RESUMO
We present a chip design allowing rapid and robust lipid bilayer (LBL) membrane formation using a Parylene coated thin silicon nitride aperture. After bilayer formation, single membrane channels can be reconstituted and characterized by electrophysiology. The ability for robust reconstitution will allow parallelization and enhanced screening of small molecule drugs acting on or permeating across the membrane channel. The aperture was realized on a microfabricated silicon nitride membrane by using standard clean-room fabrication processes. To ensure the lipid bilayer formation, the nitride membrane was coated with a hydrophobic and biocompatible Parylene layer. We tested both Parylene-C and Parylene-AF4. The contact angle measurements on both Parylene types showed very good hydrophobic properties and affinity to lipids. No precoating of the Parylene with an organic solvent is needed to make the aperture lipophilic, in contradiction to Teflon membranes. The chips can be easily placed in an array utilizing a 3D printed platform. Experiments show repetitive LBL formation and destruction (more than 6 times) within a very short time (few seconds). Through measurements we have established that the LBL layers are very thin. This allows the investigation of the fusion process of membrane proteins i.e. outer membrane protein (OmpF) in the LBL within a few minutes.
Assuntos
Bioensaio/instrumentação , Membrana Celular/metabolismo , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Perileno/química , Cinética , Permeabilidade , Impressão Tridimensional , Compostos de Silício/químicaRESUMO
Hemogenic endothelial cells (HECs) are considered to be the origin of hematopoietic stem cells (HSCs). HECs have been identified in differentiating mouse embryonic stem cells (ESCs) as VE-cadherin+ cells with both hematopoietic and endothelial potential in single cells. Although the bipotential state of HECs is a key to cell fate decision toward HSCs, the molecular basis of the regulation of the bipotential state has not been well understood. Here, we report that the CD41+ fraction of CD45- CD31+ VE-cadherin+ endothelial cells (ECs) from mouse ESCs encompasses an enriched HEC population. The CD41+ ECs expressed Runx1, Tal1, Etv2, and Sox17, and contained progenitors for both ECs and hematopoietic cells (HCs) at a high frequency. Clonal analyses of cell differentiation confirmed that one out of five HC progenitors in the CD41+ ECs possessed the bipotential state that led also to EC colony formation. A phenotypically identical cell population was found in mouse embryos, although the potential was more biased to hematopoietic fate with rare bipotential progenitors. ESC-derived bipotential HECs were further enriched in the CD41+ CXCR4+ subpopulation. Stimulation with CXCL12 during the generation of VE-cadherin+ CXCR4+ cells attenuated the EC colony-forming ability, thereby resulted in a decrease of bipotential progenitors in the CD41+ CXCR4+ subpopulation. Our results suggest that CXCL12/CXCR4 signaling negatively modulates the bipotential state of HECs independently of the hematopoietic fate. Identification of signaling molecules controlling the bipotential state is crucial to modulate the HEC differentiation and to induce HSCs from ESCs. Stem Cells 2016;34:2814-2824.
Assuntos
Células Endoteliais/metabolismo , Hemangioblastos/citologia , Hemangioblastos/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Receptores CXCR4/metabolismo , Transdução de Sinais , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Linhagem Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Hematopoese , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Células-Tronco Embrionárias Murinas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismoRESUMO
Mutations in U2AF1 are relatively common in myelodysplastic neoplasms (MDS) and are associated with an inferior prognosis, but the molecular mechanisms underlying this are not fully elucidated. Circular RNAs (circRNAs) have been implicated in cancer, but it is unknown how mutations in splicing factors may impact on circRNA biogenesis. Here, we used RNA-sequencing to investigate the effects of U2AF1 mutations on circRNA expression in K562 cells with a doxycycline-inducible U2AF1S34 mutation, in a mouse model with a doxycycline-inducible U2AF1S34 mutation, and in FACS-sorted CD34+ bone marrow cells from MDS patients with either U2AF1S34 or U2AF1Q157 mutations. In all contexts, we found an increase in global circRNA levels in the U2AF1-mutated setting, which was independent of expression changes in the cognate linear host genes. In patients, the U2AF1S34 and U2AF1Q157 mutations were both associated with an overall increased expression of circRNAs. circRNAs generated by a non-Alu-mediated mechanism generally showed the largest increase in expression levels. Several well-described cancer-associated circRNAs, including circZNF609 and circCSNK1G3, were upregulated in MDS patients with U2AF1 mutations compared to U2AF1-wildtype MDS controls. In conclusion, high circRNA expression is observed in association with U2AF1 mutations in three biological systems, presenting an interesting possibility for biomarker and therapeutic investigation.
Assuntos
Síndromes Mielodisplásicas , Neoplasias , Animais , Camundongos , RNA Circular/genética , Fator de Processamento U2AF/genética , Doxiciclina , Fatores de Processamento de RNA/genética , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Mutação , Splicing de RNARESUMO
In this work, we present a microsystem setup for performing sensitive biological membrane translocation measurements. Thin free-standing synthetic bilayer lipid membranes (BLM) were constructed in microfabricated silicon nitride apertures (<100 µm in diameter), conformal coated with Parylene (Parylene-C or Parylene-AF4). Within these BLMs, electrophysiological measurements were conducted to monitor the behavior of different pore proteins. Two approaches to integrate pore-forming proteins into the membrane were applied: direct reconstitution and reconstitution via outer membrane vesicles (OMVs) released from Gram-negative bacteria. The advantage of utilizing OMVs is that the pore proteins remain in their native lipid and lipopolysaccharide (LPS) environment, representing a more natural state compared to the usage of fused purified pore proteins. Multiple aperture chips can be easily assembled in the 3d-printed holder to conduct parallel membrane transport investigations. Moreover, well defined microfabricated apertures are achievable with very high reproducibility. The presented microsystem allows the investigation of fast gating events (down to 1 ms), pore blocking by an antibiotic, and gating events of small pores (amplitude of approx. 3 pA).
RESUMO
Somatic mutations in spliceosome genes are found in â¼50% of patients with myelodysplastic syndromes (MDS), a myeloid malignancy associated with low blood counts. Expression of the mutant splicing factor U2AF1(S34F) alters hematopoiesis and mRNA splicing in mice. Our understanding of the functionally relevant alternatively spliced target genes that cause hematopoietic phenotypes in vivo remains incomplete. Here, we demonstrate that reduced expression of H2afy1.1, an alternatively spliced isoform of the histone H2A variant gene H2afy, is responsible for reduced B cells in U2AF1(S34F) mice. Deletion of H2afy or expression of U2AF1(S34F) reduces expression of Ebf1 (early B cell factor 1), a key transcription factor for B cell development, and mechanistically, H2AFY is enriched at the EBF1 promoter. Induced expression of H2AFY1.1 in U2AF1(S34F) cells rescues reduced EBF1 expression and B cells numbers in vivo. Collectively, our data implicate alternative splicing of H2AFY as a contributor to lymphopenia induced by U2AF1(S34F) in mice and MDS.
Assuntos
Processamento Alternativo , Linfócitos B/metabolismo , Histonas/metabolismo , Linfopoese , Síndromes Mielodisplásicas/metabolismo , Fator de Processamento U2AF/metabolismo , Animais , Linfócitos B/imunologia , Sítios de Ligação , Estudos de Casos e Controles , Células HEK293 , Histonas/genética , Humanos , Células K562 , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/imunologia , Regiões Promotoras Genéticas , Transdução de Sinais , Fator de Processamento U2AF/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained WT allele is expressed, suggesting that mutant hematopoietic cells may require the residual WT allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knockout mice were similar to those in control mice, but that deletion of the WT allele in U2AF1(S34F) heterozygous mutant-expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of WT U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1(S34F)-expressing cells were also more sensitive to reduced expression of WT U2AF1 than nonmutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the WT U2af1 allele was deleted compared with when it was not deleted. These results suggest that selectively targeting the WT U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.
Assuntos
Alelos , Neoplasias Hematológicas , Heterozigoto , Leucemia , Proteínas de Neoplasias , Neoplasias Experimentais , Fator de Processamento U2AF , Animais , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Leucemia/genética , Leucemia/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Fator de Processamento U2AF/biossíntese , Fator de Processamento U2AF/genéticaRESUMO
Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. SIGNIFICANCE: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.
Assuntos
Mutação , Síndromes Mielodisplásicas/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Fator de Processamento U2AF/genética , Ciclo Celular , Linhagem Celular Tumoral , Instabilidade Cromossômica , Corantes Fluorescentes , Regulação da Expressão Gênica , Genes Reporter , Estudo de Associação Genômica Ampla , Humanos , Células K562 , Proteínas de Ligação a RNA , RNA-Seq , Ribonuclease H/metabolismo , SpliceossomosRESUMO
A novel tri metallic oxide nanocomposite La2O2CO3·CeO2·ZnO has been synthesized by a simple co-precipitation method. The nanocomposite has been characterized by XRD, SEM, EDS, FTIR and PL spectra. The crystallite size of the La2O2CO3·CeO2·ZnO was calculated using XRD data. The crystallite size of the as synthesized sample varies in the range of 16-30nm and those annealed at 950°C in the range of 26-70nm. Excitation at different wavelengths showed PL in UV and visible regions. It has been found that PL behavior of La2O2CO3·CeO2·ZnO is excitation wavelength dependent. This PL property is conflicting to well-known Kasha's rule of excitation wavelength dependence of emission spectrum. The catalyst shows better photocatalytic dye degradation efficiency in slightly alkaline pH in presence of H2O2.
Assuntos
Cério/química , Lantânio/química , Luminescência , Óxido de Zinco/química , Óxido de Zinco/síntese química , Catálise/efeitos da radiação , Precipitação Química , Violeta Genciana , Cinética , Nanocompostos/química , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
A novel mixed metal oxide, CeO2·CuAlO2 was fabricated by co-precipitation method in aqueous medium. CeO2·CuAlO2 was characterized by XRD, SEM, EDS, TEM, FTIR and PL spectra. The optical properties of the nanoparticles were studied by photoluminescence (PL) spectra. PL spectra at different excitations were recorded. The composite showed emission in UV, visible and NIR region depending on the excitation wavelength. The special spectral feature observed for this composite is that it showed six emission bands at 364, 409, 434, 448, 465 and 481 nm when excited at 298 nm. The green and red emissions observed at 512 and 669 nm are originated from cubic CeO2 phase when excited at 450 nm. The PL spectra were found to be dependent on excitation wavelength violating Kasha's rule. The X-ray diffraction reveals a cubic CeO2 phase and hexagonal CuAlO2 phase. EDS spectra revealed the presence of cerium (Ce), copper (Cu), aluminum (Al) and oxygen (O) elements. The particle size of the CeO2·CuAlO2 mixed oxide was estimated using Scherrer's formula, which was found to be in the range of 17.2-34.2 nm. The TEM image showed particles are almost uniform size of approximately 15-50 nm with spherical morphology.
Assuntos
Cério/química , Precipitação Química , Medições Luminescentes/métodos , Óxidos/química , Nanocompostos/química , Nanocompostos/ultraestrutura , Espectrometria de Fluorescência , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Difração de Raios XRESUMO
A novel multi-metal nanocomposite, NiOâ CeO2â ZnO has been prepared by co-precipitation of their carbonates from aqueous solutions of the metal nitrates following calcination and annealing 5 h at 450°C and 10 h at 950°C. NiOâ CeO2â ZnO has been characterized by XRD, SEM, EDS, IR and PL spectra. The crystallite size of the as-synthesized sample varies in the range of 14-23 nm and those of the annealed sample in the range of 17-50 nm. Emissions of NiOâ CeO2â ZnO have been observed in UV (NBE emission) and visible region at different excitations. Excitation wavelength dependent PL behavior of NiOâ CeO2â ZnO has been observed in acetone at room temperature. This PL property is in disagreement with Kasha's rule of excitation wavelength dependence of emission spectrum. Photocatalytic as well as anti-bacterial activities were studied.
Assuntos
Antibacterianos/química , Cério/química , Níquel/química , Óxido de Zinco/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Catálise , Cério/farmacologia , Humanos , Luz , Luminescência , Nanocompostos/química , Nanocompostos/ultraestrutura , Níquel/farmacologia , Difração de Raios X , Óxido de Zinco/farmacologiaAssuntos
Linfócitos B/patologia , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Haploinsuficiência , Síndromes Mielodisplásicas/patologia , Células-Tronco/patologia , Animais , Linfócitos B/metabolismo , Humanos , Camundongos , Camundongos Knockout , Síndromes Mielodisplásicas/genética , Células-Tronco/metabolismoRESUMO
A novel multi-metal nanocomposite, Co3O4·CeO2·ZnO has been prepared by co-precipitation of their carbonates from aqueous solutions of the metal nitrates following calcining and annealing 5h at 450°C and 10h at 950°C. Co3O4·CeO2·ZnO has been characterized by XRD, SEM, EDS, IR and PL spectra. The crystallite size of the as-synthesized sample varies in the range of 9-33nm and those of the annealed sample in the range of 19-42nm. Emissions of Co3O4·CeO2·ZnO were observed in UV and visible region at different excitations. Excitation wavelength dependent PL behavior of Co3O4·CeO2·ZnO has been observed in acetone. This PL property is contrary to the well-known Kasha's rule of excitation wavelength dependence of emission spectrum.
Assuntos
Cério/química , Cobalto/química , Nanoestruturas/química , Óxidos/química , Óxido de Zinco/química , Precipitação Química , Nanoestruturas/ultraestrutura , Análise EspectralRESUMO
A novel multi-metal nanocomposite, La2O2CO3·CuO·ZnO has been synthesized by co-precipitation method and characterized by XRD, SEM, EDS and PL spectra. XRD showed the presence of La2O2CO3, CuO and ZnO phases with an average particle size of 20 nm. Excitation at different wavelengths showed PL in UV and visible regions. Excitation at 220-270 nm provided UV emissions. Excitations at 298, 324 and 355 nm showed PL in the violet and blue regions. Excitation at 395 and 450 nm provided green and red luminescence, respectively. It has been found that PL behavior of La2O2CO3·CuO·ZnO is excitation wavelength dependent. This PL property is contrary to well-known Kasha's rule of excitation wavelength dependence of emission spectrum. The excitation peaks at 300-325 (broad band), 353, 371, 394, 408 nm were observed when monitored at 450 nm.