The use of the luciferase reporter system forin planta gene expression studies

The properties of the firefly luciferase (LUC) make it a very good nondestructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measuredin planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed inin planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegatedin planta luciferase activity, and we show whenin planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on thein vivo andin vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially forin vivo gene expression studies in plant research.     

THE RELIABILITY OF SKINFOLD-CALIPERS FOR MEASURING BLUBBER THICKNESS OF STELLER SEA LION PUPS (EUMETOPIAS JUBATUS)

Twelve dead Steller sea lion pups ( Eumetopias jubatus ) aged 3-14 d were recovered from rookeries in Southeast Alaska. They had a wide range of body sizes and conditions (small to large and fat to no fat). The ability of calipers to estimate the thickness of their blubber layer was assessed with a set of skinfold calipers. Average error of measurement for skin and blubber thickness was an acceptable 5.4%, but the skin and blubber of the pups were highly compressible. Skinfold thickness increased with body mass but did not necessarily reflect the development of blubber, given that pups with no blubber also showed an increase in skinfold thickness with increases in body mass. Skinfold thickness of sea lion pups appears to predict body size better than it predicts blubber thickness, making it difficult if not impossible to develop a simple index of body condition or a calculation of percent body fat for Steller sea lion pups from skinfold caliper measurements.     

Restoration of Botshol (The Netherlands) by reduction of external nutrient load: Recovery of the Characean community

The formerly rich characean community in Botshol with six species of which the rareNitellopsis obtusa andChara hispida dominated at many sites, decreased to only two species,Chara globularis andC. connivens, in the period 1980–1988. The macrophyteNajas marina also remained at some sites, and the aquatic mossFontinalis antipyretica and the filamentous algaVaucheria dichotoma predominated at many sites. These phenomena may have been due to eutrophication by the inlet of polluted water. This process of eutrophication was stopped by restoration measures in 1989, resulting in a lower phosphorus concentration (ca. 0.024 mg l^–1) and a higher water transparency. Immediately after these measures the Characeae community increased strongly in abundance and number of species. During the summer of 1990, and especially of 1991, a spectacular growth occurred ofChara connivens. Chara connivens was often accompanied byChara hispida. Other species with scattered occurrence wereChara aculeolata, C. aspera, C. contraria andC. Globularis. The reasons for the shift in dominance fromNitellopsis obtusa toChara connivens are discussed. One of the reasons may be the recent higher chloride content which is one of the consequences of the restoration measures.     

Effect of Ceramide Tail Length on the Structure of Model Stratum Corneum Lipid Bilayers

Lipid bilayers composed of non-hydroxy sphingosine ceramide (CER NS), cholesterol (CHOL), and free fatty acids (FFAs), which are components of the human skin barrier, are studied via molecular dynamics simulations. Since mixtures of these lipids exist in dense gel phases with little molecular mobility at physiological conditions, care must be taken to ensure that the simulations become decorrelated from the initial conditions. Thus, we propose and validate an equilibration protocol based on simulated tempering, in which the simulation takes a random walk through temperature space, allowing the system to break out of metastable configurations and hence become decorrelated from its initial configuration. After validating the equilibration protocol, which we refer to as random-walk molecular dynamics, the effects of the lipid composition and ceramide tail length on bilayer properties are studied. Systems containing pure CER NS, CER NS + CHOL, and CER NS + CHOL + FFA, with the CER NS fatty acid tail length varied within each CER NS-CHOL-FFA composition, are simulated. The bilayer thickness is found to depend on the structure of the center of the bilayer, which arises as a result of the tail-length asymmetry between the lipids studied. The hydrogen bonding between the lipid headgroups and with water is found to change with the overall lipid composition, but is mostly independent of the CER fatty acid tail length. Subtle differences in the lateral packing of the lipid tails are also found as a function of CER tail length. Overall, these results provide insight into the experimentally observed trend of altered barrier properties in skin systems where there are more CERs with shorter tails present.     

Young But Not Old Adult African Striped Mice Reduce Their Activity in the Dry Season When Food Availability is Low

An individual′s survival and fitness depend on its ability to effectively allocate its time between competing behaviors. Sex, social tactic, season and food availability are important factors influencing activity budgets. However, few field studies have tested their influences. The African striped mouse (Rhabdomys pumilio) lives in highly seasonal habitats in southern Africa, and individuals can adopt different social tactics. We investigated seasonal changes in activity budgets of different tactics and predicted that individuals will reduce their activity in the non‐breeding season to save energy when food availability is low and that young non‐breeding adults (‘philopatrics’) invest mainly in activities related to gaining body mass to increase survival probability. We predicted old adults (‘breeders’), which bred during the previous breeding season, to invest mainly in maintenance of their social status. We conducted 90 focal observations during the non‐breeding season and 73 during the breeding season. Activity budgets of striped mice were season and tactic specific, with philopatrics, but not breeders, reducing activity when food availability was low, possibly to decrease energy expenditure. Philopatrics of both sexes foraged and basked more in the breeding season than during the non‐breeding season. Male philopatrics gained body mass and female philopatrics maintained their body mass in both seasons. Sex‐specific differences occurred during the breeding season, when female breeders foraged more than male breeders, while male breeders chased other individuals more than female breeders. These findings indicate that individuals adopting different social tactics display distinct behaviors to fulfill tactic‐specific energetic needs .     

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 °C for 30 min. Thereafter, the milk is cooled down to 45 °C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 °C and 45 °C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 °C for 30 min, and subsequently cooled to 45 °C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 °C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.     

Near‐genomewide RNAi screening for regulators of BRAFV600E‐induced senescence identifies RASEF,a gene epigenetically silenced in melanoma

The activation of oncogenes in primary cells blocks proliferation by inducing oncogene‐induced senescence (OIS), a highly potent in vivo tumor‐suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near‐genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF^V600E‐induced arrest. With genome‐wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence‐associated (SA)‐β‐galactosidase activity, interleukins, and tumor suppressor p15^INK4B. Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.     

Autoregulation of RCO by Low-Affinity Binding Modulates Cytokinin Action and Shapes Leaf Diversity

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