Light regulates important metabolic processes in microalgal cells, which can further impact the metabolism and the accumulation of biomolecules such as lipids, carbohydrates, and proteins. Different characteristics of...Light regulates important metabolic processes in microalgal cells, which can further impact the metabolism and the accumulation of biomolecules such as lipids, carbohydrates, and proteins. Different characteristics of light have been studied on various strains of the model diatom Phaeodactylum tricornutum, but not on transconjugant cells and information on wild-type strains is still limited. Therefore, we studied the impact of different light characteristics such as spectral quality, light intensity and light shift on the growth, and the composition in lipids and fatty acids of P. tricornutum cells to provide a comprehensive context for future applications. Initially, we tested the impact of spectral quality and light intensity on P. tricornutum transformed with an episomal vector (Ptev), harboring the resistance gene Sh ble. Results indicated that Ptev cells accumulated more biomass and overall lipids in spectral quality Red 1 (R1: 34% > 600 nm > 66%) more effectively as compared to Red 2 (R2: 8% > 600 nm > 92%). It was also detected that cell granularity was higher in R1 as compared to R2. Furthermore, by testing two light intensities 65 μmol·m<sup>-2</sup>·s<sup>-1</sup> and 145 μmol·m<sup>-2</sup>·s<sup>-1</sup> light, it was observed that 145 μmol·m<sup>-2</sup>·s<sup>-1</sup> led to an increase in growth trend, total biomass and lipid content. Combining spectral qualities and light intensities, we show that the lipid accumulation raised by 2.8-fold. Studying the light intensity and spectral quality allowed us to optimize the light conditions to R1 spectral quality and light intensity 145 μmol·m<sup>-2</sup>·s<sup>-1</sup>. These initial results showed that red light R1 at 145 μmol·m<sup>-2</sup>·s<sup>-1</sup> was the best condition for biomass and total lipids accumulation in Ptev cells. Next, we further combined these two-light optimizations with a third light characteristics, i.e. light shift, where the cultures were shifted during the early stationary phase to a different light environment. We studied Red light shift (Rs) to investigate how light condition variations impacted P. tricornutum transconjugants Ptev and with an episomal vector containing the reporter gene YFP (PtYFP). We observed that Rs induced growth and fatty acid eicosapentaenoic acid (EPA) in Ptev as compared to PtYFP. Altogether, the study shows that red light shift of R1 at 145 μmol·m<sup>-2</sup>·s<sup>-1</sup> promoted biomass and total lipids accumulation in Ptev and PtYFP cells. The study provides a comprehensive approach to using different light characteristics with the aim to optimize growth and lipids, as well as to fatty acid production.展开更多
Cardiomyocytes differentiated from human induced pluripotent stem cells(iPSCs)are valuable for the understanding/treatment of the deadly heart diseases and their drug screening.However,the very much needed homogeneous...Cardiomyocytes differentiated from human induced pluripotent stem cells(iPSCs)are valuable for the understanding/treatment of the deadly heart diseases and their drug screening.However,the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging.Here,it is discovered surprisingly that Rock inhibitor(RI),used ubiquitously to improve the survival/yield of human iPSCs,induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers(i.e.,ectoderm,mesoderm,and endoderm)at the commonly used concentration(10μM).This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation.By reducing the RI to 1μM for 3D culture,the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids.Consequently,the beating efficiency of 3D cardiac differentiation can be improved to more than 95%in~7 days(compared to less than~50%in 14 days for the 10μM RI condition).Furthermore,the outset beating time(OBT)of all resultant cardiac spheroids(CSs)is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol(GelMA)hydrogel,showing high homogeneity(in terms of the OBT)in functional maturity of the CSs.Moreover,the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs.These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases.展开更多
Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral(NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high t...Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral(NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region(S/MAR) sequence(SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore,Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion,the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.展开更多
T-lymphoblastic lymphoma(T-LBL)is a rare and aggressive form of non-Hodgkin’s lymphoma and little is known about their molecular background.However,complex karyotypes were already related to this group of malignancy ...T-lymphoblastic lymphoma(T-LBL)is a rare and aggressive form of non-Hodgkin’s lymphoma and little is known about their molecular background.However,complex karyotypes were already related to this group of malignancy and associated with poor outcome.Here,we describe a 17-year-old female being diagnosed with T-LBL and a normal karyotype after standard G-banding with trypsin-Giemsa(GTG)-banding.However,further analyses including high-resolution molecular approaches,array-comparative genomic hybridization(aCGH),multiplex ligation-dependent probe amplification,fluorescence in situ hybridization and multicolor chromosome banding revealed a cryptic complex karyotype,NUP214-ABL1 gene fusion,episomes and intra-tumor genetic heterogeneity.In addition,homozygous loss of CDKN2A,as well as amplification of oncogene TLX1(HOX11)were detected.Actually,NUP214-ABL1 fusion gene replicated autonomously in this case as episomes.Overall,highly amplification of NUP214-ABL1 fusion gene defines possibly a new subgroup of T-LBL patients which accordingly could benefit from treatment with tyrosine kinase inhibitors.As episomes are missed in standard karyotyping aCGH should be performed routinely in T-LBL to possibly detect more of such cases.展开更多
文摘Light regulates important metabolic processes in microalgal cells, which can further impact the metabolism and the accumulation of biomolecules such as lipids, carbohydrates, and proteins. Different characteristics of light have been studied on various strains of the model diatom Phaeodactylum tricornutum, but not on transconjugant cells and information on wild-type strains is still limited. Therefore, we studied the impact of different light characteristics such as spectral quality, light intensity and light shift on the growth, and the composition in lipids and fatty acids of P. tricornutum cells to provide a comprehensive context for future applications. Initially, we tested the impact of spectral quality and light intensity on P. tricornutum transformed with an episomal vector (Ptev), harboring the resistance gene Sh ble. Results indicated that Ptev cells accumulated more biomass and overall lipids in spectral quality Red 1 (R1: 34% > 600 nm > 66%) more effectively as compared to Red 2 (R2: 8% > 600 nm > 92%). It was also detected that cell granularity was higher in R1 as compared to R2. Furthermore, by testing two light intensities 65 μmol·m<sup>-2</sup>·s<sup>-1</sup> and 145 μmol·m<sup>-2</sup>·s<sup>-1</sup> light, it was observed that 145 μmol·m<sup>-2</sup>·s<sup>-1</sup> led to an increase in growth trend, total biomass and lipid content. Combining spectral qualities and light intensities, we show that the lipid accumulation raised by 2.8-fold. Studying the light intensity and spectral quality allowed us to optimize the light conditions to R1 spectral quality and light intensity 145 μmol·m<sup>-2</sup>·s<sup>-1</sup>. These initial results showed that red light R1 at 145 μmol·m<sup>-2</sup>·s<sup>-1</sup> was the best condition for biomass and total lipids accumulation in Ptev cells. Next, we further combined these two-light optimizations with a third light characteristics, i.e. light shift, where the cultures were shifted during the early stationary phase to a different light environment. We studied Red light shift (Rs) to investigate how light condition variations impacted P. tricornutum transconjugants Ptev and with an episomal vector containing the reporter gene YFP (PtYFP). We observed that Rs induced growth and fatty acid eicosapentaenoic acid (EPA) in Ptev as compared to PtYFP. Altogether, the study shows that red light shift of R1 at 145 μmol·m<sup>-2</sup>·s<sup>-1</sup> promoted biomass and total lipids accumulation in Ptev and PtYFP cells. The study provides a comprehensive approach to using different light characteristics with the aim to optimize growth and lipids, as well as to fatty acid production.
基金This work was partially supported by grants from the Maryland Stem Cell Research Fund(#2021-MSCRFD-5660)National Institutes of Health(NIH R01EB023632)+1 种基金and National Science Foundation(NSF CBET-1831019)We thank Dr.Kimberly M.Stroka for generously providing us the human eiPSCs.
文摘Cardiomyocytes differentiated from human induced pluripotent stem cells(iPSCs)are valuable for the understanding/treatment of the deadly heart diseases and their drug screening.However,the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging.Here,it is discovered surprisingly that Rock inhibitor(RI),used ubiquitously to improve the survival/yield of human iPSCs,induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers(i.e.,ectoderm,mesoderm,and endoderm)at the commonly used concentration(10μM).This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation.By reducing the RI to 1μM for 3D culture,the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids.Consequently,the beating efficiency of 3D cardiac differentiation can be improved to more than 95%in~7 days(compared to less than~50%in 14 days for the 10μM RI condition).Furthermore,the outset beating time(OBT)of all resultant cardiac spheroids(CSs)is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol(GelMA)hydrogel,showing high homogeneity(in terms of the OBT)in functional maturity of the CSs.Moreover,the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs.These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases.
基金supported by the Major State Basic Research Development Program of China(2011CB965203)
文摘Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral(NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region(S/MAR) sequence(SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore,Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion,the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.
文摘T-lymphoblastic lymphoma(T-LBL)is a rare and aggressive form of non-Hodgkin’s lymphoma and little is known about their molecular background.However,complex karyotypes were already related to this group of malignancy and associated with poor outcome.Here,we describe a 17-year-old female being diagnosed with T-LBL and a normal karyotype after standard G-banding with trypsin-Giemsa(GTG)-banding.However,further analyses including high-resolution molecular approaches,array-comparative genomic hybridization(aCGH),multiplex ligation-dependent probe amplification,fluorescence in situ hybridization and multicolor chromosome banding revealed a cryptic complex karyotype,NUP214-ABL1 gene fusion,episomes and intra-tumor genetic heterogeneity.In addition,homozygous loss of CDKN2A,as well as amplification of oncogene TLX1(HOX11)were detected.Actually,NUP214-ABL1 fusion gene replicated autonomously in this case as episomes.Overall,highly amplification of NUP214-ABL1 fusion gene defines possibly a new subgroup of T-LBL patients which accordingly could benefit from treatment with tyrosine kinase inhibitors.As episomes are missed in standard karyotyping aCGH should be performed routinely in T-LBL to possibly detect more of such cases.
