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.     

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.     

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.     

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.     

Effect of time of day on the offensive capability and aerobic performance in football game

The purpose of this study was to examine the time-of-day effects on the offensive capability and aerobic performance in football game in young subjects. In a randomized order, participants realized the Yo–Yo intermittent recovery test in two test sessions and a football game situations (two 15-min games), interspersed by a verbalization sequence (3 min) at 08:00 and 17:00 h on separate days. A recovery period of 24 h was permitted between two consecutive test sessions. The results revealed diurnal variations on the maximal aerobic velocity during the Yo–Yo test (MAV) and the oral temperature with higher values in the afternoon than morning (p < 0.05). Concerning offensive capability, the numbers of scored goals were significantly higher at 17:00 h in comparison with 08:00 h (p < 0.05). However, there was no significant difference between 08:00 and 17:00 h for the kicked balls (shooting parameter). In conclusion, our findings suggest that performance was improved in the evening and the parameters (shooting and Scored goals) can be used as a model to describe the offensive capacity in football game depending on the time of day.     

Selection of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> TN627 as a new probiotic candidate based on <Emphasis Type="Italic">in vitro</Emphasis> functional properties

Nine lactobacilli previously selected for high antagonism against food borne bacterial pathogens were identified via 16S rRNA gene sequencing and screened for probiotic potential for use in poultry production. The lactobacilli were subjected to a subtractive in vitro analysis system using a certified probiotic as reference. This allowed for selection of a milk-derived Lactobacillus plantarum strain, termed TN627. This organic acid-producing bacterium was free of harmful enzymatic activity and sensitive to several antibiotics. It also showed good growth at pH 4 and in the presence of bile. L. plantarum TN627 also exhibited high efficacy of adhesion to chicken enterocytes, which correlated with detecting genes encoding the mucusbinding, adhesion-promoting proteins (Mub and MapA) and the adhesion-like factor EF-Tu, commonly involved in adherence of lactobacilli to mucosal surfaces. Taken together, our findings suggest that TN627 is a promising probiotic candidate with high potential for application as a supplement in the animal feed industry.     

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 mediates the biogenesis of isoprenyl-linked glycans and influences development, stress response, and ammonium hypersensitivity in Arabidopsis

The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans, which determines the fate and localization of the modified protein and modulates the biological functions of glycosylphosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance. This configuration is reminiscent of that observed in mammals but is distinct from the single DPMS protein catalyzing Dol-P-Man biosynthesis in bakers' yeast and protozoan parasites. Overexpression of DPMS1 in Arabidopsis thaliana results in disorganized stem morphology and vascular bundle arrangements, wrinkled seed coat, and constitutive ER stress response. Loss-of-function mutations and RNA interference-mediated reduction of DPMS1 expression in Arabidopsis also caused a wrinkled seed coat phenotype and most remarkably enhanced hypersensitivity to ammonium that was manifested by extensive chlorosis and a strong reduction of root growth. Collectively, these data reveal a previously unsuspected role of the prenyl-linked carrier pathway for plant development and physiology that may help integrate several aspects of candidate susceptibility genes to ammonium stress.