The ability to control integration, inheritance, and expression of multiple transgenes is a prerequisite for manipulating biosynthetic pathways and complex agronomic characteristics in plants. One hundred and twenty-five independent transgenic rice plants were regenerated after cobombarding embryogenic tissues with a mixture of 14 different pUC-based plasmids. Eighty-five percent of the R0 plants contained more than two, and 17% more than nine, of the target genes. Plants containing multiple transgenes displayed normal morphologies and 63% set viable seed. Multigene cotransformation efficiency was correlated with the ratio in which the plasmids were mixed with respect to the selectable marker. All target genes had an equal chance of integration, indicating that the nature of the coding region had no effect on the efficiency of integration. Three plant lines containing 11, 10, and 9 transgenes, respectively, were analyzed for patterns of integration and inheritance until the R3 generation. Integration of multiple transgenes occurred at either one or two genetic loci, with inheritance conforming to a 3:1 Mendelian ratio. Coexpression of four marker genes was investigated until the R2 generation.

Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFβ1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFβ1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.

Immunoglobulin rearrangement from variable heavy chain (V(H)) to diversity (D)-joining heavy chain (J(H)), which occurs exclusively in B lineage cells, is impaired in mice deficient for the B lineage-specific transcription factor Pax5. Conversely, ectopic Pax5 expression in thymocytes promotes the rearrangement of D(H)-proximal V(H)7183 genes. In exploring the mechanism for Pax5 regulation of V(H)-to-DJ(H) recombination, we have identified multiple Pax5 binding sites in the coding regions of human and mouse V(H) gene segments. Pax5 bound to those sites in vitro and occupied V(H) genes in early human and mouse B lineage cells. Moreover, Pax5 interacted with the recombination-activating gene 1 (RAG1)-RAG2 complex to enhance RAG-mediated V(H) recombination signal sequence cleavage and recombination of a V(H) gene substrate. These findings indicate a direct activating function for Pax5 in RAG-mediated immunoglobulin V(H)-to-DJ(H) recombination.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved-caspase 9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase 3, 8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its GPI anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1−/−) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1−/− mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.

During B lymphocyte development, Ig heavy and L chain genes are assembled by V(D)J recombination. Individual V, D, and J genes rearrange at very different frequencies in vivo, and the natural variation in recombination signal sequence does not account for all of these differences. Because a permissive chromatin structure is necessary for the accessibility of VH genes for VH to DJH recombination, we hypothesized that gene rearrangement frequency might be influenced by the extent of histone modifications. Indeed, we found in freshly isolated pro-B cells from μMT mice a positive correlation between the level of enrichment of VHS107 genes in the acetylated histone fractions as assayed by chromatin immunoprecipitation, and their relative rearrangement frequency in vivo. In the VH7183 family, the very frequently rearranging VH81X gene showed the highest association with acetylated histones, especially in the newborn. Together, our data show that the extent of histone modifications in pro-B cells should be considered as a mechanism by which accessibility and the rearrangement level of individual VH genes is regulated.

We have cloned and sequenced genomic DNA from a human library extending 1300 bp upstream the 5'-untranslated sequence of the cDNA coding for the sodium/iodide symporter. In transient transfection assays this sequence exhibited promoter activity, which could be confined to nucleotides -443 to -395 relative to the ATG start codon. This minimal promoter, including a putative GC- and TATA- box, was preferentially activated in the rat thyroid cell line FRTL-5, but was also active in non-thyroidal cells, such as COS-7 and Chinese-hamster ovary, albeit to a markedly lower extent.

Biological systems are increasingly being studied by high throughput profiling of molecular data over time. Determining the set of time points to sample in studies that profile several different types of molecular data is still challenging. Here we present the Time Point Selection (TPS) method that solves this combinatorial problem in a principled and practical way. TPS utilizes expression data from a small set of genes sampled at a high rate. As we show by applying TPS to study mouse lung development, the points selected by TPS can be used to reconstruct an accurate representation for the expression values of the non selected points. Further, even though the selection is only based on gene expression, these points are also appropriate for representing a much larger set of protein, miRNA and DNA methylation changes over time. TPS can thus serve as a key design strategy for high throughput time series experiments. Supporting Website: www.sb.cs.cmu.edu/TPSDOI: http://dx.doi.org/10.7554/eLife.18541.001

V, D, and J gene segments rearrange at very different frequencies. As with most biological systems, there are multiple levels of control of V gene recombination frequency, and here we review some of the work from our laboratory that addresses these various control mechanisms. One of the important factors that affect non-random V gene rearrangement frequency is the natural heterogeneity in recombination signal sequences (RSSs). Not only does variation in the heptamer and nonamer affect rearrangement, but variation in the spacer can also dramatically affect recombination. However, there are clearly other factors which control V gene rearrangement, as revealed by the fact that genes with identical RSSs can rearrange at different frequencies in vivo. Some of these other influences most likely affect the earliest stages of control--the change from an inaccessible state to an accessible state. Transcription factors can play a role in inducing these changes. Rearrangement of many VkappaI genes can be induced in a non-lymphoid cell line after ectopic expression of E2A, while neighboring VkappaII and VkappaIII genes do not rearrange, demonstrating that at least one level of control of induction of accessibility occurs at the level of the individual gene. Also, changes in chromatin structure can affect accessibility and might influence individual V gene rearrangement frequency.