A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos

A fast and highly efficient short-term in vitro regeneration system was developed for barley (Hordeum vulgare L.) based on readily available explants. Clumps of multiple shoots and buds suitable for transformation were obtained 9–10 weeks after culture initiation from model and current commercial cultivars. Meristematic shoot segments (MSSs) excised from mature embryo-derived seedlings and subsequently cultured on MS-based medium containing 2 mg/l Picloram and 3 mg/l thidiazuron (TDZ) differentiated up to ten multiple shoots after 3–4 weeks with no or very little callus formation. Sectors of the already multiplied shoot clumps were further multiplied on proliferation-maintenance medium containing 2 mg/l Picloram and 2.5 mg/l TDZ. Biweekly subcultures resulted in a continuous process of multiplication of these highly differentiating green sectors without any loss of morphogenic potential. The differentiated small shoots and shoot buds gave rise to normal shoots on medium with 0.1 mg/l Picloram and 1 mg/l TDZ. After rooting on basal medium with 0.5 mg/l or 1 mg/l IBA the plants were transferred to soil and showed normal growth and fertility compared to the seed-grown plants. All of the genotypes tested formed multiple shoots. The percentage of relative MSS multiplication was 63–83%, and the average number of multiplied shoots per MSS ranged from 16 to 34 among the genotypes after 9–11 weeks.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba 3,6-Dichloro-2-methoxybenzoic acid - IBA Indole-3-butyric acid - MSS Meristematic shoot segment - NAA -Naphthaleneacetic acid - Picloram 4-Amino-3,5,6-trichloropicolinic acid - TDZ Thidiazuron     

Rational and random strategies for the mimicry of discontinuous protein binding sites

The high technical standard of current peptide chemistry, which has evolved over the past three decades, has profoundly facilitated the investigation of proteins and their interactions with other molecules at the level of individual amino acids. Using currently available peptide synthesis methods, sequentially continuous protein binding sites can be readily mapped, characterized, optimized, and used as lead compounds for inhibitors of protein-ligand interactions. The mimicry of sequentially discontinuous protein binding sites, on the other hand, continues to present a challenge for peptide and organic chemists. This mini-review summarizes currently used and emerging, rational and random strategies for the design of synthetic mimetics of discontinuous protein binding sites.     

Quantification and fatty acid profiles of sulfolipids in two halophytes and a glycophyte grown under different salt concentrations

This study was aimed at understanding the role of sulfolipids in salt tolerance mechanisms of the halophytes Aster tripolium L., Compositae, and Sesuvium portulacastrum L., Aizoaceae, and of the glycophyte Arabidopsis thaliana (L.) Heynh., Brassicaceae. In Aster and Sesuvium the sulfolipid contents increased significantly under salt stress conditions (517 mM or 864 mM). In Arabidopsis, changes in sulfolipid contents were not observed (NaCl up to 100 mM). The fatty acid profile of sulfoquinovosyldiacylglycerol (SQDG) in Aster was modified with increasing NaCl concentrations. LC-MS analyses of sulfolipids from Aster and Sesuvium revealed the presence of 18:3/18:3 and 16:0/18:3 molecules. Obviously, the function of sulfolipids during salt stress differs between halophytic species and between halophytes and glycophytes where sulfolipid accumulation was not observed.     

Degradation of cyanobacterial biomass in anoxic tidal-flat sediments: a microcosm study of metabolic processes and community changes

To follow the anaerobic degradation of organic matter in tidal-flat sediments, a stimulation experiment with ¹³C-labeled Spirulina biomass (130 mg per 21 g sediment slurry) was conducted over a period of 24 days. A combination of microcalorimetry to record process kinetics, chemical analyses of fermentation products and RNA-based stable-isotope probing (SIP) to follow community changes was applied. Different degradation phases could be identified by microcalorimetry: Within 2 days, heat output reached its maximum (55 μW), while primary fermentation products were formed (in μmol) as follows: acetate 440, ethanol 195, butyrate 128, propionate 112, H₂ 127 and smaller amounts of valerate, propanol and butanol. Sulfate was depleted within 7 days. Thereafter, methanogenesis was observed and secondary fermentation proceeded. H₂ and alcohols disappeared completely, whereas fatty acids decreased in concentration. Three main degraders were identified by RNA-based SIP and denaturant gradient gel electrophoresis. After 12 h, two phylotypes clearly enriched in ¹³C: (i) Psychrilyobacter atlanticus, a fermenter known to produce hydrogen and acetate and (ii) bacteria distantly related to Propionigenium. A Cytophaga-related bacterium was highly abundant after day 3. Sulfate reduction appeared to be performed by incompletely oxidizing species, as only sulfate-reducing bacteria related to Desulfovibrio were labeled as long as sulfate was available.     

Determination of the labeling density of fluorophore-biomolecule conjugates with absorption spectroscopy

Dye-biomolecule conjugation is frequently accompanied by considerable spectral changes of the dye's absorption spectrum that limit the use of the common photometrical method for the determination of labeling densities. Here, we describe an improvement of this method using the integral absorbance of the dye instead of its absorbance at the long wavelength maximum to determine the concentration of the biomolecule-coupled dye. This approach is illustrated for three different cyanine dyes conjugated to the antibody IgG.     

An Analytical Method for Assessing Stage-Specific Drug Activity in Plasmodium vivax Malaria: Implications for Ex Vivo Drug Susceptibility Testing

The emergence of highly chloroquine (CQ) resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT), originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC(50)) values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC(50) for CQ. Our findings indicate that EC(50) values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.     

Cyanobacteria and cyanotoxins: the influence of nitrogen versus phosphorus

The importance of nitrogen (N) versus phosphorus (P) in explaining total cyanobacterial biovolume, the biovolume of specific cyanobacterial taxa, and the incidence of cyanotoxins was determined for 102 north German lakes, using methods to separate the effects of joint variation in N and P concentration from those of differential variation in N versus P. While the positive relationship between total cyanobacteria biovolume and P concentration disappeared at high P concentrations, cyanobacteria biovolume increased continually with N concentration, indicating potential N limitation in highly P enriched lakes. The biovolumes of all cyanobacterial taxa were higher in lakes with above average joint NP concentrations, although the relative biovolumes of some Nostocales were higher in less enriched lakes. Taxa were found to have diverse responses to differential N versus P concentration, and the differences between taxa were not consistent with the hypothesis that potentially N(2)-fixing Nostocales taxa would be favoured in low N relative to P conditions. In particular Aphanizomenon gracile and the subtropical invasive species Cylindrospermopsis raciborskii often reached their highest biovolumes in lakes with high nitrogen relative to phosphorus concentration. Concentrations of all cyanotoxin groups increased with increasing TP and TN, congruent with the biovolumes of their likely producers. Microcystin concentration was strongly correlated with the biovolume of Planktothrix agardhii but concentrations of anatoxin, cylindrospermopsin and paralytic shellfish poison were not strongly related to any individual taxa. Cyanobacteria should not be treated as a single group when considering the potential effects of changes in nutrient loading on phytoplankton community structure and neither should the N(2)-fixing Nostocales. This is of particular importance when considering the occurrence of cyanotoxins, as the two most abundant potentially toxin producing Nostocales in our study were found in lakes with high N relative to P enrichment.