VCAM1, and ICAM1. Significant increases in migration and tube formation were also observed in the HUVECs incubated with exosomes. Moreover, experiments with a nude mouse xenotransplantation model showed that exosomes injected near tumors could be strongly absorbed by tumor cells. The numbers of endothelial cells and blood vessels were significantly increased in tumor tissues treated with exosomes compared to control tissues. Furthermore, to reveal the mechanism underlying exosome-mediated angiogenesis in retinoblastoma, we analyzed the levels of 12 microRNAs in the exosomes. Specifically, our data showed that miR-92a-3p was enriched in RB exosomes. Accordingly, miR-92a-3p was increased in the HUVECs incubated with these exosomes. After treatment with a miR-92a-3p inhibitor, the promoting effect of exosomes on the migration and tube formation of HUVECs was significantly abrogated. The expression of the angiogenesis-related genes mentioned above was markedly decreased in HUVECs. Similarly, treatment with a microRNA mimic also demonstrated that miR-92a-3p was involved in the angiogenesis of HUVECs. More importantly, bioinformatics analysis predicted that Krüppel-like factor 2 (KLF2), a member of the KLF family of zinc-finger transcription factors, might be an active target of miR-92a-3p. Notably, this prediction was confirmed both in vitro and in vivo. Thus, our work suggests that exosomal miR-92a-3p is involved in tumor angiogenesis and might be a promising therapeutic candidate for retinoblastoma.
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Exosomas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , MicroARNs/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Comunicación Paracrina , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Exosomas/genética , Exosomas/patología , Exosomas/trasplante , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones Desnudos , MicroARNs/genética , Neoplasias de la Retina/genética , Neoplasias de la Retina/patología , Retinoblastoma/genética , Retinoblastoma/patologíaRESUMEN
In search of anti-inflammatory compounds, novel scaffolds containing isonicotinoyl motif were synthesized via an efficient strategy. The compounds were screened for their in vitro anti-inflammatory activity. Remarkably high activities were observed for isonicotinates 5-6 and 8a-8b. The compound 5 exhibits an exceptional IC50 value (1.42 ± 0.1 µg/mL) with 95.9% inhibition at 25 µg/mL, which is eight folds better than the standard drug ibuprofen (11.2 ± 1.9 µg/mL). To gain an insight into the mode of action of anti-inflammatory compounds, molecular docking studies were also performed. Decisively, further development and fine tuning of these isonicotinates based scaffolds for the treatment of various aberrations is still a wide-open field of research.
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Antiinflamatorios no Esteroideos/síntesis química , Inflamación/tratamiento farmacológico , Ácidos Isonicotínicos/síntesis química , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa 2/síntesis química , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/farmacología , Humanos , Ibuprofeno/química , Ácidos Isonicotínicos/química , Ácidos Isonicotínicos/farmacología , Simulación del Acoplamiento Molecular , Especies Reactivas de Oxígeno/química , Relación Estructura-ActividadRESUMEN
Nucleotide-binding oligomerization domain 2 (NOD2) is cytosolic surveillance receptor of the innate immune system capable of recognizing the bacterial and viral infections. Muramyl dipeptide (MDP) is the minimal immunoreactive unit of murein. NOD2 perceives MDP as pathogen-associated molecular pattern, thereby triggering an immune response with undesirable side-effects. Beneficial properties of MDP, such as pro-inflammatory characteristics for the rational design of new vaccine adjuvants, can be harnessed by strategically re-designing the molecule. In this work, a new class of amphiphilic desmuramylpeptides (DMPs) were synthesized by replacing the carbohydrate moiety (muramic acid) of the parent molecule with hydrophilic arenes. A lipophilic chain was also introduced at the C-terminus of dipeptide moiety (alanine-isoglutamine), while conserving its L-D configuration. These novel DMPs were found to set off the release of higher levels of tumour necrosis factor alpha (TNF-α) than Murabutide, which is a well-known NOD2 agonist. Molecular docking studies indicate that all these DMPs bind well to NOD2 receptor with similar dock scores (binding energy) through a number of hydrogen bonding and hydrophobic/π interactions with several crucial residues of the receptor. More studies are needed to further assess their immunomodulatory therapeutic potential, as well as the possible involvement of NOD2 activation.
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Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Péptidos/química , Péptidos/farmacología , Adyuvantes Inmunológicos/síntesis química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Proteína Adaptadora de Señalización NOD2/metabolismo , Péptidos/síntesis química , Tensoactivos/síntesis química , Tensoactivos/química , Tensoactivos/farmacología , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Annonaceous acetogenins (ACGs) are secondary metabolites produced by the Annonaceae family and display potent anticancer activity against various cancer cell lines. Squamocin and bullatacin are two examples of ACGs that show promising antitumor activity; however, preclinical data are not sufficient partly due to their being highly lipophilic and poorly soluble in water. These compounds also display high toxicity to normal cells. Due to these disadvantageous properties, the therapeutic potential of squamocin and bullatacin as antitumor agents has not been fully evaluated. METHODS: In order to enhance their water solubility and potentially improve their cancer targeting, squamocin and bullatacin were conjugated to a glucose or galactose to yield glycosylated derivatives by direct glycosylation or the Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction (the click reaction). The synthesized compounds were evaluated for their anticancer property against HeLa, A549 and HepG2 cancer cell lines using MTT assay. RESULTS: Nine glycosyl derivatives were synthesized and structurally characterized. Most of them show comparable in vitro cytotoxicity against HeLa, A549 and HepG2 cancer cell lines as their parent compounds squamocin and bullatacin. It appears that the type of sugar residue (glucose or galactose), the position at which the sugar residue is attached, and whether or not a linking spacer is present do not affect the potency of these derivatives much. The solubility of galactosylated squamocin 13 in phosphate buffer saline (PBS, pH = 7) is greatly improved (1.37 mg/mL) in comparison to squamocin (not detected in PBS). CONCLUSION: The conjugation of a glucose or galactose to squamocin and bullatacin yields glycosyl derivatives with similar level of anticancer activity in tested cell lines. Further studies are needed to demonstrate whether or not these compounds show reduced toxicity to normal cells and their therapeutic potential as antitumor agents.
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Acetogeninas/farmacología , Annonaceae/química , Antineoplásicos Fitogénicos/farmacología , Glicoconjugados/farmacología , Acetogeninas/química , Acetogeninas/aislamiento & purificación , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Glicoconjugados/síntesis química , Glicoconjugados/química , Humanos , Estructura Molecular , Solubilidad , Células Tumorales CultivadasRESUMEN
Two new compounds (9 and 10) having a camptothecin (CPT) analog conjugated to the 4ß-azido-4-deoxypodophyllotixin analog by untilizing the copper-catalyzed azide-alkyne cycloadditon (CuAAC) reaction, and were evaluated for their cytotoxicity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480) using the MTT (3-(4,5-dimethyl-thiahiazo-2-yl)-2,5-diphenyltetrazolium bromide) assay. Two novel conjugates shown weak cytotoxicity, compound 10 showed highly potent against HL-60 cell line tested, with IC50 value 17.69 ± 0.19 µM. This compound suggested its potential as anticancer agents for further development. [Formula: see text].
Asunto(s)
Antineoplásicos/química , Camptotecina/análogos & derivados , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Camptotecina/síntesis química , Camptotecina/farmacología , Línea Celular Tumoral , Reacción de Cicloadición , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Podofilotoxina/química , Relación Estructura-ActividadRESUMEN
BACKGROUND: Widespread concern of the side effects and the broad-spectrum anticancer property of podophyllotoxin as an antitumor agent highlight the need for the development of new podophyllotoxin derivatives. Although some per-butyrylated glucosides of podophyllotoxin and 4ß-triazolyl-podophyllotoxin glycosides show good anticancer activity, the per-acetylated/free of podophyllotoxin glucosides and their per-acetylated are not well studied. METHODS: A few glucoside derivatives of PPT were synthesized and evaluated for their in vitro cytotoxic activities against five human cancer cell lines, HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer), as well as the normal human pulmonary epithelial cell line (BEAS-2B). In addition, we investigated the structure-activity relationship and the physicochemical property-anticancer activity relationship of these compounds. RESULTS: Compound 6b shows the highest cytotoxic potency against all five cancer cell lines tested, with IC50 values ranging from 3.27±0.21 to 11.37±0.52 µM. We have also found that 6b displays higher selectivity than the etoposide except in the case of HL-60 cell line. The active compounds possess similar physicochemical properties: MSA > 900, %PSA < 20, ClogP > 2, MW > 700 Da, and RB > 10. CONCLUSION: We synthesized several glucoside derivatives of PPT and tested their cytotoxicity. Among them, compound 6b showed the highest cytotoxicity. Further studies including selectivity of active compounds have shown that the selectivity indexes of 6b are much greater than the etoposide except in the case of HL-60 cell line. The active compounds possessed similar physicochemical properties. This study indicates that active glucoside analogs of podophyllotoxin have potential as lead compounds for developing novel anticancer agents.