Effects of the sliaa9 Mutation on Shoot Elongation Growth of Tomato Cultivars.

Tomato INDOLE-3-ACETIC ACID9 (SlIAA9) is a transcriptional repressor in auxin signal transduction, and SlIAA9 knockout tomato plants develop parthenocarpic fruits without fertilization. We generated sliaa9 mutants with parthenocarpy in several commercial tomato cultivars (Moneymaker, Rio Grande, and Ailsa Craig) using CRISPR-Cas9, and null-segregant lines in the T1 generation were isolated by self-pollination, which was confirmed by PCR and Southern blot analysis. We then estimated shoot growth phenotypes of the mutant plants under different light (low and normal) conditions. The shoot length of sliaa9 plants in Moneymaker and Rio Grande was smaller than those of wild-type cultivars in low light conditions, whereas there was not clear difference between the mutant of Ailsa Craig and the wild-type under both light conditions. Furthermore, young seedlings in Rio Grande exhibited shade avoidance response in hypocotyl growth, in which the hypocotyl lengths were increased in low light conditions, and sliaa9 mutant seedlings of Ailsa Craig exhibited enhanced responses in this phenotype. Fruit production and growth rates were similar among the sliaa9 mutant tomato cultivars. These results suggest that control mechanisms involved in the interaction of AUX/IAA9 and lights condition in elongation growth differ among commercial tomato cultivars.

Genome editing in plants using CRISPR type I-D nuclease.

Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I-the most abundant CRISPR system in bacteria-has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

Impact of the individualization of the first-line chemotherapy for advanced colorectal cancer based on collagen gel droplet-embedded drug sensitivity test.

Leucovorin (FOL) and fluorouracil (5-FU) plus oxaliplatin (l-OHP; FOLFOX) or FOL and 5-FU plus irinotecan (SN-38; FOLFIRI) are widely used as first-line chemotherapy regimens in the treatment of advanced colorectal cancer (CRC). However, second-line chemotherapy must be abandoned in certain cases due to disease progression, adverse effects or high medical cost. Therefore, the most effective regimen should be selected as first-line chemotherapy. We reported that individualization of first-line treatment (FOLFOX/FOLFIRI/Dual/Poor responder) was possible using the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) and that individualized first-line chemotherapy with CD-DST may improve the prognosis of patients with unresectable CRC. The aim of the present prospective cohort study was to evaluate the individualization of first-line chemotherapy using CD-DST, with a focus on prognosis. Between March 2008 and December 2015, tumor specimens were obtained from 120 patients with CRC who had not received preoperative chemotherapy. CD-DST was performed and the growth inhibition rate (IR) was determined by exposure for 24 h with 5-FU and l-OHP (6.0 and 3.0 µg/ml, respectively) and 5-FU and SN-38 (6.0 and 0.2 µg/ml, respectively). The cumulative distribution of IR values under each condition was evaluated on the basis that the clinical response to FOLFOX and FOLFIRI is equivalent (~50%). The prognosis of dual responder was improved compared with that of poor responders, however this difference was identified to be significant. There was no different prognosis between patients treated with an appropriate first-line regimen and patients treated with an inappropriate first-line regimen in dual responders. However, in poor responders, there were significant differences of prognosis between patients treated with an appropriate first-line regimen and patients treated with an inappropriate first-line regimen (P=0.036). In conclusion, the results from the present study suggest that administration of the recommended first-line regimen using CD-DST for patients with unresectable CRC is important for the improvement of prognosis, particularly in poor responders.

[Pharmacokinetics characteristics of dexamethasone in Crush syndrome model rats].

Crush syndrome (CS) is characterized by ischemia/reperfusion-induced rhabdomyolysis and subsequent systemic inflammation and has a high mortality rate, even when treated with conventional therapy. In previous studies, we demonstrated that treatment of rats with acute lethal CS using dexamethasone (DEX) had therapeutic effects in laboratory findings and improved the clinical course of CS. However, because the application of DEX in CS therapy is unknown, evaluation of the pharmacokinetic parameters of DEX was considered essential to support its clinical use. Here, we investigated the pharmacokinetic characteristics of DEX in a rat model of CS. Anesthetized rats were subjected to bilateral hind limb compression using rubber tourniquets for 5 h, followed by reperfusion for 0 to 24 h. Rats were divided randomly into 4 groups: saline-treated sham (S) and CS groups and 5.0 mg/kg DEX-treated S (S-DEX) and CS (CS-DEX) groups. Blood and tissue samples were collected for HPLC analysis. In the CS-DEX group, the pharmacokinetic parameters of the area under the concentration-time curve, mean residence time, and distribution volume levels increased significantly compared to the S-DEX group, whereas total body clearance, elimination rate constant, and renal clearance levels decreased significantly. Moreover, decrease of muscle tissue DEX concentration and of CYP3A activity were observed in the CS-DEX group. These results show the pharmacokinetic characteristics of DEX in the rat CS model and support the potential use of DEX in disaster medical care.

CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms.

Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. Here, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIα weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein 1 (BMAL1). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.

Strong two-photon absorption and its saturation of a self-organized dimer of an ethynylene-linked porphyrin tandem.

The two-photon absorption properties of a self-organized dimer of a free-base and zinc(II) porphyrins tandem linked with an ethynylene group and terminated by imidazolyl and phenylethynyl groups were investigated. The self-organized dimer was found to exhibit strong two-photon absorption and furthermore the saturation of the two-photon absorption owing to the intense transition.