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BACKGROUND: Early identification of abnormal left ventricular function in children with obstructive sleep apnea (OSA) is difficult using conventional echocardiographic indices and commonly used clinical markers of myocardial damage. We sought to investigate the value of automatic function imaging and myocardial work parameters in predicting early cardiac impairment in children having OSA with preserved left heart function and thereby identifying an optimal index for assessment. PATIENTS AND METHODS: Fifty-two children who presented with symptoms of nocturnal sleep snoring and open-mouth breathing and 34 healthy controls were enrolled in this study. Clinical characteristics and conventional echocardiographic data were collected, and image analysis was performed using two-dimensional speckle-tracking echocardiography to obtain left ventricular global longitudinal strain (GLS), post-systolic index, peak strain dispersion, global work index (GWI), global constructive work (GCW), global wasted work, and global work efficiency. RESULTS: Children with OSA had significantly lower GLS, GWI, and GCW than those without (P < 0.05). Additionally, GWI (ß = -32.87, 95% CI: -53.47 to -12.27), and GCW (ß = -35.09, 95% CI: -55.35 to -14.84) were found to correlate with the disease severity in the multiple linear regression mode, with worsening values observed as the severity of the disease increased. ROC curve analysis revealed that GCW was the best predictor of myocardial dysfunction, with an AUC of 0.809 (P < 0.001), and the best cutoff point for diagnosing myocardial damage in children with OSA was 1965.5 mmHg%, with a sensitivity of 92.5% and a specificity of 58.7%. CONCLUSIONS: GLS, GWI, and GCW were identified as predictors of myocardial dysfunction in children with OSA, with GCW being the best predictor.
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Apnea Obstructiva del Sueño , Disfunción Ventricular Izquierda , Niño , Humanos , Valor Predictivo de las Pruebas , Ecocardiografía/métodos , Sístole , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/etiología , Apnea Obstructiva del Sueño/diagnóstico , Apnea Obstructiva del Sueño/diagnóstico por imagen , Función Ventricular Izquierda , Volumen SistólicoRESUMEN
Chemical transfer across the air-water interface is one of the most important geochemical processes of global significance. Quantifying such a process has remained extremely challenging due to the lack of suitable technologies to measure chemical diffusion across the air-water microlayer. Herein, we present a fluorescence optical system capable of visualizing the formation of the air-water microlayer with a spatial resolution of 10 µm and quantifying air-water diffusion fluxes using pyrene as a target chemical. We show for the first time that the air-water microlayer is composed of the surface microlayer in water (â¼290 ± 40 µm) and a diffusion layer in air (â¼350 ± 40 µm) with 1 µg L-1 of pyrene. The diffusion flux of pyrene across the air-water interface is derived from its high-resolution concentration profile without any pre-emptive assumption, which is 2 orders of magnitude lower than those from the conventional method. This system can be expanded to visualize diffusion dynamics of other fluorescent chemicals across the air-water interface and provides a powerful tool for furthering our understanding of air-water mass transfer of organic chemicals related to their global cycling.
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Monitoreo del Ambiente , Agua , Monitoreo del Ambiente/métodos , Compuestos Orgánicos , PirenosRESUMEN
Klinefelter syndrome (KS) is the most common sex chromosome abnormality, which occurs in about one in 660 newly born males, and it is the most common genetic cause of infertility in infertile men, accounting for 11%. It is rare for non-mosaic KS patients to have sperm and reproduce naturally, and there are currently no reports of KS patients with normal motile sperm. Microdissection testicular sperm extraction associated with intracytoplasmic sperm injection (micro-TESE-ICSI) is currently the main assisted reproductive method for patients with KS. In this study, we describe a patient of non-mosaic KS (47, XXY) who had given birth to a healthy girl naturally. The patient had normal male characteristics and did not have the symptoms of hypogonadism commonly seen in KS. He had high levels of serum follicle stimulating hormone and luteinizing hormone, a low level of serum testosterone, and a normal level of prolactin. Semen analysis showed that this case had normal motile sperm (total motility of 57.66% and progressive motility of 46.19%) but low sperm concentration (1.7 × 106 cells/mL). He gave birth to a boy by intracytoplasmic sperm injection (ICSI) using his ejaculated sperm purified to high density and motility by Percoll density gradient centrifugation. In conclusion, this case is a unique non-mosaic KS patient who had a normal sperm motility, experienced a natural fertility, and received a successful ICSI outcome, which enlarges our knowledges on non-mosaic KS.
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Androgen receptor gene (AR) is essential for male growth and fertility. Its mutations are responsible for androgen insensitivity syndrome (AIS) that usually shows the phenotype of azoospermia resulting in male infertility. This study reported the first case of mild AIS with complete normal serum hormones in a Chinese family. The proband referred for infertility because of azoospermia. His uncle and two cousins are both infertile and have azoospermia. Whole-exome sequencing in the genetic analyses showed that the proband carries a novel hemizygous AR missense mutation, NM_000044.6: c.2051G>C (p.Gly684Ala), in exon four within the ligand-binding domain. His mother and maternal aunt are heterozygous carriers, while his father and brother are wildtype, indicating that the mutation in the proband was inherited from his mother. This pattern is consistent with the genetic model of the X-linked recessive inheritance of AR in AIS pathogenesis. HOPE predicts that p.Gly684Ala increases the hydrophobicity of AR but does not change the AR conformation. PolyPhen-2 predicts that p.Gly684Ala is harmful. This study provides the new knowledge to understand the AR gene mutations in MAIS.
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Multiple morphological abnormalities of the sperm flagella (MMAF) is a characteristic form of severe asthenozoospermia and closely related to male infertility. However, it is not sure whether intracytoplasmic sperm injection (ICSI) allows MMAF patients reproductive success. The present study reported the first case of successful intracytoplasmic sperm injection-embryo transfer (ICSI-ET) in Chinese brothers with a novel compound heterozygous mutation of FSIP2 (fibrous sheath interacting protein 2), a newly identified MMAF-related genes. The proband and his brother were referred for MMAF because of their abnormal sperm flagellum. Through whole-exome sequencing in the genetic analyses of the proband, his brother and parents showed that the proband and his brother carry a novel compound heterozygous FSIP2 mutation (c.1750T>A and c.13600A>G), which will lead to abnormal expression of FSIP2 and loss of its function. Considering that these brothers had the MMAF phenotype, we recommended ICSI treatment. The successful outcome of ICSI indicated that a lose-function mutation of FSIP2 might not have any effect on ICSI, although the latest report showed a failed outcome of ICSI in a patient with FSIP2 mutation. This study provides new knowledge to understand the effect of MMAF caused by FSIP2 mutation on ICSI outcome.
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Infertilidad Masculina , Inyecciones de Esperma Intracitoplasmáticas , China , Transferencia de Embrión , Humanos , Infertilidad Masculina/genética , Infertilidad Masculina/terapia , Masculino , Mutación , Hermanos , Cola del Espermatozoide , EspermatozoidesRESUMEN
An epidemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. SARS-CoV-2 relies on its spike protein to invade host cells by interacting with the human receptor protein Angiotensin-Converting Enzymes 2 (ACE2). Therefore, designing an antibody or small-molecular entry blockers is of great significance for virus prevention and treatment. This study identified five potential small molecular anti-virus blockers via targeting SARS-CoV-2 spike protein by combining in silico technologies with in vitro experimental methods. The five molecules were natural products that binding to the RBD domain of SARS-CoV-2 was qualitatively and quantitively validated by both native Mass Spectrometry (MS) and Surface Plasmon Resonance (SPR). Anti-viral activity assays showed that the optimal molecule, H69C2, had a strong binding affinity (dissociation constant KD) of 0.0947 µM and anti-virus IC50 of 85.75 µM.
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Tratamiento Farmacológico de COVID-19 , Glicoproteína de la Espiga del Coronavirus , Humanos , Unión Proteica , SARS-CoV-2RESUMEN
By searching the randomized controlled trials (RCTs) of acupuncture and moxibustion from CNKI since its inception date to december 31 of 2019, the development status and hot trend of RCTs of acupuncture and moxibustion in China were summarized. The CiteSpace and VOSviewer software were used to perform keyword co-occurrence analysis, clustering analysis, time-zone analysis and citation-burst analysis, and visual map was drawn. As a result, a total of 60 995 articles were included, which were published in 1027 academic journals with 1787 keywords. The publication date was from 1975 to 2019. During the past 45 years, the publications of RCTs on acupuncture and moxibustion had shown an overall growth trend with characteristics of the times. The RCTs of manual acupuncture ranked the top, and its proportion of publications every 5 years was stable in the past 30 years. Since 1994, the hot words such as electroacupuncture, warming needling, auricular point sticking and various acupoint therapies had emerged; meanwhile, the spectrum of diseases had broadened, and an evolutionary trend corresponding to therapies and disease systems had been formed. In recent decade, the RCTs using moxibustion therapy have increased significantly, and the hot words such as "sub-health" "winter diseases being treated in summer" and "acupoint application/ moxibustion during the dog days" had indicated that acupuncture clinical research was further inclined to the field of chronic disease prevention and health services, which was in line with social development and the needs of the times.
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Terapia por Acupuntura , Acupuntura , Moxibustión , Puntos de Acupuntura , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Given the relentless renewal ability of intestinal crypt-base stem cells, small intestine in the gastrointestinal (GI) tract is more vulnerable to radiation-induced disruption. Through promoting epithelial integrity and reducing intracellular reactive oxygen species (ROS) levels, hypoxia-inducible factors (HIFs) have been proved to exhibit radioprotective effects in the GI tract. Therefore, enhancing stability or transcriptional activity of HIFs might be a therapeutic strategy for developing radioprotectors. Factor inhibiting HIF (FIH or HIF-1AN) can hamper transcriptional capacity of HIF-1α via interacting with Asn803 in its C-terminal domain. Previously, we discovered promoting HIF-1α transcriptional activity in vitro by FIH inhibitor-N-oxalyl-D-phenylalanine (NOFD) exerts radioprotection on cells. However, the radioprotective effect of FIH inhibitor on the GI tract and its competing endogenous RNA (ceRNA) regulatory network from the FIH/HIF axis has never been addressed. Here we verified radioprotection of NOFD for the GI tract by an animal model and performed whole-transcriptome analysis to fully elucidate the radioprotective mechanism from the FIH/HIF axis against GI syndrome. We identified two novel circular RNAs (circRNAs) (circRNA_2909 and circRNA_0323) and two long non-coding RNAs (lncRNAs) (NONMMUT140549.1 and NONMMUT148249.1) that promote expression of HIF1A and NOS2 in the HIF-1 pathway by sponging microRNAs (miRNAs), especially mmu-miR-92a-1-5p. The de-repression of HIF-1α transcriptional capacity by inhibiting FIH proteomic activity suggests a new therapeutic strategy in alleviating radiation-induced GI syndrome.
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BACKGROUND: The aim of this research is to further evaluate the efficacy and safety of percutaneous coronary intervention (PCI) in patients with acute myocardial infarction (AMI) complicated with chronic renal insufficiency (CRI) by meta-analysis, to provide scientific and effective medical evidence for PCI in patients with AMI complicated with CRI, and to support the clinical application of PCI. METHODS: Electronic databases will be searched, including PubMed, Cochrane Library, Embase, CNKI, CBM, VIP, and Wanfang Data. Patients with AMI complicated by renal insufficiency treated with PCI will be included. The retrieval time is from inception to January 2019. The inclusion and exclusion criteria are formulated to search only the relevant literature. Endnote software management for literature will be adopted. The literature will be independently screened by 2 researchers. Excel 2016 will be applied to extract literature data with the "Research Information Registration Form." The final selected literature will be assessed for bias risk. Stata 12.0 software will be used for the meta-analysis. RESULTS: The systematic evaluation and meta-analysis will be carried out strictly in accordance with the requirements of the Cochrane System Evaluator Manual 5.3 on meta-analyses, which will provide a high-quality evaluation of the clinical efficacy and safety of PCI in patients with AMI and CRI. ETHICS AND DISSEMINATION: This study belongs to the category of systematic reviews, not clinical trials. Therefore, it does not require ethical approval. The results of this study will be published in influential international academic journals related to this topic. CONCLUSION: PCI is an effective and safe treatment for patients with AMI and CRI. This study will provide a definite evidence-based medical conclusion and provide a scientific basis for the clinical treatment of patients with AMI and CRI. PROSPERO REGISTRATION NUMBER: CRD42019131367.
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Infarto del Miocardio/cirugía , Intervención Coronaria Percutánea/métodos , Insuficiencia Renal Crónica/etiología , Femenino , Humanos , Masculino , Infarto del Miocardio/complicaciones , Intervención Coronaria Percutánea/efectos adversos , Proyectos de Investigación , Resultado del Tratamiento , Metaanálisis como AsuntoRESUMEN
Radiation-induced damage to the mitochondrial macromolecules and electron transfer chain (ETC), causing the generation of primary and secondary reactive oxygen (ROS) species. The continuous ROS production after radiation will trigger cell oxidative stress and ROS-mediated nucleus apoptosis and autophagy signaling pathways. Scavenging radiation-induced ROS effectively can help mitochondria to maintain their physiological function and relief cells from oxidative stress. Nicotinamide is a critical endogenous antioxidant helping to neutralize ROS in vivo. In this study, we designed and synthetized a novel mitochondrial-targeted dihydronicotinamide (Mito-N) with the help of mitochondrial membrane potential to enter the mitochondria and scavenge ROS. According to experiment results, Mito-N significantly increased cell viability by 30.75% by neutralizing the accumulated ROS and resisting DNA strands breaks after irradiation. Furthermore, the mice survival rate also improved with the treatment of Mito-N, by effectively ameliorating the hematopoietic system infliction under lethal dose irradiation.
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Mitocondrias/efectos de los fármacos , Niacinamida/análogos & derivados , Protectores contra Radiación/farmacología , Animales , Antioxidantes/farmacología , Células CHO , Cricetinae , Cricetulus , Diseño de Fármacos , Masculino , Ratones Endogámicos C57BL , Niacinamida/farmacología , Estrés Oxidativo/efectos de los fármacos , Traumatismos Experimentales por Radiación/prevención & control , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pyrogenic carbonaceous matter (PCM) catalyzes the transformation of a range of organic pollutants by sulfide in water; however, the mediation mechanisms are not fully understood. In this study, we observed for the first time that the degradation of azo dyes by sulfide initially underwent a lag phase followed by a fast degradation phase. Interestingly, the presence of PCM only reduced the lag phase length of the azo dye decolorization but did not significantly enhance the reaction rate in the fast degradation phase. An analysis of the azo dye reduction and polysulfide formation indicated that PCM facilitated the transformation of sulfide into polysulfides, including disulfide and trisulfide, resulting in fast azo dye reduction. Moreover, the oxygen functional groups of the PCM, especially the quinones, may play an important role in the transformation of sulfide into polysulfides by accelerating the electron transfer. The results of this study provide a better understanding of the PCM-mediated abiotic transformation of organic pollutants by sulfide in anaerobic aqueous environments.
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Compuestos Azo , Sulfuros , Colorantes , Transporte de Electrón , Oxidación-Reducción , QuinonasRESUMEN
BACKGROUND: Previous studies have shown that miR-144-3p might be a potential biomarker in non-small cell lung cancer (NSCLC). Nevertheless, the comprehensive mechanism behind the effects of miR-144-3p on the origin, differentiation, and apoptosis of NSCLC, as well as the relationship between miR-144-3p and clinical parameters, has been rarely reported. METHODS: We investigated the correlations between miR-144-3p expression and clinical characteristics through data collected from Gene Expression Omnibus (GEO) microarrays, the relevant literature, The Cancer Genome Atlas (TCGA), and real-time quantitative real-time PCR (RT-qPCR) analyses to determine the clinical role of miR-144-3p in NSCLC. Furthermore, we investigated the biological function of miR-144-3p by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Protein-protein interaction (PPI) network was created to identify the hub genes. RESULTS: From the comprehensive meta-analysis, the combined SMD of miR-144-3p was - 0.95 with 95% CI of (- 1.37, - 0.52), indicating that less miR-144-3p was expressed in the NSCLC tissue than in the normal tissue. MiR-144-3p expression was significantly correlated with stage, lymph node metastasis and vascular invasion (all P < 0.05). As for the bioinformatics analyses, a total of 37 genes were chosen as the potential targets of miR-144-3p in NSCLC. These promising target genes were highly enriched in various key pathways such as the protein digestion and absorption and the thyroid hormone signaling pathways. Additionally, PPI revealed five genes-C12orf5, CEP55, E2F8, STIL, and TOP2A-as hub genes with the threshold value of 6. CONCLUSIONS: The current study validated that miR-144-3p was lowly expressed in NSCLC. More importantly, miR-144-3p might function as a latent tumor biomarker in the prognosis prediction for NSCLC. The results of bioinformatics analyses may present a new method for investigating the pathogenesis of NSCLC.
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Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Análisis de Secuencia de ARN/métodos , Anciano , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Bases de Datos Genéticas , Regulación hacia Abajo/fisiología , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad , Análisis por Matrices de Proteínas/métodos , Mapas de Interacción de Proteínas/fisiologíaRESUMEN
MicroRNAs (miRNAs or miRs) contribute to the development of various malignant neoplasms, including glioblastoma multiforme (GBM). The present study aimed to explore the pathogenesis of GBM and to identify latent therapeutic agents for patients with GBM, based on an in silico analysis. Gene chips that provide miRNA expression profiling in GBM were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs (DEMs) were also determined via the RobustRankAggreg algorithm. The target genes of DEMs were predicted and then intersected with GBMassociated genes that were collected from the Gene Expression Profiling Interactive Analysis. Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the overlapping genes were then performed. Simultaneously, a connectivity map (CMap) analysis was performed to screen for potential therapeutic agents for GBM. A total of 10 DEMs (hsamiR196a, hsamiR10b, hsamiR196b, hsamiR18b, hsamiR5423p, hsamiR1293p, hsamiR12245p, hsamiR8763p and hsamiR7705p) were obtained from three GEO gene chips (GSE25631, GSE42657 and GSE61710). Then, 1,720 target genes of the 10 miRNAs and 4,185 differently expressed genes in GBM were collected. By intersecting the aforementioned gene clusters, the present study identified 390 overlapping genes. GO and KEGG analyses of the 390 genes demonstrated that these genes were involved in certain cancerassociated biological functions and pathways. Eight genes [(GTPase NRas (NRAS), calcium/calmodulindependent protein kinase type II subunit Gamma (CAMK2G), plateletderived growth factor receptor alpha (PDGFRA), calmodulin 3 (CALM3), cyclindependent kinase 6 (CDK6), calcium/calmodulindependent protein kinase type II subunit beta (CAMK2B), retinoblastomaassociated protein (RB1) and protein kinase C beta type (PRKCB)] that were centralized in the glioma pathway were selected for CMap analysis. Three chemicals (W13, gefitinib and exemestane) were identified as putative therapeutic agents for GBM. In summary, the present study identified three miRNAbased chemicals for use as a therapy for GBM. However, more experimental data are needed to verify the therapeutic properties of these latent drugs in GBM.
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Neoplasias Encefálicas/tratamiento farmacológico , Descubrimiento de Drogas/métodos , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , MicroARNs/metabolismo , Androstadienos/química , Androstadienos/farmacología , Androstadienos/uso terapéutico , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Biología Computacional , Gefitinib/química , Gefitinib/farmacología , Gefitinib/uso terapéutico , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/genética , Glioblastoma/genética , Glioblastoma/patología , Humanos , Simulación del Acoplamiento Molecular , Terapia Molecular Dirigida/métodos , Sulfonamidas/química , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Transcriptoma/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Pharmacological control against ovarian serous cystadenocarcinoma has received increasing attention. The purpose of this study was to investigate multi-drug treatments as synergetic therapy for ovarian serous cystadenocarcinoma and to explore their mechanisms of action by the network pharmacology method. METHODS: Genes acting on ovarian serous cystadenocarcinoma were first collected from GEPIA and DisGeNET. Gene Ontology annotation, Kyoto Encyclopedia of Genes and Genomes pathway, Reactome pathway, and Disease Ontology analyses were then conducted. A connectivity map analysis was employed to identify compounds as treatment options for ovarian serous cystadenocarcinoma. Targets of these compounds were obtained from the Search Tool for Interacting Chemicals (STITCH). The intersections between the ovarian serous cystadenocarcinoma-related genes and the compound targets were identified. Finally, the Kyoto Encyclopedia of Genes and Genomes and Reactome pathways in which the overlapped genes participated were selected, and a correspondence compound-target pathway network was constructed. RESULTS: A total of 541 ovarian serous cystadenocarcinoma-related genes were identified. The functional enrichment and pathway analyses indicated that these genes were associated with critical tumor-related pathways. Based on the connectivity map analysis, five compounds (resveratrol, MG-132, puromycin, 15-delta prostaglandin J2, and valproic acid) were determined as treatment agents for ovarian serous cystadenocarcinoma. Next, 48 targets of the five compounds were collected. Following mapping of the 48 targets to the 541 ovarian serous cystadenocarcinoma-related genes, we identified six targets (PTGS1, FOS, HMOX1, CASP9, PPARG, and ABCB1) as therapeutic targets for ovarian serous cystadenocarcinoma by the five compounds. By analysis of the compound-target pathway network, we found the synergistic anti-ovarian serous cystadenocarcinoma potential and the underlying mechanisms of action of the five compounds. CONCLUSION: In summary, latent drugs against ovarian serous cystadenocarcinoma were acquired and their target actions and pathways were determined by the network pharmacology strategy, which provides a new prospect for medicamentous therapy for ovarian serous cystadenocarcinoma. However, further in-depth studies are indispensable to increase the validity of this study.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Cistadenocarcinoma Seroso/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Epitelial de Ovario/genética , Estudios de Cohortes , Cistadenocarcinoma Seroso/genética , Sistemas de Liberación de Medicamentos/tendencias , Femenino , Redes Reguladoras de Genes/genética , Humanos , Células MCF-7 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Resultado del TratamientoRESUMEN
Mitochondrial dysfunction plays an important role in gamma-radiation-induced mediating oxidative stress. Scavenging radiation-induced reactive oxygen species (ROS) can help mitochondria to maintain their physiological function. Rosmarinic acid is a polyphenol antioxidant that can scavenge radiation-induced ROS, but the structure prevents it from accumulating in mitochondria. In this study, we designed and synthesized a novel rosmarinic acid derivative (Mito-RA) that could use the mitochondrial membrane potential to enter the organelle and scavenge ROS. The DCFH-DA assay revealed that Mito-RA was more effective than rosmarinic acid at scavenging ROS. DNA double-strand breaks, chromosomal aberration, micronucleus and comet assays demonstrated the ability of Mito-RA to protect against radiation-induced oxidative stress in vitro. These findings demonstrate the potential of Mito-RA as an antioxidant, which can penetrate mitochondria, scavenge ROS and protect cells against radiation-induced oxidative damage.
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Cinamatos/administración & dosificación , Daño del ADN/fisiología , Depsidos/administración & dosificación , Mitocondrias/fisiología , Estrés Oxidativo/fisiología , Protectores contra Radiación/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/administración & dosificación , Células CHO , Aberraciones Cromosómicas/efectos de los fármacos , Aberraciones Cromosómicas/efectos de la radiación , Cinamatos/síntesis química , Cricetulus , Daño del ADN/efectos de los fármacos , Depsidos/síntesis química , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Potencial de la Membrana Mitocondrial/efectos de la radiación , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Protectores contra Radiación/síntesis química , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/efectos de la radiación , Ácido RosmarínicoRESUMEN
BACKGROUND: Current regulations and recommendations proposed for the production of vaccines in continuous cell lines of any origin demand that these be free of exogenous viruses, particularly retroviruses. Recently, the ultra-sensitive product-enhanced reverse transcriptase (PERT) assay can be used to detect minute of reverse transcriptase (RTase) in single retroviral particle and is 10(6) times more sensitive than the conventional RTase assays. However, coincidental with this increase in sensitivity is an increase in false-positive reactions derived from contaminating cellular DNA polymerases, which are known to have RTase-like activities. OBJECTIVES: To develop a modified single-tube one-step PERT (mSTOS-PERT) assay with improvements on decreasing significantly the level of false-positive reactions, and to evaluate the mSTOS-PERT assay for sensitivity and specificity. STUDY DESIGN: Ampliwaxtrade mark was used to compartmentalize the reverse transcription (RT) and PCR step in the same micro-tube with more efficiency and reproducibility, while maintaining the high sensitivity. The DNA amplification products were separated by 2% agarose gel electrophoresis, and then analyzed by non-isotopic Southern blot hybridization. A wide variety of cell lines used in biologicals production were detected to validate the improved mSTOS-PERT assay. RESULTS: The detection limit for the mSTOS-PERT assay was at least 10(-9)U, when using AMV-RTase as a positive control. Furthermore, heparin involvement in the RT step can eliminate completely the false-positive PERT signals which are exhibited by cellular polymerases such as DNA-dependent DNA polymerase alpha, gamma released by cell death. Most mammalian cells (MRC-5, Vero, WISH, 2BS, RK-13, MDCK, etc.) are PERT-negative in cell supernatants. Some PERT-positive signals in cell lysates were found to be introduced by the cellular DNA polymerases and could be inhibited specifically by heparin. Chick cells derived from either chick embryo fibroblasts (CEF) or allantoic fluid from SPF embryonated eggs, murine hybridoma cell SP2/0, etc., contained authentic RTase activities, which could not be inactivated by heparin. CONCLUSIONS: The improved mSTOS-PERT assay described here may distinguish the genuine RTase activity from cellular polymerases with high sensitivity and specificity, and is rapid and easy to perform to screen for the possible contamination of minute retroviruses in the cell substrates used in vaccine production.