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Background: Empagliflozin has been shown in clinical studies to lower the risk of adverse cardiovascular events. Using proteomics, the current study aims to determine whether empagliflozin reduces aortic alterations in obese mice and to investigate its molecular mechanism of action. Methods: We constructed obese mice and then treated them with empagliflozin. Changes in the weight of the mice were recorded. Blood glucose and lipid levels were measured in each group of mice, and changes in pulse wave velocity and aortic structure were recorded. In addition, changes in aortic protein expression were detected by proteomics and analyzed bioinformatically. Results: Proteomics results showed that 507 differentially expressed proteins (DEPs) were identified in the comparison of normal and obese mice, while 90 DEPs were identified in the comparison of obese and empagliflozin-treated mice. Examination of these three groups revealed that DEPs were largely associated with the digestion of unsaturated fats. Among them, empagliflozin significantly reduced the expression of fatty acid synthase (FASN), acyl-CoA desaturase 3 (SCD3), ACSL1. and ACSL5 in the aorta of obesity-induced mice, and there was a close relationship between the four. Conclusion: Empagliflozin reduced the protein expression of FASN, SCD3, ACSL1, and ACSL5 in the aorta of obese mice and improved aortic fatty acid metabolism and reduced vascular stiffness for vasoprotective effects.
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KEY MESSAGE: A 4.43-Kb structural variation in the sesame genome results in the deletion of the Siofp1 gene and induces the long capsule length trait. Capsule length (CL) has a positive effect on seed weight and yield in various agronomically important species; however, the molecular mechanism underlying long capsule trait regulation in sesame remains unknown. The inheritance analysis showed that long capsule traits (CL > 4.0 cm) were dominant over normal length (average CL = 3.0 cm) and were controlled by a single gene pair. Association mapping with a RIL population and 259 natural sesame germplasm accessions indicated that the target interval was 52,830-730,961 bp of SiChr.10 in sesame. Meanwhile, the structural variation (SV) of the association mapping revealed that only SV_414325 on chromosome 10 was significantly associated with the CL trait, with a P value of 1.1135E-19. SV_414325 represents a 4430-bp deletion from 414,325 to 418,756 bp on SiChr.10, covering Sindi_2155000 (named SiOFP1). In the normal length type, Siofp1 encodes 411 amino acids of the ovate family proteins and is highly expressed in the leaf, stem, bud, and capsule tissues of sesame. In accordance with the transcriptional repressor character, Siofp1 overexpression in transgenic Arabidopsis (T0 and T1 generations) induced a 25-39% greater shortening of silique length than the wild type (P < 0.05), as well as round cauline leaves and short carpels. These results confirm that SiOFP1 plays a key role in regulating CL trait in sesame and other flowering plants. These findings provide a theoretical and material basis for sesame capsule development and high-yield breeding research.
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Sesamum , Sesamum/genética , Mapeo Cromosómico/métodos , Fitomejoramiento , Fenotipo , Patrón de HerenciaRESUMEN
BACKGROUND Depression is a common disease worldwide, with about 280 million people having depression. The unique facial features of depression provide a basis for automatic recognition of depression with deep convolutional neural networks. MATERIAL AND METHODS In this study, we developed a depression recognition method based on facial images and a deep convolutional neural network. Based on 2-dimensional images, this method quantified the binary classification problem and distinguished patients with depression from healthy participants. Network training consisted of 2 steps: (1) 1020 pictures of depressed patients and 1100 pictures of healthy participants were used and divided into a training set, test set, and validation set at the ratio of 7: 2: 1; and (2) fully connected convolutional neural network (FCN), visual geometry group 11 (VGG11), visual geometry group 19 (VGG19), deep residual network 50 (ResNet50), and Inception version 3 convolutional neural network models were trained. RESULTS The FCN model achieved an accuracy of 98.23% and a precision of 98.11%. The Vgg11 model achieved an accuracy of 94.40% and a precision of 96.15%. The Vgg19 model achieved an accuracy of 97.35% and a precision of 98.13%. The ResNet50 model achieved an accuracy of 94.99% and a precision of 98.03%. The Inception version 3 model achieved an accuracy of 97.10% and a precision of 96.20%. CONCLUSIONS The results show that deep convolution neural networks can support the rapid, accurate, and automatic identification of depression.
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Depresión , Redes Neurales de la Computación , Depresión/diagnóstico por imagen , HumanosRESUMEN
Objectives: Glioblastoma is the most common primary malignant brain tumor in adults and can be treated with radiation therapy. However, tumor target contouring for head radiation therapy is labor-intensive and highly dependent on the experience of the radiation oncologist. Recently, autosegmentation of the tumor target has been playing an increasingly important role in the development of radiotherapy plans. Therefore, we established a deep learning model and improved its performance in autosegmenting and contouring the primary gross tumor volume (GTV) of glioblastomas through transfer learning. Methods: The preoperative MRI data of 20 patients with glioblastomas were collected from our department (ST) and split into a training set and testing set. We fine-tuned a deep learning model for autosegmentation of the hippocampus on separate MRI scans (RZ) through transfer learning and trained this deep learning model directly using the training set. Finally, we evaluated the performance of both trained models in autosegmenting glioblastomas using the testing set. Results: The fine-tuned model converged within 20 epochs, compared to over 50 epochs for the model trained directly by the same training set, and demonstrated better autosegmentation performance [Dice similarity coefficient (DSC) 0.9404 ± 0.0117, 95% Hausdorff distance (95HD) 1.8107 mm ±0.3964mm, average surface distance (ASD) 0.6003 mm ±0.1287mm] than the model trained directly (DSC 0.9158±0.0178, 95HD 2.5761 mm ± 0.5365mm, ASD 0.7579 mm ± 0.1468mm) with the same test set. The DSC, 95HD, and ASD values of the two models were significantly different (P<0.05). Conclusion: A model developed with semisupervised transfer learning and trained on independent data achieved good performance in autosegmenting glioblastoma. The autosegmented volume of glioblastomas is sufficiently accurate for radiotherapy treatment, which could have a positive impact on tumor control and patient survival.
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Transition-metal indenyl complexes usually exhibit different reactivities compared with their cyclopentadienyl analogues. Up to now, at least 10 metal-indenyl bonding modes have been reported. Because of the "indenyl effect", transition-metal indenyl complexes usually show enhanced reactivity in substitution and related reactions. This review provides an overview on the use and impact of indenyl phosphines in organometallic chemistry and transition-metal-catalysed reactions in the recent two decades. Some cationic and zwitterionic metal complexes supported by P,N-substituted indene or indenide ligands are described. They have been reported to induce the cleavage of E-H (E = H, Si and B) bonds and can be used as catalysts for addition of E-H bonds to unsaturated substrates. 2-Aryl indenyl phosphine ligands L3-L11 have been proven to be a class of versatile ligands for palladium-catalysed C-C and C-N cross-coupling reactions. Moreover, optically active tethered indenyl phosphine ligands can have better stereoselective control over the chirality arising at the metal center in the oxidative addition of their rhodium complexes with alkyl halides.
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BACKGROUND: It is known to all that the incidence of insomnia is becoming higher and higher with the increase of people's life stress. To some extent, it has brought about bad effects on people's life, work, study, and health, such as mental exhaustion, low work efficiency, and mood irritability. Now there are medications and non-medications methods for insomnia. As one of the treatments for insomnia, western medicine is to prolong the sleeping time and improve the anxious mood. However, taking western medicine to treat insomnia can also be accompanied by some adverse reactions at the same time, such as drug dependence, an allergic reaction, and so on. Traditional Chinese medicine therapy is based on syndrome differentiation and holistic concept. Shumian capsules (SM) are a kind of proprietary Chinese medicine for insomnia, which have the effect of relieving depression and calming the mind. But there are no studies on the efficacy and safety of SM in the treatment of insomnia. Therefore, I will provide a systematic review and meta-analysis to evaluate the efficacy and safety of SM for insomnia. METHODS: All the studies searched were from PubMed, EMBASE, Web of Science, Cochrane Library, Chinese National Knowledge Infrastructure, and WanFang databases, and the studies types included in the analysis were all randomized controlled trials. All the retrieval contents were completed independently by 2 researchers, and a third reviewer would be involved when there existed any disagreement. The eligible studies were screened out according to the inclusion criteria and exclusion criteria, and some useful information was extracted and made into a feature table, including the year of the included studies, the age, and disease course of the participants in the studies and intervention methods, etc. Cochrane risk-of-bias tool was used to evaluate the quality of literature and meta-analysis was conducted by RevMan 5.4 software. RESULTS: A total of 9 articles including 709 participants were included in the study after screening out. The primary outcomes of statistical analysis were cure rate and total effective rate, while the secondary outcomes included Pittsburgh sleep quality index score and incidence of adverse reactions. The results showed that Pittsburgh sleep quality index score of the SM group and Western medicine group were statistically significant (MDâ=â-0.50, 95% confidence interval [CI]â=â[-0.78, -0.22], Pâ=â.0005). The total effective rate of the SM group was slightly higher than that of the Western medicine group, but there was no statistical significance (relative risk [RR]â=â1.03, 95% CIâ=â[0.95,1.13], Pâ=â.43). CONCLUSION: This meta-analysis provides evidence for the efficacy and safety of SM in the treatment of insomnia, and provides a new idea for the clinical treatment of insomnia. But more research is needed to support further evidence.
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Medicamentos Herbarios Chinos/uso terapéutico , Medicina Tradicional China/métodos , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Cápsulas , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/efectos adversos , Humanos , Sueño , Calidad del SueñoRESUMEN
An efficient C-P coupling reaction of enantiopure tert-butylmethylphosphine-boranes with aryl and heteroaryl halides is developed by using Pd(OAc)2/dppf as a catalyst, affording a series of (S) or (R)-P-chiral phosphines in moderate to high yields and with ee values up to 99% ee. Moreover, the reaction time could be reduced from 72 h to 6 h with increased ee values under microwave irradiation.
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BACKGROUND: Acute infection leads to substantial mortality in the nonagenarian population. However, the predictive efficacies of functional status and biochemical indexes for in-hospital mortality in these patients remain to be determined. METHODS: A single-center, retrospective cohort study was performed. Consecutive nonagenarian patients who were admitted to our department from January 1, 2014 to December 31, 2016 for acute infectious diseases were included. Baseline data for medical history, functional status, and biochemical indexes were obtained on admission. The outcomes of these patients during hospitalization were recorded. Predictors of in-hospital mortality were identified via logistic regression analyses. RESULTS: A total of 162 patients were included, and 46 patients died (17.2%) during hospitalization. Univariate analysis showed that the prevalence rates of atrial fibrillation (32.1%) and malignant disease (26.5%) were higher in nonagenarian patients who died during hospitalization than in those who discharged. Multivariate logistic regression analyses identified malignant disease (odds ratio [OR] 2.73, 95% confidence interval [CI]: 1.10-6.78), ADL category (OR 0.82, 95% CI: 0.75-0.89) and serum albumin (OR 0.86, 95%CI 0.78-0.95) as independent predictors of in-hospital mortality in nonagenarian patients hospitalized for acute infection. CONCLUSIONS: Functional impairment as well as serum albumin may be independent predictors of in-hospital mortality in nonagenarian patients hospitalized for acute infectious diseases. Stratification of patients according to Barthel Index score and serum albumin is very necessary.
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Enfermedades Transmisibles/sangre , Enfermedades Transmisibles/mortalidad , Mortalidad Hospitalaria/tendencias , Albúmina Sérica Humana/metabolismo , Enfermedad Aguda , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios de Cohortes , Enfermedades Transmisibles/diagnóstico , Femenino , Hospitalización/tendencias , Humanos , Masculino , Alta del Paciente/tendencias , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Factores de RiesgoRESUMEN
To comparatively evaluate chemoradiotherapy (CRT) and chemotherapy (CT) for the treatment of locally advanced pancreatic cancer (LAPC) by meta-analysis.A literature search was performed until August 2016 to identify comparative studies assessing survival rates and complications. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined with the fixed or random effects model.Five randomized controlled trials (RCTs) met the defined inclusion criteria. A total of 593 patients were included, with 295 and 298 treated with CRT and CT, respectively. Overall survival showed no statistically significant difference in patients treated with CRT and CT at 6, 12, 18, and 24 months (respectively: ORâ=â1.13, 95% CI: 0.60-2.17; ORâ=â1.15, 95% CI: 0.53-2.52; ORâ=â1.13, 95% CI: 0.43-2.95; ORâ=â1.07, 95% CI: 0.67-1.72). Meanwhile, CRT had higher rates of grade 3 to 4 adverse events (nausea and vomiting, ORâ=â2.74, 95% CI: 1.36-5.52; diarrhea, ORâ=â4.28, 95% CI: 1.16-15.71).The data are not sufficient to change from CT to CRT in the treatment of patients with LAPC and thus clinical discretion is required until more data is accumulated.
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Antineoplásicos/uso terapéutico , Quimioradioterapia , Neoplasias Pancreáticas/terapia , Humanos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
OBJECTIVE: This study is to investigate the effect of ß-adrenergic receptor kinase inhibitor (Adeno-ßARKct) on heart failure (HF) rat model. METHODS: Male SD rats weighted 250 g undertaken ligation of the left anterior descending branch of coronary artery to establish HF model. Survival rats were randomly divided into experimental treated group (EXP) and control treated group (CONT). Additionally, pseudo-operated rats were taken as sham-ligated group (SHAM). Adeno-ßARKct particles or Adeno empty vector was injected to the rats. Multiple indicators including left ventricular ejection fraction (LVEF), index of hemodynamics, plasma and myocardial tissue catecholamine, NT-ProBNP, mRNA levels of ß1AR, ß2AR and ßARK1, and protein level of ßARK1 were measured 4 weeks later. RESULTS: Compared with rats in SHAM group, levels of LVEF, ±dp/dt max, and catecholamine in myocardial tissue were lower while plasma NT-ProBNP and plasma catecholamine were higher in rats of EXP and CONT. Additionally, ß1AR and ß2AR mRNA expressions were downregulated whereas ßARK1 mRNA and ßARK1 protein levels were upregulated in EXP and CONT. Compared with CONT, the levels of LVEF, -dp/dt max, and catecholamine in myocardial tissue were higher, while plasma NT-ProBNP and plasma catecholamine were lower in EXP. ß1AR and ß2AR mRNA expressions were upregulated, whereas ßARK1 mRNA expression and ßARK1 protein levels were downregulated in EXP. CONCLUSION: In vivo delivery of Adeno-ßARKct by caudal vein is feasible and can improve cardiac function in rats with HF after myocardial infarction.
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AIM: The study aims to compare the ability of CHA2DS2-VASc (defined as congestive heart failure, hypertension, age ≥75 years [two scores], type 2 diabetes mellitus, previous stroke, transient ischemic attack, or thromboembolism [TE] [doubled], vascular disease, age 65-74 years, and sex category) and CHADS2 (defined as congestive heart failure, hypertension, age ≥75 years, type 2 diabetes mellitus, previous stroke [doubled]) scores to predict the risk of ischemic stroke (IS) or TE among patients with nonvalvular atrial fibrillation (NVAF). METHODS: A total of 413 patients with NVAF aged ≥65 years, and not on oral anticoagulants for the previous 6 months, were enrolled in the study. The predictive value of the CHA2DS2-VASc and CHADS2 scores for IS/TE events was evaluated by the Kaplan-Meier method. RESULTS: During a follow-up period of 1.99±1.29 years, 104 (25.2%) patients died and 59 (14.3%) patients developed IS/TE. The CHADS2 score performed better than the CHA2DS2-VASc score in predicting IS/TE as assessed by c-indexes (0.647 vs 0.615, respectively; P<0.05). Non-CHADS2 risk factors, such as vascular disease and female sex, were not found to be predictive of IS/TE (hazard ratio 1.518, 95% CI: 0.832-2.771; hazard ratio 1.067, 95% CI: 0.599-1.899, respectively). No differences in event rates were found in patients with the CHADS2 scores of 1 and 2 (7.1% vs 7.8%). It was observed that patients with a CHADS2 score of ≥3 were most in need of anticoagulation therapy. CONCLUSION: In patients with NVAF aged ≥65 years, the CHADS2 score was found to be significantly better in predicting IS/TE events when compared to the CHA2DS2-VASc score. Patients with a CHADS2 score of ≥3 were associated with high risk of IS/TE events.
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Fibrilación Atrial/complicaciones , Accidente Cerebrovascular/etiología , Anciano , Femenino , Humanos , Masculino , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Medición de Riesgo , Factores de RiesgoRESUMEN
OBJECTIVES: The objective was to determine who will benefit most from oral anticoagulation (OAC) for non-valvular atrial fibrillation (NVAF) patients aged ≥ 75 years. BACKGROUND: It was unclear whether all of NVAF aged ≥ 75 should receive OAC. METHODS: We recruited NVAF ≥ 75 years without QAC who were divided into three groups according to CHA2DS-VASc scores. The clinical endpoints were ischemic stroke (IS), thromboembolism (TE), or death. RESULTS: The patients with CHA2DS2-VASc score of 6-9 were not appropriate for anticoagulation with the highest HAS-BLED scores. CHA2DS2-VASc of 2 had little risk for IS/TE. We further divided CHA2DS2-VASc of 3-5 into three subgroups with estimated glomerular filtration rate (eGFR; ml/min/1.73 m(2)): < 30, 30-60, and > 60. The patients with eGFR < 30 had the highest bleeding risk with a comparable IS/TE. CONCLUSIONS: NVAF with CHA2DS2-VASc of 3-5 and eGFR > 30 ml/min/1.73 m(2) represent the most appropriate population for anticoagulation.
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Seed coat color is an important agronomic trait in sesame, as it is associated with seed biochemical properties, antioxidant content and activity and even disease resistance of sesame. Here, using a high-density linkage map, we analyzed genetic segregation and quantitative trait loci (QTL) for sesame seed coat color in six generations (P1, P2, F1, BC1, BC2 and F2). Results showed that two major genes with additive-dominant-epistatic effects and polygenes with additive-dominant-epistatic effects were responsible for controlling the seed coat color trait. Average heritability of the major genes in the BC1, BC2 and F2 populations was 89.30%, 24.00%, and 91.11% respectively, while the heritability of polygenes was low in the BC1 (5.43%), in BC2 (0.00%) and in F2 (0.89%) populations. A high-density map was constructed using 724 polymorphic markers. 653 SSR, AFLP and RSAMPL loci were anchored in 14 linkage groups (LG) spanning a total of 1,216.00 cM. The average length of each LG was 86.86 cM and the marker density was 1.86 cM per marker interval. Four QTLs for seed coat color, QTL1-1, QTL11-1, QTL11-2 and QTL13-1, whose heritability ranged from 59.33%-69.89%, were detected in F3 populations using CIM and MCIM methods. Alleles at all QTLs from the black-seeded parent tended to increase the seed coat color. Results from QTLs mapping and classical genetic analysis among the P1, P2, F1, BC1, BC2 and F2 populations were comparatively consistent. This first QTL analysis and high-density genetic linkage map for sesame provided a good foundation for further research on sesame genetics and molecular marker-assisted selection (MAS).
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Mapeo Cromosómico , Pigmentación/genética , Sitios de Carácter Cuantitativo/genética , Semillas/genética , Sesamum/genética , Algoritmos , Análisis de Varianza , Cruzamientos Genéticos , Genética de Población , Funciones de Verosimilitud , Modelos Genéticos , Fenotipo , Carácter Cuantitativo HeredableRESUMEN
BACKGROUND: Sesame (Sesamum indicum L.) is one of the most important oil crops; however, a lack of useful molecular markers hinders current genetic research. We performed transcriptome sequencing of samples from different sesame growth and developmental stages, and mining of genic-SSR markers to identify valuable markers for sesame molecular genetics research. RESULTS: In this study, 75 bp and 100 bp paired-end RNA-seq was used to sequence 24 cDNA libraries, and 42,566 uni-transcripts were assembled from more than 260 million filtered reads. The total length of uni-transcript sequences was 47.99 Mb, and 7,324 SSRs (SSRs ≥15 bp) and 4,440 SSRs (SSRs ≥18 bp) were identified. On average, there was one genic-SSR per 6.55 kb (SSRs ≥15 bp) or 10.81 kb (SSRs ≥18 bp). Among perfect SSRs (≥18 bp), di-nucleotide motifs (48.01%) were the most abundant, followed by tri- (20.96%), hexa- (25.37%), penta- (2.97%), tetra- (2.12%), and mono-nucleotides (0.57%). The top four motif repeats were (AG/CT)n [1,268 (34.51%)], (CA/TG)n [281 (7.65%)], (AT/AT)n [215 (5.85%)], and (GAA/TTC)n [131 (3.57%)]. A total of 2,164 SSR primer pairs were identified in the 4,440 SSR-containing sequences (≥18 bp), and 300 SSR primer pairs were randomly chosen for validation. These SSR markers were amplified and validated in 25 sesame accessions (24 cultivated accessions, one wild species). 276 (92.0%) primer pairs yielded PCR amplification products in 24 cultivars. Thirty two primer pairs (11.59%) exhibited polymorphisms. Moreover, 203 primer pairs (67.67%) yielded PCR amplicons in the wild accession and 167 (60.51%) were polymorphic between species. A UPGMA dendrogram based on genetic similarity coefficients showed that the correlation between genotype and geographical source was low and that the genetic basis of sesame in China is narrow, as previously reported. The 32 polymorphic primer pairs were validated using an F2 mapping population; 18 primer pairs exhibited polymorphisms between the parents, and 14 genic-SSRs could be integrated into 9 main linkage groups. CONCLUSIONS: 2,164 genic-SSR markers have been developed in sesame using transcriptome sequencing. 276 of 300 validated primer pairs successfully yielded PCR amplicons in 24 cultivated sesame accessions. These markers increase current SSR marker resources and will greatly benefit genetic diversity, qualitative and quantitative trait mapping and marker-assisted selection studies in sesame.
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Genes de Plantas/genética , Repeticiones de Microsatélite/genética , ARN de Planta/genética , Análisis de Secuencia de ARN/métodos , Sesamum/genética , Emparejamiento Base/genética , Mapeo Cromosómico , Minería de Datos , Ecotipo , Regulación de la Expresión Génica de las Plantas , Ligamiento Genético , Marcadores Genéticos , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transcriptoma/genéticaRESUMEN
The original definition of sarcopenia refers to the age-related loss of muscle mass. The literature suggests that the prevalence of sarcopenia in 60- to 70-year-olds is in the range of 5-13%. Prevalence estimates increase to 11-50% for the population aged 80 years or older. Estimates by the World Health Organization suggest that there were 600 million people aged 60 years or older in the year 2000, and that this number will increase to 1.2 billion by the year 2025. There are, however, limited published data on serial measures of muscle mass in older subjects to establish the age-related changes in muscle mass and to relate this change with adverse health consequences. This review is focused on the definition, prevalence, symptoms, pharmacy and physical therapy of sarcopenia in older subjects with the aim of promoting the recognition and treatment of age-related sarcopenia in the clinical setting.
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Sarcopenia/epidemiología , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modalidades de Fisioterapia , Prevalencia , Sarcopenia/diagnóstico , Sarcopenia/fisiopatología , Sarcopenia/terapiaRESUMEN
The lentiviral vector is a useful tool for delivery of hairpin siRNA (shRNA) into mammalian cells. However, the efficiency of this system for carrying double-stranded siRNA (dsRNA) has not been explored. In this study we cloned the two forms of siRNA-coding sequence, a palindromic DNA with a spacer loop for shRNA and a double-stranded DNA with opposing Pol III promoters for dsRNA, into lentiviral DNA vectors, and compared their viral vector production yields. Our results indicate that sharply lower titer vector was obtained for dsRNA while much higher titer vector was produced for shRNA, posing a fundamental concern whether siRNA-carrying viral RNA itself is an inherent target of RNAi. Further experimental analyses using packaging cells that either allow or do not allow siRNA transcription indicate that the shRNA-carrying viral RNA is resistant to RNAi but the viral RNA carrier for dsRNA is not, offering a linker of RNAi bias-target secondary structure that causes shRNA vector to evade RNAi degradation. More importantly, the poor yield of dsRNA vector production was restored when a novel packaging cell line was used that blocks the antisense strand from dsRNA duplexes. This method has important implications for the RNAi field, especially for those who are using lentiviral dsRNA and dsRNA libraries for various biological discovery and therapeutic interventions.
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Técnicas Genéticas , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Viral/genética , Vectores GenéticosRESUMEN
Two types of tetracycline-controlled inducible RNAi expression systems have been developed that generally utilize multiple tetracycline operators (TetOs) or repressor fusion proteins to overcome the siRNA leakiness. Here, we report a novel system that overexpresses the tetracycline repressor (TetR) via a bicistronic construct to control siRNA expression. The high level of TetR expression ensures that the inducible promoter is tightly bound, with minimal basal transcription, allowing for regulation solely dependent on TetR rather than a TetR fusion protein via a more complicated mechanism. At the same time, this system contains only a single TetO, thus minimizing the promoter impairment occurring in existing systems due to the incorporation of multiple TetOs, and maximizing the siRNA expression upon induction. In addition, this system combines all the components required for regulation of siRNA expression into a single lentiviral vector, so that stable cell lines can be generated by a single transduction and selection, with significant reduction in time and cost. Taken together, this all-in-one lentiviral vector with the feature of TetR overexpression provides a unique and more efficient tool for conditional gene knockdown that has wide applications. We have demonstrated the high degree of robustness and versatility of this system as applied to several mammalian cells and xenograft animals.
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Regulación de la Expresión Génica , Técnicas Genéticas , Interferencia de ARN , Animales , Línea Celular Tumoral , Humanos , Ratones , Modelos Animales , Trasplante de Neoplasias , Regiones Promotoras Genéticas , ARN Interferente Pequeño/metabolismo , Tetraciclina/metabolismo , Transducción Genética , Trasplante HeterólogoRESUMEN
We report here a direct surface-grafting approach to forming DNA-containing polymer shells outside of Au nanoparticles using aqueous atom transfer radical polymerization (ATRP). In this approach, DNA molecules were immobilized on Au particles to introduce ATRP initiators on the surface. The same DNA molecules also acted as particle stabilizers through electrostatic repulsion and allowed particles to stay suspended in water. The immobilized ATRP initiators prompted polymer chain growth under certain conditions to form thick polymer shells outside of the particles. The formation of DNA-polymer hybrids outside of Au nanoparticles was characterized using absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and gel electrophoresis. The presence of thick polymer shells improved particle stability in high ionic strength media, whereas particles with the DNA coating only aggregated. A visible color difference between these two particle solutions was clearly observed, providing the basis for DNA sensing in homogeneous solutions.