Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs

The potential of RNA interference (RNAi) to inhibit virus propagation has been well established in recent years. In several studies, however, emergence of viral escape mutants after prolonged exposure to RNAi has been observed, raising a major hurdle for a possible therapeutic application of this strategy. Here, we report the design and characterisation of a vector that allows the simultaneous expression of two short hairpin RNAs (shRNAs), thereby maintaining high silencing activity even against a viral RNA bearing mutations in one of the target sites. Two short interfering RNAs (siRNAs) against the 3D-RNA dependent RNA polymerase of coxsackievirus B3 were identified that displayed efficient inhibition of virus propagation in HeLa cells and reduced the virus titre by up to 90%. We generated two expression vectors encoding these newly identified siRNAs and evaluated their silencing efficiency against the target gene in a reporter assay. Viral escape was then simulated by introducing a point mutation into either of the target sites. This substitution led to complete abrogation of silencing by the respective vector. To bypass this blockade of silencing, an siRNA double expression vector (SiDEx) was constructed to achieve simultaneous expression of both siRNAs from one plasmid. The silencing efficiency of both siRNAs generated by SiDEx was comparable to that of the individual mono-expression vectors. In contrast to the conventional expression vectors, SiDEx displayed substantial gene regulation also of the mutated target RNA. As our approach of expressing various shRNAs from one vector is based on a simple and universally applicable cloning strategy, SiDEx may be a helpful tool to achieve sustained silencing of viruses, ultimately reducing the risk of emergence of viable mutants. An additional application of SiDEx vectors will be the simultaneous knockdown of two targeted genes for functional studies.     

From function to shape: a novel role of a formin in morphogenesis of the fungus Ashbya gossypii

Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.     

β-Lactamase export across the outer membrane of Acinetobacter calcoaceticus:perturbation of the outer membrane lipopolysaccharide leaflet by enzyme overproduction

Acinetobacter calcoaceticus is able to produce a β-lactamase which was found in the periplasm and to be released into the extracellular culture medium. β-Lactamase export was dependent on enzyme over-production in a cooperative manner. Furthermore, it was accompanied by a steadily increasing release of lipopolysaccharide, an outer membrane constituent, and by an increase in the susceptibility to hydrophobic antibiotics. The data point towards a self-promoted perturbation of the outer membrane by overproduction of the enzyme, leading to a semi-selective increase in membrane permeability.     

A Robust Test for Homoscedasticity in a Two-Way Layout

This paper discusses tests for homogeneity of row-variances in a two-way classification. Parametric tests for this problem are shown to be highly sensitive to departures from the normality assumption. Levene's robust tests tend to be rather liberal when the number of rows exceeds the number of columns. A modification is suggested which improves the approximation for this situation, while retaining the desirable robustness property.     

Formation of cross-fractures in cellulose microfibril structure by an endoglucanase-cellobiohydrolase complex from Trichoderma reesei

Abstract An endoglucanase-cellobiohydrolase from Trichoderma reesei culture fluids was purified by means of preparative isoelectric focusing. The cellulase complex had a common apparent isoelectric point (p I ) of 3.8. Beyond this p I , the electrophoretic mobilities of endoglucanase and cellobiohydrolase were different under conditions of titration curves. The effect of this endoglucanase-cellobiohydronalase complex on Sinapis cellulose microfibril ultranstructure was observed by transmission electron microscopy after metal shadowing of the specimen. By the action of this cellulase complex, the microfibril structure was converted into an amorphous form of cellulose. Moreover, the hydrolase complex induced visible cross-fractures within the cellulose microfibril structure. The mean cellulose microfibril lenght of 1.2 μm was reduced to 0.9 μm in the presence (12 h) of this cellulase complex by the formation of shorter microfibril fragments.     

Efficiency of intrathymic tolerance induction in various inbred rat strains: relationship with TH1/TH2 status of the recipient?

The simultaneous transplantation and intrathymic tolerance induction (STITTI) protocol induces a longlasting state of functional tolerance in over 90% of AO (RT1^u) recipients transplanted with a fully MHC-incompatible PVG (RT1^c) cardiac allograft. Similar results are obtained when using LEWIS (RT1¹) rats as recipients of either PVG or DA (RT1^avl) grafts. However, when STITTI is performed on PVG and BN (RT1ⁿ) as recipient animals receiving spleen cells intrathymically and a cardiac allograft from respectively AO and PVG rats, this procedure results in significantly shorter graft survival (MST PVG → BN 25 ± 9 days; AO → PVG 31 ± 8 days) as compared to the combinations using AO (MST PVG → AO > 236 ± 28 days) and LEWIS (MST PVG → LEW > 366 ± 51 days; DA → LEW > 123 ± 33 days) rats as recipients. Since both PVG and BN rats are relatively deficient in their ability to produce IFNγ and intrathymic IFNγ responses are very dominant upon intrathymic injection of alloantigens, it is argued that the inability to effectively induce a longlasting state of functional tolerance in BN and PVG rats using the STITTI protocol may be related to their decreased IFNγ-production potential.     

Interspecies Variability of Plant Hormesis by the Antiauxin PCIB in a Laboratory Bioassay

Chemical hormesis constitutes an alternative possible use of herbicidal agents for crop enhancement that is, however, compromised by the apparent variability of this low-dose stimulation phenomenon. Studies demonstrating the variability are rare and, therefore, this study investigated the interspecies variability of growth stimulation induced by the auxin-inhibitor PCIB [2-(p-chlorophenoxy)-2-methylpropionic acid] to determine if hormesis is generalizable enough and sufficiently stable between species/cultivars for practical use or which implications may have to be taken into account. In 85 complete dose–response bioassays with 23 cultivars of five species, the variability of PCIB effects was evaluated. The expression of PCIB hormesis proved to depend on the species/cultivar tested, ranging from a cultivar-dependent hormetic efficacy and an occasional lack of hormesis, to a complete lack of hormetic effectiveness in certain species/cultivars. Therefore, frequency estimations, as well as the pattern of dose-dependent variability of dose–response quantities, may inevitably depend on the biological model(s) used and, thus, apply only to the specific conditions for characterization. Comparing the frequency distribution of effective doses demonstrated a risk of a previously hormetic dose causing a loss of hormesis or inhibitory effects in another species/cultivar. Therefore, selecting a dose that will induce hormesis in every species/cultivar is unrealistic. This may limit the window for practical applications to stimulants with negligible varietal differences, to cultivar selective treatments, and/or to cultivars that enable a beneficial long-term use. Hence, efficient crop enhancement by chemical hormesis needs not only a good stimulant, but also a species/cultivar able to convert a specific low-dose treatment into an economic benefit.     

Promoting Myelination in an In Vitro Mouse Model of the Peripheral Nerve System: The Effect of Wine Ingredients

Protective properties of moderate wine consumption against cancers, cardiovascular, metabolic and degenerative diseases have been reported in various clinical studies. Here, we analysed the effect of red wine (RW) and white wine (WW) on myelination using an in vitro embryonic co-culture mouse model. The total amount of myelin was found to be significantly increased after RW and WW treatment, while only RW significantly increased the number of internodes. Both types of wine increased rat Schwann cell- (rSC) expression of the NAD+-dependent deacetylase sirtuin-two-homolog 2 (Sirt2), a protein known to be involved in myelination.Detailed chemical analysis of RW revealed a broad spectrum of anthocyanins, piceids, and phenolics, including resveratrol (RSV). In our assay system RSV in low concentrations induced myelination. Furthermore RSV raised intracellular glutathione concentrations in rSCs and in co-cultures and therefore augmented antioxidant capacity.We conclude that wine promotes myelination in a rodent in vitro model by controlling intracellular metabolism and SC plasticity. During this process, RSV exhibits protective properties; however, the fostering effect on myelinaton during exposure to wine appears to be a complex interaction of various compounds.     

Relationships between proteasomes and RNA

The 20S proteasome (prosome) is a highly organized multi-protein complex with approximate molecular weight of about 700 kDa. Whilst the role of the proteasome in the processing and turnover of cellular proteins is becoming clearer, its relationship with RNA remains obscure. Over the last decade the possibility of association of proteasomes with specific RNAs or mRNPs have been particularly controversial. Proteasomes were reported to inhibit translation of viral mRNAs and to be tightly associated with RNase activity. It is possible that proteasomes are also involved in cellular RNA breakdown and RNA processing like prokaryotic RNase E.     

Cultivated plant remains of the late Neolithic Michelsberg Culture at Heilbronn-Klingenberg (southwest Germany) - a comparison of different features, find assemblages and preservation conditions relating to the representation of archaeobotanical remains

A large area investigation was undertaken of the plant remains from a Michelsberg Culture (late Neolithic) settlement. The charred macroscopic remains and imprints in pieces of daub were expected to show both the spectrum of the cultivated plants there, and also the degree of their cultivation and use. The loess covered hill-top lies in a landscape with favourable climatic and soil conditions and is blocked off by two parallel ditches running in an arc. Ditches and pits filled with different sediments were investigated. The daub, unearthed in several pits, had been deliberately mixed with chaff of the glume wheats einkorn and emmer to temper it when it was originally made. The investigated imprints and charred plant remains give hints of spatial distribution of crop processing activities. To determine the amounts of the crops that were cultivated and used, it is necessary to study the charred remains. The degree of ubiquity (frequency of occurrence) of grains in the pit sediments seems to be the best indicator of the representation of cereals. Four main cereals were found: Triticum monococcum, T. dicoccum, T. aestivum/T. durum and Hordeum vulgare var. nudum. Pisum sativum also was an important cultivated plant, much more than Lens culinaris. The role of Linum usitatissimum and Papaver somniferum is less clear.