Apolipoprotein E4 domain interaction occurs in living neuronal cells as determined by fluorescence resonance energy transfer

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer disease. Although the mechanisms remain to be determined, the detrimental effects of apoE4 in neurobiology must be based on its unique structural and biophysical properties. One such property is domain interaction mediated by a salt bridge between Arg-61 in the N-terminal domain and Glu-255 in the C-terminal domain of apoE4. This interaction, which does not occur in apoE3 or apoE2, causes apoE4 to bind preferentially to certain lipoprotein particles in vitro and in vivo. Here we used fluorescence resonance energy transfer (FRET) to determine whether apoE4 domain interaction occurs in living neuronal cells. Neuro-2a cells were transfected with constructs encoding apoE3 or apoE4 in which yellow fluorescent protein (YFP) was fused to the N terminus, and cyan fluorescent protein (CFP) was fused to the C terminus. To generate a FRET signal that can be detected by spectrum confocal microscopy, the labeled N and C termini must be in close proximity (<100 A). FRET signals occurred in cells transfected with YFP-apoE4-CFP but not in those transfected with YFP-apoE3-CFP, suggesting that the N and C termini of apoE4 are in close proximity in living cells and that those of apoE3 are not. FRET signals did not occur in cells cotransfected with YFP-apoE4 and apoE4-CFP, suggesting that the FRET in YFP-apoE4-CFP-transfected cells was intramolecular. Mutation of Arg-61 to Thr or Glu-255 to Ala in apoE4, which disrupts domain interaction, abolished FRET in Neuro-2a cells, strongly suggesting that the FRET in YFP-apoE4-CFP cells was caused by domain interaction. ApoE4-producing cells secreted less phospholipid than apoE3-producing cells, but after disruption of domain interaction in apoE4, phospholipid secretion increased to the levels seen with apoE3, suggesting that domain interaction decreases the phospholipid-binding capacity of apoE4. Thus, apoE4 domain interaction occurs in living neuronal cells and may be a molecular basis for apoE4-related neurodegeneration.     

Bio-deposition of a calcium carbonate layer on degraded limestone by Bacillus species

To obtain a restoring and protective calcite layer on degraded limestone, five different strains of the Bacillus sphaericus group and one strain of Bacillus lentus were tested for their ureolytic driven calcium carbonate precipitation. Although all the Bacillus strains were capable of depositing calcium carbonate, differences occurred in the amount of precipitated calcium carbonate on agar plate colonies. Seven parameters involved in the process were examined: calcite deposition on limestone cubes, pH increase, urea degrading capacity, extracellular polymeric substances (EPS)-production, biofilm formation, ζ-potential and deposition of dense crystal layers. The strain selection for optimal deposition of a dense CaCO₃ layer on limestone, was based on decrease in water absorption rate by treated limestone. Not all of the bacterial strains were effective in the restoration of deteriorated Euville limestone. The best calcite precipitating strains were characterised by high ureolytic efficiency, homogeneous calcite deposition on limestone cubes and a very negative ζ-potential.     

Slow-release inoculation allows sustained biodegradation of gamma-hexachlorocyclohexane

This study investigated the feasibility of a slow-release inoculation approach as a bioaugmentation strategy for the degradation of lindane (gamma-hexachlorocyclohexane [gamma-HCH]). Slow-release inoculation of Sphingomonas sp. gamma 1-7 was established in both liquid and soil slurry microcosms using open-ended silicone tubes in which the bacteria are encapsulated in a protective nutrient-rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free-living cells. Encapsulation of cells in tubes caused the removal of lindane by adsorption to the silicone tubes but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer for liquid and soil slurry microcosms, respectively, than that for inoculation with free cells. While inoculation of free-living cells led to a loss in lindane-degrading activity in limited time intervals, encapsulation in tubes allowed for a more stable actively degrading community. The loss in degrading activity was linked to the loss of the linA gene, encoding gamma-HCH dehydrochlorinase (LinA), which is involved in the initial steps of the lindane degradation pathway. This work shows that a slow-release inoculation approach using a catabolic strain encapsulated in open-ended tubes is a promising bioaugmentation tool for contaminated sites, as it can enhance pollutant removal and can prolong the degrading activity in comparison with traditional inoculation strategies.     

Cultivation of Denitrifying Bacteria: Optimization of Isolation Conditions and Diversity Study

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.     

Neuronal communication: firing spikes with spikes

Spikes of single cortical neurons can exert powerful effects even though most cortical synapses are too weak to fire postsynaptic neurons. A recent study combining single-cell stimulation with population imaging has visualized in?vivo postsynaptic firing in genetically identified target cells. The results confirm predictions from in?vitro work and might help to understand how the brain reads single-neuron activity.     

Diatomaceous earth as a protective vehicle for bacteria applied for self-healing concrete

Crack repair is crucial since cracks are the main cause for the decreased service life of concrete structures. An original and promising way to repair cracks is to pre-incorporate healing agents inside the concrete matrix to heal cracks the moment they appear. Thus, the concrete obtains self-healing properties. The goal of our research is to apply bacterially precipitated CaCO₃ to heal cracks in concrete since the microbial calcium carbonate is more compatible with the concrete matrix and more environmentally friendly relative to the normally used polymeric materials. Diatomaceous earth (DE) was used in this study to protect bacteria from the high-pH environment of concrete. The experimental results showed that DE had a very good protective effect for bacteria. DE immobilized bacteria had much higher ureolytic activity (12–17 g/l urea was decomposed within 3 days) than that of un-immobilized bacteria (less than 1 g/l urea was decomposed within the same time span) in cement slurry. The optimal concentration of DE for immobilization was 60% (w/v, weight of DE/volume of bacterial suspension). Self-healing in cracked specimens was visualized under light microscopy. The images showed that cracks with a width ranging from 0.15 to 0.17 mm in the specimens containing DE immobilized bacteria were completely filled by the precipitation. Scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS) were used to characterize the precipitation around the crack wall, which was confirmed to be calcium carbonate. The result from a capillary water absorption test showed that the specimens with DE immobilized bacteria had the lowest water absorption (30% of the reference ones), which indicated that the precipitation inside the cracks increased the water penetration resistance of the cracked specimens.