Flow cytometric analysis of Xenopus laevis and X. tropicalis blood cells using acridine orange.

Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.

A retrospective, hospital-based study to determine the incidence of rotavirus hospitalizations among children less than 5 years of age over a 10-year period (2001-2011) in Akita prefecture, Japan.

Rotavirus is the most common cause of severe gastroenteritis in children worldwide. This retrospective, cross-sectional study was undertaken in a sentinel hospital that provides the only pediatric beds for the local population with an average of 4,400 children aged <5 years and determined the incidence of rotavirus hospitalizations. Medical charts that recorded acute gastroenteritis cases occurring in children aged <5 years living in the cites of Yuri-Honjo or Nikaho, Akita, Japan between 2001 and 2011 were retrieved and examined to enumerate rotavirus antigen-positive hospitalizations. Of the 1,596 acute gastroenteritis cases retrieved, antigen detection was performed in 834 cases, and 387 were positive; hence, the crude annual incidence rate of rotavirus hospitalizations was 8.8 per 1,000 person-years. The adjusted annual incidence rate of rotavirus hospitalizations was 13.7 per 1,000 person-years when untested samples collected during the peak season were extrapolated to the same rotavirus detection proportion as the tested samples (58.9%). We confirmed a high incidence of rotavirus hospitalizations in Akita Prefecture and revealed a considerable degree of annual fluctuation in the rotavirus hospitalization rates, which exceeded the degree of stochastic fluctuation. Thus, caution must be exercised when interpreting the impact of a rotavirus vaccine on the reduction of the number of rotavirus hospitalizations.

Asymmetric tandem Michael-aldol reactions between 3-cinnamoyloxazolidine-2-thiones and aldehydes.

Reactions between chiral 3-cinnamoyl-4-methyl-5-phenyl-1,3-oxazolidine-2-thiones and aromatic aldehydes in the presence of BF3Et2O diastereoselectively produced tricyclic compounds incorporating a bridgehead carbon bound to four heteroatoms in high yields. Four stereocenters were induced during the reaction. The tricyclic products were transformed into propane-1,3-diols bearing three consecutive stereocenters by acid hydrolysis, S-methylation, and reductive removal of the chiral auxiliary.

Chalcogeno-Morita-Baylis-Hillman reaction of enones with acetals: simple alpha-alkoxyalkylation of enones.

1-[2-(Methylsulfanyl)phenyl]prop-2-en-1-one (1) and the seleno congener (2) reacted with acetals 3 and 21 in the presence of BF(3).Et(2)O to give alpha-alkoxyalkyl enones 4, 5 and 22, 23 in good yields. When the reaction mixtures were worked up with a saturated NaHCO(3) solution instead of Et(3)N, onium salts 6 and 7 were obtained together with 4 and 5. Reactions with cyclic acetal 14 gave alpha-(beta-hydroxyethoxy) enones 15 and 16 accompanied by dimeric products 17 and 18.

Chalcogenide-Lewis acid mediated reactions of electron-deficient alkynes with aldehydes.

Reactions of but-3-yn-2-one (2) with aldehydes 1 in the presence of a Lewis acid and dimethyl sulfide (3 a) predominantly gave (E)-alpha-(halomethylene)aldols 4-5 in high yields, while reactions of methyl propiolate (6 a) mainly afforded (Z)-3-halogeno-2-(hydroxymethyl)acrylates 7-8 in low to moderate yields. A reaction of dimethyl acetylenedicarboxylate (10) with 1 a in the presence of TiCl(4) and 1,1,3,3-tetramethylthiourea (3 c) produced maleate (E)-11 (40 %) and butenolide 12 (40 %). When a reaction of 6 a with 1 a was carried out in the presence of TiBr(4) and 3 a (0.2 equiv) at -20 degrees C for 60 h, 3-(methylthio)-2-(hydroxyalkyl)acrylate 9 a was obtained in an 8 % yield. Experiments were conducted in order to elucidate the formation mechanism of 9 a, and it was made clear that 9 a was formed via the processes of the Michael addition of sulfide 3 a to alkynoate 6 a and an aldol reaction with 1 a and demethylation.

Novel Perfluoroacyl Olefinations of Aldehydes Using ß-Thio-Substituted Perfluoroalkyl Enol Ethers.

α-(Methylthio)- or α-(phenylthio)-substituted perfluoroacylolefinations of nonenolizable aldehydes using the ß-lithio-ß-thio-perfluoroalkyl enol ethers 1-4 stereoselectively proceeded to give (Z)-α,ß-unsaturated perfluoroalkyl ketones 9a-e, 10a-c, 11a-c, and 12a. The α-(thio)-α,ß-unsaturated trifluoromethyl ketones were easily converted to 3-(thio)-2-(trifluoromethyl)-1,3-butadienes 21a-c and 22a,b in moderate to high yields (41-85%).