Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference.

RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.

Spin polarized tunneling at finite bias.

A mesoscopic spin valve is used to determine the dynamic spin polarization of electrons tunneling out of and into ferromagnetic (FM) transition metals at finite voltages. The dynamic polarization of electrons tunneling out of the FM slowly decreases with increasing bias but drops faster and even inverts with voltage when electrons tunnel into it. A free-electron model shows that in the former case electrons originate near the Fermi level of the FM with large polarization whereas in the latter, electrons tunnel into hot electron states for which the polarization is significantly reduced. The change in sign is ascribed to the matching of the electron wave function inside and outside the tunnel barrier.

Anthropometry and somatotype of competitive female figure skaters 11-22 years. Variation by competitive level and discipline.

AIM: Variation in anthropometric characteristics and somatotype of female figure skaters by level of competition and discipline was examined. EXPERIMENTAL DESIGN: ANCOVA with age as the covariate was used to compare the anthropometry of skaters by level (test stream, pre-elite, elite) and discipline (free, dance, pair), while MANCOVA was used to compare somatotype. SETTING: purposive sampling reflecting the skating population by level and discipline was used to recruit skaters in 4 American, and 7 Canadian figure skating clubs. PARTICIPANTS: a total of 161 competitive female figure skaters 11-22 years of age (15.7+/-2.4 years) comprised the sample. MEASURES: a battery of 15 anthropometric dimensions was taken on each skater. Several dimensions, ratios and Heath-Carter somatotypes were derived. RESULTS: Test skaters are heavier and generally larger than pre-elite skaters. Test stream skaters also have larger limb circumferences, estimated calf and arm musculature, and a thicker sum of skinfolds, and are more endomorphic than pre-elite skaters. Elite skaters are more mesomorphic than pre-elite skaters. The sitting height/stature (SH/ST) ratio is significantly lower in pre-elite skaters, while elite and test stream skaters do not differ in this indicator of proportions. Free skaters are taller and heavier; have a higher body mass index (BMI), limb circumferences and sum of skinfolds; and proportionally shorter legs than dancers and pair skaters. Free skaters, dancers and pair skaters, however, do not differ in somatotype. CONCLUSIONS: The results suggest that figure skating favors lightness, leanness, higher mesomorphy and lower endomorphy at more elite levels. Shortness, leanness and linearity of physique appear to be selective factors associated with specialization in dance and pair skating.

Current-induced magnetization reversal in high magnetic fields in Co/Cu/Co nanopillars.

Current-induced magnetization dynamics in Co/Cu/Co trilayer nanopillars (approximately 100 nm in diameter) have been studied experimentally at low temperatures for large applied fields perpendicular to the layers. At 4.2 K an abrupt and hysteretic increase in resistance is observed at high current densities for one polarity of the current, comparable to the giant magnetoresistance effect observed at low fields. A micromagnetic model that includes a spin-transfer torque suggests that the current induces a complete reversal of the thin Co layer to alignment antiparallel to the applied field--that is, to a state of maximum magnetic energy.

In vitro fermentation of swine ileal digesta containing oat bran dietary fiber by rat cecal inocula adapted to the test fiber increases propionate production but fermentation of wheat bran ileal digesta does not produce more butyrate.

This experiment evaluated three hypotheses: i) production of propionate is increased during fermentation of substrate containing oat bran (OB)(6); ii) production of butyrate is increased during fermentation of substrate containing wheat bran (WB) and iii) results of in vitro fermentations using physiological substrates and inocula agree with in vivo data. Ileal digesta collected from swine fed OB and WB were the substrates. Digesta was fermented for 0-96 h in an anaerobic in vitro system using inocula prepared from ceca of rats fed the same fiber sources. Carbohydrate and short-chain fatty acid (SCFA) contents in the fermentations were measured by gas chromatography. Fermentation of WB digesta did not produce more n-butyrate (P > 0.05) and was significantly slower (P < 0.05) than fermentation of OB digesta. OB digesta fermentation produced a significantly greater (P < 0.05) molar proportion of SCFA as propionate. Bacterial mass increased more and was maintained longer during fermentation of OB digesta than the WB digesta. Our results indicate that dilution of undigested WB fiber and not n-butyrate production is one mechanism by which WB may protect colonic mucosa; propionate production is increased during fermentation of beta-glucan in OB; and an in vitro system using physiological sources of inoculum and substrate containing WB and OB yields results that agree with in vivo findings in humans and rats.

Fermentation of carbohydrate in rat ileal excreta is enhanced with cecal inocula compared with fecal inocula.

The differential fermentative capacities of microflora from two regions of the large bowel and how fermentation was altered by prior exposure of the microflora to the substrate to be fermented were studied using an in vitro fermentation system. Bacterial inocula were prepared from cecal contents and feces from three groups of rats fed purified diets containing 100 g/kg dietary fiber from canned peas or psyllium seed husk, or a nonpurified diet containing 170 g/kg dietary fiber. The substrate for all fermentations was ileal excreta from colectomized rats fed a purified diet containing 100 g/kg dietary fiber from canned peas. Anaerobic fermentations were conducted for 0, 3, 6, 12, 24, 48 and 96 h. Sugars of the unfermented polysaccharides were measured by gas chromatography following acid hydrolysis; disappearance was the measure of fermentation. Independent of inoculum source, > 90% of the starch and arabinose and 75% of the uronic acids, but < 30% of non-starch glucose (the measure of cellulose), were fermented by 24 h. Cecal inocula fermented arabinose and uronic acids more quickly (P < 0.05) and fermented more (P < 0.05) non-starch glucose than fecal inocula. Inocula adapted to psyllium seed husk fermented starch faster (P < 0.05) and non-starch glucose, arabinose and uronic acids more slowly (P < 0.05) than inocula adapted to peas or nonpurified diet. Bacterial efficiency of carbohydrate utilization, the increase in muramic acid/mole carbohydrate fermented, was greatest (P < 0.05) with cecal inocula adapted to peas. We conclude that using cecal microflora as the inoculum source provides a more accurate index of fermentation during transit through the large bowel and that noncellulosic and storage polysaccharides of the plant cell wall are utilized before cellulose.

Rat cecal inocula produce different patterns of short-chain fatty acids than fecal inocula in in vitro fermentations.

The effects of bacterial collection site, bacterial adaption to substrate and duration of fermentation on short-chain fatty acid (SCFA) production were studied using an in vitro fermentation system. Cecal and fecal inocula from rats fed purified diets containing 100 g/kg dietary fiber from canned peas or psyllium seed husk, or a nonpurified diet containing 170 g/kg dietary fiber fermented ileal excreta from colectomized rats fed a purified diet containing 100 g/kg dietary fiber from canned peas. The SCFA concentration, measured by gas chromatography, in anaerobic fermentations of 0, 3, 6, 12, 24, 48, 72 and 96 h, increased (P < 0.05) through 72 h. Compared with fecal inocula, cecal inocula produced SCFA at greater (P < 0.05) initial rates, more (P < 0.05) total SCFA and a greater (P < 0.05) proportion as n-butyrate. Inocula from rats fed the pea or nonpurified diets produced SCFA at greater (P < 0.05) molar proportions as acetate and less (P < 0.05) as propionate than inocula from rats fed psyllium seed husk at most time points. We conclude that collection site, adaptation of bacteria to the substrate to be fermented and duration of fermentation significantly influence the results of in vitro fermentation studies.

Determination of fermentable carbohydrate from the upper gastrointestinal tract by using colectomized rats.

The primary aim of this study was to characterize the carbohydrate that would be supplied to the colon for fermentation under physiological conditions. Colectomized rats were fed fiber-free diets or diets containing 5% (wt/wt) gum arabic. Four (fucose, galactose, glucosamine, and galactosamine) of 11 analyzed sugars accounted for 77% of the total sugar in ileal excreta from colectomized rats fed fiber-free diets. The three sugars in gum arabic, rhamnose, arabinose, and galactose, accounted for 84% of the total sugars in gum arabic ileal excreta. Comparisons of the sugar compositions of the ileal excreta, the water-soluble fractions of the excreta, and three gel filtration fractions of the water-soluble material with those of the water-soluble fraction of rat mucosa, the acetone-soluble fraction of pancreas, and pancreatin suggested that the major source of endogenous carbohydrate is mucin. Gum arabic increased the daily excretion of the four mucin-derived sugars (fucose, galactose, glucosamine, and galactosamine) by the colectomized rats from 473 mumol per day to 634 mumol per day. We conclude that mucin is the major endogenous carbohydrate excreted from the upper gut and that gum arabic increases the amount of this endogenous carbohydrate.