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ObjectiveTo investigate the effects of Linggui Zhugantang on mitochondrial fission and fusion and silencing information regulator 3（Sirt3）/adenosine monophosphate dependent protein kinase （AMPK） signaling pathway in chronic heart failure （CHF） rats after myocardial infarction （MI）. MethodSD rats randomly divide into sham operation group （normal saline ，thread only without ligature）， model group （normal saline， ligation of the left anterior descending coronary artery proximal to the heart）， Linggui Zhugantang group （4.8 g·kg-1） and Captopril group （0.002 57 g·kg-1）， with 10 rats in each group. Administere drug continuously for 28 days. Echocardiography detected cardiac function parameters. Hematoxylin eosin （HE） staining observed the pathological changes of the heart. Immunofluorescence detected the levels of reactive oxygen species （ROS）. JC-1 detect mitochondrial membrane potential. Colorimetry measure adenosine triphosphate （ATP）， superoxide dismutase （SOD）， malondialdehyde （MDA）， mitochondrial respiratory chain complex activity （Ⅰ-Ⅳ）. TdT-mediated dUTP nick end labeling （TUNEL） staining detected the apoptosis rate of myocardial tissue. Western blot detected protein expression levels of Sirt3， phosphorylated AMPK （p-AMPK）， phosphorylated dynamic-related protein 1（p-Drp1）， mitochondrial fission protein 1（Fis1）， mitochondrial fission factor （MFF）， optic atrophy protein 1（OPA1）. ResultCompared to the sham group， the left ventricular end diastolic diameter （LVIDd） and left ventricular end systolic diameter （LVIDs） were significantly increased in model group （P<0.01）， while the left ventricular short axis shortening rate （LVFS） and left ventricular ejection fraction （LVEF） were significantly decreased （P<0.01）. There were inflammatory cell infiltration and obvious pathological injury in myocardial tissue. ROS， MDA levels and myocardial cell apoptosis rate were significantly increased （P<0.01）， SOD level， ATP content， and membrane potential were significantly decreased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） was significantly decreased （P<0.01）. Levels of p-Drp1， Fis1， MFF proteins were significantly up-regulated （P<0.01）， while Sirt3， p-AMPK， OPA1 proteins level were significantly down-regulated （P<0.01）. Compared with model group， LVIDd and LVIDs were significantly decreased （P<0.01）， LVEF and LVFS were significantly increased （P<0.01）. Inflammatory cell infiltration and pathological damage of myocardial tissue were significantly relieved. ROS， MDA levels and myocardial cell apoptosis rate were significantly decreased in Linggui Zhugantang group and Captopril group （P<0.01）， SOD level， ATP content， and membrane potential significantly increased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） increased significantly （P<0.01），and p-Drp1， Fis1， MFF protein levels were significantly down-regulated （P<0.01）， Sirt3， p-AMPK， OPA1 protein were significantly up-regulated （P<0.01）. ConclusionLinggui Zhugantang can alleviate oxidative stress and apoptosis damage of myocardial cells， maintain mitochondrial function stability， and its effect may be related to mitochondrial mitosis fusion and Sirt3/AMPK signaling pathway.
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ObjectiveTo investigate the effect of Gegen Qinliantang on glucose and lipid metabolism in the rat model of catch-up growth （CUG） induced by a high-fat diet and the underlying mechanism. MethodA total of 60 SD rats were randomized into a normal control group （n=18） and a modeling group （n=42）. The rat model of CUG was established with a restricted diet followed by a high-fat diet， and the changes of general status and body weight were observed. The levels of fasting blood glucose （FBG）， fasting insulin （FINS）， triglyceride （TG）， and total cholesterol （TC） were measured in 6 rats in each group at the end of the 4th and 8th week， respectively. The homeostasis model assessment of insulin resistance index （HOMA-IR） was calculated， and the insulin sensitivity and body composition changes of CUG rats were evaluated. The successfully modeled rats were assigned into 6 groups： normal control， model， high-， medium-， and low-dose Gegen Qinliantang （2.5， 5， 10 g·kg-1）， and pioglitazone （3.125 mg·kg-1）. The rats were administrated with corresponding drugs by gavage for 6 weeks， and the normal control group and model group were administrated with the same amount of normal saline. During the experiment period， the changes of body weight were recorded， and the FBG， FINS， HOMA-IR， TG， and TC were determined at the end of the experiment. Hematoxylin-eosin （HE） staining was employed to observe the pathological changes of skeletal muscle in rats. The levels of reactive oxygen species （ROS） and malondialdehyde （MDA） in the skeletal muscle were measured strictly according to the manuals of the reagent kits. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was performed to measure the mRNA levels of silencing information regulator 1 （SIRT1）， peroxisome proliferator-activated receptor-gamma coactivator1α （PGC1α）， and nuclear respiratory factor 1 （Nrf1） in the skeletal muscle. Western blot and immunohistochemistry were employed to assess the expression of SIRT1， PGC1α， and Nrf1 in the skeletal muscle. ResultCompared with the normal control group， the model group presented elevated levels of FBG， FINS， TG， and TC （P<0.05， P<0.01）， increased HOMA-IR （P<0.01）， increased diameter of muscle fibers and adipocytes between muscle cells in the skeletal muscle， rising levels of ROS and MDA in the skeletal muscle （P<0.01）， and down-regulated mRNA and protein levels of SIRT1， PGC1α， and Nrf1 （P<0.05， P<0.01）. Compared with the model group， Gegen Qinliantang （especially the medium and high doses） and pioglitazone decreased the body weight， FINS， HOMA-IR， and TG （P<0.05， P<0.01） and reduced interstitial components such as intermuscular fat in the skeletal muscles and the diameter of muscle fibers. Furthermore， the drugs lowerd the levels of ROS and MDA （P<0.05， P<0.01） and up-regulated the mRNA and protein levels of SIRT1， PGC1α， and Nrf1 （P<0.05， P<0.01） in the skeletal muscle. ConclusionGegen Qinliantang can ameliorate the glucose and lipid metabolism disorders and insulin resistance in CUG rats by regulating the SIRT1/PGC1α/Nrf1 signaling pathway.
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Quem pode falar no divã? Como a inscrição do sujeito e do sujeito do inconsciente em relações sociais de poder de classe, gênero, sexualidade, raça, idade, validade, limita o acesso a uma elaboração analítica? O reconhecimento da colonialidade, como efeito de dominação e lugar de enunciação que persiste além da colonização, tornou possível a emergência de novas formas subjetivas, culturais e epistêmicas, incentivando a psicanálise a escutar de outra forma. Este artigo propõe se debruçar sobre a incidência da raça e da branquitude na psicanálise a partir das epistemologias do posicionamento e da epistemologia da ignorância. No contexto social francês, enquanto uma parte crescente da população francesa experimenta diariamente a discriminação racial, essa é veementemente negada por uma maioria de político·as e pesquisadore·as, que recusam até o uso da palavra "raça". A partir dessa negação oficial do racismo sistémico pelo poder político e por uma maioria de estudos acadêmicos, o artigo tenta analisar a epistemologia da ignorância que prevalece na postura clínica e teórica de uma psicanálise maioritária. Trata-se de estudar a forma como uma ignorância branca provoca uma desescuta das questões de raça no divã, produz efeitos transferenciais de silenciamento, e nega vivências particulares em nome do universalismo.
Who can speak on the couch? How does the inscription of the subject and the subject of the unconscious in class, gender, sexuality, race, age and validity social power relations limit access to a psychoanalytical elaboration? The recognition of coloniality as an effect of domination and a locus of enunciation that persists beyond colonisation has made it possible for new subjective, cultural and epistemic forms to emerge, encouraging psychoanalysis to listen differently. This article looks at the impact of race and whiteness on psychoanalysis through the perspective of the Standpoint Epistemologies and the Epistemology of Ignorance. In the French social context, while a growing part of the French population experiences racial discrimination on a daily basis, it is vehemently denied by a majority of politicians and researchers, who refuse to even use the word "race". Starting from this official denial of systemic racism by the political establishment and a majority of academic studies, the article seeks to analyse the epistemology of ignorance that prevails in the clinical and theoretical stance of a majoritian psychoanalysis. The aim is to study the way in which white ignorance causes race issues to be non-listened to on the couch produces silencing transferential effects, and denies particular experiences in the name of universalism.
¿Quién puede hablar en el diván? ¿Cómo la inscripción del sujeto y del sujeto del inconsciente en las relaciones sociales de poder de clase, género, sexualidad, raza, edad, validez, limitan el acceso a una elaboración analítica? El reconocimiento de la colonialidad como un efecto de dominación y un lugar de enunciación que persiste más allá de la colonización ha posibilitado la emergencia de nuevas formas subjetivas, culturales y epistémicas, impulsionando al psicoanálisis a escuchar de otra manera. Este artículo examina el impacto de la raza y la blanquitud en el psicoanálisis desde la perspectiva de las epistemologías del posicionamiento y la epistemología de la ignorancia. En el contexto social francés, mientras que una parte creciente de la población francesa experimenta a diario la discriminación racial, ésta es negada con vehemencia por una mayoría de políticos/as e investigadores/as, que se niegan incluso a utilizar la palabra "raza". Partiendo de esta negación oficial del racismo sistémico por parte del poder político y de una mayoría de estudios académicos, el artículo intenta analizar la epistemología de la ignorancia que prevalece en la postura clínica y teórica de un psicoanálisis mayoritario. El objetivo es estudiar el modo en que la ignorancia blanca hace que las cuestiones raciales sean des-escuchadas en el diván, produce efectos transferenciales de silenciamiento y niega las experiencias particulares en nombre del universalismo.
Subject(s)
Psychoanalysis , Racial Groups , NarcissismABSTRACT
@#Abstract：Objective To improve the replication level of varicella⁃zoster virus（VZV）in human diploid cell line MRC⁃5 and increase the yield of VZV vaccine by reducing the expression of interferon（IFN）related genes via optimizing the cell line MRC⁃5. Methods Interferon receptor 1（IFNAR1）silenced MRC⁃5 cell line（MRC⁃5IFNAR1⁃）was constructed by CRISPR／Cas9 gene editing technology，which was determined for the relative expression of IFNAR1 mRNA，and for those of mRNA of IFN related genes IFNβ and OAS1 after VZV infection by qRT⁃PCR to evaluate the effect of gene silencing. Gene mutation sequences were further identified by sequencing of the silenced sites. The replication of VZV in MRC⁃5 and MRC⁃5IFNAR1⁃ cell lines was compared 168 h after VZV infection by using qRT⁃PCR and plaque formation unit（PFU）assay， to evaluate the effect of MRC⁃5IFNAR1⁃cell line on VZV replication. Results The growth status of MRC⁃5IFNAR1⁃ cell line wasconsistent with that of MRC ⁃ 5 cells，and the relative expression of IFNAR1 mRNA decreased by 73%；The relative expressions of IFNβ and OAS1 mRNA in MRC⁃5IFNAR1⁃ cell line were 61% and 90% lower than those in MRC⁃ 5 cells respectively after VZV infection；In addition，168 h after VZV infection，the level of DNA replication and the titer of VZV increased by 5. 7 folds and 4 folds respectively. Conclusion The successful establishment of MRC⁃5IFNAR1⁃ cell line may be a potential scheme to increase the yield of vaccines based on human diploid cells，and provided a reference for expanding production of VZV vaccine.
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Color is an important indicator for evaluating the ornamental traits of horticultural plants, and plant pigments is a key factor affecting the color phenotype of plants. Plant pigments and their metabolites play important roles in color formation of ornamental organs, regulation of plant growth and development, and response to adversity stress. It has therefore became a hot topic in the field of plant research. Virus-induced gene silencing (VIGS) is a vital genomics tool that specifically reduces host endogenous gene expression utilizing plant homology-dependent defense mechanisms. In addition, VIGS enables characterization of gene function by rapidly inducing the gene-silencing phenotypes in plants. It provides an efficient and feasible alternative for verifying gene function in plant species lacking genetic transformation systems. This paper reviews the current status of the application of VIGS technology in the biosynthesis, degradation and regulatory mechanisms of plant pigments. Moreover, this review discusses the potential and future prospects of VIGS technology in exploring the regulatory mechanisms of plant pigments, with the aim to further our understandings of the metabolic processes and regulatory mechanisms of different plant pigments as well as improving plant color traits.
Subject(s)
Plant Viruses/genetics , Plants/genetics , Gene Silencing , Plant Development , Gene Expression Regulation, Plant , Genetic VectorsABSTRACT
Autophagy is a highly conserved mechanism for material degradation and recycling in eukaryote cells, and plays important roles in growth, development, stress tolerance and immune responses. ATG10 plays a key role in autophagosome formation. To understand the function of ATG10 in soybean, two homologous GmATG10 genes, namely GmATG10a and GmATG10b, were silenced simultaneously by bean pod mottle virus (BPMV) induced gene silencing. The carbon starvation induced by dark treatment and Western blotting analysis of GmATG8 accumulation level indicated that concurrent silencing GmATG10a/10b resulted in the impairment of autophagy in soybean; disease resistance and kinase assays demonstrated that GmATG10a/10b participated in the immune responses by negatively regulating the activation of GmMPK3/6, indicating that GmATG10a/10b plays a negative regulatory role in immune response in soybean.
Subject(s)
Soybeans/genetics , ImmunityABSTRACT
Resumo Este artigo é um desdobramento da tese de doutorado que trata das relações e processos de subjetivação entre equipes técnicas e famílias na rede de saúde mental. Neste contexto, o trabalho aborda forças conservadoras e possibilidades de resistência ao poder. O racismo está presente no cotidiano de pessoas que convivem com situações de sofrimento mental, mas poucas vezes esse marcador social é abordado nos serviços, caracterizando o silenciamento de experiências vividas. A metodologia utilizada foi a cartografia, incluindo pesquisa de campo, permitindo rastreamento de processos e considerando o posicionamento político de quem pesquisa, com orientação para práticas comprometidas com transformações sociais. A análise dos dados produzidos associa perspectiva interseccional sobre as demandas em saúde mental à esquizoanálise, buscando a construções de saída dos impasses entre familiares e equipes. Concluímos que sustentando indagações sobre modos de nos relacionar e revisitar nossa história, podemos construir práticas coletivas antirracistas na saúde mental.
Resumen Este artículo es fruto de una tesis doctoral que aborda las relaciones y los procesos de subjetivación entre los equipos técnicos y las familias en la red de salud mental. Aborda las fuerzas conservadoras y las posibilidades de resistencia al poder. El racismo está presente en el cotidiano de las personas que viven con sufrimiento mental, pero ese marcador social raramente es abordado en los servicios, caracterizando el silenciamiento de las experiencias vividas. La metodología utilizada fue la cartografía, incluyendo la investigación de campo, permitiendo rastrear procesos y teniendo en cuenta el posicionamiento político hacia prácticas comprometidas con la transformación social. El análisis de los datos asocia perspectiva interseccional de las demandas de salud mental con el esquizoanálisis, con búsquedas de salidas a los impasses entre familiares y equipos. Concluimos que apoyando preguntas sobre los modos de relacionarnos y revisitando nuestra historia, podemos construir prácticas colectivas antirracistas en salud mental.
Abstract This article is an offshoot of a doctoral thesis that deals with the relationships and processes of subjectivation between technical teams and families in the mental health network. In this context, it deals with conservative forces and possibilities of resistance to power. Racism is present in the daily lives of people who live with situations of mental suffering, but this social marker is rarely addressed in the services, characterizing the silencing of lived experiences. Cartography was the methodology used, including field research, which allows processes to be traced and takes into account the political positioning of the researcher towards practices committed to social transformation. The analysis of the data produced associates an intersectional perspective on mental health demands with schizoanalysis, seeking ways out of the impasse between family members and teams. We conclude that by supporting questions about ways of relating and revisiting our history, we can build anti-racist collective practices in mental health.
Subject(s)
Family , Mental Health , Racism , Systemic Racism/prevention & control , Mental Health ServicesABSTRACT
Objective @# To investigate the effects of angiopoietin 4 (ANGPT4) on the odontogenic differentiation of human dental pulp stem cells. @* Methods @#This study has been reviewed and approved by the Ethics Committee, and informed consent has been obtained from patients. Human premolars were fixed, decalcified, dehydrated, embedded, and sectioned. Immunofluorescence staining was used to observe the expression and localization of ANGPT4. Human dental pulp stem cells (hDPSCs) were isolated and cultured in vitro. The growth state and morphology of hDPSCs were observed under an inverted phase contrast microscope. The expression of cell surface-related molecular markers was detected by flow cytometry. Alkaline phosphatase and alizarin red S staining were used to detect the odontogenic differentiation potential of hDPSCs. Oil-red O staining was used to detect the adipogenic differentiation potential of hDPSCs. RNA was extracted from hDPSCs at different time points after odontogenic induction, and RT-qPCR was used to analyze the mRNA expression of ANGPT4 and odontogenic-related genes during the odontogenic differentiation of hDPSCs in vitro. siRNA gene silencing technology was used to silence the expression of ANGPT4 in hDPSCs, and the silencing efficiency was detected by RT-qPCR and Western Blot. After silencing ANGPT4 in hDPSCs for 24 h, odontogenic induction was performed. Alkaline phosphatase and alizarin red S staining were performed on the 7th and 14th of induction to detect the odontogenic differentiation ability of hDPSCs after silencing ANGPT4@*Results @# Immunofluorescence staining of human premolars showed that ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone. hDPSCs were in good condition after 14 days of isolation and culture. Under the microscope, multiple cell colonies were observed, and the cell morphology was uniform and long spindle-shaped. The results of flow cytometry showed that hDPSCs expressed mesenchymal stem cell markers CD105 (90.42%) and CD90 (97.15%), but did not express hematopoietic cell markers CD45 (0.01%) and CD34 (0.08%). After odontogenic and adipogenic induction of hDPSCs, alkaline phosphatase staining, alizarin red S staining and oil red O staining were positive. The results of RT-qPCR after the odontogenic induction of hDPSCs showed that ANGPT4 was highly expressed on the 5th, 7th, 11th and 14th days of differentiation of hDPSCs (P<0.05), with the highest expression level on the 5th day. After hDPSCs were transfected with si-ANGPT4, the expression of ANGPT4 mRNA and protein was significantly down-regulated (P<0.05). The results of alkaline phosphatase staining showed that ALP staining intensity and area in the si-ANGPT4 group were significantly lower than those in the negative control. Alizarin red S staining showed that the formation of calcium nodules in the si-ANGPT4 group was significantly lower than that in the negative control.@* Conclusion@#ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone of human premolars. ANGPT4 may be a factor to promote the odontogenic differentiation of hDPSCs.
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Small interfering RNA (siRNA) is the initiator of RNA interference and inhibits gene expression by targeted degradation of specific messenger RNA. siRNA-mediated gene regulation has high efficiency and specificity and exhibits great significance in the treatment of diseases. However, the naked or unmodified siRNA has poor stability, easy to degrade by nuclease, short half-life, and low intracellular delivery. As an emerging non-viral nucleic acid delivery system, ionizable lipid nanoparticles play an important role in improving the druggability of siRNA. At present, one siRNA drug based on ionizable lipid nanoparticles has been approved for the treatment of rare disease. This review introduces the research progress in ionizable lipid nanoparticles for siRNA delivery, focusing on the effect of each component of lipid nanoparticles on the efficiency of siRNA-mediated gene silencing, which provides new references for the studies on ionizable lipid nanocarriers for siRNA delivery.
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ObjectiveTo investigate whether the effects of paeonol （Pae） on angiotensin Ⅱ （AngⅡ）-induced senescence in vascular smooth muscle cells （VSMCs） were related to angiotensinogen of silencing regulatory information factor 6 （SIRT6）/adenosine diphosphate ribose polymerase 1 （PARP1） signaling pathway in VSMCs. MethodThe model of VSMC-stress aging induced by AngⅡ （100 nmol·L-1） was established. The rats were divided into normal group， model group， low， medium， and high-concentration Pae groups （30， 60， 120 μmol·L-1）. The positive rate of cell senescence was detected by SA-β-Gal staining， the ability of cell proliferation was detected by the cell counting kit-8 （CCK-8） method， the expression of SIRT6， PARP1， p16， p21， p53， proliferating cell nuclear antigen （PCNA）， deoxyribonucleic acid （DNA）-damaged protein γ-H2AX was detected by Western blot， and VSMC proliferation was detected by EdU staining. The silenced VSMCs were prepared by siRNA-SIRT6 transfection， and the protein expressions of SIRT6， PARP1， p16， and γ-H2AX in VSMCs silenced by SIRT6 were observed. ResultThe results of SA-β-Gal staining showed that the senescence positive rate of SA-β-Gal staining in the model group was higher than that in the normal group （P<0.01）， and the positive rate of SA-β-Gal staining in the Pae group was significantly lower than that in the model group （P<0.05， P<0.01）. The results of Western blot showed that as compared with the normal group， the expression of PCNA， SIRT6， and PARP1 in the model group was down-regulated， and the expression of aging-related proteins p16， p21， p53， and γ-H2AX was up-regulated in the model group （P<0.05， P<0.01）. Compared with the model group， Pae promoted the protein expression of PCNA， SIRT6， and PARP1 and inhibited the protein expression of p16， p21， p53， and γ-H2AX in a dose-dependent manner （P<0.05， P<0.01）. The results of EdU staining showed that the number of EdU positive cells in the model group was lower than that in the normal group （P<0.01）， and the number of EdU positive cells in Pae groups was significantly higher than that in the model group （P<0.05， P<0.01）. After SIRT6 silencing， the effects of Pae on promoting SIRT6 and PARP1 and inhibiting P16 were reversed （P<0.05， P<0.01）. In addition， the addition of SIRT6 inhibitor （IN-1） promoted the occurrence of cell senescence induced by AngⅡ （P<0.05， P<0.01）. ConclusionPae can effectively inhibit the aging of VSMCs， and its mechanism may be related to the regulation of SIRT6/PARP1 signal pathway.
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Abstract Therapeutics that inhibit enzymes, receptors, ion channels, and cotransporters have long been the mainstay of cardiovascular medicine. Now, oligonucleotide therapeutics offer a modern variation on this paradigm of protein inhibition. Rather than target a protein, however, small interfering ribonucleic acids and antisense oligonucleotides target the messenger RNA (mRNA) from which a protein is translated. Endogenous, cellular mechanisms enable the oligonucleotides to bind a selected sequence on a target mRNA, leading to its degradation. The catalytic nature of the process confers an advantage over the stoichiometric binding of traditional small molecule therapeutics to their respective protein targets. Advances in nucleic acid chemistry and delivery have enabled development of oligonucleotide therapeutics against a wide range of diseases, including hyperlipidemias and hereditary transthyretin-mediated amyloidosis with polyneuropathy. While most of these therapeutics were initially designed for rare diseases, recent clinical trials highlight the potential impact of oligonucleotides on more common forms of cardiovascular disease.
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Objectives:The aim of this study was to evaluate the serum RE1 silencing transcription factor (REST) level in Alzheimer's disease (AD), mild cognitive impairment (MCI), and elderly controls by using surface plasmon resonance (SPR) technology. Materials and Methods?In this case–control study of 133 subjects, 49 patients with AD, 49 patients with MCI, and 35 elderly controls were recruited. The REST protein concentrations were evaluated by SPR. The resonance unit for each sample was recorded and the concentration of serum REST of study group was derived from the standard curve. All the experiments were done in triplicates. Statistical analysis was done and p-value of < 0.05 was considered as statistically significant. Results?A significant difference was observed in the Montreal Cognitive Assessment score, Hindi Mental State Examination scale (HMSE) score education, disease duration, and gender among the groups. A significant (p>0.0001) difference in the duration of disease between AD and MCI was observed. It was observed that the mean concentration of serum REST was not significantly (p?=?0.266) different among the groups. Conclusion?This study first time evaluated the serum levels of REST in AD, MCI and age-matched elderly controls. The rest levels were similar in all groups; however, it can provide a new direction to future blood-based biomarker studies of REST.
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Objective:To evaluate the effect of down-regulation of Sp1 expression on the radiosensitivity of glioma cells.Methods:The oligonucleotide sequence encoding shRNA was designed and synthesized, and cloned into LV3 (H1/GFP & Puro) vector to construct the recombinant. U251 and U87 cells were infected with recombinant lentivirus, then the stably-transfected cell lines were obtained by puromycin screening. The expression levels of Sp1 mRNA and protein were detected by RT-PCR and Western blot. Cell survival was detected by clonal survival assay, cell cycle was determined by flow cytometry, and DNA damage was measured by immunofluorescence assay, respectively.Results:At 72 h after infection, high expression of Sp1 lentiviral vector was observed in two cell lines under fluorescence microscope. RT-PCR and Western blot confirmed that the expression levels of Sp1 mRNA and protein were significantly down-regulated in both transfected cells (both P<0.01) and the silencing rates of Sp1 were above 90%. The sensitization enhancement ratio (SER) of shRNA-U251 and shRNA-U87 cells at 10% cell survival level were 1.39 and 1.18, respectively. After irradiation, the G 2/M phase ratio and the number of γ-H2AX foci in two Sp1 knockout groups were significantly increased. Conclusion:shRNA silencing of Sp1 increases the G 2/M phase arrest induced by X-ray, aggravates the degree of DNA double-strand breaks, and improves the radiosensitivity of glioma cells.
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Objective@#To explore the effects of long noncoding-RNA (lncRNA) taurine upregulated gene 1 (TUG1) on the proliferation and osteogenic/odontoblast differentiation of human dental pulp stem cells (hDPSCs). @*Methods @# hDPSCs were isolated and cultured. The surface antigens CD44, CD45, CD73, CD90, CD133 and STRO-1 were detected by flow cytometry. Alkaline phosphatase (ALP) staining and alizarin red staining were used to identify the ability of cells to differentiate. RNA was collected on Days 0, 7 and 14 of the osteogenic induction of hDPSCs, and qRT-PCR was used to detect the relative expression of TUG1. The hDPSCs were stably transfected with a lentiviral vector containing the TUG1-silenced pSLenti-U6-shRNA(TUG1)-CMV-EGFP-F2A-Puro-WPRE to silence TUG1. The ability of hDPSCs to proliferate was assessed with the CCK-8 method. ALP and alizarin red staining and quantitative detection were used to detect the ALP activity and formation of mineralized nodules of hDPSCs. The expression levels of dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), Runt-associated transcription factor 2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) genes and proteins were measured by qRT-PCR and Western blot.@*Results @#The hDPSCs were successfully isolated and cultured, and TUG1 expression was significantly increased during osteogenic differentiation (P<0.05). The hDPSCs proliferation was suppressed after silencing TUG1(P<0.05). After osteogenic induction, ALP and alizarin red staining showed that ALP activity and mineralized nodules were suppressed by silencing TUG1. The expression levels of the odontogenic differentiation gene DSPP and DMP-1 and the osteogenic differentiation gene Runx2, OCN and OPN were also significantly decreased (P<0.05).@*Conclusion @# Knocking down TUG1 can inhibit the proliferation and osteogenic/odontogenic differentiation of hDPSCs.
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Objective:To investigate the expression of silencing kinesin KIF4A in thyroid cancer tissues and its relationship with clinicopathological characteristics of thyroid cancer patients, and to assess the role of KIF4A in the progression of thyroid cancer.Methods:The expression of KIF4A in normal thyroid tissues and the thyroid cancer population and its relationship with disease-free survival of patients were analyzed online by gene expression interaction analysis (GEPIA) database, and the expression of KIF4A in tumor tissues and paraneoplastic tissues of thyroid cancer patients was assessed by immunohistochemical assays. The patients were divided into high- and low-expression groups according to the staining intensity, and the correlation between the expression of KIF4A and clinicopathological features was analyzed. The effect of KIF4A on the proliferation of thyroid cancer cells was explored by a clone formation assay and an MTT assay.Results:According to the analysis of the web-based database, KIF4A showed significantly high expression in human thyroid cancer tissues, and disease-free survival was significantly lower in highly expressed patients. The results of the case analysis showed that the correlation between KIF4A expression intensity and gender, age, and lymph node metastasis in thyroid cancer patients was not statistically significant (all P>0.05), and the correlation with TNM stage and intraglandular dissemination was statistically significant (all P<0.05). The results of the colony formation assay and the MTT assay showed that the expression of KIF4A promoted the proliferation of thyroid cancer cells ( P<0.05). Conclusions:KIF4A can promote the progression of thyroid cancer and has the potential to become a new therapeutic target for thyroid cancer.
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Resumen Los insectos plaga, son especies de organismos vivos que en forma constante se encuentran en poblaciones altas, ocasionando daños económicos en los cultivos. Generalmente, suele tratarse de especies puntuales, por lo general, sólo una o dos, que pueden causar gran afectación económica en el sector de la agricultura. En las últimas 3 décadas se ha venido desarrollando el concepto de un proceso biológico, detectado en eucariotas ampliamente, mediante el que se pueden silenciar genes, a partir de ARN de doble cadena (ARNdc). Esta maquinaria se ha investigado para conocer su funcionamiento y buscar potenciales aplicaciones que podrían tener en el campo de la biotecnología. En varios estudios se encontró que el silenciamiento de genes se debe a las interacciones enzimáticas intracelulares citoplasmáticas con moléculas de ARN pequeñas (ARNsi), que actúan sobre el ARN mensajero (ARNm) intracelular, impidiendo que este se traduzca a proteína. Mediante este mecanismo se busca silenciar genes específicos en insectos plaga, que sean esenciales para que el insecto pueda vivir y de esa manera evitar la proliferación de la plaga. Este artículo recopila los estudios realizados acerca del ARN de interferencia, referidos al mecanismo genético de los insectos, como alternativa para su control.
AbstractPest insects are species of living organisms that are constantly found in high populations,causing economic crops damage. Generally, it tends to be specific species, usually onlyone or two, which can cause great economic damage in the agricultural sector. In thelast 3 decades, the concept of a biological process has been developed, widely detected in eukaryotes, by which genes can be silenced, from double-stranded RNA (dsRNA). This machinery has been investigated to understand its operation and to look for potential applications that it could have in the field of biotechnology. In several studies it was found that gene silencing is due to cytoplasmic intracellular enzymatic interactions with small RNA molecules (siRNA), which act on intracellular messenger RNA (mRNA), preventing it from translating a protein. Through this mechanism, the aim is to silence specific genes in pest insects, which are essential for the insect to live and thus prevent the proliferation of the pest. This article compiles the studies carried out on RNA interference, referring to the genetic mechanism of insects, as an alternative for its control.
Subject(s)
Animals , Biotechnology , Gene Silencing , Agribusiness , InsectaABSTRACT
O campo da Psicologia produz saberes e discursos que referenciam as políticas públicas que alteram ou sedimentam o lugar social atribuído ao sujeito. Este artigo considera que há modalidades de gestão política, controle e exploração que incluem em sua estratégia de dominação promover a experiência do sofrimento, seja composta pela angústia, culpa, vergonha ou humilhação social. Tendo em vista essa premissa, formulamos as vicissitudes e especificidades da escuta de sofrimento sociopolítico advindo de posições socialmente desqualificadas devido a fatores econômicos, raciais, culturais, religiosos, de gênero, entre outros. A vicissitude clínica que nos norteia é o silenciamento derivado desse sofrimento. A escuta clínica de sujeitos sob o desamparo discursivo se ancora na constatação de que o sofrimento sociopolítico desarvora o sujeito de seu lugar de fala, promovendo o abalo narcísico e a eclosão da dimensão traumática - questão diagnóstica que deve ser levada em conta -, e pondo em jogo um possível conflito de lealdades quanto ao pacto social daquele que pratica a psicanálise, o que pode gerar sua resistência à escuta. Essas considerações clínicas não seriam possíveis sem superar uma dicotomia e um recalque: a questão da política na psicanálise. Tais concepções supõem repensar dimensões táticas, estratégicas e éticas no atendimento clínico psicanalítico. Abordaremos as artimanhas do poder que produzem o desamparo discursivo e a dimensão traumática, chegando finalmente a algumas considerações sobre o tratamento a ser dado ao sofrimento sociopolítico e as especificidades da escuta nesses contextos.(AU)
The field of psychology produces knowledge and discourses that guide public policies and alter or consolidate the social place attributed to the subject. This article considers that there are modalities of political management, control, and exploitation which include in their own strategy of domination promoting the experience of suffering, whether comprising anguish, guilt, shame or social humiliation. In view of this premise, we formulate the vicissitudes and specificities of listening to socio-political suffering arising from socially disqualified positions due to economic, racial, cultural, religious and gender factors, among others. The clinical vicissitude that will guide us will be the silencing derived from this suffering. The clinical listening to subjects under discursive helplessness is anchored on the observation that sociopolitical suffering takes the subject out of his or her place of speech, promoting narcissistic shock and the emergence of the traumatic dimension - a diagnostic issue that should be considered - and raising a possible conflict of loyalties regarding the psychoanalyst's social pact that can generate his or her resistance to listening. These clinical considerations would not be possible without overcoming a dichotomy and a repression: the question of politics in psychoanalysis. Such conceptions suppose rethinking tactical, strategic and ethical dimensions in psychoanalytic clinical care. We will approach the tricks of power that produce the discursive helplessness and the traumatic dimension, finally reaching some considerations about the treatment to be given to socio-political suffering and the specifics of listening in these contexts.(AU)
El campo de la Psicología produce conocimiento y discursos que son decisivos para las políticas públicas y para alterar o consolidar el lugar social atribuido al sujeto. Este artículo considera la existencia de modalidades de gestión política, control y explotación que incluyen, en su estrategia de dominación, promover la experiencia del sufrimiento, ya sea compuesta por angustia, culpa, vergüenza o humillación social. En vista de esta premisa, formulamos las vicisitudes y especificidades de escucha del sufrimiento sociopolítico que surge de posiciones socialmente descalificadas debido a factores económicos, raciales, culturales, religiosos y de género, entre otros. La vicisitud clínica que nos guiará es el silenciamiento derivado de este sufrimiento. La escucha clínica de los sujetos bajo impotencia discursiva está anclada en la observación de que el sufrimiento sociopolítico saca al sujeto de su lugar de discurso promoviendo el shock narcisista y la aparición de la dimensión traumática -un problema de diagnóstico que debe tenerse en cuenta-, y pone en juego un posible conflicto de lealtades con respecto al pacto social del psicoanalista, lo que puede generar su resistencia a la escucha. Estas consideraciones clínicas no serían posibles sin superar una dicotomía y una represión: la cuestión de la política en el psicoanálisis. Dichas concepciones suponen repensar las dimensiones tácticas, estratégicas y éticas en la atención clínica psicoanalítica. Se abordarán los trucos del poder para producir la impotencia discursiva y la dimensión traumática, llegando finalmente a algunas consideraciones sobre el tratamiento que se dará al sufrimiento sociopolítico y los detalles específicos de la escucha en estos contextos.(AU)
Subject(s)
Humans , Psychoanalysis , Psychological Trauma , Psychological Distress , Organization and Administration , Pain , Public Health Administration , Public Policy , Shame , Poverty Areas , Humanization of Assistance , Health Vulnerability , Frustration , Guilt , Mental Health ServicesABSTRACT
Objective:To investigate the role of integrin-linked kinase (ILK)-small interfering RNA (siRNA)-adeno-associated virus (AAV) in scar formation after glaucoma filtering surgery in rat eyes.Methods:Forty-eight Sprague Dawley rats of SPF grade, aged 8 to 9 weeks old, were selected and divided into blank control group, ILK-siRNA-AAV group, NC-siRNA-AAV group and mitomycin C (MMC) group by random number table method, with 12 rats in each group.Left eyes of the rats were taken as experimental eyes, and no intervention was administered to fellow eyes.The bulbar conjunctival filtering bleb after glaucoma filtration surgery in rats was established by anterior chamber drainage tube implantation.One day after operation, phosphate buffer solution, ILK-siRNA-AAV, and NC-siRNA-AAV were injected into the filtering bleb of blank control group, ILK-siRNA-AAV group and NC-siRNA-AAV group, 5 μl each group, respectively.Cotton tablets containing 0.4 mg/ml MMC were placed under conjunctival flap for 5 minutes during operation in MMC group.Intraocular pressure (IOP) was measured with a handheld tonometer before surgery and at 1, 2, 3, 7, 14, 21, 28 days after surgery.Formation of filtering blebs in rats was observed with a surgical microscope at 1, 2, 3, 7, 14, 21, 28 days after operation, and the bleb survival time was calculated by Kaplan-Meier survival analysis.The mRNA and protein expression levels of ILK in conjunctival and subconjunctival tissues at the surgical sites were detected by reverse transcription PCR and Western blot, respectively, on the 28th day after operation.Silencing of ILK gene was identified.Effect of ILK gene silencing on the morphology of drainage pathway was observed by hematoxylin-eosin staining.Effect of ILK gene silencing on collagen fiber deposition in the bulbar conjunctiva at filtration area was examined by Masson staining, and the percentage of positive area of collagen fiber staining in the total tissue visual field was calculated.The use and care of the animals complied with the ARVO Statement.This research protocol was approved by an Ethics Committee of Xi'an Jiaotong University (No.2013-772). Results:There were statistically significant differences in IOP at different time points between before and after surgery among four groups ( Fgroup=76.84, P<0.001; Ftime=114.49, P<0.001). The IOP of ILK-siRNA-AAV group on the 1st, 7th, 14th and 28th day after operation and the IOP of MMC group on the 2nd, 3rd, 7th, 14th and 28th day after operation were lower than those of blank control group, and the differences were statistically significant (all at P<0.05). The IOP of ILK-siRNA-AAV group was lower on 7th, 14th, 21st and 28th day after operation than those of NC-siRNA-AAV group, with statistically significant differences (all at P<0.05). The bleb survival time of blank control group, NC-siRNA-AAV group, ILK-siRNA-AAV group and MMC group was (3.50±1.51), (5.00±3.41), (31.50±3.15) and (31.33±2.46) days, respectively, with a significant difference among them ( F=395.83, P<0.05). The bleb survival time of ILK-siRNA-AAV group and MMC group was higher than that of blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of ILK mRNA and protein among four groups ( F=222.32, 752.69; both at P<0.05), and the relative expression levels of ILK mRNA and protein were significantly lower in ILK-siRNA-AAV group than blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Proliferative fibrous connective tissue and a large number of cells at surgical sites were found in blank control group and NC-siRNA-AAV group, and the fibroblasts were of a high density and grew in clumps.In ILK-siRNA-AAV group, the bulbar conjunctiva was thin, and the arrangement of fibrous connective tissue was loose, and a few proliferative fibroblasts were found.In MMC group, the conjunctival fibrous layer was loose and thin, forming cavities, and scarce cells were found.There was statistically significant difference in the percentage of collagen fiber positive staining area among four groups ( F=741.66, P<0.05). The positive staining percentage of ILK-siRNA-AAV group and MMC group was significantly lower than that of blank control group, among which there was lower positive staining percentage in ILK-siRNA-AAV group than NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Conclusions:Silencing of ILK can inhibit the scar formation after glaucoma filtering surgery and maintain low IOP in rats.
ABSTRACT
ObjectiveTo study the underlying mechanism of Liuwei Dihuangwan in inhibiting triple-negative breast cancer through mitogen-activated protein kinase kinase kinase 1 （MAPKKK1） and Krüppel-like factor 4 （KLF4）. MethodFour hundreds SPF female Kunming mice aged 11.5 months were palpated once every 3 days. The model mice of spontaneous tumors were randomly divided into a model group （normal saline）， a paclitaxel group （0.025 g·kg-1·d-1， ip， 21 days）， and high-， medium- and low-dose Liuwei Dihuangwan groups （7.2， 3.6， 1.8 g·kg-1·d-1， ig）. Tumor tissues were separated until the moribund stage. The tumor volume and weight were measured， and the tumor inhibition rate and the survival time of the tumor mice were calculated （after 6 months， tumor-free mice were assigned into the normal group）. SPF SD rats were selected to prepare serum samples containing Liuwei Dihuangwan of different concentrations for cell culture， and MAPKKK1 in MDA-MB-231 cells was silenced. The protein expression of MAPKKK1 and KLF4 was detected by immunofluorescence and Western blot. ResultThe in vivo experimental results showed that compared with the conditions of the normal group， the protein expression of MAPKKK1 and KLF4 in tumor tissues of the model group dropped （P<0.01）. Compared with the model group， all medication groups showed reduced tumor volume and weight （P<0.05， P<0.01）， increased tumor inhibition rate， prolonged survival time of tumor mice （P<0.05）， and increased protein expression of MAPKKK1 （P<0.01）. Additionally， the paclitaxel group and the high-dose Liuwei Dihuangwan group exhibited increased protein expression of KLF4 （P<0.01）. The in vitro experiments showed that compared with the conditions of the normal group， the fluorescence intensities of MAPKKK1 and KLF4 in MDA-MB-231 cells in all medication groups were potentiated， and the protein expression of MAPKKK1 in the paclitaxel group and the high- and medium-dose Liuwei Dihuangwan groups， and the protein expression of KLF4 in the paclitaxel group and high-dose Liuwei Dihuangwan group increased （P<0.01）. After silencing of MAPKKK1， compared with the conditions of the negative plasmid group （unsilenced MAPKKK1）， the fluorescence intensities of MAPKKK1 and KLF4 and the protein expression decreased in the RNAi-27 positive plasmid group （silenced MAPKKK1） （P<0.05， P<0.01）. Compared with the RNAi-27 positive plasmid group， all medication groups had enhanced fluorescence intensities of MAPKKK1 and KLF4 and protein expression to different degrees （P<0.05， P<0.01）. ConclusionLiuwei Dihuangwan can inhibit the growth of triple-negative breast cancer， and the underlying molecular mechanism is related to the up-regulation of MAPKKK1 and activation of KLF4 expression.
ABSTRACT
ObjectiveTo explore the neuroprotective effect of Buyang Huanwutang （BYHW） on diabetic peripheral neuropathy （DPN） rats by regulating SIRT1/p53 pathway and to clarify the mechanism and the dosage of astragalus in the prescription. MethodA total of 90 SD rats were randomized into control group， DPN group， DPN + BYHW containing 120 g Astragalus （at 15 g·kg-1·d-1） （BYHW120 group）， DPN + BYHW containing 60 g Astragalus （at 8.75 g·kg-1·d-1） （BYHW60 group）， DPN + BYHW containing 30 g Astragalus （at 5.625 g·kg-1·d-1） （BYHW30 group）， and DPN + α-lipoic acid （at 60 mg·kg-1·d-1） （ALA group）. Standard diet was given to rats in the control group and high-carbohydrate/high-fat diet and streptozotocin （ip） were used to induce diabetes in rats in other groups. The administration lasted 12 weeks. After the intervention， mechanical pain threshold and nerve conduction velocity were detected. The L4-5 dorsal root ganglions were stained with haematoxylin-eosin （HE） and toluidine blue to observe the pathological changes， and the apoptosis of nerve cells was detected by terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling （TUNEL） assay. Cleaved cysteinyl aspartate-specific proteinase-3 （Caspase-3）， and the main proteins in the SIRT1/p53 pathway， such as silencing information regulator 2 related enzyme 1 （SIRT1）， acetyl-p53， dynamin-related protein 1 （Drp1）， Bcl-2 associated X protein （Bax）， and B-cell lymphoma-2 （Bcl-2）， were detected by immunohistochemistry and Western blot. ResultCompared with the control group， the DPN group presented increase in blood glucose （P<0.01）， decrease in nerve conduction velocity and mechanical pain threshold （P<0.01）， rise of the percentage of positive cells （TUNEL assay， the same below） and the expression of cleaved Caspase-3 （P<0.01）， drop in the expression of SIRT1 （P<0.01）， and elevation of acetyl-p53， Drp1， and Bax/Bcl-2 ratio （P<0.01）. Cleaved Caspase-3， acetyl-p53， Drp1， and Bax/Bcl-2 ratio in each administration group decreased as compared with those in the DPN group （P<0.01）. Nerve conduction velocity， mechanical pain threshold （P<0.05， P<0.01）， and the percentage of positive cells （P<0.05， P<0.01） increased in the administration groups as compared with those in the DPN group except for the BYHW30 group， and BYHW120 group and ALA group showed the increase in SIRT1 （P<0.05， P<0.01）. Nerve conduction velocity， mechanical pain threshold， and SIRT1 expression were lower （P<0.05， P<0.01） and expression of cleaved Caspase-3 was higher （P<0.01） in the BYHW60 and BYHW30 groups than in the BYHW120 group. The percentage of positive cells and the expression of acetyl-p53 were higher in the BYHW30 group than in the BYHW120 group （P<0.01）. ConclusionBYHW inhibits apoptosis and exerts therapeutic effect on DPN by regulating the SIRT1/p53 pathway. The therapeutic effect is related to the dosage of Astragalus in the prescription. BYHW containing 120 g Astragalus suppresses p53-dependent apoptosis more significantly than Buyang Huanwutang containing 60 g and 30 g of Astragalus.