Aiming at the issues of controlling the translocation speed of DNA through a solid-state nanopore and enlarging the signal-to-noise ratio of ionic current modulation, which are challenges for the application of nanopo...Aiming at the issues of controlling the translocation speed of DNA through a solid-state nanopore and enlarging the signal-to-noise ratio of ionic current modulation, which are challenges for the application of nanopore technology in DNA detection, salt concentration gradients are applied across the nanopore to investigate their influence on the DNA translocation time and signal-to-noise ratio. Experimental data demonstrates that, in symmetric concentration conditions, both the current blockade and dwell time for A-DNA translocation through a solid-state nanopore increase along with potassium chloride concentration. When the concentration in the trans chamber is decreased from 1 to 0.1 mol/L, keeping the concentration of the cis chamber at 1 mol/L, the normalized current blockade is found to be increased by one order. The increased dwell time and enhanced signal-to-noise ratio are achieved with salt gradients across the nanopore, which can improve the sensitivity when detecting DNA samples.展开更多
Objectives To construct the cancellous bone explant model and a method of culturing these bone tissues in vitro, and to investigate the effect of mechanical load on growth of cancellous bone tissue in vtro. Methods C...Objectives To construct the cancellous bone explant model and a method of culturing these bone tissues in vitro, and to investigate the effect of mechanical load on growth of cancellous bone tissue in vtro. Methods Cancellous bone were extracted from rabbit femoral head and cut into I-ram-thick and 8-ram-diameter slices under sterile conditions. HE staining and scanning electron microscopy were employed to identify the histomorphology of the model after being cultured with a new dynamic load and circulating perfusion bioreactor system for 0, 3, 5, and 7 days, respectively. We built a three-dimensional model using microCT and analyzed the loading effects using finite element analysis. The model was subjected to mechanical load of 1000, 2000, 3000, and 4000 με respectively for 30 minutes per day. After 5 days of continuous stimuli, the activities of alkaline phosphatase （AKP） and tartrate-resistant acid phosphatase （TRAP） were detected. Apoptosis was analyzed by DNA ladder detection and caspase-3/8/9 activity detection. Results After being cultured for 3, 5, and 7 days, the bone explant model grew well. HE staining showed the apparent nucleus in cells at the each indicated time, and electron microscope revealed the living cells in the bone tissue. The activities of AKP and TRAP in the bone explant model under mechanical load of 3000 and 4000 με were significantly lower than those in the unstressed bone tissues （all P〈0.05）. DNA ladders were seen in the bone tissue under 3000 and 4000με mechanical load. Moreover, there was significant enhancement in the activities of caspase-3/8/9 in the mechanical stress group of 3000 and 4000 με （all P〈0.05）. Conclusions The cancellous bone explant model extracted from the rabbit femoral head could be alive at least for 7 days in the dynamic load and circulating perfusion bioreactor system, however, pathological mechanical load could affect the bone tissue growth by apoptosis in vitro. The differentiation of osteobiasts and osteoclasts might be inhibited after the model is stimulated by mechanical load of 3000 and 4000 με.展开更多
We study the effects of electrolyte temperature on DNA molecule translocation experimentally without and with a temperature gradient across nanopore membranes.The same temperatures on both electrolyte chambers are fir...We study the effects of electrolyte temperature on DNA molecule translocation experimentally without and with a temperature gradient across nanopore membranes.The same temperatures on both electrolyte chambers are first considered.The DNA molecule translocation time is measured to be 2.44 ms at 2°C in both chambers,which is 1.57 times longer than at 20°C.Then the temperature difference effect is characterized in both chambers.The results show that the DNA translocation speed can be slowed down as long as one side temperature is lowered,irrespective of the temperature gradient direction.This indicates that the thermophoretic driving force generated by a temperature gradient has no obvious effect on the threading speed of DNA molecules,while the main reason for the slowed DNA translocation speed is the increased viscosity.Interestingly,the capture rate of DNA molecules is enhanced under a temperature gradient condition,and the capture rate during DNA translocation from hot side at 21°C to cold one at 2°C is 1.7 times larger than that under the condition of both chambers at 20°C.Finally,an optimized configuration is proposed to acquire higher capture rates and lower DNA translocation speeds.展开更多
The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, str...The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, structures as well as pro- tein/DNA composition of isolated nucleoids were then compared under various growth phases. The nucleoid structures were found to undergo changes during the cell growth; i. e., the nucleoid structure in the stationary phase was more tightly com- pacted than that in the exponential phase. In addition to factor for inversion stimulation （Fis）, histone-like nucleoid structuring protein （H-NS）, heat-unstable nucleoid protein （HU） and integration host factor （IHF） here we have identified, three new can- didates of E. coli nucleoid, namely DNA-binding protein from starved cells （Dps）, host factor for phage QJ3 （Hfq） and sup- pressor of taC phenotype A （StpA）. Our results reveal that the major components of exponential phase nucleoid are Fis, HU, H-NS, StpA and Hfq, while Dps occupies more than half of the stationary phase nucleoid. It has been known for a while that Dps is the main nucleoid-associated protein at stationary phase. From these results and the prevailing information, we propose a model for growth phase dependent changes in the structure and protein composition of nucleoid in E. coli.展开更多
基金The National Natural Science Foundation of China(No.51435003,51375092)Fundamental Research Funds for the Central Universities+1 种基金the Innovative Project for Graduate Students of Jiangsu Province(No.KYLX_0100)the Scientific Research Foundation of Graduate School of Southeast University(No.YBJJ1540)
文摘Aiming at the issues of controlling the translocation speed of DNA through a solid-state nanopore and enlarging the signal-to-noise ratio of ionic current modulation, which are challenges for the application of nanopore technology in DNA detection, salt concentration gradients are applied across the nanopore to investigate their influence on the DNA translocation time and signal-to-noise ratio. Experimental data demonstrates that, in symmetric concentration conditions, both the current blockade and dwell time for A-DNA translocation through a solid-state nanopore increase along with potassium chloride concentration. When the concentration in the trans chamber is decreased from 1 to 0.1 mol/L, keeping the concentration of the cis chamber at 1 mol/L, the normalized current blockade is found to be increased by one order. The increased dwell time and enhanced signal-to-noise ratio are achieved with salt gradients across the nanopore, which can improve the sensitivity when detecting DNA samples.
基金Supported by grants from the National Natural Science Foundation Key Project of China(10832012)the National Natural Science Foundation of China(31370942 and 11072266)
文摘Objectives To construct the cancellous bone explant model and a method of culturing these bone tissues in vitro, and to investigate the effect of mechanical load on growth of cancellous bone tissue in vtro. Methods Cancellous bone were extracted from rabbit femoral head and cut into I-ram-thick and 8-ram-diameter slices under sterile conditions. HE staining and scanning electron microscopy were employed to identify the histomorphology of the model after being cultured with a new dynamic load and circulating perfusion bioreactor system for 0, 3, 5, and 7 days, respectively. We built a three-dimensional model using microCT and analyzed the loading effects using finite element analysis. The model was subjected to mechanical load of 1000, 2000, 3000, and 4000 με respectively for 30 minutes per day. After 5 days of continuous stimuli, the activities of alkaline phosphatase （AKP） and tartrate-resistant acid phosphatase （TRAP） were detected. Apoptosis was analyzed by DNA ladder detection and caspase-3/8/9 activity detection. Results After being cultured for 3, 5, and 7 days, the bone explant model grew well. HE staining showed the apparent nucleus in cells at the each indicated time, and electron microscope revealed the living cells in the bone tissue. The activities of AKP and TRAP in the bone explant model under mechanical load of 3000 and 4000 με were significantly lower than those in the unstressed bone tissues （all P〈0.05）. DNA ladders were seen in the bone tissue under 3000 and 4000με mechanical load. Moreover, there was significant enhancement in the activities of caspase-3/8/9 in the mechanical stress group of 3000 and 4000 με （all P〈0.05）. Conclusions The cancellous bone explant model extracted from the rabbit femoral head could be alive at least for 7 days in the dynamic load and circulating perfusion bioreactor system, however, pathological mechanical load could affect the bone tissue growth by apoptosis in vitro. The differentiation of osteobiasts and osteoclasts might be inhibited after the model is stimulated by mechanical load of 3000 and 4000 με.
基金supported by the National Basic Research Program of China("973"Project)(Grant Nos.2011CB707601 and 2011CB707605)the National Natural Science Foundation of China(Grants Nos.50925519 and 51375092)+1 种基金supported by the Scientific Research Foundation of Graduate School of Southeast University(Grant No.YBJJ1004)supported by the Fundamental Research Funds for the Central Universities and the Innovative Project for Graduate Students of Jiangsu Province(Grant No.KYLX_0100)
文摘We study the effects of electrolyte temperature on DNA molecule translocation experimentally without and with a temperature gradient across nanopore membranes.The same temperatures on both electrolyte chambers are first considered.The DNA molecule translocation time is measured to be 2.44 ms at 2°C in both chambers,which is 1.57 times longer than at 20°C.Then the temperature difference effect is characterized in both chambers.The results show that the DNA translocation speed can be slowed down as long as one side temperature is lowered,irrespective of the temperature gradient direction.This indicates that the thermophoretic driving force generated by a temperature gradient has no obvious effect on the threading speed of DNA molecules,while the main reason for the slowed DNA translocation speed is the increased viscosity.Interestingly,the capture rate of DNA molecules is enhanced under a temperature gradient condition,and the capture rate during DNA translocation from hot side at 21°C to cold one at 2°C is 1.7 times larger than that under the condition of both chambers at 20°C.Finally,an optimized configuration is proposed to acquire higher capture rates and lower DNA translocation speeds.
基金supported by Grants-in-Aid from the Ministry of Education,Science and Culture of Japan,and Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation
文摘The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, structures as well as pro- tein/DNA composition of isolated nucleoids were then compared under various growth phases. The nucleoid structures were found to undergo changes during the cell growth; i. e., the nucleoid structure in the stationary phase was more tightly com- pacted than that in the exponential phase. In addition to factor for inversion stimulation （Fis）, histone-like nucleoid structuring protein （H-NS）, heat-unstable nucleoid protein （HU） and integration host factor （IHF） here we have identified, three new can- didates of E. coli nucleoid, namely DNA-binding protein from starved cells （Dps）, host factor for phage QJ3 （Hfq） and sup- pressor of taC phenotype A （StpA）. Our results reveal that the major components of exponential phase nucleoid are Fis, HU, H-NS, StpA and Hfq, while Dps occupies more than half of the stationary phase nucleoid. It has been known for a while that Dps is the main nucleoid-associated protein at stationary phase. From these results and the prevailing information, we propose a model for growth phase dependent changes in the structure and protein composition of nucleoid in E. coli.
