Efficient and multiplexable genome editing using Platinum TALENs in oleaginous microalga, Nannochloropsis oceanica NIES-2145.

Algae accumulate large amounts of lipids produced by photosynthesis, and these lipids are expected to be utilized as feedstocks for sustainable new energies, known as biodiesels. Nannochloropsis species are eukaryotic microalgae that produce high levels of lipids. However, since the production costs of algal biodiesels are higher than those of fossil fuels, the improved productivity of algal lipids by molecular breeding of algae is required for practical use. In the present study, we developed a highly efficient genome-editing system involving Platinum transcription activator-like effector nucleases (TALENs) in Nannochloropsis oceanica. Platinum TALENs codon-optimized for N. oceanica were synthesized, and their DNA-binding activity was confirmed by single-strand annealing assays in human HEK293T cells. All-in-one expression vectors for Platinum TALEN targeting the nitrate reductase gene, NoNR, and acyltransferase gene, LPAT1, were transfected into Nannochloropsis species. The introduction of each Platinum TALEN revealed high genome-editing efficiency with no detectable off-target mutations at the candidate sites in N. oceanica. By simultaneously introducing TALENs targeting two genes, we obtained double mutant strains. The loss-of-function phenotype of NoNR was also confirmed. These findings will provide an essential technology for molecular breeding in Nannochloropsis species.

Dai Nippon Printing Co., Ltd Medi·Ca CC for Enumeration of Coliform Bacteria.

A ready-made dry medium method for coliform count, the Medi·Ca CC method, was compared to the Violet Red Bile Agar method (Bacteriological Analytical Manual, Chapter 4, Enumeration of Escherichia coli and the Coliform Bacteria, Section G) for nine raw foods from four food categories: raw ground pork, raw lamb, raw ground chicken, raw tuna fillet, raw salmon fillet, raw shrimp, fresh peeled banana, fresh cut pineapple, and fresh cut apple. The 95% confidence interval for the mean difference between the two methods at each contamination level for seven matrixes from all four categories fell within the range of -0.50 to 0.50, and no statistical difference was observed at all three contamination levels for four matrixes from three categories. These results demonstrated that the Medi·Ca CC method is a reasonable alternative to the reference method for raw meat, raw poultry, raw fish, and fresh fruits.

Synthesis of optically active regioregular polythiophenes and their self-organization at the air-water interface.

Regioregular polythiophenes containing an optically active substituent in the third position of the thiophene ring, head-to-tail poly(3-[2-((S)-1-methyloctyloxy)ethyl]thiophene)s (HT-P(S)MOETs), were synthesized using highly reactive zinc. For comparison, HT-P(R)MOET and achiral HT-P(±)MOET also were synthesized from R-type monomers and racemic monomers, respectively. The HT-PMOET possessed greater than 95% head-to-tail coupling with a weight-average molecular weight (Mw) between 1.96 × 10(4) and 2.94 × 10(4). The polymers were characterized using (1)H and (13)C NMR, optical rotatory power measurements, circular dichroism (CD), and UV-vis spectroscopy. X-ray diffraction patterns of the cast films demonstrated that regioregular HT-PMOET possessed a strong tendency to self-assemble into highly ordered, crystalline structures. The HT-P(S)MOET and HT-P(R)MOET showed strong Cotton effects, while HT-P(±)MOET showed very weak Cotton effects. The presence of a circular dichroism effect indicated that the side chain chirality induced optical activity in poly(thiophene) main chains. The monolayer formation of HT-PMOET spread on the water surface was characterized using a pressure-area (π-A) isotherm. The molecular areas of HT-P(S)MOET and HT-P(R)MOET molecules on the water surface were 33.5 and 32.9 Å(2), respectively, at 10 °C, which were larger than that of HT-P(±)MOET (27.9 Å(2)), suggesting that optically active HT-PMOET expanded because of the chiral repulsion between side chains. Multilayer films of HT-PMOET were prepared by repeating horizontal deposition of the monolayer on the water surface. The multilayer films of optically active HT-PMOET obtained showed stronger Cotton effects than did the cast films. In addition, electrical conductivities of HT-PMOET multilayer films were superior to those of spin-coated films. Head-to-tail poly(3-[2-((S)-1-methylpropyloxy)ethyl]thiophene) (HT-P(S)MPET), which contained shorter side chain lengths compared to HT-P(S)MOET, also was synthesized. The CD intensities of HT-P(S)MPET multilayer films were smaller than those of HT-P(S)MOET multilayer films, suggesting that the optically active side-chain length is critically important to the optically active self-assembly.

Genome editing with removable TALEN vectors harboring a yeast centromere and autonomous replication sequence in oleaginous microalga.

Algal lipids are expected to become a basis for sustainable fuels because of the highly efficient lipid production by photosynthesis accompanied by carbon dioxide assimilation. Molecular breeding of microalgae has been studied to improve algal lipid production, but the resultant gene-modified algae containing transgenes are rarely used for outdoor culture because the use of genetically modified organisms (GMOs) is strictly restricted under biocontainment regulations. Recently, it was reported that plasmids containing yeast centromere and autonomous replication sequence (CEN/ARS) behaved as episomes in Nannochloropsis species. We previously reported that the Platinum TALEN (PtTALEN) system exhibited high activity in Nannochloropsis oceanica. Therefore, we attempted to develop a genome editing system in which the expression vectors for PtTALEN can be removed from host cells after introduction of mutations. Using all-in-one PtTALEN plasmids containing CEN/ARS, targeted mutations and removal of all-in-one vectors were observed in N. oceanica, suggesting that our all-in-one PtTALEN vectors enable the construction of mutated N. oceanica without any transgenes. This system will be a feasible method for constructing non-GMO high-performance algae.

A homolog of Arabidopsis SDP1 lipase in Nannochloropsis is involved in degradation of de novo-synthesized triacylglycerols in the endoplasmic reticulum.

Organisms of the microalgal genus Nannochloropsis produce high levels of triacylglycerols (TAGs), an efficient raw material for biofuels. A complete understanding of the TAG-breakdown pathway is critical for improving the productivity of TAGs to meet future needs. Among a number of lipases annotated as TAG lipase in the genomes of every organism, Arabidopsis SUGAR-DEPENDENT 1 (AtSDP1) lipases are characterized as a type of crucial TAG lipase in plants, similar to ScTgl3-5 in Saccharomyces cerevisiae. Homologs of the AtSDP1 TAG lipases are universally found in the genomes of plants, fungi, and algae. Here we identified two homologs of AtSDP1 TAG lipases in the oleaginous microalga species Nannochloropsis oceanica, NoTGL1 and NoTGL2. We generated single- and double-knockout strains for these lipases by homologous recombination. Whereas overall TAG content in the NoTGL2 single-knockout mutant was identical to that of wild type, the NoTGL1 knockout showed a two-fold increase in TAG content per cell in early log phase under nutrient-sufficient conditions without affecting growth. Homologs of AtSDP1 in S. cerevisiae are localized to the surface of lipid droplets, and AtSDP1 is transported from peroxisomes to the surface of lipid droplets. In contrast, NoTGL1 localized to the endoplasmic reticulum in both Nannochloropsis and yeast. We suggest that homologs of AtSDP1 lipases in Nannochloropsis modulate de novo TAG biosynthesis in the endoplasmic reticulum, unlike the roles of these lipases in other organisms. These results provide important insights into the mechanisms of TAG metabolism catalyzed by homologs of AtSDP1 lipase, which are highly conserved across species.

[A case of hepatic eosinophilic granuloma, which needs distinction with metastatic liver cancer].

A 68-year-old man was referred to our hospital because of eosinophilia in peripheral blood and pancreatic tumor on abdominal US. He was accustomed to eating the raw flesh of wild boar and keeping wild boar, and under medical treatment for Diabetes. Pancreatic tumor was diagnosed to the pancreatic ductal cancer by the imaging examination and endoscopic transpapillary brushing cytology for pancreatic duct. The diagnosis of hepatic eosinophilic granuloma was done by aspiration biopsy for hepatic multiple small nodules. Because of the strong positive finding for nematose in the assay of multi dot-ELISA for parasite, hepatic eosinophilic granuloma caused by visceral larva migrans was accidentally complicated by pancreatic cancer, and operation for the pancreatic cancer was done. To bear this disease in mind and to research his life history, is important to diagnose hepatic multiple nodules with eosinophilia.

Validation of Medi·Ca EC Method for AOAC Research Institute Performance Tested MethodsSM Certification.

A ready-made dry medium method for Escherichia coli and coliform count, the Medi·Ca EC method, was compared with the most probable number (MPN) method using Brilliant Green Lactose Bile broth and E. coli broth (AOAC INTERNATIONAL Official MethodSM 966.24) for seven food matrixes: raw beef, raw pork, raw frozen pork, raw lamb, raw salmon, frankfurter sausage, and cooked ham. The mean difference between the two methods at each contamination level for each matrix was <0.5 log10, and the 95% confidence intervals for the mean differences fell within the range of -0.5 to 0.5, with the exception of a few cases in the independent laboratory study. sr and RSDr values of the Medi·Ca EC method were generally lower than those of the MPN method, and r2 ranged from 0.91 to 0.99. Product consistency and stability studies showed little variability between production lots and the shelf-life of 20 months. An incubation time within the range of 22-26 h did not adversely affect the results; however, variations in sample volume did affect final counts. These results showed that the Medi·Ca EC method is a reasonable alternative to the reference method for the selected food matrixes and makes it possible to simultaneously detect and enumerate E. coli and coliform in only 24 h.

[Arterial infusion chemotherapy for patients with advanced pancreatic cancer].

Although various therapies have been tried to improve advanced nonresectable pancreatic cancer, a sufficient consensus has not yet been obtained about the treatment. We have performed arterial infusion chemotherapy for pancreatic cancer in order to maintain QOL. The response rate was 17.3%, the mean survival time 282.1+/-204.7 days, median survival time 243.0+/-84.7 days, and many patients were continuously treated on an outpatient basis. It is thus expected that survival time and maintenance of QOL can be extended by self-sustaining arterial infusion chemotherapy.

MRI findings of hemolytic uremic syndrome with encephalopathy: widespread symmetrical distribution.

The authors report the magnetic resonance imaging (MRI) findings in a 22-year-old woman with hemolytic uremic syndrome and encephalopathy secondary to verotoxin-producing Escherichia coli. Multiple lesions in the midbrain, cerebellum, occipital lobe, and basal ganglia showed high signal intensity on T2-weighted images with widespread symmetrical distribution. Most of these findings showed remarkable reduction on MRI images obtained 70 days after the onset. It is suggested that edema induced by local breakdown of blood-brain barrier might play an important role in the patient.