On-demand droplet sorting is extensively applied for the efficient manipulation and genome-wide analysis of individual cells.However,state-of-the-art microfluidic chips for droplet sorting still suffer from low sortin...On-demand droplet sorting is extensively applied for the efficient manipulation and genome-wide analysis of individual cells.However,state-of-the-art microfluidic chips for droplet sorting still suffer from low sorting speeds,sample loss,and labor-intensive preparation procedures.Here,we demonstrate the development of a novel microfluidic chip that integrates droplet generation,on-demand electrostatic droplet charging,and high-throughput sorting.The charging electrode is a copper wire buried above the nozzle of the microchannel,and the deflecting electrode is the phosphate buffered saline in the microchannel,which greatly simplifies the structure and fabrication process of the chip.Moreover,this chip is capable of high-frequency droplet generation and sorting,with a frequency of 11.757 kHz in the drop state.The chip completes the selective charging process via electrostatic induction during droplet generation.On-demand charged microdroplets can arbitrarilymove to specific exit channels in a three-dimensional(3D)-deflected electric field,which can be controlled according to user requirements,and the flux of droplet deflection is thereby significantly enhanced.Furthermore,a lossless modification strategy is presented to improve the accuracy of droplet deflection or harvest rate from 97.49% to 99.38% by monitoring the frequency of droplet generation in real time and feeding it back to the charging signal.This chip has great potential for quantitative processing and analysis of single cells for elucidating cell-to-cell variations.展开更多
BACKGROUND： Previous studies have demonstrated that brain-derived neurotrophic factor （BDNF） promotes neural differentiation. However, the mechanisms involved in cell cycle-related protein regulation, which highly ...BACKGROUND： Previous studies have demonstrated that brain-derived neurotrophic factor （BDNF） promotes neural differentiation. However, the mechanisms involved in cell cycle-related protein regulation, which highly correlates to neural proliferation and apoptosis, remain poorly understood. OBJECTIVE： To investigate the effects of various concentrations of BDNF on cycle-related protein mRNA expression in induce-differentiated SH-SY5Y cells in vitro prior to and following G2 phase, and to analyze the neuroprotective effects of BDNF. DESIGN, TIME AND SETTING： A comparison, observational study, based on cell biology, was performed at the Department of Biochemistry, Medical College of Tongji University, from March 2005 to October 2006. MATERIALS： SH-SY5Y cells were provided by Shanghai Institute of Cytology, Chinese Academy of Science; BDNF by Alomone Labs, Israel; all-trans retinoic acid （ATRA） by Sigma-Aldrich, USA. METHODS： SH-SY5Y cells were randomly divided into three groups： blank control [cells were treated in Insulin-Transferrin-Selenium （ITS） solution for 7 days], ATRA （cells were treated with ITS solution containing 10 μmol/L ATRA for 7 days）, and BDNF （cells were treated identical to the ATRA group for 5 days, and then respectively treated in ITS solution containing 1, 10, and 100 μg/L BDNF for 2 days）. The experiment was repeated three times for each group. MAIN OUTCOME MEASURES： mRNA expression levels of cyclin A1, B1, B2, cyclin-dependent kinase 1, and 5 were detected using quantitative real-time RT-PCR; percentage of cells in G1, S, and G2 phases were detected using fluorescence-activated cell sorting. RESULTS： mRNA expression levels of cyclin A1 in the high-dose BDNF group was significantly less than the ATRA group （P 〈 0.05）.mRNA expression levels of cyclin B1 was significantly less in the different BDNF concentration groups compared with the control and ATRA groups （P 〈 0.05 or P 〈 0.01）. mRNA expression levels of cyclin B2 and cyclin-dependent kinase 1 were significantly decreased in the high-dose BDNF group （P 〈 0.05 or P 〈 0.01）. Cyclin-dependent kinase 5 mRNA expression was significantly greater in the low-dose and moderate-dose BDNF groups compared with the ATRA group （P 〈 0.05）. The percentage of cells in G1 phase was significantly greater in the different BDNF concentration groups compared with the ATRA and control groups （P 〈 0.01）. Moreover, the percentage of cells in S phase was significantly less in the three BDNF groups compared with the ATRA group （P 〈 0.01）. However, the percentage of cells in S phase was significantly less in the low-dose and high-dose BDNF groups compared with the control group （P 〈 0.01）. CONCLUSION： BDNF enhanced the percentage of cells in G1 phase, but did not alter mRNA expression of cell cycle-related proteins prior to or following G2 phase. These results suggested that BDNF was not a risk factor for inducing apoptosis.展开更多
Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed i...Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets （LD）, whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum （ER）. Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin- green fluorescent protein （GFP） peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein （BiP）, were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.展开更多
基金The authors acknowledge the financial support from the NationalNatural Science Foundation ofChina(No.52275562)the Technology Innovation Fund of Huazhong University of Science and Technology(No.2022JYCXJJ015).
文摘On-demand droplet sorting is extensively applied for the efficient manipulation and genome-wide analysis of individual cells.However,state-of-the-art microfluidic chips for droplet sorting still suffer from low sorting speeds,sample loss,and labor-intensive preparation procedures.Here,we demonstrate the development of a novel microfluidic chip that integrates droplet generation,on-demand electrostatic droplet charging,and high-throughput sorting.The charging electrode is a copper wire buried above the nozzle of the microchannel,and the deflecting electrode is the phosphate buffered saline in the microchannel,which greatly simplifies the structure and fabrication process of the chip.Moreover,this chip is capable of high-frequency droplet generation and sorting,with a frequency of 11.757 kHz in the drop state.The chip completes the selective charging process via electrostatic induction during droplet generation.On-demand charged microdroplets can arbitrarilymove to specific exit channels in a three-dimensional(3D)-deflected electric field,which can be controlled according to user requirements,and the flux of droplet deflection is thereby significantly enhanced.Furthermore,a lossless modification strategy is presented to improve the accuracy of droplet deflection or harvest rate from 97.49% to 99.38% by monitoring the frequency of droplet generation in real time and feeding it back to the charging signal.This chip has great potential for quantitative processing and analysis of single cells for elucidating cell-to-cell variations.
文摘BACKGROUND： Previous studies have demonstrated that brain-derived neurotrophic factor （BDNF） promotes neural differentiation. However, the mechanisms involved in cell cycle-related protein regulation, which highly correlates to neural proliferation and apoptosis, remain poorly understood. OBJECTIVE： To investigate the effects of various concentrations of BDNF on cycle-related protein mRNA expression in induce-differentiated SH-SY5Y cells in vitro prior to and following G2 phase, and to analyze the neuroprotective effects of BDNF. DESIGN, TIME AND SETTING： A comparison, observational study, based on cell biology, was performed at the Department of Biochemistry, Medical College of Tongji University, from March 2005 to October 2006. MATERIALS： SH-SY5Y cells were provided by Shanghai Institute of Cytology, Chinese Academy of Science; BDNF by Alomone Labs, Israel; all-trans retinoic acid （ATRA） by Sigma-Aldrich, USA. METHODS： SH-SY5Y cells were randomly divided into three groups： blank control [cells were treated in Insulin-Transferrin-Selenium （ITS） solution for 7 days], ATRA （cells were treated with ITS solution containing 10 μmol/L ATRA for 7 days）, and BDNF （cells were treated identical to the ATRA group for 5 days, and then respectively treated in ITS solution containing 1, 10, and 100 μg/L BDNF for 2 days）. The experiment was repeated three times for each group. MAIN OUTCOME MEASURES： mRNA expression levels of cyclin A1, B1, B2, cyclin-dependent kinase 1, and 5 were detected using quantitative real-time RT-PCR; percentage of cells in G1, S, and G2 phases were detected using fluorescence-activated cell sorting. RESULTS： mRNA expression levels of cyclin A1 in the high-dose BDNF group was significantly less than the ATRA group （P 〈 0.05）.mRNA expression levels of cyclin B1 was significantly less in the different BDNF concentration groups compared with the control and ATRA groups （P 〈 0.05 or P 〈 0.01）. mRNA expression levels of cyclin B2 and cyclin-dependent kinase 1 were significantly decreased in the high-dose BDNF group （P 〈 0.05 or P 〈 0.01）. Cyclin-dependent kinase 5 mRNA expression was significantly greater in the low-dose and moderate-dose BDNF groups compared with the ATRA group （P 〈 0.05）. The percentage of cells in G1 phase was significantly greater in the different BDNF concentration groups compared with the ATRA and control groups （P 〈 0.01）. Moreover, the percentage of cells in S phase was significantly less in the three BDNF groups compared with the ATRA group （P 〈 0.01）. However, the percentage of cells in S phase was significantly less in the low-dose and high-dose BDNF groups compared with the control group （P 〈 0.01）. CONCLUSION： BDNF enhanced the percentage of cells in G1 phase, but did not alter mRNA expression of cell cycle-related proteins prior to or following G2 phase. These results suggested that BDNF was not a risk factor for inducing apoptosis.
文摘Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets （LD）, whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum （ER）. Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin- green fluorescent protein （GFP） peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein （BiP）, were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.
