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Drug-eluting microspheres are commonly used as a local drug delivery system for interventional therapy. However, current drug-eluting microspheres have poor X-ray visibility, which can hinder tracking and postembolization evaluation. In the current study, X-ray-visible poly(acrylic acid) drug-eluting beads loaded with iodized oil (IO-PAA-DEBs) ranging from 100-300 µm were prepared and evaluated both in vitro and in vivo. Iodized oil served as the radiopaque agent, and X-ray and computed tomography scanning confirmed that the microspheres exhibited excellent X-ray-visible properties. The drug-loading capacities of bleomycin hydrochloride, doxorubicin hydrochloride, and oxaliplatin were also investigated. IO-PAA-DEBs exhibited sustained drug release properties, accompanied by a cumulative drug release rate that reached approximately 60% after 120 h. In vitro and in vivo experiments revealed that IO-PAA-DEBs had good biocompatibility. Collectively, these results demonstrated that IO-PAA-DEBs could facilitate transarterial embolization and sustained drug delivery.
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Advances in chemotherapeutic strategies are urgently required to improve antitumor efficiency. Herein, a carboxylated pillar[6]arene (CP6A) was employed to load chemotherapy medication, nitrogen mustard (NM), via forming a direct host-guest complex, as this helps to decrease the cytotoxicity of NM on normal mammary epithelial cells. Attributed to the stronger complexation ability of CP6A for endogenous spermine (SPM) than for NM, the complexed NM could be competitively released from the CP6A cavity via replacement with SPM. This chemotherapy strategy performed well in vitro and in vivo for SPM-overexpressed cancers. In comparison with free NM, antitumor efficiency of NM/CP6A was significantly enhanced, which originated from the synergistic effect of competitive release of NM and simultaneous trapping of SPM. This strategy might guide expansion to other first-line antitumor agents to improve therapeutic efficacy and decrease side effects, thereby replenishing the possibilities of supramolecular chemotherapy.
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Background and Purpose: Accumulating evidence suggests that circular RNAs (circRNAs) are involved in immune and inflammatory processes after acute ischemic stroke (AIS). However, the roles of circRNA-mediated competing endogenous RNA (ceRNA) in modulating immune inflammation of AIS have not yet been determined. This study aimed to construct a circRNA-mediated immune-related ceRNA network and identify novel circRNAs in AIS. Methods: Microarray data were downloaded from the GEO database and further analysed by R software. Then, we constructed a circRNA-mediated ceRNA network based on interaction information from the bioinformatics database. A topological property analysis of the ceRNA network was conducted to screen novel circRNAs. Finally, we further applied quantitative real-time polymerase chain reaction (qRT-PCR) to two independent sets. Results: We constructed an AIS immune-related ceRNA (AISIRC) network containing immune-related genes (IRGs), miRNAs, and circRNAs. Additionally, we extracted the subnetwork from the AISIRC network and screened six immune-related circRNAs. After identification and validation, we finally confirmed that plasma levels of circPTP4A2 and circTLK2 were significantly increased in AIS patients compared with both healthy control subjects (HCs) and transient ischemic attack (TIA) patients. Logistic regression and receiver-operating characteristic (ROC) curve analyses demonstrated that these two circRNAs may function as predictive and discriminative biomarkers for AIS. We also confirmed that plasma levels of circPTP4A2 were elevated in TIA patients compared with HCs and might be an independent risk factor for predicting TIA. Longitudinal analysis of circRNA expression up to 90 days after AIS indicated that the ability of circPTP4A2 and circTLK2 to monitor AIS dynamics was highly desirable. Conclusion: In summary, the circRNA-mediated immune-related ceRNA network was successfully constructed, and two circulating circRNAs (circPTP4A2 and circTLK2) improved sensitivity for the diagnosis of AIS and could be considered diagnostic biomarkers.
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OBJECTIVE: To study the distributional characteristics of allergens in children with allergic diseases in Shaanxi province. METHODS: A total of 4 622 children diagnosed with allergic diseases in the Asthma Center, Department of Pediatrics, Xijing Hospital from March 2015 to February 2019 were selected. Serum allergen-specific immunoglobulin E (sIgE) of 19 common kinds of allergens were examined with enzyme-linked immunosorbent assay (ELISA). The children were divided into different groups according to sex, age and geographical regions, and the distributional characteristics of allergens of the different groups were compared. RESULTS: The overall positive rate for the 19 allergens of the 4 622 children was 62.8%. The ranking of the positive rates for individual allergens from high to low were as follows: 24.2% for milk, 18.0% for mold mix, 16.7% for dog dander, 16.4% for house dust mite, 11.7% for cat dander, 10.7% for cashew, 10.6% for weed pollen, 8.8% for egg white, 7.8% for house dust, 7.7% for tree pollen, 5.6% for amaranth, 4.9% for mulberry tree, 3.6% for mango, 3.2% for beef, 2.8% for cockroach, 2.1% for crab, 1.5% for shrimp, 0.8% for pineapple, and 0.3% for shellfish. Analysis based on sex showed that the allergen positive rates in boys were higher than those in girls. Analysis by age difference showed that generally the positive rates for inhaled allergens increased along with the increase in patient age, while the positive rates for ingested allergens decreased along with the increase in patient age. Analysis by geographical regions showed that the positive rate of house dust mite in the patients from the southern part of Shaanxi, the positive rate of weed pollen in the patients from the northern part of Shaanxi and the positive rates of milk and egg white in the patients from the central part of Shaanxi were higher than those in other areas. The cluster analysis and correlation analysis showed that the 19 allergens could be roughly divided into 4 categories. There were moderate correlations among tree pollen, mulberry tree and amaranth. There were moderate correlations among mulberry tree, mango and amaranth. There was moderate correlation between shrimp and crab, and there were mild or weak correlations among most of the other allergens. CONCLUSION: Among the 4 622 children with allergic diseases in Shaanxi Province who were treated in the Asthma Center, Department of Pediatrics, Xijing Hospital, male patients showed higher sensitivity to allergens. The positive rates of inhaled allergens increased, while the positive rates of ingested allergens decreased with increase in patient age. There were regional differences in the distribution of allergens. Some allergens were correlated with each other, which may be related to cross-reaction.
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Asma , Hipersensibilidad , Alérgenos , Animales , Asma/epidemiología , Gatos , Bovinos , Niño , Perros , Polvo , Humanos , Inmunoglobulina E , MasculinoRESUMEN
Phonon-assisted single-photon upconversion, which was not previously reported in organic materials, has been demonstrated in the 6-pentaceneone crystal through the linear pumping power dependent anti-Stokes photoluminescence (ASPL), nanoseconds PL lifetime and quenched ASPL at low temperature. Furthermore, the 6-pentaceneone crystal can be mechanically exfoliated to ultrathin flakes and it exhibits thickness-dependent photoluminescence.
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A metal-free photocatalytic protocol for efficient construction of difluoro-containing dibenzazepines is developed. The transformation is redox-neutral and environmentally benign, has a wide range of substrate scope, proceeds at room temperature under irradiation of visible light, and furnishes dibenzazepine derivatives in mild to excellent yield.
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A series of inexpensive, easily tunable, and highly reducing organic photocatalysts (PCs) that exhibit strong absorption in the visible region are described. Time-dependent density functional theory calculations were applied to corroborate the experimental observations and explain the relationship between the structure and property. The photocatalytic efficiency of these PCs was demonstrated by catalyzing several challenging reactions and the radical cascade cyclization of 1,6-diyne with superior photocatalytic efficiency to other organic photocatalysts.
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This study aimed to estimate the loss of health-related quality of life (HRQOL) associated with common chronic conditions in older Chinese population.Weitang Geriatric Diseases Study is a community-based health survey on 5557 Chinese aged 60 years or older. The study population was measured with EQ-5D-3L for HRQOL assessment. We also assessed the coexisting chronic conditions of depression, cognitive dysfunction, stroke, heart disease, diabetes, hypertension, obesity, hyperlipidemia and visual impairment based on self-completed questionnaires, biomarkers and medical records. Ordinary Least Square regression was performed to model the relationship between the conditions and the EQ-5D-3L index scores. The robust standard error (RSE) estimator was adopted to calculate 95% confidence interval (CI) for coefficients. The mean EQ-5D-3L representing overall HRQOL was 0.954 (standard deviation: 0.081) with 70% of participants reporting full health. After controlling for socio-demographic characteristics and comorbidities, depression, stroke, heart disease and cognitive dysfunction had significantly adverse impact on the EQ-5D index score. The respective coefficients (95% CI) of each condition were -0.191 (-0.233, -0.150), -0.052 (-0.086, -0.019), -0.019 (-0.029, -0.010), and -0.016 (-0.024, -0.008). Chronic conditions were found to contribute to HRQOL loss in older Chinese population. The utility and utility decrement estimated can be used for quality-adjusted life-year calculation.
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Enfermedad Crónica/epidemiología , Estado de Salud , Encuestas Epidemiológicas/métodos , Calidad de Vida/psicología , Población Rural , Anciano , Anciano de 80 o más Años , China/epidemiología , Enfermedad Crónica/psicología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Morbilidad/tendenciasRESUMEN
Herein, for the first time, the typical porous Covalent Organic Frameworks (COFs) CTpBD with superior chemical stability and large surface area were applied as sorbents for solid phase extraction of trace ions via flow injection followed by inductively coupled plasma mass spectrometry (ICP-MS) detection. The well-prepared and fully-characterized CTpBD COFs were filled in solid phase extraction cartridge as novel and robust adsorbents for element analysis. Separation and enrichment of Cr (III), Mn (II), Co (II), Ni (II), Cd (II), V (V), Cu (II), As (III), Se (IV), and Mo (VI) was then carried out, and the contents were measured by ICP-MS. Owing to the large surface area and instinctive porous structure of CTpBD, preconcentration of the target trace elements via COF-filled on-line SPE column has achieved low detection limits of 2.1-21.6ngL-1 along with a wide linearity range at 0.05-25µgL-1 for all target ions. The relative standard deviations (RSD) of 1.2%-4.3% obtained via 11 parallel determinations at the sample concentration of 100ngL-1 revealed excellent repeatability of the developed methods Our proposed methods have been successfully utilized for trace element analysis in environmental and food samples.
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BACKGROUND: This study investigated whether quercetin could alleviate vascular calcification in experimental chronic renal failure rats induced by adenine. METHODS: 32 adult male Wistar rats were randomly divided into 4 groups fed normal diet, normal diet with quercetin supplementation (25 mg/kg·BW/d), 0.75% adenine diet, or adenine diet with quercetin supplementation. All rats were sacrificed after 6 weeks of intervention. Serum renal functions biomarkers and oxidative stress biomarkers were measured and status of vascular calcification in aorta was assessed. Furthermore, the induced nitric oxide synthase (iNOS)/p38 mitogen activated protein kinase (p38MAPK) pathway was determined to explore the potential mechanism. RESULTS: Adenine successfully induced renal failure and vascular calcification in rat model. Quercetin supplementation reversed unfavorable changes of phosphorous, uric acid (UA) and creatinine levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity in serum and the increases of calcium and alkaline phosphatase (ALP) activity in the aorta (P < 0.05) and attenuated calcification and calcium accumulation in the medial layer of vasculature in histopathology. Western blot analysis showed that iNOS/p38MAPK pathway was normalized by the quercetin supplementation. CONCLUSIONS: Quercetin exerted a protective effect on vascular calcification in adenine-induced chronic renal failure rats, possibly through the modulation of oxidative stress and iNOs/p38MAPK pathway.
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Fallo Renal Crónico/inducido químicamente , Fallo Renal Crónico/tratamiento farmacológico , Estrés Oxidativo , Quercetina/uso terapéutico , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/patología , Adenina , Fosfatasa Alcalina/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/patología , Aorta/fisiopatología , Biomarcadores/sangre , Calcio/sangre , Riñón/efectos de los fármacos , Riñón/fisiopatología , Fallo Renal Crónico/patología , Fallo Renal Crónico/fisiopatología , Masculino , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fósforo/sangre , Quercetina/farmacología , Ratas Wistar , Calcificación Vascular/sangre , Calcificación Vascular/fisiopatología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Immunoglobulin A nephropathy (IgAN) is the most frequent form of glomerulonephritis, which is characterized by glomerular proliferation and renal inflammation. Icariin is a flavonoid from the Chinese herb Epimedium, and its anti-inflammatory effect has been reported. This study aimed to investigate the effects of icariin on the renal damage in IgAN rats and the mechanisms behind these effects. IgAN model was established in Sprague-Dawley rats by oral and intravenous immunization with bovine gamma-globulin for 12 weeks, and rats were treated with icariin from 12 to 18 weeks. At the end of experimental period, kidneys, urine, and blood samples were collected for further analysis. Our results showed that icariin ameliorated the increase in the levels of proteinuria, serum creatinine, and urea nitrogen without severe side effects. IgAN rats exhibited significantly increased IgA deposition, mesangial matrix expansion, and glomerular fibrosis, while icariin treatment markedly attenuated these alterations. Moreover, treatment with icariin also dramatically blocked nuclear factor kappa b (NF-κB) nuclear translocation and Nlrp3 inflammasome activation in IgAN rats, leading to reduced downstream proinflammatory cytokines production. Mechanistically, we found that icariin treatment inhibited IKKß and IκBα phosphorylation and IκBα degradation in IgAN rats. Our data demonstrate that icariin ameliorates renal damage in IgAN rats via inhibition of NF-κB-mediated Nlrp3 inflammasome activation. These findings provide insight into an application of icariin for the treatment of IgAN disease, and represent a novel mechanism behind these effects.
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Within the cell, several mechanisms exist to maintain homeostasis of the endoplasmic reticulum (ER). One of the primary mechanisms is the unfolded protein response (UPR). In this review, we primarily focus on the latest signal webs and regulation mechanisms of the UPR. The relationships among ER stress, apoptosis, and cancer are also discussed. Under the normal state, binding immunoglobulin protein (BiP) interacts with the three sensors (protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1α (IRE1α)). Under ER stress, misfolded proteins interact with BiP, resulting in the release of BiP from the sensors. Subsequently, the three sensors dimerize and autophosphorylate to promote the signal cascades of ER stress. ER stress includes a series of positive and negative feedback signals, such as those regulating the stabilization of the sensors/BiP complex, activating and inactivating the sensors by autophosphorylation and dephosphorylation, activating specific transcription factors to enable selective transcription, and augmenting the ability to refold and export. Apart from the three basic pathways, vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR)-phospholipase C-γ (PLCγ)-mammalian target of rapamycin complex 1 (mTORC1) pathway, induced only in solid tumors, can also activate ATF6 and PERK signal cascades, and IRE1α also can be activated by activated RAC-alpha serine/threonine-protein kinase (AKT). A moderate UPR functions as a pro-survival signal to return the cell to its state of homeostasis. However, persistent ER stress will induce cells to undergo apoptosis in response to increasing reactive oxygen species (ROS), Ca2+ in the cytoplasmic matrix, and other apoptosis signal cascades, such as c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription 3 (STAT3), and P38, when cellular damage exceeds the capacity of this adaptive response.
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Factor de Transcripción Activador 6/metabolismo , Endorribonucleasas/metabolismo , Regulación de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismo , Animales , Apoptosis , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Homeostasis , Humanos , Inmunoglobulinas/química , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/metabolismo , Dominios Proteicos , Pliegue de Proteína , Especies Reactivas de Oxígeno/metabolismo , Ribosomas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
A new chalcogenide crystal, (NH4)4Ag12Sn7Se22 (FJSM-STS), has been solvothermally synthesized. The crystal structure, which is composed of arrays of [Sn3Se9]n(6n-) chains interconnecting [SnAg6Se10]n(10n-) and [Ag3Se4]n(5n-) layers, is unprecedented among the reported A/Ag/Sn/Q (A = cation; Q = S, Se, and Te) compounds. Optical absorption together with theoretical calculations of the band structure indicate a direct band gap of 1.21 eV for FJSM-STS, which is close to the ideal band gap to maximize the photoconversion efficiency proposed by Shockley and Queisser. The toxic-metal-free crystal of FJSM-STS exhibits obvious photosensitivity in the near-infrared range. The variates of power and temperature on the photosensitivity have been studied.
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2D semiconducting metal phosphorus trichalcogenides, particularly the bulk crystals of MPS3 (M = Fe, Mn, Ni, Cd and Zn) sulfides and MPSe3 (M = Fe and Mn) selenides, have been synthesized, crystallized and exfoliated into monolayers. The Raman spectra of monolayer FePS3 and 3-layer FePSe3 show the strong intralayer vibrations and structural stability of the atomically thin layers under ambient condition. The band gaps can be adjusted by element choices in the range of 1.3-3.5 eV. The wide-range band gaps suggest their optoelectronic applications in a broad wavelength range. The calculated cleavage energies of MPS3 are smaller than that of graphite. Therefore, the monolayers used for building of heterostructures by van der Waals stacking could be considered as the candidates for artificial 2D materials with unusual ferroelectric and magnetic properties.
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CdS synthesis by plasma-enhanced microwave physical vapor transport (PMPVT) has been developed in this work. The photoluminescence (PL), absorbance, Raman spectra and the mechanism of CdS crystal growth have been investigated. Furthermore, plasma-enhanced microwave chemical vapour transport (PMCVT) synthesis of CdS with additional chemical transport agents has been explored. In addition, other II-VI chalcogenides were also synthesized by PMPVT.
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During embryonic development of the mammalian cerebral cortex, postmitotic cortical neurons migrate radially from the ventricular zone to the cortical plate. Proper migration involves the correct orientation of migrating neurons and the transition from a multipolar to a mature bipolar morphology. Herein, we report that the 2 isoforms of Myosin-10 (Myo10) play distinct roles in the regulation of radial migration in the mouse cortex. We show that the full-length Myo10 (fMyo10) isoform is located in deeper layers of the cortex and is involved in establishing proper migration orientation. We also demonstrate that fMyo10-dependent orientation of radial migration is mediated at least in part by the netrin-1 receptor deleted in colorectal cancer. Moreover, we show that the headless Myo10 (hMyo10) isoform is required for the transition from multipolar to bipolar morphologies in the intermediate zone. Our study reveals divergent functions for the 2 Myo10 isoforms in controlling both the direction of migration and neuronal morphogenesis during radial cortical neuronal migration.
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Movimiento Celular/genética , Corteza Cerebral/citología , Corteza Cerebral/embriología , Miosinas/metabolismo , Neuronas/fisiología , Análisis de Varianza , Animales , Células Cultivadas , Receptor DCC , Electroporación , Embrión de Mamíferos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Antígeno Ki-67/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Miosinas/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Isoformas de Proteínas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The fibroblast growth factors (FGFs) are important for embryo development, wound healing, hematopoiesis, and angiogenesis. FGF-1, a member of FGF family, is involved in both receptor-dependent pathways and an intracrine pathway. Studies have recently shown that FGF-1 is overexpressed in the early stages of several kinds of cancer. Thus, FGF-1 is a candidate for cancer immunotargeting. To study the potential use of therapeutic antibodies against FGF-1, a monoclonal hybridoma 1C9 secreting monoclonal antibody specific for FGF-1 was developed. Then, a single-chain variable fragment (scFv) antibody was genetically engineered from hybridama 1C9. The binding of the scFv1C9 to the antigen FGF-1 was demonstrated by ELISA and immunoprecipitation assays. Functional analysis showed that the overexpressed scFv1C9 in MCF-7 cells targeted endogenous FGF-1 and prevented the translocation of FGF-1 into the nucleus, resulting in the blockade of the intracrine pathway of FGF-1, which caused the G1 arrest by p21 up-regulation. These results suggest that the generated scFv1C9 is an effective inhibitor of the intracrine pathway of FGF-1 and has a potential application as anti-tumoral agent in breast cancer.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Anticuerpos de Cadena Única/farmacología , Animales , Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Secuencia de Bases , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Escherichia coli , Femenino , Factor 1 de Crecimiento de Fibroblastos/genética , Expresión Génica , Humanos , Hibridomas/química , Hibridomas/metabolismo , Inmunomodulación , Ratones , Datos de Secuencia Molecular , Terapia Molecular Dirigida/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéutico , Regulación hacia ArribaRESUMEN
Myosin X (Myo X), an unconventional myosin with a tail homology 4-band 4.1/ezrin/radixin/moesin (MyTH4-FERM) tail, is expressed ubiquitously in various mammalian tissues. In addition to the full-length Myo X (Myo X FL), a headless form is synthesized in the brain. So far, little is known about the function of this motor-less Myo X. In this study, the role of the headless Myo X was investigated in immortalized gonadotropin-releasing hormone (GnRH) neuronal cells, NLT. NLT cells overexpressing the headless Myo X formed fewer focal adhesions and spread more slowly than the wild-type NLT cells and GFP-expressing NLT cells. In chemomigration assays, the NLT cells overexpressing the headless Myo X migrated shorter distances and had fewer migratory cells compared with the control NLT cells.
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Movimiento Celular/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Miosinas/metabolismo , Animales , Adhesión Celular/fisiología , Línea Celular , Ratones , Miosinas/genética , Neuronas/citología , Neuronas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
A system of virus-induced post-transcriptional gene silencing for studying rbcS gene function was established and optimized using tobacco rattle virus vector and Nicotiana benthamiana as experimental materiaes. The following analyses were conducted: phenotypic characterization of rbcS gene silenced plants, transcription levels of rbcS gene by RT-PCR; protein levels of rbcS by the antibodies of rbcS and rbcL and photosynthetic pigments wntents in rbcS silenced plants by HPLC method. The results showed that the seedlings at 21-24-day-old and Agrobacterium concentration at OD600 = 1-1.5 gave the best results for gene silencing. The expression level of rbcL was very likely regulated by rbcS, and rbcS gene did not relate to the collection of photosynthetic energy. Probability analysis showed that the tobacco rattle virus vector system is a useful and effective technique to study rbcS gene function via post-transcriptional gene silencing.
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Agrobacterium tumefaciens/genética , Nicotiana/genética , Proteínas de Plantas/genética , Interferencia de ARN , Ribulosa-Bifosfato Carboxilasa/genética , Western Blotting , Clorofila/análisis , Cromatografía Líquida de Alta Presión , ADN Complementario/genética , Vectores Genéticos , Luteína/análisis , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Virus de Plantas/genética , Plásmidos , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Plantones/genética , Plantones/metabolismo , Nicotiana/metabolismoRESUMEN
The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived alpha-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved the quantity and quality of alpha-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.