Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects. Tomato (Solanum lycopersicum L.) is among the most widely cultivated and consumed vegetables in the world and contains higher levels of GABA than other major crops. Increasing these levels can further enhance the blood pressure-lowering function of tomato fruit. Glutamate decarboxylase (GAD) is a key enzyme in GABA biosynthesis; it has a C-terminal autoinhibitory domain that regulates enzymatic function, and deleting this domain increases GAD activity. The tomato genome has five GAD genes (SlGAD1–5), of which two (SlGAD2 and SlGAD3) are expressed during tomato fruit development. To increase GABA content in tomato, we deleted the autoinhibitory domain of SlGAD2 and SlGAD3 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 technology. Introducing a stop codon immediately before the autoinhibitory domain increased GABA accumulation by 7 to 15 fold while having variable effects on plant and fruit size and yield. This is the first study describing the application of the CRISPR/Cas9 system to increase GABA content in tomato fruits. Our findings provide a basis for the improvement of other types of crop by CRISPR/Cas9-based genetic modification

Simple chest closure of open window thoracostomy for postpneumonectomy empyema: a case report

Abstract Background Management of postpneumonectomy empyema requires comprehensive strategies, especially when the condition is associated with large bronchopleural fistulae. We report a case involving the simple chest closure of open window thoracostomy with remaining residual space. Case presentation We performed open window thoracostomy for empyema with a huge bronchial stump dehiscence after right pneumonectomy for a large lung cancer. We definitively closed the chest window infected with chronic persistent Pseudomonas aeruginosa via a simple chest closure technique with the remaining residual space, after repairing the bronchial dehiscence using an omental flap and the appearance of healthy granulation tissue throughout the cavity. The patient died of recurrent cancer 10 months after the definitive chest closure. Until the patient died, there were no symptoms or signs suggestive of recurrent empyema. Conclusion This simple chest closure technique allows “silent empyema” to be observed carefully, is less invasive, and can even be applied to cases of recurrent cancer

Artificial Conversion of the Mating-Type of <i>Saccharomyces cerevisiae</i> without Autopolyploidization

Crossbreeding is a classical yeast hybridization procedure, where the mating of haploid cells of opposite mating-type, <i>MAT</i>a and <i>MATα</i> cells, produces a new heterozygous diploid. Here, we describe a method to generate haploid <i>MAT</i>a and <i>MATα</i> cells using mating-type conversion caused by expression of the <i>HO</i> gene, which encodes an endonuclease. Importantly, our method prevents the autopolyploidization that typically arises during artificial mating-type conversion. This facilitates isolation of the desired mating-type of yeast cells with simple and easy procedure. In the current study, we designed a suitable genetic circuit for each haploid cell and converted <i>MAT</i>α haploid cells into <i>MAT</i>a haploid cells and vice versa, demonstrating the utility of constructed artificial regulation network to prevent autopolyploidization. Via forced expression of the a1 gene in <i>MAT</i>α haploid cells or of α2 in <i>MAT</i>a haploid cells, the undesirable mating ability of yeast cells was completely suppressed. We confirmed the success in prevention of autopolyploidization by ploidy analysis. This new approach provides a reliable and versatile tool for yeast crossbreeding, so that it will be useful for scientific research and industrial applications of yeast

Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates.

We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times

Synthetic PAMPS gel activates BMP/Smad signaling pathway in ATDC5 cells, which plays a significant role in the gel-induced chondrogenic differentiation

The purposes of this study were to identify signaling pathways that were specifically activated in ATDC5 cells cultured on poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel in insulin-free maintenance medium and to evaluate the significance of the determined signaling pathways in the chondrogenic differentiation induced by this gel. In this study, ATDC5 cells cultured on PAMPS gel using the maintenance medium without insulin (PAMPS Culture) were compared with cells cultured on polystyrene using the differentiation medium containing insulin (PS-I Culture). The microarray analysis, Western blot analysis, and real-time PCR analysis demonstrated that the TGF-β/BMP signaling pathway was significantly enhanced at Days 1, 2, and 3 in the PAMPS Culture when compared with the PS-I Culture. Inhibition of the BMP type-I receptor reduced the phosphorylation level of Smad1/5 and expression of type-2 collagen and aggrecan mRNA in the cells accompanied by a reduction in cell aggregation at Day 13 in the PAMPS Culture. The inhibition of the TGF-β/BMP signaling pathway significantly inhibited the chondrogenic differentiation induced by the PAMPS gel. The present study demonstrated that synthetic PAMPS gel activates the TGF-β/BMP/Smad signaling pathway in the ATDC5 cells in the absence of insulin, and that this activation plays a significant role in the chondrogenic differentiation induced by PAMPS gel