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The transforming growth factor-ß (TGF-ß) superfamily signaling pathway contains two general branches, known as TGF-ß and bone morphogenetic protein (BMP), that regulate development in animals. It is well known that TGF-ß superfamily signaling participates in the regulation of dauer (lifespan extension) in Caenorhabditis elegans, but little is known about the molecular mechanisms of lifespan extension in the pathway. Diapause, a programmed developmental arrest in insects, is similar to dauer in C. elegans. In this study, we find that TGF-ß superfamily signaling regulates Helicoverpa armigera diapause via a novel mechanism. Both TGF-ß and BMP signals are weaker in the brains of diapause-destined pupae than in nondiapause-destined pupae, and the levels of p-Smad1, POU, TFAM, and mitochondrial activity are decreased in diapause pupae. Development in nondiapause pupae is delayed by an injection of TGF-ß or BMP receptor inhibitors. Both TGF-ß and BMP signals can activate a common target, Smad1. ChIP and EMSA assays indicate that Smad1 can bind to the POU promoter to regulate its expression. POU can improve the transcription of TFAM, which regulates mitochondrial activity. This is the first report showing that both TGF-ß and BMP signals regulate development or diapause through the Smad1-POU-TFAM-mitochondrial activity in insects.
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Proteínas Morfogenéticas Ósseas/metabolismo , Mariposas/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Encéfalo/metabolismo , Diapausa de Inseto , Proteínas de Insetos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fatores do Domínio POU/genética , Regiões Promotoras Genéticas , Proteínas Smad/genética , Fatores de Transcrição/genéticaRESUMO
PURPOSE: The goal of this article is to evaluate the efficacy and the safety of the percutaneous vertebroplasty (PVP) versus percutaneous kyphoplasty (PKP) in dealing with the osteoporotic vertebral compression fracture (OVCF). METHODS: In July 2014, a comprehensive systematic computer-based online search was performed by using the databases of PubMed, EMBASE, Cochrane Library, Web of Science, Wan Fang, and the China Biological Medicine. Only prospective comparative trials (PCT) and randomized controlled trials (RCT) that compared PVP with PKP were included. Trials were screened based on the inclusion and exclusion criteria previously formed. The Cochrane collaboration guidelines were also used to assess the quality of these included studies. The primary data of these studies [volume of the cement, postoperative vertebral height, visual analog scale (VAS) score and Oswestry Disability Index (ODI) score after the surgery, and so on] were carefully abstracted and processed by Revman 5.2.0 software The publication bias of the main results (cement leakage and adjacent-level fracture) were examined by Stata 12.0 (Begg and Egger test). Furthermore, the stability of the main results were also detected by sensitivity and cumulative analyses. RESULTS: Six RCT and 14 PCT studies involving 1,429 patients met our criteria and were included finally. Comparing these two methods, the PKP group took more operation time [SMD = 0.66, 95 % CI (0.28, 1.03), p = 0.0006] with higher anterior vertebral body height [SMD = 1.40, 95 % CI (0.49, 2.32), p = 0.003], greatly reduced Cobb angle in the long run [SMD = -0.61, 95 % CI (-1.04, -0.19), p = 0.005] and had lower risk of cement leakage. However, in VAS scores and ODI scores after the surgery whether for the short-term efficacy (no more than 1 week after the surgery) or long-term efficacy (more than six months), Cobb angle in the short run and new fracture in the adjacent level, no statistically differences were found between the two groups. CONCLUSIONS: Based on current evidence, PVP takes less time in the operation, while it has greater risk of cement leakage, was inferior in reducing Cobb angle in the long term and results in lower anterior vertebral body height after the surgery. For pain relief, which is the main desire of the patients, both procedures provide significant improvement in VAS and ODI pain scores. PVP is still an effective procedure.
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Fraturas por Compressão/cirurgia , Cifoplastia/métodos , Fraturas por Osteoporose/cirurgia , Fraturas da Coluna Vertebral/cirurgia , Vertebroplastia/métodos , Idoso , Cimentos Ósseos/efeitos adversos , Cimentos Ósseos/uso terapêutico , Humanos , Cifoplastia/efeitos adversos , Masculino , Dor/cirurgia , Manejo da Dor , Complicações Pós-Operatórias , Estudos Prospectivos , Coluna Vertebral/cirurgia , Resultado do Tratamento , Vertebroplastia/efeitos adversosRESUMO
TDP-43 (TAR DNA-binding protein of 43 kDa) is a major deposited protein in amyotrophic lateral sclerosis and frontotemporal dementia with ubiquitin. A great number of genetic mutations identified in the flexible C-terminal region are associated with disease pathologies. We investigated the molecular determinants of TDP-43 aggregation and its underlying mechanisms. We identified a hydrophobic patch (residues 318-343) as the amyloidogenic core essential for TDP-43 aggregation. Biophysical studies demonstrated that the homologous peptide formed a helix-turn-helix structure in solution, whereas it underwent structural transformation from an α-helix to a ß-sheet during aggregation. Mutation or deletion of this core region significantly reduced the aggregation and cytoplasmic inclusions of full-length TDP-43 (or TDP-35 fragment) in cells. Thus, structural transformation of the amyloidogenic core initiates the aggregation and cytoplasmic inclusion formation of TDP-43. This particular core region provides a potential therapeutic target to design small-molecule compounds for mitigating TDP-43 proteinopathies.
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Amiloide/metabolismo , Proteínas de Ligação a DNA/metabolismo , Corpos de Inclusão/metabolismo , Amiloide/genética , Animais , Caenorhabditis elegans , Proteínas de Ligação a DNA/genética , Desenho de Fármacos , Células HeLa , Sequências Hélice-Volta-Hélice , Humanos , Interações Hidrofóbicas e Hidrofílicas , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Estrutura Terciária de Proteína , Proteinopatias TDP-43/tratamento farmacológico , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/metabolismo , Proteinopatias TDP-43/patologiaRESUMO
PURPOSE: The objective of this study was to compare the safety and efficacy of minimally invasive discectomy (MID) with standard discectomy (SD) and determine whether the use of the MID technique could decrease the recurrence of lumbar disc herniation (LDH) after the surgery. METHODS: In February 2014, a comprehensive search was performed in PubMed, EMBASE, Web of Science, Cochrane Library and the Chinese Biological Medicine Database. Only randomised controlled trials (RCT) that compared MID with SD for the surgical management of LDH were included. These trials were carefully picked out following the inclusion and exclusion criteria. Using the Cochrane Collaboration guidelines, two authors independently extracted data and assessed these trials' quality. The age of the patients, size of incision, surgical time, blood loss, visual analogue scale (VAS) score after the surgery, hospital stay, disc herniation recurrence, X-ray exposure and surgical costs in these studies were abstracted and synthesised by a meta-analysis with RevMan 5.2.0 software, and the main results (VAS score after the surgery and disc herniation recurrence) of publication bias were examined by Stata 12.0. RESULTS: Overall, 16 trials involving 2,139 patients meeting our criteria were included and analysed. Comparing MID and SD, the former was more likely to increase disc herniation recurrence [relative risk (RR) = 1.95, 95 % confidence interval (CI) 1.19-3.19, p = 0.008], and it involved a smaller size of incision [mean difference (MD) = -1.91, 95 % CI -3.33 to -0.50, p = 0.008], shorter hospital stay, longer operating time (MD = 11.03, 95 %C I 6.62-15.44, p < 0.00001) and less blood loss (MD = -13.56, 95 % CI -22.26 to -4.87, p = 0.002), while no statistical difference appeared with regard to the age of the patients, VAS score after the surgery, X-ray exposure, hospital stay and surgical costs. CONCLUSIONS: Based on available evidence, MID results in less suffering for patients during the hospital course with a similar clinical efficacy compared to SD. This makes MID a promising procedure for patients with LDH; however, to popularise it greater effort is required to reduce disc herniation recurrence.
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Discotomia/métodos , Deslocamento do Disco Intervertebral/cirurgia , Vértebras Lombares , Humanos , Deslocamento do Disco Intervertebral/prevenção & controle , Procedimentos Cirúrgicos Minimamente Invasivos , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Recidiva , Resultado do TratamentoRESUMO
In the Gram-negative bacterium of Escherichia coli, eight genes organized as a ccm operon (ccmABCDEFGH) are involved in the maturation of c-type cytochromes. The proteins encoded by the last three genes ccmFGH are believed to form a lyase complex functioning in the reduction of apocytochrome c and haem attachment. Among them, CcmH is a membrane-associated protein; its N-terminus is a catalytic domain with the active CXXC motif and the C-terminus is predicted as a TPR-like domain with unknown function. By using SCAM (scanning cysteine accessibility mutagenesis) and Gaussia luciferase fusion assays, we provide experimental evidence for the entire topological structure of E. coli CcmH. The mature CcmH is a periplasm-resident oxidoreductase anchored to the inner membrane by two transmembrane segments. Both N- and C-terminal domains are located and function in the periplasmic compartment. Moreover, the N-terminal domain forms a monomer in solution, while the C-terminal domain is a compact fold with helical structures. The NMR solution structure of the catalytic domain in reduced form exhibits mainly a three-helix bundle, providing further information for the redox mechanism. The redox potential suggests that CcmH exhibits a strong reductase that may function in the last step of reduction of apocytochrome c for haem attachment.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Domínio Catalítico , Proteínas de Escherichia coli/química , Modelos Moleculares , Oxirredução , Dobramento de ProteínaRESUMO
Reactive oxygen species (ROS) are considered a major cause of ageing and ageing-related diseases through protein carbonylation. Little is known about the molecular mechanisms that confer protection against ROS. Here, we observed that, compared with nondiapause-destined pupae, high protein carbonyl levels are present in the brains of diapause-destined pupae, which is a 'non-ageing' phase in the moth Helicoverpa armigera. Protein carbonyl levels respond to ROS and decrease metabolic activity to induce diapause in order to extend lifespan. However, protein carbonylation in the brains of diapause-destined pupae still occurs at a physiological level compared to young adult brains. We find that ROS activate Akt, and Akt then phosphorylates the transcription factor CREB to facilitate its nuclear import. CREB binds to the promoter of carbonyl reductase 1 (CBR1) and regulates its expression. High CBR1 levels reduce protein carbonyl levels to maintain physiological levels. This is the first report showing that the moth brain can naturally control protein carbonyl levels through a distinct ROS-Akt-CREB-CBR1 pathway to extend lifespan.
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Mariposas , Proteínas Proto-Oncogênicas c-akt , Animais , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Carbonil Redutase (NADPH) , Longevidade/fisiologia , Carbonilação Proteica , Mariposas/genética , Mariposas/metabolismo , Pupa/metabolismoRESUMO
A novel piezochromic fluorescent (PCF) compound with aggregation-induced emission (AIE) effect and morphology-alterable emission property was developed. The amorphous and crystalline aggregates were obtained, and their spectroscopic properties and morphological structures were reversibly and repeatedly exhibited upon pressing (fuming) or annealing. The piezochromic fluorescent nature was generated through crystalline-amorphous phase transformation. It was proposed that AIE compounds existing a twisted propeller-shaped conformation will exhibit PCF activity. The common relationship betweeen AIE and PCF established will guide researchers in identifying and synthesizing more piezochromic fluorescent materials.
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Compostos de Anilina/química , Etilenos/química , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Modelos Moleculares , Estrutura MolecularRESUMO
Aspongopus chinensis is a Chinese traditional edible and medicinal insect, which is in great demand in the society. This insect reproduces once a year which is caused by reproductive diapause resulting in insufficient production in wild resources. However, the mechanism of diapause in A. chinensis is still unclear. In this study, we focus on the relationship between juvenile hormones (JHs) and A. chinensis diapause. The results showed that JHIII concentration in diapause adult individuals was significantly lower than that in diapause termination adult individuals. When exogenous JHâ ¢ was injected into diapause adults, the rate of mating was increased significantly, development of the reproductive systems was accelerated, consumption of fat intensified, the expression of juvenile hormone acid o-methyl-transferase (JHAMT) was upregulated, and juvenile hormone epoxide hydrolase (JHEH) and fatty acid synthase (FAS) gene expressions were downregulated. In addition, RNAi of JHAMT decreased JH concentration, delayed the development of reproductive systems, slowed down fat consumption, and delayed the mean mating occurrence time significantly. Conversely, RNAi of JHEH resulted in an increased concentration of JH, development of reproductive systems was accelerated, consumption of fat was intensified, and mean mating occurrence time advanced significantly. Taken together, these findings uncovered that JH plays an important role in regulating reproductive diapause in A. chinensis and, thus, could provide a theoretical basis for further research on the diapause of A. chinensis.
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BACKGROUND: Nucleus pulposus mesenchymal stem cells (NPMSCs) transplantation is a promising treatment for intervertebral disc degeneration (IVDD). However, the transplanted NPMSCs exhibited weak cell proliferation, high cell apoptosis, and a low ability to resist the harsh microenvironment of the degenerated intervertebral disc. There is an urgent need to explore feasible methods to enhance the therapeutic efficacy of NPMSCs transplantation. OBJECTIVE: To identify the optimal concentration for NPMSCs pretreatment with hydrogen peroxide (H2O2) and explore the therapeutic efficacy of NPMSCs transplantation using H2O2 pretreatment in IVDD. METHODS: Rat NPMSCs were pretreated with different concentrations (range from 25 to 300 µM) of H2O2. The proliferation, reactive oxygen species (ROS) level, and apoptosis of NPMSCs were detected by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, and flow cytometry in vitro. The underlying signalling pathways were explored utilizing Western blotting. A rat needle puncture-stimulated IVDD model was established. X-ray, histological staining, and a multimode small animal live imaging system were used to evaluate the therapeutic effect of H2O2-pretreated NPMSCs in vivo. RESULTS: NPMSCs pretreated with 75 µM H2O2 demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway (P < 0.01). Meanwhile, 75 µM H2O2-pretreated NPMSCs exhibited significantly enhanced antioxidative stress ability (P < 0.01), which is related to downregulated Brd4 and Keap1 and upregulated Nrf2. NPMSCs pretreated with 75 µM H2O2 also exhibited distinctly decreased apoptosis (P < 0.01). In vivo experiments verified that 75 µM H2O2-pretreated NPMSCs-transplanted rats exhibited an enhanced disc height index (DHI% = 90.00 ± 4.55, P < 0.01) and better histological morphology (histological score = 13.5 ± 0.5, P < 0.01), which means 75 µM H2O2-pretreated NPMSCs can better adapt to the environment of degenerative intervertebral discs and promote the repair of IVDD. CONCLUSIONS: Pretreatment with 75 µM H2O2 was the optimal concentration to improve the proliferation, antioxidative stress, and antiapoptotic ability of transplanted NPMSCs, which is expected to provide a new feasible method to improve the stem cell therapy efficacy of IVDD.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Células-Tronco Mesenquimais , Núcleo Pulposo , Animais , Apoptose , Peróxido de Hidrogênio/farmacologia , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Proteína 1 Associada a ECH Semelhante a Kelch , Células-Tronco Mesenquimais/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Núcleo Pulposo/metabolismo , RatosRESUMO
New aggregation-induced emission (AIE) compounds derived from triphenylethylene were synthesized. The thermal, photophysical, electrochemical and aggregation-induced emissive properties were investigated. All the compounds had strong blue light emission capability and good thermal stability. Their maximum fluorescence emission wavelengths were between 443 to 461 nm in solid states, while their glass transition temperatures ranged from 86 to 129 °C. The decomposition temperatures of the synthesized compounds were in the range of 432-534 °C. The synthesized compounds possessed aggregation-induced emission properties, namely exhibited enhanced fluorescence emission in aggregated states. The highest occupied molecular orbital (HOMO) energy levels estimated from the oxidation potentials were between 5.61 and 5.66 eV and the lowest unoccupied molecular orbital/highest occupied molecular orbital (LUMO/HOMO) energy gap values were found to be in the range of 3.18-3.22 eV. The compounds 4-(4-(2,2-bis(4-(naphthalen-1-yl)phenyl)vinyl)phenyl) dibenzothiophene [(BN)(2)Bt] and 4-(4-(2,2-di(biphenyl-4-yl)vinyl)phenyl) dibenzothiophene [(BB)(2)Bt] exhibited vibronic fine-structure photoluminescence spectra when the water fraction was less than 70%.
Assuntos
Fluorescência , Estilbenos/química , Estilbenos/síntese química , Medições Luminescentes , Estrutura Molecular , Teoria Quântica , EstereoisomerismoRESUMO
Two cinnamic acid derivatives (CPA and CPC) containing carbazolyl triphenylethylene moiety have been synthesized and characterized. The two derivatives possessed aggregation-induced emission property. They exhibited different and interesting responsive behaviors to solvents, water and metal ions. Considering the structural differences between the two derivatives resulting in different interactions between their molecules and the various media was proposed as a possible explanation for these observations. The intermolecular interactions of CPC were much stronger than those of CPA, which promoted molecular association through intermolecular hydrogen bonding to form multimers. It was found that CPC and CPA exhibited high sensitivity to K(+) and Mn(2+), respectively. It is suggested that the derivatives have potential technological applications in chemosensor fields.
Assuntos
Carbazóis/química , Cinamatos/química , Estilbenos/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
New aggregation-induced emission materials derived from diphenylcarbazole triphenylethylene were prepared. The thermal, photophysical, electrochemical and aggregation-induced emissive properties were investigated. All the compounds had strong blue light emission capability and excellent thermal stability. Their maximum fluorescence emission wavelengths were between 450 to 460 nm in TLC plates, while their glass transition temperatures ranged from 162.2 to 182.4 °C. The decomposition temperatures of the synthesized compounds were all well over 500 °C. The synthesized compounds possessed aggregation-induced emission (AIE) properties, which exhibited enhanced fluorescence emissions in aggregation states or in solid states. The HOMO energy levels estimated from the oxidation potentials were found in the range from 5.49 to5.52 eV. The lowest unoccupied molecular orbital/highest occupied molecular orbital (LUMO/HOMO) energy gaps (ΔEg) for the compounds were estimated from the onset absorption wavelengths of UV absorption spectra and ranged from 3.04 to 3.20 eV.
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Objective: Nucleus pulposus cells (NPCs) are cells extracted from the intervertebral disc and are important for research into intervertebral disc degeneration (IVDD). NPCs live in an avascular and relatively hypoxic environment. Cobalt chloride (CoCl2) has been used in many cell studies to mimic hypoxia. The objective of this study was to explore the possibility of using CoCl2 to induce mimetic-hypoxia for NPCs and the comparison with hypoxia (1% O2) in vitro. Materials and methods: Rat nucleus pulposus cells of Passage 3-5 were used in this research. Cell viability, rate of cell apoptosis, ROS (reactive oxygen species) generation, cell migration, extracellular pH and extracellular matrix metabolism were determined to compare the influence of hypoxia (1% O2) and CoCl2 on NPCs. Results: We found that the effects of CoCl2 on NPCs was dose-dependent. At the proper concentration, CoCl2 could be used to elicit chemical hypoxia for nucleus pulposus cells in vitro and many biological effects, analogous to physical hypoxia (1% O2), could be achieved such as enhanced cell viability, decreased apoptosis and activated extracellular matrix metabolism. On the other hand, CoCl2 mimetic-hypoxia did not affect NPCs glycolysis and migration compared to physical hypoxia. In addition, high concentration of CoCl2 (>200 µM) is harmful to NPCs with high rates of apoptosis and ECM (extracellular matrix) degradation. Conclusions: It is feasible and convenient to use CoCl2 to induce chemical mimetic hypoxia for culturing NPCs on the premise of appropriate concentration. But in aspects of cell migration and glycolysis, CoCl2 could not achieve similar results with physical hypoxia. This study may provide a convenient method and enlightenment to induce mimetic-hypoxia for researchers studying NPCs and IVVD.
Assuntos
Degeneração do Disco Intervertebral , Núcleo Pulposo , Animais , Apoptose , Células Cultivadas , Cobalto/toxicidade , Hipóxia , RatosRESUMO
Intervertebral disc (IVD) degenerative diseases are a common problem in the world, and they cause substantial social and economic burdens for people. The current methods for treating IVD degenerative diseases mainly include surgery and conservative treatment, which cannot fundamentally restore the normal structure of the disc. With continuous research on the mechanism of degeneration and the development of regenerative medicine, rapid progress has been made in the field of regenerative medicine regarding the use of stem cell-derived exosomes, which are active biological substances used in intercellular communication, because they show a strong effect in promoting tissue regeneration. The study of exosomes in the field of IVD degeneration has just begun, and many surprising achievements have been made. This paper mainly reviews the biological characteristics of exosomes and highlights the current status of exosomes in the field of IVD degeneration, as well as future developments regarding exosomes.
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Stem cell therapies for intervertebral disc degeneration have been demonstrated as a promising strategy. Previous studies have shown that human nucleus pulposus cell- (NPC-) derived exosomes can induce the differentiation of mesenchymal stem cells (MSCs) into NP-like cells in vitro. However, the mechanism of MSC differentiation into NP-like cells with the induction of NPC exosomes is still unclear. Here, we verified the induction effects of NPC exosomes on the differentiation of MSCs into NP-like cells. In addition, the Notch1 pathway was downregulated in this process. Then, DAPT and soluble Jagged1 (SJAG) were applied to inhibit or enhance the expression of the Notch1 pathway, respectively, resulting in the upregulation or downregulation of collagen II, aggrecan, and Sox9 in MSCs. Knocking down of Notch1 protein facilitated the effects of NPC exosomes on the differentiation of MSCs into NP-like cells. NPC exosomes were more effective than an indirect coculture system in terms of the differentiation of MSCs into NP-like cells. Inhibition of NPC exosome secretion with Rab27a siRNA prevented the induction effects of an indirect coculture system on the differentiation of MSCs into NP-like cells. Transwell migration assays revealed that NPC exosomes could promote the migration of MSCs. Taken together, the Notch1 pathway was negatively associated with the differentiation of MSCs into NP-like cells with the treatments of NPC exosomes. Inhibition of the Notch1 pathway facilitates NPC exosome-induced differentiation of MSCs into NP-like cells in vitro. NPC exosomes play a key role in the differentiation of MSCs into NP-like cells in an indirect coculture system of NPCs and MSCs.
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Akt, which is a key kinase in the insulin signaling pathway, plays important roles in glucose metabolism, cell proliferation, transcription and cell migration. Our previous studies indicated that low insulin levels and high p-Akt levels are present in diapause-destined individuals. Here, we show that PI3K, which is upstream of Akt, is low in diapause-destined pupal brains but high in p-Akt levels, implying that p-Akt is modified by factors other than the insulin signaling pathway. Protein phosphatase 2A (PP2A), which is a key regulator in the TGF-ß signaling pathway, can directly bind to and dephosphorylate Akt. Low PP2A expression and activity in diapause-destined individuals suggest that a weak Akt dephosphorylation contributes to p-Akt accumulation. In addition, transforming growth factor-ß receptor I (TßRI), which is upstream of PP2A, increases the activity of PP2A and decreases the p-Akt levels. These results show that TGF-ß signaling decreases p-Akt levels by increasing the activity of PP2A. This is the first report showing that TGF-ß signaling negatively regulates the insulin pathway in insect development or diapause.
Assuntos
Diapausa de Inseto/fisiologia , Mariposas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Animais , Encéfalo/metabolismo , Mariposas/crescimento & desenvolvimento , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pupa/enzimologia , Pupa/crescimento & desenvolvimento , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: The stem cell-based therapies for intervertebral disc degeneration have been widely studied. However, the mechanisms of mesenchymal stem cells interacting with intervertebral disc cells, such as nucleus pulposus cells (NPCs), remain unknown. Exosomes as a vital paracrine mechanism in cell-cell communication have been highly focused on. The purpose of this study was to detect the role of exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) and NPCs in their interaction with corresponding cells. METHODS: The exosomes secreted by BM-MSCs and NPCs were purified by differential centrifugation and identified by transmission electron microscope and immunoblot analysis of exosomal marker proteins. Fluorescence confocal microscopy was used to examine the uptake of exosomes by recipient cells. The effects of NPC exosomes on the migration and differentiation of BM-MSCs were determined by transwell migration assays and quantitative RT-PCR analysis of NPC phenotypic genes. Western blot analysis was performed to examine proteins such as aggrecan, sox-9, collagen II and hif-1α in the induced BM-MSCs. Proliferation and the gene expression profile of NPCs induced by BM-MSC exosomes were measured by Cell Counting Kit-8 and qRT-PCR analysis, respectively. RESULTS: Both the NPCs and BM-MSCs secreted exosomes, and these exosomes underwent uptake by the corresponding cells. NPC-derived exosomes promoted BM-MSC migration and induced BM-MSC differentiation to a nucleus pulposus-like phenotype. BM-MSC-derived exosomes promoted NPC proliferation and healthier extracellular matrix production in the degenerate NPCs. CONCLUSION: Our study indicates that the exosomes act as an important vehicle in information exchange between BM-MSCs and NPCs. Given a variety of functions and multiple advantages, exosomes alone or loaded with specific genes and drugs would be an appropriate option in a cell-free therapy strategy for intervertebral disc degeneration.
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Diferenciação Celular , Exossomos/metabolismo , Células-Tronco Mesenquimais/citologia , Núcleo Pulposo/citologia , Agrecanas/genética , Agrecanas/metabolismo , Movimento Celular , Células Cultivadas , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Núcleo Pulposo/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
TDP-43 (TAR DNA binding protein of 43 kDa) and its C-terminal fragments are thought to be linked to the pathologies of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Here, we demonstrate that the aggregates or inclusions formed by its 35-kDa fragment (namely TDP-35) sequester full-length TDP-43 into cytoplasmic inclusions; and this sequestration is mediated by binding with RNA that is enriched in the cytoplasmic inclusions. RNA recognition motif 1 (RRM1) of TDP-43/TDP-35 plays a dominant role in nucleic-acid binding; mutation in this moiety abrogates formation of the TDP-35 inclusions and its RNA-assisted association with TDP-43. Thus, TDP-35 is able to sequester TDP-43 from nuclear localization into cytoplasmic inclusions, and RNA binding plays an essential role in this process.
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Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fragmentos de Peptídeos/fisiologia , RNA/metabolismo , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Mutação , Fragmentos de Peptídeos/genética , Reação em Cadeia da Polimerase , Ligação Proteica , RNA/genéticaRESUMO
STUDY DESIGN: Human ligamentum flavum (LF)-derived stem cells (LFSCs) and nucleus pulposus cells (NPCs) were cocultured under normoxia or hypoxia. OBJECTIVE: To isolate and identify human LFSCs and determine whether they can differentiate into NPCs when cocultured with NPCs under hypoxia. SUMMARY OF BACKGROUND DATA: Mesenchymal stem cell (MSC)-based therapies have been proposed as a biological treatment for intervertebral disc degeneration. MSCs derived from various tissues are leading candidates for cell-based therapies, but such cells have not been reported in LF. METHODS: LF cells were isolated from patient samples and cultured using culture flasks coated with fibronectin, and their identity was confirmed using flow cytometry. The cells were induced to differentiate into osteoblasts, chondrocytes, and adipocytes, and their morphology, immunophenotype, cell proliferation capacity, cell cycle, and expression of stem cell-specific genes were compared with those of bone marrow-MSCs (BM-MSCs) derived from the same patients. NPCs and LFSCs were cocultured in 1-µm-pore-size insert transwell-culture systems under hypoxia (2% O2) or normoxia. CD24 expression was measured by flow cytometry and confocal microscopy assay. On day 14, reverse transcription-polymerase chain reaction was used for comparing the expression of chondrogenic genes (Sox-9, collagen-II, aggrecan) and novel marker genes (KRT19, CA12, FOXF1, HIF-1α) between the 2 groups. RESULTS: LFSCs were obtained using the fibronectin differential-adhesion assay. The morphology of LFSCs was altered, and their immunophenotype, multilineage induction, cell proliferation capacity, cell cycle, and stem cell-specific gene expression were closely related-but not identical-to BM-MSCs, CD24 expression was highly significant in the differentiated LFSCs. RT/Real-time polymerase chain reaction revealed that compared with LFSCs grown under normoxia, hypoxia-treated LFSCs expressed higher levels of Sox-9, collagen-II, aggrecan, KRT19, CA12, and HIF-1α genes except FOXF1. CONCLUSION: Stem cells were identified in human LF, and LFSCs cocultured with NPCs were successfully differentiated into NP-like cells under hypoxia. This potentially provides new cell candidates for cell-based regenerative medicine and tissue engineering. LEVEL OF EVIDENCE: N/A.
Assuntos
Comunicação Celular , Diferenciação Celular , Condrogênese , Técnicas de Cocultura , Disco Intervertebral/patologia , Ligamento Amarelo/patologia , Células-Tronco Mesenquimais/patologia , Adipogenia , Adulto , Idoso , Ciclo Celular , Diferenciação Celular/genética , Hipóxia Celular , Proliferação de Células , Separação Celular , Forma Celular , Células Cultivadas , Condrogênese/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Humanos , Disco Intervertebral/metabolismo , Ligamento Amarelo/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Fenótipo , Engenharia TecidualRESUMO
Correct localization and transmembrane topology are crucial for the proteins residing and functioning in the endoplasmic reticulum (ER). We have developed a rapid and convenient assay, based on the redox-sensitive luciferase from Gaussia princeps (Gluc) and green fluorescence protein (GFP), to determine the localization or topology of ER proteins. Using the tandem Gluc-GFP reporter fused to different positions of a target protein, we successfully characterized the topologies of two ER transmembrane proteins Herp and HRD1 that are involved in the ER quality control system. This assay method may also be applicable to the proteins in secretory pathway, plasma membrane, and other compartments of cells.