Sativene Sesquiterpenoids from the Plant Endophytic Fungus Bipolaris victoriae S27 and Their Potential as Plant-Growth Regulators.

Thirteen new sativene sesquiterpenoids (1 and 3-14), one new natural product (2), and 16 known compounds (15-30) were isolated from the endophytic fungus Bipolaris victoriae S27. Their structures were elucidated by extensive spectroscopic analysis, NMR and ECD calculations, and X-ray crystal diffractions. Compound 1 represented the first example of sativene sesquiterpenoids with a 6/5/3/5-caged tetracyclic ring system. All obtained compounds were evaluated for their plant-growth regulatory activity. The results showed that 1, 3, 4, 6, 8, 11, 12, 17, 19, 26, and 27 could suppress the growth of Arabidopsis thaliana, while 2, 5, 13, 15, 18, and 25 showed promoting effects. Among them, compound 3 showed the most potent plant-growth inhibitory activity, which is obviously superior to that of the marked herbicide glyphosate.

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells.

BACKGROUND: T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS: T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). RESULTS: SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CONCLUSIONS: CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. TRIAL REGISTRATION: Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. FUNDING: National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

The doublesex splicing enhancer components Tra2 and Rbp1 also repress splicing through an intronic silencer.

The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression.

Concentration dependent selection of targets by an SR splicing regulator results in tissue-specific RNA processing.

The splicing factor Transformer-2 (Tra2) is expressed almost ubiquitously in Drosophila adults, but participates in the tissue-specific regulation of splicing in several RNAs. In somatic tissues Tra2 participates in the activation of sex-specific splice sites in doublesex and fruitless pre-mRNAs. In the male germline it affects splicing of other transcripts and represses removal of the M1 intron from its own pre-mRNA. Here we test the hypothesis that the germline specificity of M1 repression is determined by tissue-specific differences in Tra2 concentration. We find that Tra2 is expressed at higher levels in primary spermatocytes of males than in other cell types. Increased Tra2 expression in other tissues reduces viability in a manner consistent with known dose-dependent effects of excessive Tra2 expression in the male germline. Somatic cells were found to be competent to repress M1 splicing if the level of Tra2 transcription was raised above endogenous concentrations. This suggests not only that M1 repression is restricted to the germline by a difference in Tra2 transcription levels but also that the protein's threshold concentration for M1 regulation differs from that of doublesex and fruitless RNAs. We propose that quantitative differences in regulator expression can give rise to cell-type-specific restrictions in splicing.

Analysis of a null mutation in the Drosophila splicing regulator Tra2 suggests its function is restricted to sexual differentiation.

Tra2 is a regulator of pre-mRNA splicing and a key component of the Drosophila somatic sex determination pathway. Functional orthologs of this protein are thought to perform nonsex-specific functions essential for viability in both vertebrates and nematodes. Although Drosophila Tra2 is expressed throughout the soma of both sexes, studies on it have focused only on the sex-specific phenotypes of known viable alleles. Here we show that that widely used tra2 mutant alleles have residual activity and are not suitable for evaluating its effect on viability. To test whether Tra2 has an essential role in development, we generated a transposon-induced deletion in critical coding sequences. We find that tra2 deletion adults can survive as well as their heterozygous siblings. Thus, in contrast to other organisms, Tra2 is not required in Drosophila for general viability under laboratory conditions.