Functional dissection of the TBK1 molecular network

TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification-mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I∶C) stimulation, we found that TBK1 activation was strictly dependent on the integrity of the TBK1/TANK interaction.     

Brain Serum Amyloid P Levels are Reduced in Individuals that Lack Dementia While Having Alzheimer’s Disease Neuropathology

The neuropathological signs of Alzheimer’s disease (AD) include beta amyloid plaques and neurofibrillary tangles. There is a significant population of individuals that have these key hallmarks but show no signs of cognitive impairment, termed non-demented with AD neuropathology (NDAN). The protective mechanism allowing these individuals to escape dementia is unknown. Serum amyloid P (SAP) is a serum protein associated with wound repair that is elevated in the brains of Alzheimer’s patients and binds to amyloid plaques. Using immunoblotting and immunohistochemistry, we evaluated SAP levels in postmortem samples of hippocampus and frontal cortex in age-matched controls, AD, and NDAN individuals. AD individuals had significantly increased SAP levels compared to normal controls, while NDAN samples had no significant difference in SAP levels compared to normal controls. Our results suggest that low levels of SAP in plaques marks the brains of individuals that escape dementia despite the presence of beta amyloid plaques and tangles.     

Genomic regions associated with multiple sclerosis are active in B cells

More than 50 genomic regions have now been shown to influence the risk of multiple sclerosis (MS). However, the mechanisms of action, and the cell types in which these associated variants act at the molecular level remain largely unknown. This is especially true for associated regions containing no known genes. Given the evidence for a role for B cells in MS, we hypothesized that MS associated genomic regions co-localized with regions which are functionally active in B cells. We used publicly available data on 1) MS associated regions and single nucleotide polymorphisms (SNPs) and 2) chromatin profiling in B cells as well as three additional cell types thought to be unrelated to MS (hepatocytes, fibroblasts and keratinocytes). Genomic intervals and SNPs were tested for overlap using the Genomic Hyperbrowser. We found that MS associated regions are significantly enriched in strong enhancer, active promoter and strong transcribed regions (p = 0.00005) and that this overlap is significantly higher in B cells than control cells. In addition, MS associated SNPs also land in active promoter (p = 0.00005) and enhancer regions more than expected by chance (strong enhancer p = 0.0006; weak enhancer p = 0.00005). These results confirm the important role of the immune system and specifically B cells in MS and suggest that MS risk variants exert a gene regulatory role. Previous studies assessing MS risk variants in T cells may be missing important effects in B cells. Similar analyses in other immunological cell types relevant to MS and functional studies are necessary to fully elucidate how genes contribute to MS pathogenesis.     

Translocation of stream-dwelling salmonids in headwaters: insights from a 15-year reintroduction experience

Translocation programs are a common strategy to increase the number of viable populations of threatened freshwater fishes. Yet, only in a minority of cases the success or failure of translocations has been assessed through a quantitative analysis of demographic traits, compensatory responses, life-histories and population dynamics of the threatened species. A paradigmatic case a translocation program combining both management- and research-oriented activities is represented by the Marble Trout Conservation Program, which started in 1993 in the upper reaches of the Soca, Idirjca and Baca river basins (Slovenia) for the conservation of stream-dwelling marble trout Salmo marmoratus. In order to enhance the viability of the species, two new populations were created in 1996 by stocking 500 marble trout aged 1+ in previously fishless streams (Gorska and Zakojska) within the core habitat of the species. The new populations have been systematically monitored for 15 years by individually tagging and sampling marble trout. Our analyses show that deterministic extinction of marble trout populations are unlikely and that high-magnitude environmental stochasticity (i.e., severe floods) is the only main cause of local population extinction, despite the high resilience to flood-induced massive mortalities exhibited by marble trout through compensatory mechanisms (e.g., relaxation of density-dependent body growth and survival at low densities). Fishless headwaters, probably characterized by a history of recurrent severe floods, should not be considered as candidate sites for the creation of new populations. Fewer individuals than originally reintroduced (i.e., 500 fish aged 1+ in each stream) might be sufficient to establish viable populations, since compensatory mechanisms are likely to regulate population size around stream carrying capacity in a few years. Besides enhancing the species viability, translocation programs can provide an excellent framework for the estimation of ecological traits (e.g., life-histories, demography, population dynamics etc.), identify potential vulnerabilities and thus guide well-formed management actions for the threatened species.     

Tracing the role of R-bodies in the killer trait: Absence of toxicity of R-body producing recombinant E. coli on paramecia

R-bodies are coiled proteinaceous ribbons produced by Paramecium endosymbionts belonging to the genus Caedibacter. These intracellular bacteria confer upon their hosts a phenomenon called the killer trait. It is the ability to kill symbiont-free competitors called sensitives. The R-body is the crucial element of this process, but despite many efforts, the actual role of R-bodies in killing sensitive paramecia is still not satisfactory clarified. The open question is whether the R-body acts as transmitter for a yet unidentified toxin or whether it directly kills sensitive paramecia having intrinsic cytotoxic effects. In the present study, this problem is addressed by heterologous expression of Caedibacter taeniospiralis R-body in Escherichia coli followed by a detailed analysis of its potential intrinsic toxic effect on feeding sensitive Paramecium tetraurelia. Using this approach, we can exclude any eventual effects of additional, unidentified factors produced by C. taeniospiralis and thus observe the impact of the recombinant R-body itself. No cytotoxic effects of recombinant R-bodies were detected following this approach, strengthening the hypothesis that R-bodies act as releasing system for an unidentified C. taeniospiralis toxin.     

The Origin of the 5S Ribosomal RNA Molecule Could Have Been Caused by a Single Inverse Duplication: Strong Evidence from Its Sequences

The secondary structure of the 5S ribosomal RNA (5S rRNA) molecule shows a high degree of symmetry. In order to explain the origin of this symmetry, it has been conjectured that one half of the 5S rRNA molecule was its precursor and that an indirect duplication of this precursor created the other half and thus the current symmetry of the molecule. Here, we have subjected to an empirical test both the indirect duplication model, analysing a total of 684 5S rRNA sequences for complementarity between the two halves of the 5S rRNA, and the direct duplication model analysing in this case the similarity between the two halves of this molecule. In intra- and inter-molecule and intra- and inter-domain comparisons, we find a high statistical support to the hypothesis of a complementarity relationship between the two halves of the 5S rRNA molecule, denying vice versa the hypothesis of similarity between these halves. Therefore, these observations corroborate the indirect duplication model at the expense of the direct duplication model, as reason of the origin of the 5S rRNA molecule. More generally, we discuss and favour the hypothesis that all RNAs and proteins, which present symmetry, did so through gene duplication and not by gradualistic accumulation of few monomers or segments of molecule into a gradualistic growth process. This would be the consequence of the very high propensity that nucleic acids have to be subjected to duplications.     

Transfection efficiency boost of cholesterol-containing lipoplexes

Most lipid formulations require cholesterol for successful transfection, but the precise reason remains to be more clearly understood. Here, we have studied the effect of cholesterol on the transfection efficiency (TE) of lipoplexes in vitro. Addition of cholesterol to highly effective DC-Chol-DOPE/DNA lipoplexes increases TE, with 40mol% cholesterol yielding about 10-fold improvement. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by combining dynamic light scattering, synchrotron small angle X-ray scattering, laser scanning confocal microscopy and transfection efficiency measurements. Our results revealed that cholesterol-containing lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for efficient endosomal escape. We also found evidence that formulations with high cholesterol content are not specifically targeted to metabolic degradation. These studies will contribute to rationally design novel delivery systems with superior transfection efficiency.     

Oxidative stress and antioxidant therapy in cystic fibrosis

Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conductance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes such as reduced glutathione. This defect produces an increased viscosity of secretions together with other metabolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by pancreatic and liver insufficiency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is associated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the biochemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exacerbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main contributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Observed defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that further and more rigorous trials are needed together with new generations of pharmacological tools to a more effective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Optomotorische Reaktionen der Fliege Musca Domestica

Summary The torque exerted by the housefly Musca domestica during fixed flight was used as a measure of the optomotor reaction of the insect elicited by the rotation of cylindrical patterns with periodic distributions of surface brightness. Measurements were made of the dependence of the reaction on the wave length, speed of rotation, contrast, and mean brightness of the stimulus patterns. The effect on the reaction of modulation of the light illuminating the stimulus pattern was examined. Further experiments indicated that stimulation of only one of the two complex eyes is sufficient to elicit an optomotor reaction, and that there is overlap between the visual fields of neighboring photoreceptor units in the complex eye. Estimates of the rates of absorption of light quanta by individual ommatidia in the complex eye indicated that these rates are low enough that the Poisson statistics of the light quanta results in a significant level of noise in the light signals received by the photoreceptors, when the brightness of the stimulus pattern is low but still sufficient to elicit a measurable reaction. The contrast that is required of a rotating stimulus pattern in order to elicit a just-measurable reaction was found to depend upon the mean brightness of the pattern in a manner that is consistent with the hypothesis that the noise due to the statistics of the light quanta absorbed by the photoreceptors in the complex eye is a principle cause of the breakdown of the optomotor reaction at low values of the contrast and mean brightness of the stimulus pattern.

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Mit Unterstützung durch ein Fellowship des National Institute of Neurological Diseases and Blindness, US Public Health Service und ein Fellowship der National Science Foundation, USA.

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Herrn Freiberg danken wir für das Anfertigen der Abbildungen.