CRISPR-KRISPR: A Method to Identify On-Target and Random Insertion of Donor DNAs and their Characterization in Knock-In Mice

CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding

Hepatocyte apoptosis is enhanced after ischemia/reperfusion in the steatotic liver

Liver steatosis is associated with organ dysfunction after hepatic resection and transplantation which may be caused by hepatic ischemia/reperfusion injury. The aim of the current study was to determine the precise mechanism leading to hepatocyte apoptosis after steatotic liver ischemia/reperfusion. Using a murine model of partial hepatic ischemia for 90 min, we examined the levels and pathway of apoptosis, and the peroxynitrite expression, serum alanine aminotransferase levels, and liver histology 1 and 4 h after reperfusion. In the steatotic liver, the peroxynitrite expression increased after ischemia/reperfusion. Significant hepatocyte apoptosis in the steatotic liver was seen after reperfusion, caused by upregulation of cleaved caspases 9 and 3, but not caspase 8. Serum alanine aminotransferase levels were elevated and histological examination revealed severe liver injury in the steatotic liver 4 h after reperfusion. In mice treated with aminoguanidine, ischemia/reperfusion-induced increases in serum alanine aminotransferase levels and apoptosis were significantly reduced in steatotic liver compared with mice treated with phosphate buffered saline. Survival of mice with steatotic livers significantly improved by treatment with aminoguanidine. Our data suggested that the steatotic liver is vulnerable to hepatic ischemia/reperfusion, leading to significant hepatocyte apoptosis by the mitochondrial permeability transition, and thereby resulting in organ dysfunction

Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling

Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs

千葉県君津市川谷地域に露出する中部更新統柿ノ木台層から産出する冷湧水化石群集: その時空分布と共産する自生炭酸塩

金沢大学国際基幹教育院 GS教育系冷湧水性群集が房総半島の中部更新統柿ノ木台層の陸棚相から産出する．群集は，化学合成二枚貝類から排他的になり，著しく13Cに枯渇した自生炭酸塩と共産することから，AOM（嫌気的メタン酸化）に依存していたと考えられる．自生炭酸塩は巣穴壁面と巣穴周囲の堆積物中に沈殿し，巣穴からスナモグリ類の爪化石と糞化石が産出することから，これらはスナモグリ類の巣穴であると考えられる．スナモグリ類はメタン生成帯まで巣穴を堀り，海水を巣穴深部へ供給し，AOMを活性化させることによって巣穴中の硫化水素イオン濃度を上昇させた．溶存酸素濃度が高い巣穴浅部では，硫黄酸化菌が繁茂し，スナモグリ類の食糧となった．巣穴深部では，浮遊する生物源炭酸塩などを核とした針状アラゴナイトが重力方向に沈下して炭酸塩ジオペタル状構造を形成し，巣穴周囲の堆積物中では，リン酸イオン濃度の上昇により高Mgカルサイトが，また硫酸イオンの枯渇によりドロマイトが沈殿した．Cold-seep-dependent molluscan assemblages occur in the outer-shelf facies of the middle Pleistocene Kakinokidai Formation of the Kazusa Group, a forearc basin-fill sequence on the Pacific side of central Japan, in strata corresponding to the interval 707.6-667.0 ka. The assemblages consist exclusively of chemosymbiotic bivalves (lucinids, thyasirids, and solemyids) and are associated with 13C-depleted authigenic carbonates (δ13C = −61.60‰ to −10.96‰ VPDB), which suggest that their main carbon source was anaerobic oxidation of methane (AOM). Authigenic carbonate precipitates are common on burrow walls (mainly acicular aragonite) and the surrounding sediments (mainly micritic high-Mg calcite and dolomite). The burrows are cylindrical, 1.5-3.0 cm in diameter, and >1 m long. Callianassid claws and the trace fossil Palaxius (probable callianassid fecal pellets) in the burrow carbonates suggest that the burrows were produced by sediment-dwelling callianassid decapods.\nWe propose the following formation mechanism of burrows and their related authigenic carbonates. Firstly, callianassids produced deep burrows, penetrating the AOM zone and reaching the methanogenic zone. Methane then seeped into the burrows and AOM occurred in its deeper parts, promoted by a supply of seawater via callianassid activity, resulting in an increase in the concentration of hydrogen sulfide ions. Thiobacteria flourished in the shallower parts of the burrows, which were enriched in dissolved oxygen, and provided a source of food for the callianassids. In the deeper parts of the burrows, acicular aragonite precipitated around suspended carbonate nuclei and sank to the bottoms of the burrows, forming geopetal-like carbonate structures. In the surrounding sediment, high-Mg calcite precipitated in response to an increase in the concentration of phosphate ions (due to the decomposition of organic matter), and dolomite precipitated in response to decreasing concentrations of sulfate ions (caused by active AOM)

Production and Properties of Nano-scale Oxide Dispersion Strengthened (ODS) 9Cr Martensitic Steel Claddings

The 9Cr-ODS martensitic steel claddings were developed by cold-rolling and subsequent heat-treatment. The standard chemical composition is Fe-0.13C-9Cr-2W-0.2Ti-0.35Y2O3. The substantially elongated grains formed by cold-rolling turned out to be into equi-axied grains by ferrite to austenite phase transformation at the final heat-treatment. The produced claddings have the tempered martensitic structure and excess oxygen of 0.060 mass%. The superior tensile and creep rupture strength were shown in the produced cladding, compared with conventional ferritic (PNC-FMS) and even austenitic (PNC316) claddings at higher temperature and extended time. The strength improvement is attributed to finely distributed nano-scale complex oxide. The coarser ferrite grains produced by slow cooling make further improvement in the tensile and creep rupture strength beyond those of tempered martensite at high temperature and longer testing time. The higher excess oxygen content of 0.137 mass% prevents fine distribution of the oxide particles that lead to inferior high temperature tensile and creep strength

Examination of the lifestyle of head injury patients using the Frenchay Activities Index

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Search of optimum gas mixture ratio as gas insulating medium by genetic algorithm

Genetic algorithm is applied to find the optimum gas mixture ratio as gas insulating medium substituting pure SF/sub 6/. Genetic algorithm is very useful to find the optimum solution from vast searching possibilities. Testing each gas mixture by experiment requires a long time. The present method is very efficient to preselect the candidates of gas mixtures before more thorough but time-consuming investigation via experiment is carried out. The gas mixture ratio is coded as a series of bits simulating a genetic sequence of a life form. Two-term Boltzmann equation is used to calculate the effective ionization coefficient of each gas mixture that is used to evaluate the degree of adaptation of each individual representing one set of mixture ratios. Two types of degree of adaptation are used to evaluate each individual, the effective ionization coefficient at the critical ratio of the electric field to the gas density of SF/sub 6/ of 359.3/spl times/10/sup 21/Vm/sup 2/, and the global warming potential. Based on the degree of adaptation, better individuals can be selected as parents of the next generation, leaving their genes to future generations. After some generations, the group of individuals converges into the optimum with the best degree of adaptation