RESUMO
RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.
Assuntos
Biotina/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Virais/metabolismo , Zika virus/metabolismo , Bacillus subtilis/metabolismo , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Neurônios/citologia , Neurônios/metabolismo , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Zika virus/genéticaRESUMO
Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Motores/citologia , Esclerose Lateral Amiotrófica/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologiaRESUMO
To observe the clinical effect of Yisui decoction plus western medicine in treating multiple system atrophy patients, totally 65 patients from China-Japan Friendship hospital during 2008-2012 with complete clinical data and received consecutive traditional Chinese medicine and western medicine treatment for more than 3 months were observed changes of traditional Chinese medicine symptom score, part 1 of unified multiple system atrophy rating scale, orthostatic hypotension before treatment and after 3 months treatment. After 3 months treatment, total effective rate of traditional Chinese medicine symptom was 70.8%. Compared with before treatment, score of part 1 of unified multiple system atrophy rating scale was obviously reduced after 3 month treatment (P < 0.001). Ex- cept swallow function without significant improvement, the remaining projects of unified multiple system atrophy rating scale were im- proved obviously (P < 0.05), of which the most obvious differences were orthostatic symptoms, falls and intestinal function (P < 0.001). Orthostatic hypotension after 1 month treatment and 3 month treatment was obviously better than before treatment (P < 0.001). There was no significant difference in orthostatic hypotension between 1 month treatment and 3 month treatment. The research results show that Yisui decoction plus western medicine has a certain effect on improving clinical symptoms of multiple system atrophy patients, especially has a significant effect on orthostatic hypotension, and can maintain a stable clinical effect in a certain period of time.
Assuntos
Medicina Tradicional Chinesa/métodos , Atrofia de Múltiplos Sistemas/tratamento farmacológico , Adulto , Idoso , Humanos , Hipotensão Ortostática/tratamento farmacológico , Masculino , Medicina Tradicional Chinesa/efeitos adversos , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
In this study, we demonstrate that BMSCs (bone marrow mesenchymal stem cells) can be successfully differentiated into type II alveolar epithelial cells in vitro under mimic pulmonary microenvironment. BMSCs were co-cultured with MRC-5 cells in modified SAGM (small airway growth medium). The BMSC-derived type II alveolar epithelial cells morphologically resemble human lung epithelial cells. They began to appear after 10 days in co-culture and became morphologically dominant after day 15. Correspondingly, SPC (surfactant protein C), a specific functional marker of human type II alveolar epithelial cells, was detected in differentiated cells by RT-PCR (reverse transcription-PCR) analysis after day 15. Immunostaining analysis revealed the present of scattered SPC-positive cells with a differentiation efficiency of 2.43-4.21%. Our study further showed that the SPC gene expression level in differentiated cells was related to the ratio of BMSCs to MRC-5 cells and the components of modified SAGM.
Assuntos
Células Epiteliais Alveolares/citologia , Células da Medula Óssea/citologia , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Proteína C Associada a Surfactante Pulmonar/genética , Células Epiteliais Alveolares/metabolismo , Células da Medula Óssea/metabolismo , Técnicas de Cocultura , Humanos , Células-Tronco Mesenquimais/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismoRESUMO
Induced pluripotent stem (iPS) cells can be generated from somatic cells of individuals by retrodifferentiation using defined transcription factors. Similar to embryonic stem (ES) cells, iPS cells can be differentiated into a variety of specific cell types. However, to date, no detailed hepatic differentiation of mouse iPS cells has been reported. In this study, we successfully developed a stepwise protocol to induce hepatic differentiation of iPS cells reprogrammed from mouse tail tip fibroblasts. At day 25 of differentiation, the iPS cell-derived hepatocytes morphologically resemble mouse primary hepatocytes with a distinct polygonal shape. Immunostaining and reverse transcription-polymerase chain reaction analysis revealed expression of specific hepatic markers including alpha-fetoprotein, albumin and alpha-1-anti-trypsin. In addition, these iPS cell-derived hepatocytes successfully demonstrated mature liver cell functions in vitro. Furthermore, in vivo assays revealed that the mouse iPS cell-derived hepatocytes successfully engrafted into the recipient livers with typical hepatic morphology. Thus, iPS cell-derived hepatocytes may hold great promise as a unique system for basic liver research and liver regeneration in the near future.
Assuntos
Células-Tronco Embrionárias/fisiologia , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Regeneração Hepática , Animais , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , alfa-Fetoproteínas/metabolismoRESUMO
Microdeletions and microduplications of the 16p11.2 chromosomal locus are associated with syndromic neurodevelopmental disorders and reciprocal physiological conditions such as macro/microcephaly and high/low body mass index. To facilitate cellular and molecular investigations into these phenotypes, 65 clones of human induced pluripotent stem cells (hiPSCs) were generated from 13 individuals with 16p11.2 copy number variations (CNVs). To ensure these cell lines were suitable for downstream mechanistic investigations, a customizable bioinformatic strategy for the detection of random integration and expression of reprogramming vectors was developed and leveraged towards identifying a subset of 'footprint'-free hiPSC clones. Transcriptomic profiling of cortical neural progenitor cells derived from these hiPSCs identified alterations in gene expression patterns which precede morphological abnormalities reported at later neurodevelopmental stages. Interpreting clinical information-available with the cell lines by request from the Simons Foundation Autism Research Initiative-with this transcriptional data revealed disruptions in gene programs related to both nervous system function and cellular metabolism. As demonstrated by these analyses, this publicly available resource has the potential to serve as a powerful medium for probing the etiology of developmental disorders associated with 16p11.2 CNVs.
Assuntos
Deleção de Genes , Células-Tronco Pluripotentes Induzidas/fisiologia , Transtorno do Espectro Autista/genética , Transtorno Autístico , Deleção Cromossômica , Transtornos Cromossômicos , Cromossomos Humanos Par 16 , Variações do Número de Cópias de DNA , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Neurônios/fisiologia , TranscobalaminasRESUMO
We have successfully developed both spontaneous and inductive cardiomyocyte differentiation of iPS cells reprogrammed from human foreskin fibroblasts. The reprogrammed iPS cells morphologically resemble human cardiomyocytes which can beat. RT-PCR and immunostaining show that cardiac markers are expressed that are comparable to the differentiation pattern of authentic human embryonic stem cells, indicating the existence of both immature and mature differentiated cardiomyocytes. 5-Azacytidine greatly enhanced the efficiency of cardiomyocyte differentiation, whereas dimethylsulfoxide had no effect. Low serum and bone morphogenetic protein-2 marginally improved differentiation efficiency. iPS cell-derived cardiomyocytes changed their beat frequency in response to cardiac drugs, which included ion channel blockers and alpha/beta adrenergic stimulators. Derived cardiomyocytes look promising as an in vitro system for potential drug screen and/or toxicity, making this system closer to practical use in the near future.
Assuntos
Diferenciação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Dimetil Sulfóxido/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Fibroblastos/fisiologia , Prepúcio do Pênis/citologia , Sequestradores de Radicais Livres/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Masculino , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos/efeitos dos fármacos , Receptores Adrenérgicos/fisiologia , Transcrição GênicaRESUMO
OBJECTIVE: To screen differentially expressed genes of liver tissue in rat sepsis model and analyze them in terms of functions. METHODS: Thirty male Wistar rats were randomly divided into model group and blank control group with 15 rats in each group. Cecal ligation and puncture (CLP) was used to reproduce rat sepsis model, gene expression profile microarray that contains 4 096 rat cDNA clones was used to detect the change in gene expression pattern of rat liver tissue 24 hours after CLP, then differentially expressed genes that high correlated to sepsis were screened, and the functions of these genes were analyzed by means of related computer software. RESULTS: Compared to the controls, gene expression of 522 genes in rat sepsis model were changed 24 hours after CLP, accounting for 12.7%, among them 244 gene expression down-regulated, and 278 gene expression up-regulated. CONCLUSION: Multiple organ dysfunction syndrome (MODS) induced by sepsis involves to a series of gene differential expressions, such as cell cycle and control related genes, cell apoptosis genes, immunity related genes, genes concerning energy metabolism, blood system related genes, cancer related genes, growth factor genes, acute stress reaction related genes etc. Gene microarray technique can be used to comprehensively study gene expression profile in rat sepsis model, in order to find new research objectives and gene therapy strategies for sepsis.
Assuntos
Fígado/metabolismo , Sepse/metabolismo , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Fígado/fisiopatologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Wistar , Sepse/fisiopatologiaRESUMO
BACKGROUND: Ambient aerosol fine particulate matter (PM2.5) is associated with male reproductive toxicity in experiments and may have adverse effects in the female. However, studies evaluating the protective effects and precise mechanisms of aspirin, Vitamin C, Vitamin E, or ozone against toxic effects of PM2.5are sparse. This study was conducted to investigate the possible protective effects and mechanisms of aspirin, Vitamin C, Vitamin E, or ozone on fertility in female mice treated with PM2.5. METHODS: Eighty-four ICR mice were divided into six groups: control group, PM2.5group, PM2.5 + aspirin group, PM2.5 + Vitamin C group, PM2.5 + Vitamin E group, and PM2.5 + ozone group. PM2.5was given by intratracheal instillation every 2 days for 3 weeks. Aspirin, Vitamin C, and Vitamin E were given once a day by oral gavage for 3 weeks, and ozone was administered by intraperitoneal injection once a day for 3 weeks. The levels of anti-Müllerian hormone (AMH), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured using enzyme-linked immunosorbent assay. Western blotting analysis was used to analyze the expressions of Bcl-2, Bax, and caspase-3 in ovaries. Changes in histological structure were examined by light microscope and electron microscopy was used to detect ultramicrostructure. RESULTS: The results demonstrated that PM2.5 decreased AMH levels (P < 0.001); however, aspirin (P < 0.001), Vitamin C (P < 0.001), Vitamin E (P = 0.001), and ozone (P = 0.002) alleviated the decrease. Changes of IL-6, TNF-α, 8-OHdG, Bax/Bcl-2, and caspase-3 in PM2.5group were increased compared to control group (P < 0.001), while in PM2.5 + aspirin, PM2.5 + Vitamin C, PM2.5 + Vitamin E, and PM2.5 + ozone groups, they were statistically decreased compared to PM2.5group (P < 0.001 or P< 0.05). CONCLUSIONS: PM2.5cause the damage of ovaries, and aspirin, Vitamin C, Vitamin E, and ozone antagonizes the damage. The protective mechanism is probably due to its ability to blunt the inflammatory and oxidative stress caused by PM2.5, which subsequently suppressing the expression of apoptotic regulatory protein and reducing the incidence of ovary apoptosis.
Assuntos
Poluição do Ar/efeitos adversos , Ovário/efeitos dos fármacos , Material Particulado/toxicidade , Animais , Ácido Ascórbico/uso terapêutico , Aspirina/uso terapêutico , Feminino , Infertilidade Feminina/induzido quimicamente , Infertilidade Feminina/prevenção & controle , Masculino , Camundongos , Ovário/fisiopatologia , Ozônio/toxicidade , Vitamina E/uso terapêuticoRESUMO
Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.
Assuntos
Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/patologia , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/deficiência , Neurônios/fisiologia , Telencéfalo/fisiologia , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologiaRESUMO
We have examined the myogenic potential of human embryonic stem (hES) cells in a xeno-transplantation animal model. Here we show that precursors differentiated from hES cells can undergo myogenesis in an adult environment and give rise to a range of cell types in the myogenic lineage. This study provides direct evidences that hES cells can regenerate both muscle and satellite cells in vivo and are another promising cell type for treating muscle degenerative disorders in addition to other myogenic cell types.
Assuntos
Células-Tronco Embrionárias/fisiologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Células Satélites de Músculo Esquelético/fisiologia , Transplante Heterólogo , Animais , Biomarcadores/metabolismo , Cardiotoxinas/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/citologia , Condicionamento Pré-TransplanteRESUMO
Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.
Assuntos
Blastocisto/citologia , Linhagem Celular , Clonagem de Organismos/métodos , Células-Tronco Embrionárias/fisiologia , Fertilização , Técnicas de Transferência Nuclear , Partenogênese , Animais , Blastocisto/fisiologia , Diferenciação Celular , Meios de Cultura Livres de Soro , Células-Tronco Embrionárias/citologia , Humanos , Cariotipagem , Proteínas Luminescentes/metabolismo , Camundongos , Repetições de Microssatélites , Coelhos , Teratoma/metabolismo , Proteína Vermelha FluorescenteRESUMO
Human embryonic stem cells (hESCs) are pluripotent. They have the potential to differentiate into every cell type of an organism. Since many human somatic cell types have the ability to support the growth of hESCs, cells differentiated from hESCs may also be able to support the growth of themselves. We tested this hypothesis by growing hESCs on feeders derived from themselves and demonstrated that such feeders did constitute an environment suitable for the derivation and long-term growth of hESCs. hESCs maintained in this system expressed all the markers indicative of the undifferentiated state and gave rise to cell types representative of all three primary germ layers upon differentiation. By modifying the genome of hESCs, feeders with special features can be derived and mass produced. The system will facilitate large-scale production of hESCs in a standardized animal pathogen-free environment.
Assuntos
Células-Tronco Pluripotentes/citologia , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Meios de Cultura , Meios de Cultivo Condicionados , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Imuno-Histoquímica , Cariotipagem , Camundongos , Microscopia de Contraste de Fase , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.