Intervertebral disc degeneration is the main contributor to low back pain,now the leading cause of disability worldwide.Gene transfer,either in a therapeutic attempt or in basic research to understand the mechanisms o...Intervertebral disc degeneration is the main contributor to low back pain,now the leading cause of disability worldwide.Gene transfer,either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration,is a fascinating and promising tool to manipulate the complex physiology of the disc.Viral vectors based on the adeno-associated virus(AAV)have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism,transduction efficiency,and relative toxicity have not yet been performed in the disc in vivo.Herein,we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes,injection volumes,titers,promoters,and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus(NP)cells in vivo.We find that AAV6 using a CAG promoter to drive transgene expression,delivered into the NP of murine caudal discs at a titer of 1011 GC/mL,provides excellent transduction efficiency/kinetics and low toxicity in vivo.We also show,for the first time,that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides.Finally,to our knowledge,we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc,which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery.Taken together,these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.展开更多
D-psicose exits in an extremely small amount in nature and is difficult to be chemically synthesized.Only three bacteria have been used in the biotransformation of D-psicose from D-fructose.In this paper,another bacte...D-psicose exits in an extremely small amount in nature and is difficult to be chemically synthesized.Only three bacteria have been used in the biotransformation of D-psicose from D-fructose.In this paper,another bacterium which could convert D-fructose to D-psicose was isolated and identified as Rhodobacter sphaeroides.The process parameters of D-psicose production using permeabilized cells of Rhodobacter sphaeroides SK011 were optimized,including the permeabilization procedure:0.1%(w/v)CTAB,10 min,and reaction conditions:cell concentration,30 g dry cell wt/L;concentration of substrate,50 g/L;40℃,pH 9.0;reaction time,8 h.Under the optimized conditions,the permeabilized cells produced approximately 6.5 g/L D-psicose with a Dpsicose productivity of 0.82 g·L^(-1)·h^(-1).This is the first report of bioproduction of D-psicose using permeabilized cells of Rhodobacter sphaeroides.展开更多
基金This work was supported by a Veteran Affairs Career Development Award(IK2-BX003845).
文摘Intervertebral disc degeneration is the main contributor to low back pain,now the leading cause of disability worldwide.Gene transfer,either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration,is a fascinating and promising tool to manipulate the complex physiology of the disc.Viral vectors based on the adeno-associated virus(AAV)have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism,transduction efficiency,and relative toxicity have not yet been performed in the disc in vivo.Herein,we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes,injection volumes,titers,promoters,and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus(NP)cells in vivo.We find that AAV6 using a CAG promoter to drive transgene expression,delivered into the NP of murine caudal discs at a titer of 1011 GC/mL,provides excellent transduction efficiency/kinetics and low toxicity in vivo.We also show,for the first time,that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides.Finally,to our knowledge,we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc,which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery.Taken together,these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.
基金supported financially by the National High Technology Research and Development Program of China(Grant No.2006AA10Z334)the Research Program of Sate Key Laboratory of Food Science and Technology,Jiangnan University(SKLF-MB-200804 and SKLF-TS-200805).
文摘D-psicose exits in an extremely small amount in nature and is difficult to be chemically synthesized.Only three bacteria have been used in the biotransformation of D-psicose from D-fructose.In this paper,another bacterium which could convert D-fructose to D-psicose was isolated and identified as Rhodobacter sphaeroides.The process parameters of D-psicose production using permeabilized cells of Rhodobacter sphaeroides SK011 were optimized,including the permeabilization procedure:0.1%(w/v)CTAB,10 min,and reaction conditions:cell concentration,30 g dry cell wt/L;concentration of substrate,50 g/L;40℃,pH 9.0;reaction time,8 h.Under the optimized conditions,the permeabilized cells produced approximately 6.5 g/L D-psicose with a Dpsicose productivity of 0.82 g·L^(-1)·h^(-1).This is the first report of bioproduction of D-psicose using permeabilized cells of Rhodobacter sphaeroides.
