Protoplast isolation from Dictyopteris pacifica and Scytosiphon lomentaria, using a simple commercial enzyme preparation

Background Protoplasts (i.e., naked plant cells) can be used for in vitro manipulations and genetic improvement in cultivars with economic value. During the last decade, protoplast research in economic brown algae has been scarce, and it is usually hampered by the use of non-commercial enzymes or crude extracts for isolating protoplasts. Dictyopteris pacifica is part of a brown algal genus well known by its wide chemical diversity and biological properties. Scytosiphon lomentaria is an edible brown seaweed with antioxidant, antitumor, and antiviral properties. So far, there are no protoplast isolation protocols using commercial enzymes for these two economic brown algae. In this study, we obtained protoplasts from cultured samples of D. pacifica and S. lomentaria using commercially available enzymes. Additionally, we investigated the effects of Driselase inclusion and Ca-chelation pre-treatment on protoplast yields in order to optimize the conditions for protoplast preparations. Results Protoplasts were isolated from Dictyopteris pacifica and Scytosiphon lomentaria using the commercially available Cellulase Onozuka RS (1%) and Alginate lyase (4 U mL−1), and short incubation time (4 h). Driselase did not show significant effects on protoplast production in both species. Ca-chelation pre-treatment only increased the number of protoplasts in D. pacifica. Under optimal conditions, the protoplast yields from D. pacifica and S. lomentaria were 4.83 ± 2.08 and 74.64 ± 32.49 × 106 protoplasts g−1 fresh weight, respectively. The values obtained for S. lomentaria were 2–3 orders of magnitude higher than previously reported. Conclusions Our results show that high protoplast yields can be obtained from D. pacifica and S. lomentaria using a simple mixture of commercial enzymes (Cellulase RS and Alginate lyase) and short incubation time (4 h). This work also represents the first report of protoplast isolation in D. pacifica. The method proposed here can help to expand protoplast technology in more brown algal species.

The Effect of the Nanoscale Structure of Nanobioceramics on TheirIn VitroBioactivity and Cell Differentiation Properties

The effect of the nanoscale structure of bioceramics on theirin vitrobioactivity and capacity to osteogenically differentiate stem cell is studied. Nanoparticles of hydroxyapatite (nHA), bioactive glass (nBG), nanoporous bioactive glass (MBG), and nanoporous bioactive glass nanospheres (nMBG) are investigated. The nanometric particle size of bioceramics seems to be more determining in controlling the ability to induce bone-like apatite as compared to the nanoporous structure. At short incubation time, nBG also produces a bioactive extracellular medium capable of upregulating key osteogenic markers involved in the development of a mineralizing phenotype in DPSCs. The bioactive properties of nBG are promissory for accelerating the bone regeneration process in tissue engineering applications.

Thermodynamic Aspects of Tropical Cyclone Formation

The thermodynamic aspects of tropical cyclone (TC) formation near the center of the wave pouch, a region of approximately closed Lagrangian circulation within the wave critical layer, are examined through diagnoses of a high-resolution numerical simulation and dropsonde data from a recent field campaign. It is found that the meso-β area near the pouch center is characterized by high saturation fraction, small difference in equivalent potential temperature θe between the surface and the middle troposphere, and a short incubation time scale. Updrafts tend to be more vigorous in this region, presumably because of reduced dry air entrainment, while downdrafts are not suppressed. The thermodynamic conditions near the pouch center are thus critically important for TC formation. The balanced responses to convective and stratiform heating at the pregenesis stage are examined using the Sawyer–Eliassen equation. Deep convection is concentrated near the pouch center. The strong radial and vertical gradients of latent heat release effectively force the transverse circulation and spin up a surface protovortex near the pouch center. Stratiform heating induces modest midlevel inflow and very weak low-level outflow, which contributes to the midlevel spinup without substantially spinning down the low-level circulation. The analysis of dropsonde data shows that the midlevel θe increases significantly near the pouch center one to two days prior to genesis but changes little away from the pouch center. This may indicate convective organization and the impending TC genesis. It also suggests that the critical information of TC genesis near the pouch center may be masked out if a spatial average is taken over the pouch scale.

PrPSc detection and infectivity in semen from scrapie-infected sheep

A scrapie-positive ewe was found in a flock that had been scrapie-free for 13 years, but housed adjacent to scrapie-positive animals, separated by a wire fence. Live animal testing of the entire flock of 24 animals revealed seven more subclinical scrapie-positive ewes. We hypothesized that they may have contracted the disease from scrapie-positive rams used for breeding 4 months prior, possibly through the semen. The genotypes of the ewe flock were highly scrapie-susceptible and the rams were infected with the ‘Caine’ scrapie strain having a short incubation time of 4.3–14.6 months in sheep with 136/171 VQ/VQ and AQ/VQ genotypes. PrPSc accumulates in a variety of tissues in addition to the central nervous system. Although transmission of prion diseases, or transmissible spongiform encephalopathies, has been achieved via peripheral organ or tissue homogenates as well as by blood transfusion, neither infectivity nor PrPSc have been found in semen from scrapie-infected animals. Using serial protein misfolding cyclic amplification followed by a surround optical fibre immunoassay, we demonstrate that semen from rams infected with a short-incubation-time scrapie strain contains prion disease-associated-seeding activity that generated PrPSc in sPMCA (serial protein misfolding cyclic amplification). Injection of the ovinized transgenic mouse line TgSShpPrP with semen from scrapie-infected sheep resulted in PrPSc-seeding activity in clinical and, probably as a result of the low titre, non-clinical mouse brain. These results suggest that the transmissible agent, or at least the seeding activity, for sheep scrapie is present in semen. This may be a strain-specific phenomenon.

An approach to the investigation of CO₂ uptake by soil microorganisms

Sequestration of CO2 via biological sinks is a matter of great scientific importance due to their potential to lower atmospheric CO2 levels. In this study a custom built incubation chamber was used to cultivate a soil microbial community, under ideal conditions, to investigate soil chemoautotrophy. The internal atmospheric CO2 concentrations were monitored and used to estimate the mass of CO2 uptake. It was found after careful background corrections that 256.4 μg CO2 kg−1 dry soil was removed from the chamber atmosphere over 16 h. Comparisons were made to photosynthetic controls (i.e. grass and soil algae) whereupon it was observed that the chemoautotrophic fraction sequestered 2.6 and 5.4 % of that taken up by grass and soil algae respectively. Using isotopically labelled 13CO2 and GCMS-IRMS it was also possible to extract and identify labelled fatty acids after a short incubation time, hence confirming the CO2 uptake potential of the soil slurry. Provided with favourable conditions, chemoautotrophic soil bacteria have the potential to make a significant impact on inorganic carbon sequestration within the environment. The results of this in vivo study have provided ground work for future studies intending to mimic the in situ environment by providing a reliable method for investigating CO2 uptake by soil microorganisms.