Yi Li

To test, in vivo, the hypothesis that exosomes from multipotent mesenchymal stromal cells (MSCs) mediate microRNA 133b (miR-133b) transfer which promotes neurological recovery from stroke, we used knockin and knockdown technologies to upregulate or downregulate the miR-133b level in MSCs (miR-133b(+) MSCs or miR-133b(-) MSCs) and their corresponding exosomes, respectively. Rats were subjected to middle cerebral artery occlusion (MCAo) and were treated with naïve MSCs, miR-133b(+) MSCs, or miR-133b(-) MSC at 1 day after MCAo. Compared with controls, rats receiving naïve MSC treatment significantly improved functional recovery and exhibited increased axonal plasticity and neurite remodeling in the ischemic boundary zone (IBZ) at day 14 after MCAo. The outcomes were significantly enhanced with miR-133b(+) MSC treatment, and were significantly decreased with miR-133b(-) MSC treatment, compared to naïve MSC treatment. The miR-133b level in exosomes collected from the cerebral spinal fluid was significantly increased after miR-133b(+) MSC treatment, and was significantly decreased after miR-133b(-) MSC treatment at day 14 after MCAo, compared to naïve MSC treatment. Tagging exosomes with green fluorescent protein demonstrated that exosomes-enriched extracellular particles were released from MSCs and transferred to adjacent astrocytes and neurons. The expression of selective targets for miR-133b, connective tissue growth factor and ras homolog gene family member A, was significantly decreased in the IBZ after miR-133b(+) MSC treatment, while their expression remained at similar elevated levels after miR-133b(-) MSC treatment, compared to naïve MSC treatment. Collectively, our data suggest that exosomes from MSCs mediate the miR-133b transfer to astrocytes and neurons, which regulate gene expression, subsequently benefit neurite remodeling and functional recovery after stroke.

Nature Communications 5, Article number: 3619 (2014); Published 10 April 2014; Updated 14 October 2015 In Figure 7 of this Article, western blots presenting GAPDH loading controls in panels c and h were inadvertently duplicated from those in Fig. 6a and the upper section of Fig. 2a, respectively. The correct version of Figure 7 appears below.

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences inArabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease. A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences inArabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease. UDP-glycosyltransferase open reading frame polymerase chain reaction Glycosyltransferases are found in all living organisms, catalyzing the transfer of a glycosyl moiety from an activated donor to an acceptor molecule, forming a glycosidic bond. These glycosyl transfer reactions have been highlighted as the most important biotransformation on earth, since in quantitative terms they account for the assembly and degradation of the bulk of biomass (1Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (624) Google Scholar).A unique signature motif has been identified in the amino acid sequence of many of these glycosyltransferases, leading to their classification into a single UDP-glycosyltransferase (UGT)1 superfamily (2Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournel-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Louisot P. Magdalou J. Chowdhury J.R. Ritter J.K. Schachter H. Tephly T.R. Tipton K.F. Nebert D.W. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (992) Google Scholar). Of these, the mammalian UGTs using UDP-glucuronic acid have attracted considerable attention in pharmaceutical and clinical research due to their central role in the metabolism and detoxification of foreign chemicals such as carcinogens and hydrophobic drugs (3de Wildt S.N. Kearns G.L. Leeder J.S. van den Anker J.N. Clin. Pharmacokinet. 1999; 36: 439-452Crossref PubMed Scopus (353) Google Scholar, 4Nebert D.W. Biochem. Pharmacol. 1994; 47: 25-37Crossref PubMed Scopus (208) Google Scholar).Plant UGTs are involved in a parallel range of activities, the majority of which use UDP-glucose in the transfer reaction. These reactions are known to have a number of important consequences. First, compounds can be activated or inactivated by their conjugation to glucose. For example, glucose esters are high energy compounds that are known to act as biosynthetic intermediates for further reactions involving the aglycones (5Mock H. Strack D. Phytochemistry. 1993; 32: 575-579Crossref Scopus (70) Google Scholar). In contrast, many of the plant hormones are known to be inactivated following glucosylation (6Sembdner G. Atzorn R. Schneider G. Plant Mol. Biol. 1994; 26: 1459-1481Crossref PubMed Scopus (102) Google Scholar, 7Kleckowski K. Schell J. Crit. Rev. Plant Sci. 1995; 14: 283-298Crossref Scopus (86) Google Scholar). Second, glucosylation alters the solubility of compounds by increasing their hydrophilic properties and providing access to active membrane transport systems that recognize the glucosides but not the aglycones (8Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar).As a consequence of these events, glucosylation plays a crucial role in the maintenance of cellular homeostasis in plants through regulating the level, activity, and location of key cellular metabolites. Despite this general importance and the likely large number of these enzymes, given the diversity of substrates, plant UGTs have not been studied systematically. Rather, individual UGTs have been purified on the basis of a particular catalytic activity (9–20). The disadvantage of this approach is that the relationships of different UGTs cannot be defined easily, and, in consequence, predictions of catalytic activities based on structure-function relatedness cannot be made.Genome sequencing programs offer a new route into understanding multigene families both within a single species and across different species. In this study, we have used the data available from theArabidopsis genome sequencing program to start to build a foundation for understanding the UGT multigene family. This analysis focuses on the phylogeny and evolution of UGTs and complements parallel investigations into substrate specificity using recombinant proteins corresponding to known UGT sequences (21Lim E.-K. Li Y. Parr A. Jackson R. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4344-4349Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 22Jackson R.G. Lim E.-K. Li Y. Kowalczyk M. Sandberg G. Hoggett J. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4350-4356Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar).Note Added in ProofAn additional 18 complete UGT sequences have been identified in Arabidopsis genome data base subsequent to the submission of this manuscript. These do not change the composition of the groups defined in this manuscript, and they add an additional two groups to the tree. Glycosyltransferases are found in all living organisms, catalyzing the transfer of a glycosyl moiety from an activated donor to an acceptor molecule, forming a glycosidic bond. These glycosyl transfer reactions have been highlighted as the most important biotransformation on earth, since in quantitative terms they account for the assembly and degradation of the bulk of biomass (1Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (624) Google Scholar). A unique signature motif has been identified in the amino acid sequence of many of these glycosyltransferases, leading to their classification into a single UDP-glycosyltransferase (UGT)1 superfamily (2Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournel-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Louisot P. Magdalou J. Chowdhury J.R. Ritter J.K. Schachter H. Tephly T.R. Tipton K.F. Nebert D.W. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (992) Google Scholar). Of these, the mammalian UGTs using UDP-glucuronic acid have attracted considerable attention in pharmaceutical and clinical research due to their central role in the metabolism and detoxification of foreign chemicals such as carcinogens and hydrophobic drugs (3de Wildt S.N. Kearns G.L. Leeder J.S. van den Anker J.N. Clin. Pharmacokinet. 1999; 36: 439-452Crossref PubMed Scopus (353) Google Scholar, 4Nebert D.W. Biochem. Pharmacol. 1994; 47: 25-37Crossref PubMed Scopus (208) Google Scholar). Plant UGTs are involved in a parallel range of activities, the majority of which use UDP-glucose in the transfer reaction. These reactions are known to have a number of important consequences. First, compounds can be activated or inactivated by their conjugation to glucose. For example, glucose esters are high energy compounds that are known to act as biosynthetic intermediates for further reactions involving the aglycones (5Mock H. Strack D. Phytochemistry. 1993; 32: 575-579Crossref Scopus (70) Google Scholar). In contrast, many of the plant hormones are known to be inactivated following glucosylation (6Sembdner G. Atzorn R. Schneider G. Plant Mol. Biol. 1994; 26: 1459-1481Crossref PubMed Scopus (102) Google Scholar, 7Kleckowski K. Schell J. Crit. Rev. Plant Sci. 1995; 14: 283-298Crossref Scopus (86) Google Scholar). Second, glucosylation alters the solubility of compounds by increasing their hydrophilic properties and providing access to active membrane transport systems that recognize the glucosides but not the aglycones (8Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar). As a consequence of these events, glucosylation plays a crucial role in the maintenance of cellular homeostasis in plants through regulating the level, activity, and location of key cellular metabolites. Despite this general importance and the likely large number of these enzymes, given the diversity of substrates, plant UGTs have not been studied systematically. Rather, individual UGTs have been purified on the basis of a particular catalytic activity (9–20). The disadvantage of this approach is that the relationships of different UGTs cannot be defined easily, and, in consequence, predictions of catalytic activities based on structure-function relatedness cannot be made. Genome sequencing programs offer a new route into understanding multigene families both within a single species and across different species. In this study, we have used the data available from theArabidopsis genome sequencing program to start to build a foundation for understanding the UGT multigene family. This analysis focuses on the phylogeny and evolution of UGTs and complements parallel investigations into substrate specificity using recombinant proteins corresponding to known UGT sequences (21Lim E.-K. Li Y. Parr A. Jackson R. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4344-4349Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 22Jackson R.G. Lim E.-K. Li Y. Kowalczyk M. Sandberg G. Hoggett J. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4350-4356Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Note Added in ProofAn additional 18 complete UGT sequences have been identified in Arabidopsis genome data base subsequent to the submission of this manuscript. These do not change the composition of the groups defined in this manuscript, and they add an additional two groups to the tree. An additional 18 complete UGT sequences have been identified in Arabidopsis genome data base subsequent to the submission of this manuscript. These do not change the composition of the groups defined in this manuscript, and they add an additional two groups to the tree. We thank the Arabidopsis Biological Resource Center for providing all expressed sequence tag clones. We also thank Dr. Joe Ross for critical reading of the manuscript and helpful discussions. Supplementary Material Download .pdf (.05 MB) Help with pdf files Download .pdf (.05 MB) Help with pdf files

Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.