In vitro propagation, histochemistry, and analysis of essential oil from conventionally propagated and in vitro-propagated plants of Varronia curassavica Jacq

Varronia curassavica is cultivated for the production of an essential oil useful in the pharmaceutical industry for its strong anti-inflammatory effect. Despite a growing demand, only a few studies have evaluated alternative sources of obtaining plantlets or ways to increase the yield of essential oil from this species. Therefore, this study aimed to optimize the in vitro multiplication rate and analyze the histochemistry and sesquiterpene production potential of conventionally propagated V. curassavica plants, in vitro shoots, and acclimatized plants derived from in vitro shoots. For axillary bud proliferation, Murashige and Skoog medium was supplemented with 6-benzyladenine and thidiazuron alone or in combination with naphthalene acetic acid. Axillary bud proliferation was obtained from culture of nodal or apical segments on medium containing half-strength Murashige and Skoog salts without growth regulators. After 35 d of culture, an average of five buds developed per explant. Elongation and rooting of shoots also occurred in this medium. After the transfer of rooted plants to ex vitro conditions, 100% of the plantlets survived. Histochemical analysis of leaf tissue showed the presence of lipids, acidic lipids, essential oil, phenols, and flavonoids. The essential oils from conventionally propagated and acclimatized plants were extracted by hydrodistillation and analyzed using gas chromatography. The essential oil from acclimatized plants had a similar profile to that from ex vitro plants, but with a higher concentration of the anti-inflammatory compound alpha-humulene.     

Marionettes as Recreational Art Activities in the Chicago Parks

Rembrandt Drawings: The Overlook Press New York City 10022 $12.95. Reviewed by B. Haak

The Decoupage Workshop: Doubleday &; Company, Inc. New York City 10017 $12.95. Reviewed by Leslie Linsley

A Cabinetmaker's Notebook: Van Nostrand Reinhold Company New York City 10001 $13.50. Reviewed by James Krenov

Alkema's Scrap Magic: Sterling Publishing Co., Inc. New York City 10016 $12.95. Reviewed by Chester Jay Alkema

Things to Do in a Day Macmillan Publishing Co., Inc. $8.95 cloth New York City 10022 $5.95 paper.     

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression.     

Development and Validation of a Discriminative Dissolution Test for Betamethasone Sodium Phosphate and Betamethasone Dipropionate Intramuscular Injectable Suspension

The intramuscular administration of the injectable suspension betamethasone sodium phosphate (BSP) and betamethasone dipropionate (BD) has immediate therapeutic activity due to solubilized BSP and prolonged activity resulting from the slow release of BD micro-crystals. The purpose of this study was to develop and validate a dissolution method for BD in intramuscular injectable suspensions with detection by high-performance liquid chromatography (HPLC) method. Five commercial products presented a distribution of particle sizes, ranging between 7.43 and 40.25 μm as measured by laser diffraction. It was also found that particle sizes differed between batches of the same product. The different products were tested using the paddle apparatus, with stirring speeds of 25 and 50 rpm in 300 mL of phosphate buffer; simulated body fluid, muscle fluid, and synovial fluid were used as biorelevant dissolution media at 37 ± 0.5°C. It was verified that not only does average particle size affect the dissolution rate, but also the mode and the polydispersity index of the particles. Discriminatory power was obtained using the in vitro dissolution method with 0.1 M sodium phosphate buffer pH 7.4 containing 0.1% sodium lauryl sulfate and a stirring speed of 50 rpm. The HPLC-method is linear, precise, selective, and accurate for the quantification of BSP and BD in dissolution profile testing. This dissolution method can be utilized as a method to control the quality of these injectable suspensions.Key words: dipropionate betamethasone, dissolution test, intramuscular injectable suspensions, simulated muscular fluid, sodium phosphate betamethasone     

Bacterially Produced Recombinant Influenza Vaccines Based on Virus-Like Particles

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein''s neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.     

High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal,Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.     

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.^1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.³ BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.⁴ A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.^5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.     

Clicking in Shallow Rivers: Short-Range Echolocation of Irrawaddy and Ganges River Dolphins in a Shallow,Acoustically Complex Habitat

Toothed whales (Cetacea, odontoceti) use biosonar to navigate their environment and to find and catch prey. All studied toothed whale species have evolved highly directional, high-amplitude ultrasonic clicks suited for long-range echolocation of prey in open water. Little is known about the biosonar signals of toothed whale species inhabiting freshwater habitats such as endangered river dolphins. To address the evolutionary pressures shaping the echolocation signal parameters of non-marine toothed whales, we investigated the biosonar source parameters of Ganges river dolphins (Platanista gangetica gangetica) and Irrawaddy dolphins (Orcaella brevirostris) within the river systems of the Sundarban mangrove forest. Both Ganges and Irrawaddy dolphins produced echolocation clicks with a high repetition rate and low source level compared to marine species. Irrawaddy dolphins, inhabiting coastal and riverine habitats, produced a mean source level of 195 dB (max 203 dB) re 1 µPa_pp whereas Ganges river dolphins, living exclusively upriver, produced a mean source level of 184 dB (max 191) re 1 µPa_pp. These source levels are 1–2 orders of magnitude lower than those of similar sized marine delphinids and may reflect an adaptation to a shallow, acoustically complex freshwater habitat with high reverberation and acoustic clutter. The centroid frequency of Ganges river dolphin clicks are an octave lower than predicted from scaling, but with an estimated beamwidth comparable to that of porpoises. The unique bony maxillary crests found in the Platanista forehead may help achieve a higher directionality than expected using clicks nearly an octave lower than similar sized odontocetes.     

Epithelial Uptake of Flagella Initiates Proinflammatory Signaling

The airway epithelium serves multiple roles in the defense of the lung. Not only does it act as a physical barrier, it acts as a distal extension of the innate immune system. We investigated the role of the airway epithelium in the interaction with flagella, an important virulence factor of the pathogen Pseudomonas aeruginosa, a cause of ventilator associated pneumonia and significant morbidity and mortality in patients with cystic fibrosis. Flagella were required for transmigration across polarized airway epithelial cells and this was a direct consequence of motility, and not a signaling effect. Purified flagella did not alter the barrier properties of the epithelium but were observed to be rapidly endocytosed inside epithelial cells. Neither flagella nor intact P. aeruginosa stimulated epithelial inflammasome signaling. Flagella-dependent signaling required dynamin-based uptake as well as TLR5 and primarily led to the induction of proinflammatory (Tnf, Il6) as well as neutrophil (Cxcl1, Cxcl2, Ccl3) and macrophage (Ccl20) chemokines. Although flagella are important in invasion across the epithelial barrier their shedding in the airway lumen results in epithelial uptake and signaling that has a major role in the initial recruitment of immune cells in the lung.     

Variable δ15N Diet-Tissue Discrimination Factors among Sharks: Implications for Trophic Position,Diet and Food Web Models

The application of stable isotopes to characterize the complexities of a species foraging behavior and trophic relationships is dependent on assumptions of δ¹⁵N diet-tissue discrimination factors (∆¹⁵N). As ∆¹⁵N values have been experimentally shown to vary amongst consumers, tissues and diet composition, resolving appropriate species-specific ∆¹⁵N values can be complex. Given the logistical and ethical challenges of controlled feeding experiments for determining ∆¹⁵N values for large and/or endangered species, our objective was to conduct an assessment of a range of reported ∆¹⁵N values that can hypothetically serve as surrogates for describing the predator-prey relationships of four shark species that feed on prey from different trophic levels (i.e., different mean δ¹⁵N dietary values). Overall, the most suitable species-specific ∆¹⁵N values decreased with increasing dietary-δ¹⁵N values based on stable isotope Bayesian ellipse overlap estimates of shark and the principal prey functional groups contributing to the diet determined from stomach content analyses. Thus, a single ∆¹⁵N value was not supported for this speciose group of marine predatory fishes. For example, the ∆¹⁵N value of 3.7‰ provided the highest percent overlap between prey and predator isotope ellipses for the bonnethead shark (mean diet δ¹⁵N = 9‰) whereas a ∆¹⁵N value < 2.3‰ provided the highest percent overlap between prey and predator isotope ellipses for the white shark (mean diet δ¹⁵N = 15‰). These data corroborate the previously reported inverse ∆¹⁵N-dietary δ¹⁵N relationship when both isotope ellipses of principal prey functional groups and the broader identified diet of each species were considered supporting the adoption of different ∆¹⁵N values that reflect the predators’ δ¹⁵N-dietary value. These findings are critical for refining the application of stable isotope modeling approaches as inferences regarding a species’ ecological role in their community will be influenced with consequences for conservation and management actions.