NF2 mutation screening by denaturing high-performance liquid chromatography and high-resolution melting analysis

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder caused by mutations in the NF2 gene and predisposing to the development of nervous system. Identification of germline mutations is essential to provide appropriate genetic counseling in NF2 patients, but it represents an extremely challenging task because the vast majority of mutations are unique and spread over the entire coding sequence. Moreover, about 30% of de novo patients are indeed mosaic, and direct sequencing can undetect mutated alleles present in a minority of cells. As most screening techniques do not meet the requirements for efficient NF2 testing, we have developed a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a multiplex ligation-dependent probe amplification approach to screen for gene rearrangements. In addition, we have evaluated high-resolution melting analysis (HRMA) as an exon scanning procedure to identify point mutations in the NF2 gene. The results obtained in 92 NF2 patients expand the NF2 mutational spectrum and indicate DHPLC and HRMA as good systems to screen for point mutations in diseases with a heterogeneous spectrum of alterations.     

Room temperature microspectrofluorimetry as a useful tool for studying the assembly of the PSII chlorophyll-protein complexes in single living cells of etiolated Euglena gracilis Klebs during the greening process

The assembly kinetics of the PSII chlorophyll-protein complexes was followed during the greening of Euglena gracilis by microspectrofluorimetry in vivo, at room temperature, on single living cells. The study was correlated to micro- and submicroscopic events accompanying the proplastid to chloroplast transformation and with the immunolocalization of the LHCPII. Etiolated cells of Euglena gracilis were grown in darkness in Mego's heterotrophic liquid medium under shaking at 25+/-1 degrees C. At the stationary phase of growth, they were exposed to continuous light (330 micromol m(-2) s(-1)) for 72 h. The analyses were carried out on samples collected at different times of illumination. Microspectrofluorimetric data were recorded in the 620-780 nm range (excitation at 436 nm) and were resolved into Gaussian components corresponding to the reaction centres (RCII) and the inner antennae (CP(43-47)) of the PSII and LHCPII. From the RCII/CP(43-47) and LHCPII/PSII ratios, it was inferred that (1) a disconnection between RCII and CP(43-47) syntheses occurs during the lag phase of chloroplast differentiation, RCII being synthesized before the inner antennae. This results in the accumulation of uncoupled PSII Chl-protein complexes; (2) after lag phase, the RCII and CP(43-47) syntheses are connected one to another; (3) the freshly synthesized LHCPII complexes are immediately assembled with the PSII, suggesting that the outer antennae always maintain the form bound to PSII. Micro- and submicroscopical observations and LHCPII immunolocalization were in agreement. These data suggest that microspectrofluorimetry may constitute a useful non-destructive tool for studying the assembly kinetics of PSII, under fully physiological life conditions.     

The peroxisome proliferator BR931 kills FaO cells by p53-dependent apoptosis

Although suppression of apoptosis has been implicated as a mechanism for the hepatocarcinogenicity of peroxisome proliferators (PPs), they can also induce cell death in rat AH130 and human HepG2 hepatoma cells. To study how PPs induce cell death and to characterize the molecular events involved, we administered the hypolipidemic BR931, a peroxisome proliferator, to rat hepatoma FaO cells. Treatment with increasing concentrations of BR931 (0.015 to 0.6 mM) reduced cell viability in a dose- and time-dependent manner, associated with DNA fragmentation and morphological changes characteristic of apoptosis. BR931 also caused phosphorylation of p53 within 3 hours, translocation of the pro-apoptotic Bax protein to mitochondria, release of cytochrome-c into the cytosol, and activation of caspase-9 and -3. These results indicated that BR931 activated the intrinsic caspase cascade. Pretreatment with three different antioxidants, N-acetylcysteine, Vitamin C and Trolox, reduced apoptosis, suggesting that reactive oxygen species (ROS) plays a role in BR931-induced apoptosis. In support of this hypothesis, BR931 produced increased levels of 8-hydroxy-deoxy-guanosine, a marker of DNA oxidative damage. Antioxidants prevented the p53 phosphorylation, up-regulation of Bax and BR931-induced apoptosis. These results suggest that BR931 can increase generation of ROS, leading to DNA damage and p53 phosphorylation, which, in turn, induces the activation of Bax, release of cytochrome-c from mitochondria and activation of caspases, culminating in cell death.     

In pea stipules a functional photosynthetic electron flow occurs despite a reduced dynamicity of LHCII association with photosystems

The flexible association of the light harvesting complex II (LHCII) to photosystem (PS) I and PSII to balance their excitation is a major short-term acclimation process of the thylakoid membrane, together with the thermal dissipation of excess absorbed energy, reflected in non-photochemical quenching of chlorophyll fluorescence (NPQ). In Pisum sativum, the leaf includes two main photosynthetic parts, the basal stipules and the leaflets. Since the stipules are less efficient in carbon fixation than leaflets, the adjustments of the thylakoid system, which safeguard the photosynthetic membrane against photodamage, were analysed. As compared to leaflets, the stipules experienced a decay in PSII photochemical activity. The supramolecular organization of photosystems in stipules showed a more conspicuous accumulation of large PSII-LHCII supercomplexes in the grana, but also a tendency to retain the PSI-LHCI-LHCII state transition complex and the PSI-LHCI-PSII-LHCII megacomplexes probably located at the interface between appressed and stroma-exposed membranes. As a consequence, stipules had a lower capacity to perform state transitions and the overall thylakoid architecture was less structurally flexible and ordered than in leaflets. Yet, stipules proved to be quite efficient in regulating the redox state of the electron transport chain and more capable of inducing NPQ than leaflets. It is proposed that, in spite of a relatively static thylakoid arrangement, LHCII interaction with both photosystems in megacomplexes can contribute to a regulated electron flow.     

Morphophysiological analyses of Neochloris oleoabundans (Chlorophyta) grown mixotrophically in a carbon-rich waste product

Neochloris oleoabundans is considered one of the most promising oil-rich microalgae because of its ability to store lipids under nitrogen starvation. However, high biomass densities, required for applications on medium to large scale, are not reached in this condition of growth. As previous studies on other microalgae have shown that mixotrophy allows to obtain higher biomass in comparison to autotrophic cultures, we performed morphophysiological analyses in order to test the mixotrophic growth capability of N. oleoabundans. A carbon-rich manure derived from the apple vinegar production (AWP) was added to the medium. Cells were also cultivated under nutrient starvation (tap water), to observe the expected lipids accumulation, and combining AWP to water, to test the potential of this waste in a low-cost culture system. The results highlighted that AWP in the medium allowed to obtain the highest final cell density. Moreover, starch granules were stored inside chloroplast at the beginning of the experiment. The presence of AWP did not induce variations on light harvesting complex II (LHCII)–photosystem II (PSII) assembly, even if an interesting promotion of pigment synthesis in cells was observed. On the other hand, in starved cells, chloroplast degeneration, pigment content decrease, altered LHCII–PSII assembly and accumulation of high amount of lipid globules were observed, irrespective of the presence of AWP. The results suggest that mixotrophy promotes growth in N. oleoabundans and open up the possibility of using waste products from agri-food industries for this purpose. After growth, cells could be transferred under nutrient starvation to induce lipid accumulation.     

Phase studies of model biomembranes: complex behavior of DSPC/DOPC/cholesterol

We have undertaken a series of experiments to examine the behavior of individual components of cell membranes. Here we report an initial stage of these experiments, in which the properties of a chemically simple lipid mixture are carefully mapped onto a phase diagram. Four different experimental methods were used to establish the phase behavior of the 3-component mixture DSPC/DOPC/chol: (1) confocal fluorescence microscopy observation of giant unilamellar vesicles, GUVs; (2) FRET from perylene to C20:0-DiI; (3) fluorescence of dilute dyes C18:2-DiO and C20:0-DiI; and (4) wide angle X-ray diffraction. This particular 3-component mixture was chosen, in part, for a high level of immiscibility of the components in order to facilitate solving the phase behavior at all compositions. At 23 degrees C, a large fraction of the possible compositions for this mixture give rise to a solid phase. A region of 3-phase coexistence of {Lalpha+Lbeta+Lo} was detected and defined based on a combination of fluorescence microscopy of GUVs, FRET, and dilute C20:0-DiI fluorescence. At very low cholesterol concentrations, the solid phase is the tilted-chain phase Lbeta'. Most of the phase boundaries have been determined to be within a few percent of the composition. Measurements of the perturbations of the boundaries of this accurate phase diagram could serve as a means to understand the behaviors of a range of added lipids and proteins.     

Growth and lipid synthesis promotion in mixotrophic Neochloris oleoabundans (Chlorophyta) cultivated with glucose

In the recent years, the studies concerning the cultivation of Neochloris oleoabundans for biofuel purposes have increased, in relation to its capability to accumulate lipids when grown under nutrient starvation. Unfortunately, this cultivation mode does not allow to reach high biomass densities, which are required to improve the feasibility of the process. Increasing knowledge of the microalgal physiology is necessary to obtain new useful information for the improvement of culture performance in the perspective of large-scale cultivation. In this work, the mixotrophic cultivation of N. oleoabundans in a brackish medium added with different glucose concentrations has been tested under shaking, with the aim of stimulating growth alongside lipid accumulation inside cells. Cell morphology, glucose consumption, photosynthetic pigment content and photosynthetic efficiency were also investigated. Among all tested glucose concentrations (0–30 g L^?1), it was observed that 2.5 g L^?1 was the optimal concentration, allowing to obtain the best compromise between glucose supplement, biomass production and lipid accumulation. Growth was highly enhanced in mixotrophic cultures, linked to the release of cells from sporocysts. A unique feature characterising mixotrophy in N. oleoabundans was the promotion of the maximum quantum yield of Photosystem II. Moreover, when mixotrophic cells entered the stationary phase, high lipid accumulation was induced. This study shows that the addition of glucose to N. oleoabundans remarkably increases the production of biomass enriched in lipids and represents an advancement for the cultivation of this microalga for applied purposes.     

Projected gains and losses of wildlife habitat from bioenergy‐induced landscape change

Domestic and foreign renewable energy targets and financial incentives have increased demand for woody biomass and bioenergy in the southeastern United States. This demand is expected to be met through purpose‐grown agricultural bioenergy crops, short‐rotation tree plantations, thinning and harvest of planted and natural forests, and forest harvest residues. With results from a forest economics model, spatially explicit state‐and‐transition simulation models, and species–habitat models, we projected change in habitat amount for 16 wildlife species caused by meeting a renewable fuel target and expected demand for wood pellets in North Carolina, USA. We projected changes over 40 years under a baseline ‘business‐as‐usual’ scenario without bioenergy production and five scenarios with unique feedstock portfolios. Bioenergy demand had potential to influence trends in habitat availability for some species in our study area. We found variation in impacts among species, and no scenario was the ‘best’ or ‘worst’ across all species. Our models projected that shrub‐associated species would gain habitat under some scenarios because of increases in the amount of regenerating forests on the landscape, while species restricted to mature forests would lose habitat. Some forest species could also lose habitat from the conversion of forests on marginal soils to purpose‐grown feedstocks. The conversion of agricultural lands on marginal soils to purpose‐grown feedstocks increased habitat losses for one species with strong associations with pasture, which is being lost to urbanization in our study region. Our results indicate that landscape‐scale impacts on wildlife habitat will vary among species and depend upon the bioenergy feedstock portfolio. Therefore, decisions about bioenergy and wildlife will likely involve trade‐offs among wildlife species, and the choice of focal species is likely to affect the results of landscape‐scale assessments. We offer general principals to consider when crafting lists of focal species for bioenergy impact assessments at the landscape scale.