RNAi activity of siRNAs modified with 2'-aminoalkyl-substituted fluorinated nucleobases

We recently reported that a 1'-deoxy-1'-(4,6-difluoro-1H-benzimidazol-1-yl)-2'-(beta-aminoethyl)-beta-d-ribofuranose nucleoside appears to be a universal nucleoside which does not differentiate between the four natural nucleosides A, C, G, and U in duplexes. Moreover, ribozymes modified with this nucleoside analog showed a better or at least equal catalytic activity relative to Watson-Crick mismatches.[1] Due to these data, we investigated the ability of this compound to tolerate Watson-Crick mismatches in order to avoid HIV escape mutations in RNA interference. The influence of this nucleoside analog on siRNA efficiency was analyzed with a proven siRNA targeting GFP.     

Murine norovirus increases atherosclerotic lesion size and macrophages in Ldlr(-/-) mice

Murine norovirus (MNV) is prevalent in rodent facilities in the United States. Because MNV has a tropism for macrophages and dendritic cells, we hypothesized that it may alter phenotypes of murine models of inflammatory diseases, such as obesity and atherosclerosis. We examined whether MNV infection influences phenotypes associated with diet-induced obesity and atherosclerosis by using Ldlr(-/-) mice. Male Ldlr(-/-) mice were maintained on either a diabetogenic or high-fat diet for 16 wk, inoculated with either MNV or vehicle, and monitored for changes in body weight, blood glucose, glucose tolerance, and insulin sensitivity. Influence of MNV on atherosclerosis was analyzed by determining aortic sinus lesion area. Under both dietary regimens, MNV-infected and control mice gained similar amounts of weight and developed similar degrees of insulin resistance. However, MNV infection was associated with significant increases in aortic sinus lesion area and macrophage content in Ldlr(-/-) mice fed a high-fat diet but not those fed a diabetogenic diet. In conclusion, MNV infection exacerbates atherosclerosis in Ldlr(-/-) mice fed a high-fat diet but does not influence obesity- and diabetes-related phenotypes. Increased lesion size was associated with increased macrophages, suggesting that MNV may influence macrophage activation or accumulation in the lesion area.     

Striking denervation of neuromuscular junctions without lumbar motoneuron loss in geriatric mouse muscle

Reasons for the progressive age-related loss of skeletal muscle mass and function, namely sarcopenia, are complex. Few studies describe sarcopenia in mice, although this species is the mammalian model of choice for genetic intervention and development of pharmaceutical interventions for muscle degeneration. One factor, important to sarcopenia-associated neuromuscular change, is myofibre denervation. Here we describe the morphology of the neuromuscular compartment in young (3 month) compared to geriatric (29 month) old female C57Bl/6J mice. There was no significant difference in the size or number of motoneuron cell bodies at the lumbar level (L1-L5) of the spinal cord at 3 and 29 months. However, in geriatric mice, there was a striking increase (by ～2.5 fold) in the percentage of fully denervated neuromuscular junctions (NMJs) and associated deterioration of Schwann cells in fast extensor digitorum longus (EDL), but not in slow soleus muscles. There were also distinct changes in myofibre composition of lower limb muscles (tibialis anterior (TA) and soleus) with a shift at 29 months to a faster phenotype in fast TA muscle and to a slower phenotype in slow soleus muscle. Overall, we demonstrate complex changes at the NMJ and muscle levels in geriatric mice that occur despite the maintenance of motoneuron cell bodies in the spinal cord. The challenge is to identify which components of the neuromuscular system are primarily responsible for the marked changes within the NMJ and muscle, in order to selectively target future interventions to reduce sarcopenia.     

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.     

Genetic mapping and comparative analysis of seven mutants related to seed fiber development in cotton

Mapping of genes that play major roles in cotton fiber development is an important step toward their cloning and manipulation, and provides a test of their relationships (if any) to agriculturally-important QTLs. Seven previously identified fiber mutants, four dominant (Li ₁, Li ₂, N ₁ and Fbl) and three recessive (n ₂, sma-4(h _a), and sma-4(fz)), were genetically mapped in six F₂ populations comprising 124 or more plants each. For those mutants previously assigned to chromosomes by using aneuploids or by linkage to other morphological markers, all map locations were concordant except n ₂, which mapped to the homoeolog of the chromosome previously reported. Three mutations with primary effects on fuzz fibers (N ₁, Fbl, n ₂) mapped near the likelihood peaks for QTLs that affected lint fiber productivity in the same populations, perhaps suggesting pleiotropic effects on both fiber types. However, only Li ₁ mapped within the likelihood interval for 191 previously detected lint fiber QTLs discovered in non-mutant crosses, suggesting that these mutations may occur in genes that played early roles in cotton fiber evolution, and for which new allelic variants are quickly eliminated from improved germplasm. A close positional association between sma-4(h _a ), two leaf and stem-borne trichome mutants (t ₁ , t ₂), and a gene previously implicated in fiber development, sucrose synthase, raises questions about the possibility that these genes may be functionally related. Increasing knowledge of the correspondence of the cotton and Arabidopsis genomes provides several avenues by which genetic dissection of cotton fiber development may be accelerated.     

Seroprevalence and risk factors for Helicobacter pylori infection in Greenlanders

BACKGROUND: In contrast to most populations worldwide, the incidence of gastric cancer increases among Inuit in Greenland. Contributing factors to this increase are unknown, but Helicobacter pylori may be involved. However, little is known regarding the epidemiology of H. pylori in Arctic communities. With the aim of determining age-specific prevalence, risk factors, and association with clinical conditions of H. pylori infection, we carried out a population-based study of H. pylori in Sisimiut, the second biggest town of Greenland. MATERIALS AND METHODS: A population-based sample of 685 persons had serum drawn that was analyzed for H. pylori IgG antibodies using enzyme-linked immunosorbent assay (ELISA). Risk factors analyses were carried out using multivariate logistic regression models. RESULTS: The seroprevalence was lowest among children aged 0-4 years (6%), but increased rapidly thereafter. In persons aged 15-87 years the seroprevalence had stabilized around 58%. Total number of children in household, number of older, but not younger, siblings and narrow age gap to closest older sibling were associated with H. pylori seropositivity. In contrast, number of adults in household and socioeconomic status did not influence serostatus. CONCLUSIONS: The age-specific prevalence pattern in Greenland is intermediate between that of developing and developed countries. The risk factor pattern indicates crowding and older siblings in particular to be key elements in risk of infection.     

Differential oxidation of protein-tyrosine phosphatases

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTPalpha-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTPalpha-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTPalpha-D1 and RPTPalpha-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPmu-D1, with a similar conformation as RPTPalpha-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H(2)O(2) concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.     

Laboratory tests for controlling poultry red mites (Dermanyssus gallinae) with predatory mites in small ‘laying hen’ cages

To assess their potential to control poultry red mites (Dermanyssus gallinae), we tested selected predaceous mites (Androlaelaps casalis and Stratiolaelaps scimitus) that occur naturally in wild bird nests or sometimes spontaneously invade poultry houses. This was done under laboratory conditions in cages, each with 2–3 laying hens, initially 300 poultry red mites and later the release of 1,000 predators. These small-scale tests were designed to prevent mite escape from the cages and they were carried out in three replicates at each of three temperature regimes: 26, 30 (constant day and night) and 33–25?°C (day-night cycle). After 6?weeks total population sizes of poultry red mites and predatory mites were assessed. For the temperature regimes of 26 and 33/25?°C S. scimitus reduced the poultry red mite population relative to the control experiments by a factor 3 and 30, respectively, and A. casalis by a factor of 18 and 55, respectively. At 30?°C the predators had less effect on red mites, with a reduction of 1.3-fold for S. scimitus and 5.6-fold for A. casalis. This possibly reflected hen manure condition or an effect of other invertebrates in the hen feed. Poultry red mite control was not negatively affected by temperatures as high as 33?°C and was always better in trials with A. casalis than in those with S. scimitus. In none of the experiments predators managed to eradicate the population of poultry red mites. This may be due to a prey refuge effect since most predatory mites were found in and around the manure tray at the bottom of the cage, whereas most poultry red mites were found higher up in the cage (i.e. on the walls, the cover, the perch, the nest box and the food box). The efficacy of applying predatory mites in the poultry industry may be promoted by reducing this refuge effect, boosting predatory mite populations using alternative prey and prolonged predator release devices. Biocontrol success, however, will strongly depend on how the poultry is housed in practice (free range, cage or aviary systems) and on which chemicals are applied to disinfect poultry houses and to control other pests.