Multiplex Genome Editing in Tomato

Several expression systems for multiple guide RNA (gRNAs) have been developed in the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to induce multiple-gene modifications in plants. Here, we evaluated mutation efficiencies in the tomato genome using multiplex CRISPR/Cas9 vectors consisting of various Cas9 expression promoters with multiple gRNA expression combinations. In transgenic tomato calli induced with these vectors, mutation patterns varied depending on the promoters used to express Cas9. By using the tomato ELONGATION FACTOR-1α (SlEF1α) promoter to drive Cas9, occurrence of various types of mutations with high efficiency was detected in the tomato genome. Furthermore, sequence analysis showed that the majority of mutations using the multiplex system with the SlEF1α promoter corresponded to specific mutation pattern of deletions produced by self-ligation at two target sites of CRISPR/Cas9 with low mosaic mutations. These results suggest that optimizing the Cas9 expression promoter used in CRISPR/Cas9-mediated mutation improves multiplex genome editing, and could be used effectively to disrupt functional domains precisely in the tomato genome

Tyrosine kinase, phosphatidylinositol 3-kinase, and protein kinase C regulate insulin-stimulated NaCl absorption in the thick ascending limb

Tyrosine kinase, phosphatidylinositol 3-kinase, and protein kinase C regulate insulin-stimulated NaCl absorption in the thick ascending limb. We have previously shown a direct stimulatory effect of insulin on NaCl absorption in the medullary thick ascending limb of Henle's loop (mTAL). To further investigate the signal transduction involved, we determined whether tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase), and/or protein kinase C (PKC) regulate insulin-stimulated NaCl absorption in the mTAL by in vitro microperfusion methods. In control experiments, insulin increased transepithelial voltage (Vte) and net lumen-to-bath Cl− flux (JCl). Genistein and methyl 2,5-dihydroxycinnamate, two specific tyrosine kinase inhibitors, abolished the effects of insulin. Wort-mannin, a specific PI3-kinase inhibitor, inhibited the action of insulin. The effects of insulin also were inhibited by staurosporin and calphostin C, which are dissimilar inhibitors of PKC. These results indicate that insulin stimulates NaCl absorption in the mTAL through tyrosine kinase, PI3-kinase, and PKC-mediated mechanisms. Moreover, because we have reported previously that insulin causes no detectable change in cytosolic free Ca2+ in the mTAL cells, the present results also suggest that insulin-induced PKC activation is not related to inositol 1,4,5-triphosphate (IP3) production

SlIAA9 Controls Tomato Elongation

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

Excretion of Taurine and Sulfate in Rats Fed with a Low Protein Diet

The effects of a low protein diet on the excretion of sulfate and taurine, major metabolites of L-cysteine in mammals, were studied in rats fed with synthetic 10% (group A) and 25% (group B) casein diets. The average excretions of total taurine (taurine plus hypotaurine) and total sulfate (free plus ester sulfate) (mumol/kg of body weight per day after the adaptation to the synthetic diet) in group A were 14.2 +/- 13.4 and 122.3 +/- 39.6, respectively, which were very low compared with 280.4 +/- 93.8 and 943.2 +/- 144.8, respectively, in group B. The taurine/sulfate ratio in group A was 0.12 +/- 0.11, which was significantly lower than that (0.30 +/- 0.08) in group B. A single intraperitoneal injection of 5 mmol of L-cysteine per kg of body weight in group A resulted in an increase in average taurine and sulfate excretion to 693.4 +/- 195.6 and 2440.6 +/- 270.0, respectively, and thus the average taurine/sulfate ratio increased to 0.29. These increases were transient and low taurine excretion resumed again 24 h after the L-cysteine administration. L-Cysteine injection in group B resulted in a similar increase in taurine and sulfate excretion, but the ratio changed only slightly (0.28). The present results suggest that in vivo production of taurine is reduced preferentially over sulfate production when sulfur amino acid supply is limited. </p

Excretion of Sulfate and Taurine in Rats Fed with a High Protein Diet

Sulfate and taurine are the main metabolites of L-cysteine in mammals and are excreted in the urine. The effect of a high protein diet on the ratio of sulfate to taurine excretion was studied in rats using synthetic 25% (standard protein diet group, group A) and 40% (high protein diet group, group B) casein diets. Average taurine and sulfate excretions (mumol/kg of body weight per day) were 280.4 +/- 93.8 and 943.2 +/- 144.8 in group A and 553.4 +/- 124.5 and 2675.0 +/- 390.9 in group B, respectively. Thus, the average taurine/sulfate ratio in group A was 0.30 +/- 0.08. By a single administration of 5 mmol of L-cysteine/kg of body weight in group A, the average taurine and sulfate excretions increased to 1127.5 +/- 120.2 and 4043.0 +/- 305.6, respectively, but the taurine/sulfate ratio changed only slightly (0.28). The average taurine/sulfate ratio in group B was 0.22 +/- 0.07, a significantly lower ratio than that in group A, which means that daily intake of a high protein diet resulted in more sulfate excretion. The taurine/sulfate ratio in group B was affected only slightly (0.19) by the cysteine administration as well. These results suggest that the ratio of taurine and sulfate production was determined by dietary protein content and that the increase in sulfate production is larger than that of taurine production when the intake of dietary protein is increased.</p

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

Solitary Asymptomatic Thyroid Metastasis from Hepatocellular Carcinoma Detected by FDG-PET/CT

Thyroid metastases from hepatocellular carcinoma (HCC) seldom occur and are often difficult to diagnose because of their asymptomatic clinical course. We evaluated a very rare case of solitary thyroid metastasis from HCC that showed high uptake of fluorine-18 fluorodeoxyglucose (FDG), when imaged using fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT). The patient was a 74-year-old man and presented with a remarkably elevated des-gamma-carboxy prothrombin level of 1,157 mAU/ml 22 months after hepatic lobectomy. FDG-PET/CT imaging revealed a hypodense tumor with high FDG uptake, with a maximum standardized uptake value of 5.2 in the thyroid left lobe. Solitary thyroid metastasis from HCC was suspected and subsequent fine needle aspiration did indeed reveal HCC. The patient received left thyroidectomy with left regional lymph node dissection. Two months after left thyroidectomy, contrast-enhanced computed tomography showed local recurrence, and the patient received ongoing radiotherapy treatment. To our knowledge, the present study is the first to demonstrate the feasibility of FDG-PET/CT in the diagnosis and management of clinically diagnosed, asymptomatic, solitary thyroid metastasis from HCC