A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has emerged as a superb model species in plant biology. Although the alga is easily transformable, the low efficiency of transgene expression from the Chlamydomonas nuclear genome has severely hampered functional genomics research. For example, poor transgene expression is held responsible for the lack of sensitive reporter genes to monitor gene expression in vivo, analyze subcellular protein localization or study protein–protein interactions. Here, we have tested the luciferase from the marine copepod Gaussia princeps (G-Luc) for its suitability as a sensitive bioluminescent reporter of gene expression in Chlamydomonas. We show that a Gaussia luciferase gene variant, engineered to match the codon usage in the Chlamydomonas nuclear genome, serves as a highly sensitive reporter of gene expression from both constitutive and inducible algal promoters. Its bioluminescence signal intensity greatly surpasses previously developed reporters for Chlamydomonas nuclear gene expression and reaches values high enough for utilizing the reporter as a tool to monitor responses to environmental stresses in vivo and to conduct high-throughput screenings for signaling mutants in Chlamydomonas

In vivo multiphoton imaging reveals gradual growth of newborn amyloid plaques over weeks

The kinetics of amyloid plaque formation and growth as one of the characteristic hallmarks of Alzheimer’s disease (AD) are fundamental issues in AD research. Especially the question how fast amyloid plaques grow to their final size after they are born remains controversial. By long-term two-photon in vivo imaging we monitored individual methoxy-X04-stained amyloid plaques over 6 weeks in 12 and 18 months old Tg2576 mice. We found that in 12 months old mice, newly appearing amyloid plaques were initially small in volume and subsequently grew over time. The growth rate of plaques was inversely proportional to their volume; thus amyloid plaques that were already present at the first imaging time point grew over time but slower compared to new plaques. Additionally, we analyzed 18 months old Tg2576 mice in which we neither found newly appearing plaques nor a significant growth of pre-existing plaques over 6 weeks of imaging. In conclusion, newly appearing amyloid plaques are initially small in size but grow over time until plaque growth can not be detected anymore in aged mice. These results suggest that drugs that target plaque formation should be most effective early in the disease, when plaques are growing

Multiple Events Lead to Dendritic Spine Loss in Triple Transgenic Alzheimer's Disease Mice

The pathology of Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) peptide, hyperphosphorylated tau protein, neuronal death, and synaptic loss. By means of long-term two-photon in vivo imaging and confocal imaging, we characterized the spatio-temporal pattern of dendritic spine loss for the first time in 3xTg-AD mice. These mice exhibit an early loss of layer III neurons at 4 months of age, at a time when only soluble Aβ is abundant. Later on, dendritic spines are lost around amyloid plaques once they appear at 13 months of age. At the same age, we observed spine loss also in areas apart from amyloid plaques. This plaque independent spine loss manifests exclusively at dystrophic dendrites that accumulate both soluble Aβ and hyperphosphorylated tau intracellularly. Collectively, our data shows that three spatio-temporally independent events contribute to a net loss of dendritic spines. These events coincided either with the occurrence of intracellular soluble or extracellular fibrillar Aβ alone, or the combination of intracellular soluble Aβ and hyperphosphorylated tau

Pneumonitis as A Consequence of (Peg)Interferon-Ribavirin Combination Therapy for Hepatitis C: a Review of the Literature

Combination of peginterferon and ribavirin is the current therapy for chronic hepatitis C infection (HCV). Interstitial pneumonitis is a rare side-effect of HCV therapy and is an important cause of dose reduction or discontinuation, impairing success of antiviral therapy. We performed a review of the literature in order to present diagnostic modalities and possible treatments for pneumonitis and to offer guidelines. We searched for cases where pneumonitis as a side-effect of HCV treatment was documented. First we performed a literature search via PubMed and Web of Science interface and second we searched three drug toxicity databases. We systematically analyzed all case reports with respect to clinical manifestations, type of treatment, and outcome. A literature search revealed 19 articles, containing 25 case descriptions, while we traced 33 cases from the drug toxicity databases. Pneumonitis presented with any of the combination of fever, dyspnea, and cough and can arise with any type of (conventional or pegylated) interferon. Mortality secondary to pneumonitis was seen in 7% of cases, exclusively with peginterferon α-2b. In most cases therapy was discontinued and steroids were started. Interferon-induced pneumonitis during HCV treatment is a severe complication and should be recognized in order to prevent further pulmonary damage and/or death

A change in the optical polarization associated with a gamma-ray flare in the blazar 3C 279

It is widely accepted that strong and variable radiation detected over all accessible energy bands in a number of active galaxies arises from a relativistic, Doppler-boosted jet pointing close to our line of sight. The size of the emitting zone and the location of this region relative to the central supermassive black hole are, however, poorly known, with estimates ranging from light-hours to a light-year or more. Here we report the coincidence of a gamma-ray flare with a dramatic change of optical polarization angle. This provides evidence for co-spatiality of optical and gamma-ray emission regions and indicates a highly ordered jet magnetic field. The results also require a non-axisymmetric structure of the emission zone, implying a curved trajectory for the emitting material within the jet, with the dissipation region located at a considerable distance from the black hole, at about 10^5 gravitational radii.Comment: Published in Nature issued on 18 February 2010. Corresponding authors: Masaaki Hayashida and Greg Madejsk

Organotypic Culture of Physiologically Functional Adult Mammalian Retinas

BACKGROUND: The adult mammalian retina is an important model in research on the central nervous system. Many experiments require the combined use of genetic manipulation, imaging, and electrophysiological recording, which make it desirable to use an in vitro preparation. Unfortunately, the tissue culture of the adult mammalian retina is difficult, mainly because of the high energy consumption of photoreceptors. METHODS AND FINDINGS: We describe an interphase culture system for adult mammalian retina that allows for the expression of genes delivered to retinal neurons by particle-mediated transfer. The retinas retain their morphology and function for up to six days— long enough for the expression of many genes of interest—so that effects upon responses to light and receptive fields could be measured by patch recording or multielectrode array recording. We show that a variety of genes encoding pre- and post-synaptic marker proteins are localized correctly in ganglion and amacrine cells. CONCLUSIONS: In this system the effects on neuronal function of one or several introduced exogenous genes can be studied within intact neural circuitry of adult mammalian retina. This system is flexible enough to be compatible with genetic manipulation, imaging, cell transfection, pharmacological assay, and electrophysiological recordings

Fatty Acid and Peptide Profiles in Plasma Membrane and Membrane Rafts of PUFA Supplemented RAW264.7 Macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers

Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential

The draft genome of Dietzia cinnamea strain P4 was determined using pyrosequencing. In total, 428 supercontigs were obtained and analyzed. We here describe and interpret the main features of the draft genome. The genome contained a total of 3,555,295 bp, arranged in a single replicon with an average G+C percentage of 70.9%. It revealed the presence of complete pathways for basically all central metabolic routes. Also present were complete sets of genes for the glyoxalate and reductive carboxylate cycles. Autotrophic growth was suggested to occur by the presence of genes for aerobic CO oxidation, formate/formaldehyde oxidation, the reverse tricarboxylic acid cycle and the 3-hydropropionate cycle for CO2 fixation. Secondary metabolism was evidenced by the presence of genes for the biosynthesis of terpene compounds, frenolicin, nanaomycin and avilamycin A antibiotics. Furthermore, a probable role in azinomycin B synthesis, an important product with antitumor activity, was indicated. The complete alk operon for the degradation of n-alkanes was found to be present, as were clusters of genes for biphenyl ring dihydroxylation. This study brings new insights in the genetics and physiology of D. cinnamea P4, which is useful in biotechnology and bioremediation

Role of Fractalkine/CX3CR1 Interaction in Light-Induced Photoreceptor Degeneration through Regulating Retinal Microglial Activation and Migration

Background: Excessive exposure to light enhances the progression and severity of some human retinal degenerative diseases. While retinal microglia are likely to be important in neuron damage associated with these diseases, the relationship between photoreceptor damage and microglial activation remains poorly understood. Some recent studies have indicated that the chemokine fractalkine is involved in the pathogenesis of many neurodegenerative diseases. The present study was performed to investigate the cross-talk between injured photoreceptors and activated retinal microglia, focusing on the role of fractalkine and its receptor CX3CR1 in light-induced photoreceptor degeneration. Methodology/Principal Findings: Both in vivo and in vitro experiments were involved in the research. In vivo, Sprague– Dawley rats were exposed to blue light for 24 hours. In vitro, the co-culture of primary retinal microglia and a photoreceptor cell line (661W cell) was exposed to blue light for five hours. Some cultures were pretreated by the addition of anti-CX3CR1 neutralizing antibody or recombinant fractalkine. Expression of fractalkine/CX3CR1 and inflammatory cytokines was detected by immunofluorescence, real-time PCR, Western immunoblot analysis, and ELISA assay. TUNEL method was used to detect cell apoptosis. In addition, chemotaxis assay was performed to evaluate the impact of soluble fractalkine on microglial migration. Our results showed that the expression of fractalkine that was significantly upregulated after exposure to light, located mainly at the photoreceptors. The extent of photoreceptor degeneration and microglial migratio