Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.     

Obesity, insulin resistance and diabetes in the sand rat exposed to a hypercaloric diet; possible protective effect for IL1-β

It is well established that, upon changing their natural desert low caloric (succulent halophilic plants) to a regular laboratory high caloric diet, sand rats undergo various phenotypic changes depending on their genetic background and including obesity and various degrees of insulin resistance. Our aim was to investigate the acute effects of Interleukin-1β (IL-1β) and Interferon-γ (IFN-γ) on glucose-induced insulin secretion in normal lean sand rats maintained on their natural diet and in obese insulin resistant normoglycemic or type 2 diabetic animals after a 9-month high caloric diet. Animals were fed either a low or a high caloric diet; after 9 months, pancreatic islets were isolated and incubated in the presence of increasing cytokine concentrations. At the end of the high-energy diet, animals were all over-weight, and probably due to a different genetic background, they displayed either insulin resistance, hyperinsulinemia and normoglycemia or a marked type-2 diabetic state. Pancreatic islets from obese insulin resistant normoglycemic animals were much more sensitive and responsive to IL-1β when compared to lean controls. The cytokine was inefficient in diabetic islets. In conclusion, the markedly increased insulinotropic effect of IL-1β in obese diabetes-resistant sand rat could participate and be involved in pancreatic β-cell hyperactivity that compensates for insulin resistance and thereby prevent the development of type 2 diabetes in these animals.     

Ultrafast excited state dynamics of the protonated Schiff base of all-trans retinal in solvents

We present a comparative study of the ultrafast photophysics of all-trans retinal in the protonated Schiff base form in solvents with different polarities and viscosities. Steady-state spectra of retinal in the protonated Schiff base form show large absorption-emission Stokes shifts (6500-8100 cm(-1)) for both polar and nonpolar solvents. Using a broadband fluorescence up-conversion experiment, the relaxation kinetics of fluorescence is investigated with 120 fs time resolution. The time-zero spectra already exhibit a Stokes-shift of approximately 6000 cm(-1), indicating depopulation of the Franck-Condon region in < or =100 fs. We attribute it to relaxation along skeletal stretching. A dramatic spectral narrowing is observed on a 150 fs timescale, which we assign to relaxation from the S(2) to the S(1) state. Along with the direct excitation of S(1), this relaxation populates different quasistationary states in S(1), as suggested from the existence of three distinct fluorescence decay times with different decay associated spectra. A 0.5-0.65 ps decay component is observed, which may reflect the direct repopulation of the ground state, in line with the small isomerization yield in solvents. Two longer decay components are observed and are attributed to torsional motion leading to photo-isomerization. The various decay channels show little or no dependence with respect to the viscosity or dielectric constant of the solvents. This suggests that in the protein, the bond selectivity of isomerization is mainly governed by steric effects.     

Synthesis and biological evaluation of some alpha-[6-(1'-carbamoylalkylthio)-1 H-pyrazolo[3,4-D]pyrimidin-4-yl]thioalkylcarboxamide acyclonucleosides

The reaction of 1H-pyrazolo[3,4-d]pyrimidin-4,6-dithione 11 with compounds 12a-c produces ethyl alpha-[6-(1'-carboethoxyalkylthio)-1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylates 13a-c, respectively. These heterocycles were alkylated, separately, with alkylating agents 14, 15, and 16 to afford, predominately, the N(1)-acyclic nucleosides (17-19)a-c, which were deprotected to give the desired products (20-22)a-c. All synthetic compounds were characterized on the basis of their physical and spectroscopic properties. The acyclic nucleosides (20-22)a-c were evaluated for their inhibitory effects against the replication of varicella-zoster virus, human cytomegalovirus and M. tuberculosis. No marked biological activity was found.     

The Processing of Leaves of Trees and Aquatic Macrophytes in the Network of the River Rhone

Breakdown rate of terrestrial and aquatic leaves was found to depend on leaf species and on the biotope considered. As the leaves started decaying at different dates and had different breakdown rates, consumable allochthonous or autochthonous organic matter was fed to the aquatic system throughout the year, from one summer to the next. A comparison with former results on the main channel confirms the existence of a transversal gradient in the fluvial network: under the influence of fungal and invertebrate colonization, breakdown was fastest in the channel of the river, slower in an open side-arm, and slower still in a more lateral and closed side arm, in which organic matter accumulated in hypoxic conditions.     

Development of an optical 3-D position measuring system for simultaneous detection of 6 degrees of freedom]

A few years ago it became possible to carry out complicated surgical procedures in humans with the required precision by combining medical imaging (MRI, CT) with minimally invasive surgery. The confined space within these imaging systems and the resulting inaccuracies associated with the manual use of instruments increasingly make necessary the help of aids ranging from positioning systems to robotic devices, which themselves must be position controlled. A position sensor has been developed for a medical robotic system allowing the image-controlled insertion of injection needles and the simultaneous administration of different drugs. The 3D position coordinates are determined by a noncontact optical principle, which also enables simultaneous determination of all 6 basic degrees of freedom of the robotic system (3 translational, 3 rotational). On the basis of an area image sensor and the measurement of a geometrically defined structure in the path of the rays between sensor and light source, the position coordinates are calculated almost real time. Special emphasis was placed on designing the sensor system to cover a sufficiently large workspace to enable it to cover the entire intervention area. The sensor described herein determines the position coordinates in a volume of 10 x 10 x 10 cm at a resolution of up to 1 mm for translations and 1 degree for rotations.     

Purification and characterization of two extracellular peroxidases from Streptomyces sp. strain AM2, a decolorizing actinomycetes responsible for the biodegradation of natural humic acids

Two extracellular humic acids peroxidases called HaP1 and HaP2 were isolated from the Streptomyces sp. strain AM2 and, based on MALDI-TOF MS analysis. The purified enzymes were determined as monomers with molecular masses of 40,351.11 and 25,175.19 Da, respectively. The N-terminal amino acid sequences of HaP1 and HaP2 were identified, and their optimum pH values were determined as 6 and 7.5, respectively. Standard 2,4-dichlorophenol (2,4-DCP) assays showed that both enzymes had maximal activity at 55 °C. HaP2 was stable at 55 °C for more than 24 h and had a half-life of 90 min at 65 °C. Although the catalytic properties of HaP1 and HaP2 were nearly identical, their stabilities and Reinheitzahl (RZ) values were substantially different. Both peroxidases were found to be heme proteins that catalyzed the oxidation of a wide range of substrates in the presence of hydrogen peroxide (H₂O₂), with HaP2 exhibiting a broader range of substrate specificity. The characterization of peroxidase activity revealed activity against humic acids, guiacol, 2,4-DCP, l-3,4-dihydroxyphenylalanine, and 2,4,5-trichlorophenol as well as other chlorophenols in the presence of H₂O₂. However, the inhibition of peroxidase activity by the addition of potassium cyanide and sodium azide also indicated the presence of heme components in the tertiary structure of these enzymes.     

Sulfide-binding hemoglobins: Effects of mutations on active-site flexibility

The dynamics of Hemoglobin I (HbI) from the clam Lucina pectinata, from wild-type sperm whale (SW) myoglobin, and from the L29F/H64Q/V68F triple mutant of SW, both unligated and bound to hydrogen sulfide (H2S), have been studied in molecular dynamics simulations. Features that account for differences in H2S affinity among the three have been examined. Our results verify the existence of an unusual heme rocking motion in unligated HbI that can promote the entrance of large ligands such as H2S. The FQF-mutant partially reproduces the amplitude and relative orientation of the motion of HbI's heme group. Therefore, besides introducing favorable electrostatic interactions with H2S, the three mutations in the distal pocket change the dynamic properties of the heme group. The active-site residues Gln-64(E7), Phe-43(CD1), and His-93(F8) are also shown to be more flexible in unligated HbI than in FQF-mutant and SW. Further contributions to H2S affinity come from differences in hydrogen bonding between the heme propionate groups and nearby amino acid residues.     

Screening and preliminary characterization of biosurfactants produced by Ochrobactrum sp. 1C and Brevibacterium sp. 7G isolated from hydrocarbon-contaminated soils

Biosurfactant-producing bacteria were isolated from a crude-oil-contaminated soil in Hassi Messaoud (southern Algeria). Isolates were screened for biosurfactant/bioemulsifier production on different carbon sources (glucose, olive oil, and hexadecane) on the basis of their ability to reduce the surface tension. The highest number of positive isolates (19) was obtained on a medium containing hexadecane as carbon source. Culture broth from all 19 isolates emulsified motor oil and 14 exhibited high emulsion-stabilizing capacity, maintaining 50% of the original emulsion volume for 48 h. The cell-free culture broth of the two best-performing isolates (identified as Ochrobactrum sp. 1C and Brevibacterium sp. 7G) reduced the surface tension below 31.5 mN m⁻¹. Biosurfactant produced by strains 1C and 7G exhibited tolerance, with slight variations for heat, pH, and salinity, and based on spectral features, they were glycolipids. Furthermore, they exhibited antimicrobial activities against Pseudomonas aeruginosa and Staphylococcus aureus.