Impact of the guanidinium group on hybridization and cellular uptake of cationic oligonucleotides

The grafting of cationic groups to synthetic oligonucleotides (ONs) in order to reduce the charge repulsion between the negatively charged strands of a duplex or triplex, and consequently to increase a complex's stability, has been extensively studied. Guanidinium groups, which are highly basic and positively charged over a wide pH range, could be an efficient ON modification to enhance their affinity for nucleic acid targets and to improve cellular uptake. A straightforward post-synthesis method to convert amino functions attached to ONs (on sugar, nucleobase or backbone) into guanidinium tethers has been perfected. In comparison to amino groups, such cationic groups anchored to alpha-oligonucleotide phosphoramidate backbones play important roles in duplex stability, particularly with RNA targets. This high affinity could be explained by dual recognition resulting from Watson-Crick or Hoogsteen base pairing combined with cationic/anionic backbone recognition between strands involving H-bond formation and salt bridging. Molecular-dynamics simulations corroborate interactions between the cationic backbones of the alpha-ONs and the anionic backbones of the nucleic acid targets. Moreover, ONs with guanidinium modification increased cellular uptake relative to negatively charged ONs. The cellular localization of these new cationic phosphoramidate ONs is mainly cytoplasmic. The uptake of these ON analogues might occur through endocytosis.     

3-hydroxybenzene 1,2,4-trisphosphate, a novel second messenger mimic and unusual substrate for type-I myo-inositol 1,4,5-trisphosphate 5-phosphatase: Synthesis and physicochemistry

3-Hydroxybenzene 1,2,4-trisphosphate 4 is a new myo-inositol 1,4,5-trisphosphate analogue based on the core structure of benzene 1,2,4-trisphosphate 2 with an additional hydroxyl group at position-3, and is the first noninositol based compound to be a substrate for inositol 1,4,5-trisphosphate 5-phosphatase. In physicochemical studies on 2, when three equivalents of protons were added, the (31)P NMR spectrum displayed monophasic behaviour in which phosphate-1 and phosphate-2 behaved independently in most of the studied pH range. For compound 4, phosphate-2 and phosphate-4 interacted with the 3-OH group, which does not titrate at physiological pH, displaying complex biphasic behaviour which demonstrated co-operativity between these groups. Phosphate-1 and phosphate-2 strongly interacted with each other and phosphate-4 experienced repulsion because of the interaction of the 3-OH group. Benzene 1,2,4-trisphosphate 2 is resistant to inositol 1,4,5-trisphosphate type I 5-phosphatase catalysed dephosphorylation. However, surprisingly, 3-hydroxybenzene 1,2,4-trisphosphate 4 was dephosphorylated by this 5-phosphatase to give the symmetrical 2,3-dihydroxybenzene 1,4-bisphosphate 16. The extra hydroxyl group is shown to form a hydrogen bond with the vicinal phosphate groups at -15 degrees C, and (1)H NMR titration of the ring and hydroxyl protons in 4 shows the OH proton to be strongly stabilized as soon as the phosphate groups are deprotonated. The effect of the phenolic 3-OH group in compound 4 confirms a critical role for the 6-OH group of the natural messenger in the dephosphorylation mechanism that persists even in radically modified analogues.     

Pigeon peas as a supplement for lactating dairy cows fed corn silage-based diets

Holstein rumen-cannulated cows [n = 7; initial body weight (BW) 640.56 ± 71.43 kg] were fed a corn silage basal diet with 1 of 3 concentrates (C = control; P10 = 10% pigeon peas; P20 = 20% pigeon peas). Cows were randomly assigned to treatments in a replicated 3 × 3 Latin square and individually fed using Calan gates. Each experimental period was 21 d with 7 d for adaption and 14 d for sample collection. Ruminal fluid samples were taken the last day of each experimental period and analyzed for pH, ammonia, long-chain fatty acids, and volatile fatty acids (VFA). Consecutive a.m. and p.m. milk samples were taken during the last 2 wk of the 21-d period and analyzed for fat, protein, long-chain fatty acids, and somatic cell count. Dry matter intake (kg/d) was reduced during the second period and was greater for P10 diets. Milk protein was greater for cows fed P20 compared with P10. Energy-corrected milk was greater for cows fed the control diet compared with P10. Treatment had no effect on milk yield. Ruminal fluid pH decreased over sampling times; however, pH remained at or above 5.5. Diets did not affect ruminal fluid pH; however, pH was different for sampling periods. Ruminal ammonia decreased until 8 h postfeeding at which time it peaked consistent with changes in ammonia concentrations that usually peak 3 to 5 h postfeeding on diets high in plant proteins. Dietary treatments altered ruminal fluid VFA with reduced concentrations of acetate and greater concentrations of propionate for control diet, resulting in reduced acetate:propionate ratio. Isobutyrate exhibited an hour by treatment interaction, in which isobutyrate decreased until 8 h postfeeding and then tended to be greater for P10 than for other treatments. Animals fed the P10 diet had greater concentrations of ruminal isovalerate. Ruminal cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid (CLA) isomers were not affected by dietary treatments. The P10 diet had greatest ruminal synthesis of cis-9,trans-11, but control cows had greatest ruminal synthesis of trans-10,cis-12. Milk CLA isomers were similar among treatments. Trends were observed for greater cis-9,trans-11 and trans-10,cis-12 for the P10 diet. Pigeon peas may be used as a protein supplement in dairy diets without affecting milk production, dry matter intake, or ruminal environment when they replace corn and soybean meal.     

Effect of feeding alfalfa hay or Tifton 85 bermudagrass haylage with or without a cellulase enzyme on performance of Holstein cows

Forty-four lactating Holstein cows (173 ± 30 DIM, 42.5 ± 6.8 kg of milk, 4.03 ± 0.69% fat, 674 ± 78 kg of body weight) were used in an 8-wk, completely randomized trial with a 2 × 2 factorial arrangement of treatments to determine the effect of forage source and supplemental cellulase enzyme on performance. Treatments included 2 forage combinations (corn silage plus 12.2% dry matter, DM, from either alfalfa hay or Tifton 85 bermudagrass haylage) with or without a commercial cellulase enzyme applied to the total mixed ration at the rate of 4 g/head per day (Promote N.E.T.-L, Cargill Animal Nutrition, Minneapolis, MN). Experimental diets were formulated to provide similar concentrations of protein (16.5% of DM), energy (1.63 Mcal of net energy for lactation/kg of DM), and neutral detergent fiber (41.7% of DM) and were fed once daily as a total mixed ration behind Calan doors for ad libitum intake. The cellulase enzyme provided 1,200 cellulase units of activity/g of product and was applied to the total mixed ration and allowed to mix for 5 min before feeding. Before beginning the trial, all cows were trained to use Calan (American Calan, Northwood, NH) doors and then fed the alfalfa hay-based diet for 2 wk. Data collected during wk 2 were used as a covariate in the statistical analysis. At the beginning of the 6-wk experimental period, cows were assigned randomly to 1 of the 4 experimental diets. No interactions were observed between forage and enzyme for any measures. Daily DM intake; milk yield; concentrations of milk fat, true protein, lactose, and solids not fat; energy-corrected milk yield; and dairy efficiency were not different among alfalfa or Tifton 85 bermudagrass rations with or without cellulase enzyme supplementation. The results of this trial indicate that Tifton 85 bermudagrass haylage can replace alfalfa hay in diets fed to high-producing, lactating dairy cows without depressing DM intake or milk yield when rations are balanced for NDF. Although supplemental cellulase enzymes have been shown to improve ration digestibility and animal performance in previous trials, no advantages were observed in the current trial.     

An empirical model and scenario analysis of nitrous oxide emissions from a fertilised and grazed grassland site in Ireland

This paper describes an empirical model of soil-evolved nitrous oxide emissions inferred from data gathered in a 2-year rotational grazing experiment investigating emissions from a fertilised and grazed grassland site. The model was used to simulate daily and annual emissions for the 9-year period between 1994 and 2002 under different possible fertiliser application scenarios. As the model only requires a small number of commonly available site-specific data inputs, this facilitates its use at different geographical locations. This is the first empirical modelling of daily fluxes to give estimates of annual emissions. Results reveal a high interannual variability of emissions that increases with the amount of nitrogen fertiliser applied. Simulation using the model for the period 1994–2002 indicated that higher soil moisture status on the day of fertiliser N application increased the average annual nitrous oxide emissions.     

Chemical synthesis and characterization of POSS-functionalized poly[3-hydroxyalkanoates]

A polyhedral oligomeric silsesquioxane containing seven isobutyl groups and one mercaptopropyl group (POSS-SH) was linked via a free radical addition reaction to the side-chain double bonds of bacterial poly[3-hydroxyalkanoate-co-3-hydroxyalkenoate], (PHAE). PHAE with 11.5, 55, 78, and 97 mol% of double bonds were used to produce a series of inorganic-organic hybrid materials, PHAE-POSS, with increasing amounts of POSS. The reactions proceeded with high yields and limited side reactions, confirmed with NMR and GPC experiments. X-ray diffraction and ¹³C solid-state NMR analysis of these nanocomposite materials suggested the favoured crystallization of POSS, with PHAE functioning as a non-crystalline combining matrix. This is in contrast to blends of POSS-SH and PHAE, where incompatibility occurred and the two components crystallized independently. The covalent links between PHAE and POSS imposed spatial constraint which resulted in disordered crystalline POSS domains and limited crystal sizes. With increasing POSS content, the appearance of these POSS-based biopolyesters changed from non-sticky and elastic to brittle and glass-like. The covalent linking of POSS-SH to PHAE increased the heat stability such as glass transitions, and melting points could be tailored between 48 and 120 °C.     

Individuals taking a French driving licence points recovery course: Their attitudes towards violations

This paper sets out to describe the drivers attending driving licence points recovery courses (PRC), in order to modify the psychologist’s approach during the sessions.The paper describes a questionnaire-based survey of a representative sample of 853 drivers taking a “course designed to raise awareness about the causes and consequences of road traffic accidents”. The data are analyzed in part by applying a multiple correspondence analysis.Among those taking part in PRC, recognizing whether they commit violations or not seems to be independent from the perceived probability of sanctioning and the perceived danger. The group of course-takers is divided into two main clusters (“offenders” and “victims”) which require a completely different approach on the part of the training staff.     

Attention paid to feedback provided by a computer-based assessment for learning on information literacy

Three studies are presented on attention paid to feedback provided by a computer-based assessment for learning on information literacy. Results show that the attention paid to feedback varies greatly. In general the attention focuses on feedback of incorrectly answered questions. In each study approximately fifty percent of the respondents paid attention to feedback of incorrect answers only. Approximately another twenty-five percent did not pay attention to feedback at all. Results suggest that differences in attention paid to feedback are influenced by task difficulty and test length. Supervision, however, does not seem to influence the average attention paid to feedback. On the other hand, results show that indirect and direct supervision lead to a greater impact of feedback provided by a computer-based assessment for learning as the number increases of students taking the test and, as a consequence, paying attention to feedback.