Analysis of bacterial diversity in river biofilms using 16S rDNA PCR-DGGE: methodological settings and fingerprints interpretation

Reliability of bacterial diversity assessment using polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA fragments was evaluated for a particular complex microbial assemblage: river epilithic biofilm. By comparing 3 routine protocols on replicates of one river biofilm sample, we found that common DNA extraction procedures gave comparable diversity (from 28.0 to 30.7 bands detected) and community composition (> 75% of homology) despite differences in the total amount of extracted DNA (from 0.9 to 4.2 microg). Therefore methodological improvements only concerned electrophoretic separation of DNA fragments (range of denaturing gradient from 35% to 70% and migration time=18h) and standardisation of DNA amounts used (PCR-template=50 ng, gel loading=700 ng). Using such a standardised methodology we found a good reproducibility of all steps of the procedure. When an Escherichia coli strain was introduced as a contaminant in a biofilm sample, we were able to recover ribotypes from the strain. As concerns fields sampling, a satisfactory repeatability of banding patterns from neighbouring pebbles (sampling point) allowed discriminating between the biofilm intrasite variability (various points from a cross-profile). These trials confirmed that PCR-DGGE is suitable to assess a reliable genetic fingerprint of epilithic biofilms in the river. Phylogenetic analysis of 40 partial sequences of 16S rDNA from DGGE gels of two sets of river biofilms samples proved evidences for the retrieval of DNA fragments related to phototroph Eukarya. However, in both cases plastidial 16S rDNA represented less than 25% of the analysed operational taxonomic units. Taking into account that Cyanobacteria, as members of the Bacteria, were also detected, sequence analysis of relevant bands from the pattern is required to target "bacteria", i.e. the functional group of prokaryotic microorganisms to which one commonly refers as a key component in sustaining the nutrient turnover.     

Carbon nanotubes in macrophages: imaging and chemical analysis by X-ray fluorescence microscopy

X-ray fluorescence microscopy (microXRF) is applied for the first time to study macrophages exposed to unpurified and purified single-walled (SW) and multiwalled (MW) carbon nanotubes (CNT). Investigating chemical elemental distributions allows one to (i) image nanotube localization within a cell and (ii) detect chemical modification of the cell after CNT internalization. An excess of calcium is detected for cells exposed to unpurified SWCNT and MWCNT and related toxicological assays are discussed.     

Effects of Size and Slenderness on Ductility of Fracturing Structures

The ductility of an elastic structure with a growing crack may be defined as the ratio of the additional load-point displacement that is caused by the crack at the moment of loss of stability under displacement control to the elastic displacement at no crack at the moment of peak load. The stability loss at displacement control is known to occur when the load-deflection curve of the whole structural system with the loading device (characterized by a spring) reaches a snapback point. Based on the known stress intensity factor as a function of crack length, the well-known method of linear elastic fracture mechanics is used to calculate the load-deflection curve and determine the states of snapback and maximum loads. An example of a notched three-point bend beam with a growing crack is analyzed numerically. The ductility is determined and its dependence of the structure size, slenderness, and stiffness of the loading device is clarified. The family of the curves of ductility versus structure size at various loading device stiffnesses is found to exhibit at a certain critical stiffness a transition from bounded single-valued functions of D to unbounded two-valued functions of D. The method of solution is general and is applicable to cracked structures of any shape. The flexibility (force) method can be adapted to extend the ductility analysis to structural assemblages provided that the stress intensity factor of the cracked structural part considered alone is known. This study leads to an improved understanding of ductility, which should be useful mainly for design against dynamic loads.     

Plasmonic near-electric field enhancement effects in ultrafast photoelectron emission: correlated spatial and laser polarization microscopy studies of individual ag nanocubes

Electron emission from single, supported Ag nanocubes excited with ultrafast laser pulses (λ = 800 nm) is studied via spatial and polarization correlated (i) dark field scattering microscopy (DFM), (ii) scanning photoionization microscopy (SPIM), and (iii) high-resolution transmission electron microscopy (HRTEM). Laser-induced electron emission is found to peak for laser polarization aligned with cube diagonals, suggesting the critical influence of plasmonic near-field enhancement of the incident electric field on the overall electron yield. For laser pulses with photon energy below the metal work function, coherent multiphoton photoelectron emission (MPPE) is identified as the most probable mechanism responsible for electron emission from Ag nanocubes and likely metal nanoparticles/surfaces in general.     

Modulation of Starch Digestibility in Breakfast Cereals Consumed by Subjects with Metabolic Risk: Impact on Markers of Oxidative Stress and Inflammation during Fasting and the Postprandial Period

Scope : Decreasing postprandial glycaemic excursions may have a beneficial effect on inflammatory and oxidative stress profiles. In this study, we investigated the impact of carbohydrate digestibility modulation per se, as a means of reducing the glycaemic response, on metabolic and inflammatory responses in subjects with metabolic risk factors. Methods and results : Twenty healthy subjects with metabolic risk consumed a cereal product either high in Slowly Digestible Starch (HSDS) or low in SDS (LSDS) at breakfast daily for 3 weeks, in a cross‐over design. Following each 3‐week session, postprandial glycaemia, insulinaemia, the lipid profile, inflammation and oxidative stress markers were assessed and compared to those induced by ingestion of a glucose solution (as a reference). The 2‐h glycaemic and insulinaemic responses were significantly lower following the HSDS breakfast compared with the LSDS breakfast or glucose. No significant differences between the products were observed in terms of the lipid profile, C‐reactive protein, IL‐6 and tumour necrosis factor alpha. We observed a slight increase in fasting lipid peroxidation markers, including an increase in plasma malondialdehyde (MDA) and a decrease in whole blood glutathione (GSH), without significant alteration of urinary F2‐isoprostanes or plasma glutathione peroxidase (GPx) activity. Conclusion : Consumption of HSDS products for 3 weeks significantly altered both postprandial glycaemia and insulinaemia, but was not sufficient to modify the inflammatory profile. Consumption of both cereal products was associated with a slightly higher fasting oxidative stress profile.     

Theoretical analysis of the optical excitation spectra of silver and gold nanowires

The excitation spectra of linear atomic chains of silver and gold with various sizes have been calculated using time-dependent density functional theory. Silver chains show longitudinal and transverse peaks as well as a low-intensity d-band. The longitudinal peak, corresponding to the HOMO-LUMO transition (along the main axis of the chain), shifts linearly to the red as the length of the system increases, consistent with the particle-in-a-box model. The transverse peak remains at approximately constant energy for all systems studied and corresponds to ∑(m)→Π(m) transitions in the xy plane perpendicular to the chain. As the chain grows, transitions arising from d orbitals contribute to the transverse peak, which affects its oscillator strength. Contrary to silver, gold chains display a strong d-band that converges to a distinct pattern at a chain length of about twelve atoms. The transitions involved in the d-band originate from localized d-orbitals with a d(z(2)) character since they have the right symmetry to give transitions into the LUMO, LUMO + 1, …, which have ∑ symmetry. Transitions arising from these localized d-orbitals also affect the position of the longitudinal peak and generate a wide transverse band. Although the majority of the transitions involved in the transverse band have a d∑→Π or dΠ→∑ character, they are hidden by much stronger excitations of dΠ→Π character in gold nanowires.     

A putative glucan synthase gene dps detected in exopolysaccharide-producing Pediococcus damnosus and Oenococcus oeni strains isolated from wine and cider

Some lactic acid bacteria can induce viscosity in wine, beer and cider by production of exopolysaccharides (EPS). A polymerase chain reaction (PCR) assay was previously described for the detection of ropy Pediococcus damnosus strains in wine [J. Appl. Microbiol. 90 (2001) 535]. The primers used in that study, PF5 and PF6, are investigated in addition to new primers which broaden the range of spoiling agents detectable by PCR. Primers PF1 and PF8 allow the amplification of DNA from ropy P. damnosus strains isolated from wine, as was observed with PF5 and PF6. In addition, PF1 and PF8, unlike PF5 and PF6, are able to generate an amplicon using template DNA from a ropy P. damnosus strain isolated from cider and a ropy Oenococcus oeni strain isolated from champagne. Two different ropy Lactobacillus species were also isolated, but their DNA was not amplified using primers PF1 and PF8. The new primers PF1 and PF8 were chosen from the sequence of gene dps, a putative glucan synthase gene, found across all the ropy P. damnosus strains isolated, from both wine or cider, and also in a ropy O. oeni strain. To our knowledge, this is the first time that an EPS-producing O. oeni strain is described. Glucan biosynthesis was assessed by agglutination tests done with Streptococcus pneumoniae type 37-specific antibodies, which specifically detect glucan-producing cells. The results show that there is a direct correlation between glucan production and detection of gene dps. Therefore, Dps is considered a candidate for the glucan synthase enzyme responsible for EPS production by ropy strains of P. damnosus and O. oeni.     

New huprine derivatives functionalized at position 9 as highly potent acetylcholinesterase inhibitors

A series of 24 huprine derivatives diversely functionalized at position 9 have been synthesized and evaluated for their inhibitory activity against human recombinant acetylcholinesterase (AChE). These derivatives were prepared in one to five steps from huprine 1 bearing an ester function at position 9. Ten analogues ( 1 , 2 , 6 – 9 , 13 – 15 , and 23 ) are active in the low nanomolar range (IC₅₀ <5 nM ), very close to the parent compound huprine X. Compounds 2 , 6 , and 7 show a very good selectivity for AChE, with AChE inhibitory activities 700–1160‐fold higher than those for butyrylcholinesterase (BChE). The inhibitory potency of these compounds decreases with the steric bulk of the substituents at position 9. According to docking simulations, small substituents fit into the acyl‐binding pocket, whereas the larger ones stick out of the active site gorge of AChE. Determination of the kinetic parameters of three of the most potent huprines ( 2 , 6 , and 7 ) showed that most of the difference in K_D is accounted by a decrease in k_on, which is correlated to the increase of the substituent size. A first in vivo evaluation has been performed in mice for the most active compound 2 (IC₅₀=1.1 nM ) and showed a rather weak toxicity (LD₅₀=40 mg kg^?1) and an ability to cross the blood–brain barrier with doses above 15 mg kg^?1.