RESUMEN
Blood-brain barrier (BBB) models are important tools for studying CNS drug delivery, brain development, and brain disease. In vitro BBB models have been obtained from animals and immortalized cell lines; however, brain microvascular endothelial cells (BMECs) derived from them have several limitations. Furthermore, obtaining mature brain microvascular endothelial-like cells (BME-like cells) from human pluripotent stem cells (hPSCs) with desirable properties for establishing BBB models has been challenging. Here, we developed an efficient method for differentiating hPSCs into BMECs that are amenable to the development and application of human BBB models. The established conditions provided an environment similar to that occurring during BBB differentiation in the presence of the co-differentiating neural cell population by the modulation of TGF-ß and SHH signaling. The developed BME-like cells showed well-organized tight junctions, appropriate expression of nutrient transporters, and polarized efflux transporter activity. In addition, BME-like cells responded to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance. Moreover, the BME-like cells exhibited an immune quiescent property of BBB endothelial cells by decreasing the expression of adhesion molecules. Therefore, our novel cellular platform could be useful for drug screening and the development of brain-permeable pharmaceuticals.
RESUMEN
Particulate matter ≤ 2.5 µm (PM2.5) poses health risks related to various diseases and infections. However, the interactions between PM2.5 and cells such as uptake and cell responses have not been fully investigated despite advances in bioimaging techniques, because the heterogeneous morphology and composition of PM2.5 make it challenging to employ labeling techniques, such as fluorescence. In this work, we visualized the interaction between PM2.5 and cells using optical diffraction tomography (ODT), which provides quantitative phase images by refractive index distribution. Through ODT analysis, the interactions of PM2.5 with macrophages and epithelial cells, such as intracellular dynamics, uptake, and cellular behavior, were successfully visualized without labeling techniques. ODT analysis clearly shows the behavior of phagocytic macrophages and nonphagocytic epithelial cells for PM2.5. Moreover, ODT analysis could quantitatively compare the accumulation of PM2.5 inside the cells. PM2.5 uptake by macrophages increased substantially over time, but uptake by epithelial cells increased only marginally. Our findings indicate that ODT analysis is a promising alternative approach to visually and quantitatively understanding the interaction of PM2.5 with cells. Therefore, we expect ODT analysis to be employed to investigate the interactions of materials and cells that are difficult to label.
Asunto(s)
Material Particulado , Tomografía Óptica , Material Particulado/toxicidad , Imagenología Tridimensional/métodos , Tomografía Óptica/métodos , Células Epiteliales , MacrófagosRESUMEN
Particulate matter (PM) contributes to human diseases, particularly lung disease; however, the molecular mechanism of its action is yet to be determined. Herein, we found that prolonged PM exposure induced the cellular senescence of normal lung fibroblasts via a DNA damage-mediated response. This PM-induced senescence (PM-IS) was only observed in lung fibroblasts but not in A549 lung adenocarcinoma cells. Mechanistic analysis revealed that reactive oxygen species (ROS) activate the DNA damage response signaling axis, increasing p53 phosphorylation, ultimately leading to cellular senescence via an increase in p21 expression without affecting the p16-pRB pathway. A549 cells, instead, were resistant to PM-IS due to the PM-induced ROS production suppression. Water-soluble antioxidants, such as vitamin C and N-Acetyl Cysteine, were found to alleviate PM-IS by suppressing ROS production, implying that antioxidants are a promising therapeutic intervention for PM-mediated lung pathogenesis.
RESUMEN
Particulate matter is an air pollutant composed of various components, and has adverse effects on the human body. Particulate matter is known to induce cell death by generating an imbalance in the antioxidant system; however, the underlying mechanism has not been elucidated. In the present study, we demonstrated the cytotoxic effects of the size and composition of particulate matter on small intestine cells. We found that particulate matter 2.5 (PM2.5) with extraction ion (EI) components (PM2.5 EI), is more cytotoxic than PM containing only polycyclic aromatic hydrocarbons (PAHs). Additionally, PM-induced cell death is characteristic of ferroptosis, and includes iron accumulation, lipid peroxidation, and reactive oxygen species (ROS) generation. Furthermore, ferroptosis inhibitor as liproxstatin-1 and iron-chelator as deferiprone attenuated cell mortality, lipid peroxidation, iron accumulation, and ROS production after PM2.5 EI treatment in human small intestinal cells. These results suggest that PM2.5 EI may increase ferroptotic-cell death by iron accumulation and ROS generation, and offer a potential therapeutic clue for inflammatory bowel diseases in human small intestinal cells. [BMB Reports 2023; 56(2): 96-101].
Asunto(s)
Antineoplásicos , Ferroptosis , Humanos , Material Particulado , Hierro , Antioxidantes , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: This study evaluated whether an addition of bevacizumab to erlotinib improves clinical outcomes in patients with advanced EGFR-mutated non-small cell lung cancer (NSCLC). METHODS: This is an open-label, multicenter, randomized Phase 2 study in South Korea. Chemonaïve patients with Stage IIIB/IV NSCLC with EGFR 19 deletion or L858R mutation were eligible. Asymptomatic brain metastasis (BM) was enrolled without local treatment. Patients received either erlotinib plus bevacizumab or erlotinib. RESULTS: Between December 2016 and March 2019, 127 patients were randomly assigned to receive erlotinib plus bevacizumab (n = 64) or erlotinib (n = 63). Fifty-nine (46.5%) patients had baseline BM. Fewer patients in the erlotinib plus bevacizumab arm received radiotherapy for BM than in the erlotinib arm (10.3% vs. 40.0%). A trend toward longer progression-free survival (PFS) was observed in the erlotinib plus bevacizumab arm compared with the erlotinib alone arm; however, it was not statistically significant (median PFS, 17.5 months vs. 12.4 months; hazard ratio [HR], 0.74; 95% CI, 0.51-1.08; p = .119). The unplanned subgroup analysis showed a longer PFS with erlotinib plus bevacizumab in patients with BM (median PFS, 18.6 months vs. 10.3 months; HR, 0.54; 95% CI, 0.31-0.95; p = .032). Grade 3 or worse adverse events occurred in 56.6% of the erlotinib plus bevacizumab arm and 20.6% of the erlotinib arm. CONCLUSIONS: Although it was not statistically significant, a trend to improvement in PFS was observed in patients with erlotinib plus bevacizumab compared to erlotinib alone. PLAIN LANGUAGE SUMMARY: A randomized Phase 2 study compared erlotinib with or without bevacizumab in previously untreated patients with advanced non-small cell lung cancer with EGFR mutation. The erlotinib plus bevacizumab failed to improve median progression-free survival compared with the erlotinib alone. However, the progression-free survival benefit from erlotinib plus bevacizumab was found in patients with brain metastasis with no severe hemorrhagic adverse effects.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Clorhidrato de Erlotinib , Bevacizumab/efectos adversos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Receptores ErbB/genética , Mutación , Inhibidores de Proteínas Quinasas/efectos adversos , Supervivencia sin EnfermedadRESUMEN
AIMS: The nuclear factor-κB (NF-κB) signalling pathway plays a critical role in the pathogenesis of multiple vascular diseases. However, in endothelial cells (ECs), the molecular mechanisms responsible for the negative regulation of the NF-κB pathway are poorly understood. In this study, we investigated a novel role for protein tyrosine phosphatase type IVA1 (PTP4A1) in NF-κB signalling in ECs. METHODS AND RESULTS: In human tissues, human umbilical artery ECs, and mouse models for loss of function and gain of function of PTP4A1, we conducted histological analysis, immunostaining, laser-captured microdissection assay, lentiviral infection, small interfering RNA transfection, quantitative real-time PCR and reverse transcription-PCR, as well as luciferase reporter gene and chromatin immunoprecipitation assays. Short hairpin RNA-mediated knockdown of PTP4A1 and overexpression of PTP4A1 in ECs indicated that PTP4A1 is critical for inhibiting the expression of cell adhesion molecules (CAMs). PTP4A1 increased the transcriptional activity of upstream stimulatory factor 1 (USF1) by dephosphorylating its S309 residue and subsequently inducing the transcription of tumour necrosis factor-alpha-induced protein 3 (TNFAIP3/A20) and the inhibition of NF-κB activity. Studies on Ptp4a1 knockout or transgenic mice demonstrated that PTP4A1 potently regulates the interleukin 1ß-induced expression of CAMs in vivo. In addition, we verified that PTP4A1 deficiency in apolipoprotein E knockout mice exacerbated high-fat high-cholesterol diet-induced atherogenesis with upregulated expression of CAMs. CONCLUSION: Our data indicate that PTP4A1 is a novel negative regulator of vascular inflammation by inducing USF1/A20 axis-mediated NF-κB inactivation. Therefore, the expression and/or activation of PTP4A1 in ECs might be useful for the treatment of vascular inflammatory diseases.
Asunto(s)
Células Endoteliales , FN-kappa B , Vasculitis , Animales , Humanos , Ratones , Proteínas de Ciclo Celular/metabolismo , Células Endoteliales/metabolismo , Inflamación/genética , Inflamación/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal , Factores Estimuladores hacia 5'/metabolismo , Vasculitis/genética , Vasculitis/metabolismoRESUMEN
Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.
Asunto(s)
Transición Epitelial-Mesenquimal , Factor de Crecimiento Similar a EGF de Unión a Heparina , Macrófagos , Metástasis de la Neoplasia , Material Particulado , Animales , Ratones , Línea Celular Tumoral , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Material Particulado/efectos adversosRESUMEN
Macrophage activation has long been implicated in a myriad of human pathophysiology, particularly in the context of the dysregulated capacities of an unleashing intracellular or/and extracellular inflammatory response. A growing number of studies have functionally coupled the macrophages' inflammatory capacities with dynamic metabolic reprogramming which occurs during activation, albeit the results have been mostly interpreted through classic metabolism point of view; macrophages take advantage of the rewired metabolism as a source of energy and for biosynthetic precursors. However, a specific subset of metabolic products, namely immune-modulatory metabolites, has recently emerged as significant regulatory signals which control inflammatory responses in macrophages and the relevant extracellular milieu. In this review, we introduce recently highlighted immuno-modulatory metabolites, with the aim of understanding their physiological and pathological relevance in the macrophage inflammatory response. [BMB Reports 2022; 55(11): 519-527].
Asunto(s)
Activación de Macrófagos , Macrófagos , Humanos , Activación de Macrófagos/fisiología , Macrófagos/metabolismo , Inmunomodulación , Inflamación/metabolismoRESUMEN
Marine microplastics (MPs) pose a risk to human health through accumulation in maricultural organisms, particularly bivalves. Various studies have reported the presence of MP particles in Pacific oysters (Crasostrea gigas). In this study, we investigated the size-specific ingestion and egestion of polystyrene (PS) MPs by Pacific oysters. The cultivation density of C. gigas was maintained at 1 L of filtered seawater per oyster (n = 5) during the MP ingestion and egestion experiments. On exposure to 300 n/L of PS MP fragments for 7 d, 60.4% of the PS was ingested within 6 h (7.25 × 102 ± 1.36 × 102 n/indv.), and the ingestion was saturated at 12 h (1.2 × 103 ± 2.2 × 102 n/indv.) in C. gigas. The maximum MP ingestion capacity (Igmax) of a single Pacific oyster was 73.0 ± 16.3 n/g wet weight. Further, 62.9% of the PS MP particles were egested for 7 d from the saturated single C. gigas. Ingestion and egestion varied according to the PS MP size. In the case of <50 µm PS MP, ingestion rate was low but MP amount and net-ingestion efficiency was significantly higher than other PS MP sizes. In addition, egestion, egestion rate, and net-egestion efficiency for <50 µm PS MPs were significantly higher than other PS MP sizes. Therefore, smaller MPs (<50 µm) normally exhibit the highest ingestion and egestion rates; therefore, the 50-300 µm size fraction exhibited the highest residual possibility (particles >1000 µm were excluded). Additionally, considering the net-egestion efficiency, the most economical and efficient depuration period was 24 h. This study clarifies the size-specific MP accumulation in oysters, and the egestion results suggest that the potential risk of MPs to human health through the intake of maricultural products could be reduced by depuration.
Asunto(s)
Crassostrea , Contaminantes Químicos del Agua , Animales , Ingestión de Alimentos , Humanos , Microplásticos , Plásticos , Poliestirenos , Contaminantes Químicos del Agua/análisisRESUMEN
Probiotics are often infused into functional foods or encapsulated in a supplement form to maintain a healthy balance between the gut microbiota and their host. Because there are milk-based functional foods such as yogurt and cheese on the market, it has been suggested that milk-based probiotics could be incorporated into skim milk proteins in a liquid capsule. Skim milk is mainly composed of casein and whey protein, which create a strong natural barrier and can be used to encapsulate probiotics. In this study, we compared the encapsulated probiotics prepared with milkbased concentrated cell mixtures using commercial probiotics. Probiotic capsules were emulsified with skim milk proteins using vegetable oil to form a double coating layer. The product was heatstable when tested using a rheometer. The survival rate of the milk-based probiotic cells in the lower gastric environment with bile was significantly higher than commercial probiotics. Thus, milkencapsulated probiotics exhibited greater efficacy in the host than other types of probiotics, suggesting that the former could be more viable with a longer shelf life under harsh conditions than other form of probiotics. Our findings suggested that, compared with other types of probiotics, milkbased probiotics may be a better choice for producers and consumers.
Asunto(s)
Cápsulas/química , Proteínas de la Leche/química , Probióticos/química , Reología , Animales , Bilis , Caseínas , Queso , Alimentos Funcionales , Leche , Temperatura , Termotolerancia , Yogur/microbiologíaRESUMEN
Distal gastrectomy with Billroth I or II reconstruction may cause duodenogastroesophageal reflux (DGER), thereby resulting in digestive or respiratory symptoms. The mainstay of treatment is medication with proton pump inhibitors. However, these drugs may have limited effects in DGER. Laparoscopic fundoplication has been proven to be highly effective in treating gastroesophageal reflux disease (GERD), but it cannot be performed optimally for GERD that develops after gastrectomy. We report the case of a 72-year-old man with a history of distal gastrectomy and Billroth I anastomosis due to early gastric cancer. GERD due to bile reflux occurred after surgery and was refractory to medical therapy. The patient underwent Roux-en-Y conversion from Billroth I gastroduodenostomy and hiatal hernia repair with only cruroplasty. Fundoplication was not performed. His symptoms improved significantly after the surgery. Therefore, laparoscopic hiatal hernia repair and Roux-en-Y conversion can be an effective surgical procedure to treat medically refractory DGER after Billroth I gastrectomy.
RESUMEN
It has been shown that aerobic exercise improves atopic dermatitis (AD), although the mechanism is not clear. Here, we propose a hypothesis that moderate-intensity aerobic exercise improves AD in a mouse model through modulating allergic inflammation. The DNCB-treated mouse model for eczema was divided into 3 groups: (a) not subjected to aerobic exercise, (b) subjected to continuous aerobic exercise and (c) subjected to accumulated aerobic exercise. After given exercise using a treadmill device either 30 min/d or 10 min × 3/day at a speed of 16 m/min, for 9 days, respectively, dermatitis symptom score, thickness of epidermis/dermis and eosinophil infiltration were decreased in the 2 exercise groups compared to the sedentary living group. The serum levels of IgE, MCP-1 and MDC showed a significant decrease both in the continuous or accumulated exercise groups. Moderate-intensity aerobic exercise ameliorates dermatitis symptoms through immune modulation in the DNCB-treated mouse model for eczema.
Asunto(s)
Citocinas/sangre , Dermatitis Atópica/terapia , Eccema/inmunología , Eccema/terapia , Condicionamiento Físico Animal/fisiología , Animales , Quimiocina CCL2/sangre , Quimiocina CCL22/sangre , Dermatitis Atópica/inmunología , Dinitroclorobenceno , Eccema/sangre , Eccema/inducido químicamente , Femenino , Inmunoglobulina E/sangre , Ratones , Condicionamiento Físico Animal/métodos , Índice de Severidad de la EnfermedadRESUMEN
Malignant melanoma is a fatal disease that rapidly spreads to the whole body. Treatments have limited efficiency owing to drug resistance and various side effects. Pseudomonas syringae pv. tomato (Pto) is a model bacterial pathogen capable of systemic infection in plants. Pto injects the effector protein HopQ into the plant cytosol via a type III secretion machinery and suppresses the host immunity. Intriguingly, host plant proteins regulated by HopQ are conserved even in humans and conferred in tumor metastasis. Nevertheless, the potential for HopQ to regulate human cancer metastasis was unknown. In this study, we addressed the suitability of HopQ as a possible drug against melanoma metastasis. In melanoma cells, overexpressed HopQ is phosphorylated and bound to 14-3-3 through its N-terminal domain, resulting in stronger interaction between HopQ and vimentin. The binding of HopQ to vimentin allowed for degradation of vimentin via p62-dependent selective autophagy. Attenuation of vimentin expression by HopQ inhibited melanoma motility and in vivo metastasis. These findings demonstrated that HopQ directly degraded vimentin in melanoma cells and could be applied to an inhibitor of melanoma metastasis.
Asunto(s)
Melanoma/tratamiento farmacológico , Vimentina/uso terapéutico , Animales , Autofagia , Movimiento Celular , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Fosforilación , Transfección , Vimentina/farmacologíaRESUMEN
Interactions between pathogenic microorganisms and their hosts are varied and complex, encompassing open-field scale interactions to interactions at the molecular level. The capacity of plant pathogenic bacteria and fungi to cause diseases in human and animal systems was, until recently, considered of minor importance. However, recent evidence suggests that animal and human infections caused by plant pathogenic fungi, bacteria and viruses may have critical impacts on human and animal health and safety. This review analyses previous research on plant pathogens as causal factors of animal illness. In addition, a case study involving disruption of type III effector-mediated phagocytosis in a human cell line upon infection with an opportunistic phytopathogen, Pseudomonas syringae pv. tomato, is discussed. Further knowledge regarding the molecular interactions between plant pathogens and human and animal hosts is needed to understand the extent of disease incidence and determine mechanisms for disease prevention.
Asunto(s)
Infecciones Bacterianas/transmisión , Hongos/patogenicidad , Micosis/transmisión , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/patogenicidad , Animales , Proteínas Bacterianas/metabolismo , Hongos/metabolismo , Humanos , Solanum lycopersicum/microbiología , Pseudomonas syringae/metabolismoRESUMEN
In host defense, it is crucial to maintain the acidity of the macrophage phagosome for effective bacterial clearance. However, the mechanisms governing phagosomal acidification upon exposure to gram-negative bacteria have not been fully elucidated. In this study, we demonstrate that in macrophages exposed to Escherichia coli, the thioredoxin-interacting protein (TXNIP)-associated inflammasome plays a role in pH modulation through the activated caspase-1-mediated inhibition of NADPH oxidase. While there was no difference in early-phase bacterial engulfment between Txnip knockout (KO) macrophages and wild-type (WT) macrophages, Txnip KO macrophages were less efficient at destroying intracellular bacteria in the late phase, and their phagosomes failed to undergo appropriate acidification. These phenomena were associated with reactive oxygen species production and were reversed by treatment with an NADPH oxidase inhibitor or a caspase inhibitor. In line with these results, Txnip KO mice were more susceptible to both intraperitoneally administered E. coli and sepsis induced by cecum ligation and puncture than WT mice. Taken together, this study suggests that the TXNIP-associated inflammasome-caspase-1 axis regulates NADPH oxidase to modulate the pH of the phagosome, controlling bacterial clearance by macrophages.
Asunto(s)
Proteínas Portadoras/inmunología , Caspasa 1/inmunología , Infecciones por Escherichia coli/inmunología , Inflamasomas/inmunología , Macrófagos/inmunología , Fagosomas/química , Tiorredoxinas/inmunología , Animales , Activación Enzimática/inmunología , Escherichia coli/inmunología , Concentración de Iones de Hidrógeno , Macrófagos/química , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasas/inmunología , Fagosomas/inmunologíaRESUMEN
The disruption of the retinal pigment epithelium (RPE), for example, through oxidative damage, is a common factor underlying age-related macular degeneration (AMD). Aberrant autophagy also contributes to AMD pathology, as autophagy maintains RPE homeostasis to ensure blood-retinal barrier (BRB) integrity and protect photoreceptors. Thioredoxin-interacting protein (TXNIP) promotes cellular oxidative stress by inhibiting thioredoxin reducing capacity and is in turn inversely regulated by reactive oxygen species levels; however, its role in oxidative stress-induced RPE cell dysfunction and the mechanistic link between TXNIP and autophagy are largely unknown. Here, we observed that TXNIP expression was rapidly downregulated in RPE cells under oxidative stress and that RPE cell proliferation was decreased. TXNIP knockdown demonstrated that the suppression of proliferation resulted from TXNIP depletion-induced autophagic flux, causing increased p53 activation via nuclear localization, which in turn enhanced AMPK phosphorylation and activation. Moreover, TXNIP downregulation further negatively impacted BRB integrity by disrupting RPE cell tight junctions and enhancing cell motility by phosphorylating, and thereby activating, Src kinase. Finally, we also revealed that TXNIP knockdown upregulated HIF-1α, leading to the enhanced secretion of VEGF from RPE cells and the stimulation of angiogenesis in cocultured human retinal microvascular endothelial cells. This suggests that the exposure of RPE cells to sustained oxidative stress may promote choroidal neovascularization, another AMD pathology. Together, these findings reveal three distinct mechanisms by which TXNIP downregulation disrupts RPE cell function and thereby exacerbates AMD pathogenesis. Accordingly, reinforcing or restoring BRB integrity by targeting TXNIP may serve as an effective therapeutic strategy for preventing or attenuating photoreceptor damage in AMD.
Asunto(s)
Barrera Hematorretinal/metabolismo , Proteínas Portadoras/genética , Degeneración Macular/genética , Estrés Oxidativo/genética , Animales , Autofagia/genética , Barrera Hematorretinal/patología , Movimiento Celular/genética , Regulación de la Expresión Génica/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Degeneración Macular/patología , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Retina/metabolismo , Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Tiorredoxinas/genética , Uniones Estrechas/genética , Uniones Estrechas/patología , Proteína p53 Supresora de Tumor/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Snail is a key regulator of epithelial-mesenchymal transition (EMT), which is a major step in tumor metastasis. Although the induction of Snail transcription precedes EMT, posttranslational regulation, especially phosphorylation of Snail, is critical for determining Snail protein levels or stability, subcellular localization, and the ability to induce EMT. To date, several kinases are known that enhance the stability of Snail by preventing its ubiquitination; however, the molecular mechanism(s) underlying this are still unclear. Here, we identified p38 MAPK as a crucial posttranslational regulator that enhances the stability of Snail. p38 directly phosphorylated Snail at Ser107, and this effectively suppressed DYRK2-mediated Ser104 phosphorylation, which is critical for GSK3ß-dependent Snail phosphorylation and ßTrCP-mediated Snail ubiquitination and degradation. Importantly, functional studies and analysis of clinical samples established a crucial role for the p38-Snail axis in regulating ovarian cancer EMT and metastasis. These results indicate the potential therapeutic value of targeting the p38-Snail axis in ovarian cancer. SIGNIFICANCE: These findings identify p38 MAPK as a novel regulator of Snail protein stability and potential therapeutic target in ovarian cancer.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Neoplasias Ováricas/patología , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química , Serina/metabolismo , Factores de Transcripción de la Familia Snail/química , Factores de Transcripción de la Familia Snail/genética , Ubiquitinación , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas con Repetición de beta-Transducina/metabolismo , Quinasas DyrKRESUMEN
Accurate and rapid diagnosis of influenza infection is essential to enable early antiviral treatment and reduce the mortality associated with seasonal and epidemic infections. Immunochromatography is one of the most common methods used for the diagnosis of seasonal human influenza; however, it is less effective in diagnosing pandemic influenza virus. Currently, rapid diagnostic kits for pandemic influenza virus rely on the detection of nucleoprotein (NP) or hemagglutinin (HA). NP detection shows higher specificity and is more sensitive than HA detection. In this study, we time-dependently screened expression conditions, and herein report optimal conditions for the expression of recombinant nucleoprotein (rNP), which was 48 h after infection. In addition, we report the use of the expressed rNP in a rapid influenza diagnostic test (SGT i-flex Influenza A&B Test). We constructed expression vectors that synthesized rNP (antigen) of influenza A and B in insect cells (Sf9 cells), employed the purified rNP to the immunoassay test kit, and clearly distinguished NPs of influenza A and influenza B using this rapid influenza diagnostic kit. This approach may improve the development of rapid test kits for influenza using NP.
Asunto(s)
Antígenos Virales/análisis , Antígenos Virales/genética , Virus de la Influenza A/genética , Virus de la Influenza B/genética , Nucleoproteínas/análisis , Nucleoproteínas/genética , Animales , Antígenos Virales/aislamiento & purificación , Expresión Génica , Humanos , Inmunoensayo , Virus de la Influenza A/inmunología , Virus de la Influenza B/inmunología , Gripe Humana/diagnóstico , Gripe Humana/inmunología , Gripe Humana/virología , Límite de Detección , Nucleoproteínas/aislamiento & purificación , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Células Sf9 , Spodoptera/genéticaRESUMEN
Certain animal and plant pathogenic bacteria have developed virulence factors including effector proteins that enable them to overcome host immunity. A plant pathogen, Pseudomonas syringae pv. tomato (Pto) secretes a large repertoire of effectors via a type III secretory apparatus, thereby suppressing plant immunity. Here, we show that Pto causes sepsis in mice. Surprisingly, the effector HopQ1 disrupted animal phagocytosis by inhibiting actin rearrangement via direct interaction with the LIM domain of the animal target protein LIM kinase, a key regulator of actin polymerization. The results provide novel insight into animal host-plant pathogen interactions. In addition, the current study firstly demonstrates that certain plant pathogenic bacteria such as Pto evade phagocytosis by animal cells due to cross-kingdom suppression of host immunity.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Bacterianas/fisiología , Fagocitosis , Pseudomonas syringae/patogenicidad , Factores de Virulencia/fisiología , Animales , Bacteriemia/microbiología , Proteínas Bacterianas/inmunología , Interacciones Huésped-Patógeno , Quinasas Lim/metabolismo , Solanum lycopersicum/inmunología , Solanum lycopersicum/microbiología , Ratones , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Pseudomonas syringae/inmunología , Factores de Virulencia/inmunologíaRESUMEN
Shiga toxins (Stxs) are the main virulence factors expressed by the pathogenic Stx-producing bacteria, namely, Shigella dysenteriae serotype 1 and certain Escherichia coli strains. These bacteria cause widespread outbreaks of bloody diarrhea (hemorrhagic colitis) that in severe cases can progress to life-threatening systemic complications, including hemolytic uremic syndrome (HUS) characterized by the acute onset of microangiopathic hemolytic anemia and kidney dysfunction. Shiga toxicosis has a distinct pathogenesis and animal models of Stx-associated HUS have allowed us to investigate this. Since these models will also be useful for developing effective countermeasures to Stx-associated HUS, it is important to have clinically relevant animal models of this disease. Multiple studies over the last few decades have shown that mice injected with purified Stxs develop some of the pathophysiological features seen in HUS patients infected with the Stx-producing bacteria. These features are also efficiently recapitulated in a non-human primate model (baboons). In addition, rats, calves, chicks, piglets, and rabbits have been used as models to study symptoms of HUS that are characteristic of each animal. These models have been very useful for testing hypotheses about how Stx induces HUS and its neurological sequelae. In this review, we describe in detail the current knowledge about the most well-studied in vivo models of Stx-induced HUS; namely, those in mice, piglets, non-human primates, and rabbits. The aim of this review is to show how each human clinical outcome-mimicking animal model can serve as an experimental tool to promote our understanding of Stx-induced pathogenesis.