Discovery of a dsRNA virus infecting the marine photosynthetic protist Micromonas pusilla

AbstractWe report the isolation of the first double-stranded (ds) RNA virus in the family Reoviridae that infects a protist (microalga Micromonas pusilla, Prasinophyceae). The dsRNA genome was composed of 11 segments ranging between 0.8 and 5.8 kb, with a total size of approximately 25.5 kb. The virus (MpRNAV-01B) could not be assigned to the genus level because host type, genome size, and number of segments smaller than 2 kb did not correspond to either of the two existing 11-segmented dsRNA genera Rotavirus and Aquareovirus. MpRNAV-01B has a particle size of 65–80 nm, a narrow host range, a latent period of 36 h, and contains five major proteins (120, 95, 67, 53, and 32 kDa). MpRNAV-01B was stable to freeze–thawing, resistant to chloroform, ether, nonionic detergents, chelating and reducing agents. The virus was inactivated at temperatures above 35 °C and by ionic detergent, ethanol, acetone, and acidic conditions (pH 2–5)

Offshore Wind Power Technology Development and Testing of a Downscaled Pitch Controlled Wind Turbine

Masteroppgave i mekatronikk - Universitetet i Agder, 2015The energy demand of the world increases day by day, and at the same time people become more aware of the impact fossil energy sources have on the climate. This leads to an increased focus on renewable energy sources, such as wind power, which has been pointed out as an important contributor to the production of renewable energy. In recent years the focus has shifted from land based wind farms towards the possibilities of the large energy potential that exist in the open ocean along the coast line. The background of the thesis is motivated by the increased focus on offshore wind power technology. State of the art technology in the field of floating offshore wind power was reviewed in the first part in addition to relevant background theory regarding design and operation of wind turbines. The main objective has been to develop and test a downscaled pitch controlled wind turbine. A pitch mechanism was designed to turn the blade pitch angle to a given angle with a stepper motor. The stepper motor transfers rotational movement with a leading screw to translational movement of a sliding mechanism. The sliding mechanism rotates the angle of the blade shaft to the optimal calculated angle. Control of the stepper motor is achieved by a PLC step drive and a ladder diagram describing the desired functionality. Optimized pitch angles are calculated and implemented as a function with the current wind speed as input. The wind speed is measured with an anemometer and connected to the PLC as an analogue signal. From the PLC the signals are logged with an OPC server and processed in Excel. The wind turbine was tested on the campus roof top. The cut-in speed was as expected, and the turbine require between 3 and 5 m/s to start to rotate depending on the angle of the wind, while cut-out speed is around 17 m/s, due to high risk of malfunction and destruction of parts or components. The voltage was measured to be around 150-250 mV at optimal wind speed 9 m/s and the maximum obtained shaft speed generated about 280 mV. More extensive testing should be performed in order to obtain more reliable results and complete verify the model. Some improvements has been discussed, including implementing a gearbox, redesigning the blades with a different profile as well as material. A proper test site with more easily controllable wind speeds is preferable

Differential toxicity of bioorthogonal non-canonical amino acids (BONCAT) in Escherichia coli

Single-cell methods allow studying the activity of single bacterial cells, potentially shedding light on regulatory mechanisms involved in services like biochemical cycling. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising method for studying bacterial activity in natural communities, using the methionine analogues L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG) to track protein production in single cells. Both AHA and HPG have been deemed non-toxic, but recent findings suggest that HPG affects bacterial metabolism. In this study we examined the effect of AHA and HPG on Escherichia coli with respect to acute toxicity and growth. E. coli exposed to 5.6–90 μM HPG showed no growth, and the growth rate was significantly reduced at >0.35 μM HPG, compared to the HPG-free control. In contrast, E. coli showed growth at concentrations up to 9 mM AHA. In assays where AHA or HPG were added during the exponential growth phase, the growth sustained but the growth rate was immediately reduced at the highest concentrations (90 μM HPG and 10 mM AHA). Prolonged incubations (20h) with apparently non-toxic concentrations suggest that the cells incorporating NCAAs fail to divide and do not contribute to the next generation resulting in the relative abundance of labelled cells to decrease over time. These results show that HPG and AHA have different impact on the growth of E. coli. Both concentration and incubation time affect the results and need to be considered when designing BONCAT experiments and evaluating results. Time course incubations are suggested as a possible way to obtain more reliable results.publishedVersio

Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms

In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments.publishedVersio

Flow Cytometric Analysis of Bacterial Protein Synthesis: Monitoring Vitality After Water Treatment

Bacterial vitality after water disinfection treatment was investigated using bio-orthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM). Protein synthesis activity and DNA integrity (BONCAT–SYBR Green) was monitored in Escherichia coli monocultures and in natural marine samples after UV irradiation (from 25 to 200 mJ/cm2) and heat treatment (from 15 to 45 min at 55°C). UV irradiation of E. coli caused DNA degradation followed by the decrease in protein synthesis within a period of 24 h. Heat treatment affected both DNA integrity and protein synthesis immediately, with an increased effect over time. Results from the BONCAT method were compared with results from well-known methods such as plate counts (focusing on growth) and LIVE/DEAD™ BacLight™ (focusing on membrane permeability). The methods differed somewhat with respect to vitality levels detected in bacteria after the treatments, but the results were complementary and revealed that cells maintained metabolic activity and membrane integrity despite loss of cell division. Similarly, analysis of protein synthesis in marine bacteria with BONCAT displayed residual activity despite inability to grow or reproduce. Background controls (time zero blanks) prepared using different fixatives (formaldehyde, isopropanol, and acetic acid) and several different bacterial strains revealed that the BONCAT protocol still resulted in labeled, i.e., apparently active, cells. The reason for this is unclear and needs further investigation to be understood. Our results show that BONCAT and FCM can detect, enumerate, and differentiate bacterial cells after physical water treatments such as UV irradiation and heating. The method is reliable to enumerate and explore vitality of single cells, and a great advantage with BONCAT is that all proteins synthesized within cells are analyzed, compared to assays targeting specific elements such as enzyme activity.publishedVersio

How microbial food web interactions shape the arctic ocean bacterial community revealed by size fractionation experiments

In the Arctic, seasonal changes are substantial, and as a result, the marine bacterial community composition and functions differ greatly between the dark winter and light-intensive summer. While light availability is, overall, the external driver of the seasonal changes, several internal biological interactions structure the bacterial community during shorter timescales. These include specific phytoplankton–bacteria associations, viral infections and other top-down controls. Here, we uncover these microbial interactions and their effects on the bacterial community composition during a full annual cycle by manipulating the microbial food web using size fractionation. The most profound community changes were detected during the spring, with ‘mutualistic phytoplankton’—Gammaproteobacteria interactions dominating in the pre-bloom phase and ‘substrate-dependent phytoplankton’—Flavobacteria interactions during blooming conditions. Bacterivores had an overall limited effect on the bacterial community composition most of the year. However, in the late summer, grazing was the main factor shaping the community composition and transferring carbon to higher trophic levels. Identifying these small-scale interactions improves our understanding of the Arctic marine microbial food web and its dynamics

Marine mimivirus relatives are probably large algal viruses

<p>Abstract</p> <p>Background</p> <p><it>Acanthamoeba polyphaga </it>mimivirus is the largest known ds-DNA virus and its 1.2 Mb-genome sequence has revealed many unique features. Mimivirus occupies an independent lineage among eukaryotic viruses and its known hosts include only species from the <it>Acanthamoeba </it>genus. The existence of mimivirus relatives was first suggested by the analysis of the Sargasso Sea metagenomic data.</p> <p>Results</p> <p>We now further demonstrate the presence of numerous "mimivirus-like" sequences using a larger marine metagenomic data set. We also show that the DNA polymerase sequences from three algal viruses (CeV01, PpV01, PoV01) infecting different marine algal species (<it>Chrysochromulina ericina</it>, <it>Phaeocystis pouchetii</it>, <it>Pyramimonas orientalis</it>) are very closely related to their homolog in mimivirus.</p> <p>Conclusion</p> <p>Our results suggest that the numerous mimivirus-related sequences identified in marine environments are likely to originate from diverse large DNA viruses infecting phytoplankton. Micro-algae thus constitute a new category of potential hosts in which to look for new species of <it>Mimiviridae</it>.</p

Open-Label, Multi-Dose, Pilot Safety Study of Injection of OnabotulinumtoxinA Toward the Otic Ganglion for the Treatment of Intractable Chronic Cluster Headache

BACKGROUND: The otic ganglion (OG) provides parasympathetic innervation to the cerebral circulation and cranial structures and may be involved in the pathophysiology of trigeminal autonomic headaches. This structure has never been targeted in any headache disorder. OBJECTIVE: To investigate the safety of injecting onabotulinumtoxin A (BTA) toward the OG in 10 patients with intractable chronic cluster headache and to collect efficacy data. METHODS: A total of 10 patients with chronic cluster headache were enrolled in this open-label, multi-dose pilot safety study. All patients were recruited and treated on an out-patient basis at St Olav's University Hospital (Norway). In 5 patients each, the OG was the injection target with 12.5 IU of BTA or 25 IU, respectively. The primary outcome measure was adverse events (AEs) and the main secondary outcome was the number of attacks per week measured at baseline and in the second month following injection. RESULTS: For the primary endpoint, we analyzed data for all 10 patients. There were a total of 17 AEs in 6 of the 10 patients. All AEs were considered mild and disappeared by the end of follow-up. The median number of attacks per week at baseline was 17.0 [7.8 to 25.8] vs 14.0 [7.3 to 20.0] in the second month following injection; difference: 3 (95%CI: -0.3 to 7.9), P = .063. CONCLUSIONS: Injection with BTA toward the OG appears to be safe. We did not find a statistically significant reduction in the number of attacks per week at month 2 after injection compared to the baseline. This study suggests that the OG is not an important target for the treatment of chronic cluster headache. A future study employing more precise targeting of the OG may be indicated