Successful Production of Piglets Derived from Expanded Blastocysts Vitrified Using a Micro Volume Air Cooling Method without Direct Exposure to Liquid Nitrogen

This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN₂). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN₂ for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN₂.     

Serotypes and virulence factors of Escherichia coli strains isolated from dogs and cats

E. coli strains isolated from urine of dogs and cats with urinary tract infections (UTI) and from feces of healthy one's were serotyped, and the serotypes were correlated with uropathogenic virulence factors. The most prevalent O-serotypes, O4 and O6, were isolated from dogs and cats with UTI. In contrast, O11 and O102 strains were the most frequently found from feces of healthy dogs and cats. Most of type O4 and O6 strains possessed such virulence factors as pil, pap, sfa, hly, and cnf1, while most type O11 and O102 strains pil only or pil and aer. All strains of type O75 possessed afaI and aer. K1 antigen was negative in all strains obtained from UTI.     

The genetic landscape of the childhood cancer medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.     

Characterization of vitamin B<Subscript>12</Subscript> compounds from marine foods

Vitamin B₁₂ is synthesized by only certain bacteria and archaea but not by animals or plants. In marine environments, bacterial vitamin B₁₂ is transferred and concentrated into fish and shellfish bodies by plankton in the marine food chain. Moreover, marine macrophytic red algae, Porphyra spp. specifically contain substantial amounts of vitamin B₁₂, due to microbial interaction. Although some meats or viscera of edible fish and shellfish are excellent sources of biologically active vitamin B₁₂, an inactive corrinoid, pseudovitamin B₁₂, was found in some edible shellfish using liquid chromatography/electrospray ionization–tandem mass spectrometry. To prevent elderly people from developing vitamin B₁₂ deficiency due to food protein-bound vitamin B₁₂ malabsorption, we present a survey of marine foods containing free vitamin B₁₂. The results of our study suggest that bonito and clam extracts (or soup stocks), which contain considerable amounts of free vitamin B₁₂ are useful not only as seasonings and flavorings but also as excellent sources of free vitamin B₁₂.     

Suppression of mRNAs for lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR) in pea reduces sensitivity to the phytotoxin coronatine and disease development by Mycosphaerella pinodes

Using a recently developed model pathosystem involving Medicago truncatula and Mycosphaerella pinodes, causal agent of Mycosphaerella blight on pea to understand host molecular response to a fungal suppressor, we applied the suppressor to leaves of M. truncatula and identified 151 nonredundant cDNA fragments as newly expressed genes. These included genes encoding lipoxygenase (LOX) and enoyl-CoA hydratase, which are presumably involved in jasmonic acid (JA) synthesis. Potential genes encoding plastidic enzymes, including allene oxide synthase (AOS) and allene oxide cyclase (AOC), and other peroxisomal enzymes involved in β-oxidation were predicted from the Medicago Gene Index EST database and tested for altered expression by semiquantitative RT-PCR. The coordinated expression of genes encoding both plastidic and peroxisomal enzymes showed that the suppressor likely conditions certain cellular process(es) through the JA synthesis in M. truncatula. To explore the role of JA or JA-regulated cellular process(es) in conditioning susceptibility, we used an Apple latent spherical virus (ALSV)-based virus-induced gene silencing (VIGS) technology to silence pea genes including LOX, AOS, AOC and 12-oxo-phytodienoic acid reductase (OPR). In LOX-, AOS-, AOC- or OPR-silenced pea plants, disease development induced by M. pinodes was remarkably reduced. Similarly, silencing of mRNA for LOX, AOS, AOC or OPR reduced the sensitivity to a phytotoxin, coronatine, which is believed to act through a JA-dependent process. On the basis of these results, it is conceivable that M. pinodes has evolved a strategy to condition susceptibility by manipulating the physiology of host cells, in particular JA-regulated cellular process(es), to promote disease development in pea.     

Glycosylation of flagellin from Pseudomonas syringae pv. tabaci 6605 contributes to evasion of host tobacco plant surveillance system

Pseudomonas syringae pv. tabaci (Pta) possesses a genetic region composed of two open reading frames (ORFs), fgt1 and fgt2, that are involved in glycosylation of flagellin. The deletion mutant Δfgt1 produced non-glycosylated flagellin, and exhibited reduced ability to cause disease in the host tobacco plant. Flagellin is known to induce plant defense responses, and the recognition of flagellin by Arabidopsis thaliana is mediated by a conserved N-terminal region, flg22, in flagellin and a leucine-rich repeat domain in the FLS2 receptor. Because flg22 localizes inside the flagellum, polymerized flagellum needs to be dissociated to be recognized. Therefore, the effect of glycosylation on flagella stability was investigated. The polymerized flagella from glycosylated flagellins were more resistant to heat treatment than those from non-glycosylated flagellins, suggesting that the glycosylation of flagellin contributes to the structural stability of flagella and prevents exposure of the flg22 region. Polymerized flagella from Pta Δfgt1 flagellin and depolymerized and glycosylated flagellin from Pta wild type induced cell death and callose deposition, and inhibited seedling growth in tobacco more effectively, whereas polymerized flagella from Pta wild-type flagellin caused a low level of these responses. These results suggest Pta might have evolved the flagellin glycosylation system to evade detection and defense response of a host by increasing flagella stability and suppressing their dissociation.     

Effect of Growing Environment of Soft Wheats on Amylose Content and Its Relationship with Cookie and Sponge Cake Quality and Solvent Retention Capacity

The effect of growing environments of soft wheat on amylose content and its relationship with baking quality and solvent retention capacities (SRC) was investigated. Near‐isogenic soft wheat lines of Norin 61 differing in granule‐bound starch synthase (Wx protein) activity and grown in three different regions of Japan: Hokkaido (spring‐sown) for 2006 and 2007, Kanto (autumn‐sown), and Kyushu (autumn‐sown) for 2007 were evaluated. Spring‐sown samples produced grains of greater protein content (10.9–12.4%) than autumn‐sown samples (7.3–9.1%). In contrast, spring‐sown samples of 2007 with higher maturing temperature had lower amylose content (25.5% for Norin 61) compare to the autumn‐sown and spring‐sown samples of 2006 (27.6–28.4% for Norin 61). Amylose content was strongly correlated to sugar snap cookie (SSCD) diameter (r = 0.957–0.961; n = 10, all samples; P ≤ 0.001, r = 0.701–0.976; n = 7 partial waxy and nonwaxy samples; and Japanese sponge cake (JSCV) volume r = 0.971–0.993; n = 10; P≤ 0.001, r = 0.764–0.922; n = 7 partial waxy and nonwaxy samples), regardless of seeding season and growing conditions. The strength of the JSVC‐amylose relationship (slope) was similar among the three regions, whereas the strength of the SSCD‐amylose relationship was slightly weaker for spring‐sown samples and slightly stronger for partial waxy and nonwaxy autumn‐sown samples. Among of the four solvents (water, solutions of sodium carbonate, sucrose, or lactic acid), water‐SRC showed the greatest correlation to amylose content (r = –0.969 to –0.996; n = 10; P ≤ 0.001, r = –0.629 to –0.983; n = 7 partial waxy and nonwaxy samples), indicated that amylose content can be accurately estimated from the water‐SRC within the samples from the same grown environment.