Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

Syk-mediated translocation of PI3Kdelta to the leading edge controls lamellipodium formation and migration of leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in beta(2) integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during beta2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I(A). Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110delta of PI3K class I(A) as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110delta to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of beta2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110delta signaling for beta2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.     

Serum level of soluble 70-kD heat shock protein is associated with high mortality in patients with colorectal cancer without distant metastasis

Many findings indicate that measuring the serum concentration of soluble 70-kD heat shock protein (soluble HSP70) may provide important information in cardiovascular, inflammatory, and pregnancy-related diseases; however, only scarce data are available in cancer. Therefore, using a commercial ELISA kit, we measured soluble HSP70 concentration in the sera of 179 patients with colorectal cancer. We investigated the relationship between soluble HSP70 concentration and mortality, during 33.0 (24.4–44.0) months long follow-up. High (>1.65 pg/ml, median concentration) soluble HSP70 level was a significant (hazard ratio: 1.88 (1.20–2.96, p = 0.005) predictor of mortality during the follow-up period. When we compared the soluble HSP70 levels in patients with non-resected primary tumors as compared to those who were recruited into the study 4–6 weeks after the tumor resection they were found to be significantly (p = 0.020) higher in the former group. Since the patients with non-resected primary tumors had also distant metastasis and died early, we limited the further analysis to 142 patients with no distant metastasis at the beginning of the follow-up. This association remained significant even after multiple Cox-regression analysis had been performed to adjust the data for age and sex (p = 0.028); age, sex, and TNM-T stage (p = 0.041); age, sex, and TNM-N stage (p = 0.021); age, sex, and histological grade (p = 0.023); or age, sex, and tumor localization (p = 0.029). Further analysis showed that the significant association between high HSP70 levels and poor survival is in the strongest in the group of <70-year-old female patients (HR: 5.52 (2.02-15.15), p = 0.001), as well as in those who were in a less advanced stage of the disease at baseline. These novel findings indicate that the serum level of soluble HSP70 might prove a useful, stage-independent prognostic marker in colorectal cancer without distant metastasis.     

How the Visual Cortex Handles Stimulus Noise: Insights from Amblyopia

Adding noise to a visual image makes object recognition more effortful and has a widespread effect on human electrophysiological responses. However, visual cortical processes directly involved in handling the stimulus noise have yet to be identified and dissociated from the modulation of the neural responses due to the deteriorated structural information and increased stimulus uncertainty in the case of noisy images. Here we show that the impairment of face gender categorization performance in the case of noisy images in amblyopic patients correlates with amblyopic deficits measured in the noise-induced modulation of the P1/P2 components of single-trial event-related potentials (ERP). On the other hand, the N170 ERP component is similarly affected by the presence of noise in the two eyes and its modulation does not predict the behavioral deficit. These results have revealed that the efficient processing of noisy images depends on the engagement of additional processing resources both at the early, feature-specific as well as later, object-level stages of visual cortical processing reflected in the P1 and P2 ERP components, respectively. Our findings also suggest that noise-induced modulation of the N170 component might reflect diminished face-selective neuronal responses to face images with deteriorated structural information.     

Biological denitrification in a continuous-flow pilot bioreactor containing immobilized Pseudomonas butanovora cells

Pseudomonas butanovora, a novel denitrifying bacterium, was immobilized in composite beads and filled into a reactor system. The pilot bioreactor average denitrification activity was at ethanol-C:nitrate-N ratios of 3:1 and 1.5:1 0.88 and 0.54 kg NO3(-)-Nm(-3) d(-1), respectively. The denitrification was stable in spite of the relatively low hydraulic retention times of 2.47 and 3 h. The nitrate content of the influent was almost completely reduced at the first level of the bioreactor and the nitrite formed underwent reduction in the upper part of the reactor. The experimentally determined optimum ethanol-C:nitrate-N ratio was 1.41 +/- 0.41. In consequence of the aerobic conditions, the acetic acid produced by the oxygenation of ethanol was also detectable in the reactor effluent. The pH of the effluent (7.58) never exceeded the acceptable maximum (8.5). The nitrate removal efficiency of the cells was nearly 1000% at both C:N ratios, and the nitrite content of the effluent was around the prescribed limit throughout the continuous operation. This continuous-flow pilot bioreactor containing immobilized P. butanovora cells proved an efficient denitrification system with a relatively low retention time.     

3-(3,4,5-Trimethoxyphenyl)-1-oxo-2-propene: a novel pharmacophore displaying potent multidrug resistance reversal and selective cytotoxicity

This study revealed that various alicyclic and acyclic compounds containing the 3-(3,4,5-trimethoxyphenyl)-2-propenoyl group displayed potent MDR reversal properties. In particular, a concentration of 4 microg/ml of 2,5-bis(3,4,5-trimethoxyphenylmethylene)cyclopentanone was 31 times more potent than verapamil as a MDR revertant. In general, they were selectively toxic to malignant rather than normal cells. Two representative compounds induced apoptosis in human HL-60 cells and markedly activated caspase-3.     

Cool Headed Individuals Are Better Survivors: Non-Consumptive and Consumptive Effects of a Generalist Predator on a Sap Feeding Insect

Non-consumptive effects (NCEs) of predators are part of the complex interactions among insect natural enemies and prey. NCEs have been shown to significantly affect prey foraging and feeding. Leafhopper''s (Auchenorrhyncha) lengthy phloem feeding bouts may play a role in pathogen transmission in vector species and also exposes them to predation risk. However, NCEs on leafhoppers have been scarcely studied, and we lack basic information about how anti-predator behaviour influences foraging and feeding in these species. Here we report a study on non-consumptive and consumptive predator-prey interactions in a naturally co-occurring spider–leafhopper system. In mesocosm arenas we studied movement patterns during foraging and feeding of the leafhopper Psammotettix alienus in the presence of the spider predator Tibellus oblongus. Leafhoppers delayed feeding and fed much less often when the spider was present. Foraging movement pattern changed under predation risk: movements became more frequent and brief. There was considerable individual variation in foraging movement activity. Those individuals that increased movement activity in the presence of predators exposed themselves to higher predation risk. However, surviving individuals exhibited a ‘cool headed’ reaction to spider presence by moving less than leafhoppers in control trials. No leafhoppers were preyed upon while feeding. We consider delayed feeding as a “paradoxical” antipredator tactic, since it is not necessarily an optimal strategy against a sit-and-wait generalist predator.     

Predictability of the distribution of human-biting mosquitoes using large-scale habitat patterns

Hydrobiologia - Mosquitoes, as vectors, are important focal insects of disease ecology. Therefore, it is essential to develop predictive models showing the rules in the distribution of mosquito...