Structural changes of minor group human rhinoviruses during uncoating

Humane Rhinoviren (HRVs) der „minor“ Rezeptorgruppe, Erreger der gewöhnlichen Erkältung, werden durch Rezeptor vermittelte Endozytose in die Zelle aufgenommen. Der niedrige pH-Wert in Endosomen (<pH 5.8) induziert den Uncoating-Prozess. Das interne Kapsidprotein VP4 und der amphipathische N-Terminus von VP1 werden externalisiert, wodurch das hydrophobe A-Partikel generiert wird, das an die endosomale Membran bindet. Der darauffolgende trans-Membran RNA-Transfer resultiert in leere Kapside oder B-Partikel, die in den intakten Endosomen zurückbleiben. In der vorliegenden Studie wurde die Struktur von A- und B-Partikeln von HRV2 von cryo-EM Daten bis zu unter 10 Å Auflösung rekonstruiert. Durch Vergleiche miteinander und Docking der Röntgenstruktur von nativem Virus wurden die Strukturänderungen während des rhino-viralen Uncoating-Prozesses untersucht. A-Partikel expandierten im Vergleich zu nativem Virus um 4.7 % und Poren öffneten sich an den 2- und 5-fachen Symmetrieachsen. Gleichzeitig wurde VP4 freigesetzt und der VP1 N-Terminus an der pseudo-3-fachen Achse externalisiert. Das Genom wies einen höheren Ordnungsgrad auf und eine interne RNA-Hülle wurde rekonstruiert, die das Kapsid an den 2- und pseudo-3-fachen Achsen berührte. Säure induzierte B-Partikel unterschieden sich von A-Partikeln in der RNA-Freisetzung und im VP1 N-Terminus, der durch seine hohe, konformative Flexibilität nicht rekonstruiert werden konnte. Vergleiche mit der publizierten Struktur von Hitze induzierten B-Partikeln bewiesen die Äquivalenz von erhöhten Temperaturen und Säure als Auslöser des Uncoating-Prozesses. Um Uncoating im Zusammenhang mit Membranen zu untersuchen, wurden Liposome mit einem rekombinanten Rezeptorfragment vom VLDLR dekoriert und HRV2 wurde spezifisch daran gebunden. Virale Strukturänderungen wurden durch Ansäuerung induziert und durch EM-Präparationstechniken angehalten. Dadurch konnten neue Zwischenstadien des Uncoating mit partiell freigesetzter RNA und membrangebundene, leere Kapside visualisiert werden. Die Kinetik des RNA-Transfers war schneller als in Lösung, was auf eine mögliche, katalytische Aktivität der Membrane oder des pH-Gradienten über diese auf die RNA-Freisetzung hinweist. Die vorliegenden Daten beschreiben die Strukturänderungen von HRVs der „minor“ Rezeptorgruppe während des Uncoating-Prozesses. Sie untermauern außerdem die Wichtigkeit, rhino-viralen RNA-Transfer im Zusammenhang mit Membranen zu untersuchen.Minor group human rhinoviruses (HRVs), causative agents of the common cold, are internalized by receptor-mediated endocytosis. The low endosomal pH (<pH 5.8) triggers the uncoating process. The innermost capsid protein VP4 is released and the amphipathic N-terminus of VP1 is externalized, generating hydrophobic A-particles that interact with the endosomal membrane. Trans-membrane RNA release into the cytoplasm results in empty capsids or B-particles, retained in intact endosomes. In the current study, the 3D structures of A- and B-particles of HRV2 were solved from cryo-EM data to resolutions below 10 Å. By mutual comparison and docking of the published X-ray coordinates of native HRV2, the main structural changes during rhino-viral uncoating were investigated. A-particles expanded by 4.7 % compared to native virus and pores opened at the 2- and 5-fold axes. Concomitantly, VP4 was expelled and the VP1 N-terminus was externalized via the pseudo-3-fold axis. The genome became more ordered and an inner RNA shell could be resolved, contacting the capsid at the 2- and pseudo-3-fold axes. Acid-triggered B-particles differed from A-particles only in the released RNA genome and the VP1 N-terminus that became too disordered to be resolved. By comparison to the published EM map of heat-induced B-particles, equivalence of heating and acidification as triggers for rhino-viral uncoating was demonstrated. For studying uncoating in the context of membranes, liposomes were decorated with a recombinant receptor fragment of VLDLR and HRV2 was specifically attached. Viral conversion was triggered by acidification and halted at different stages by EM preparation techniques. New intermediate particles with partially externalized RNA were visualized and empty capsids remained membrane-associated. The kinetics of uncoating was faster than in solution, suggesting a catalytic effect of the membrane or of the pH gradient across it on RNA release. The current data depict the main conformational changes minor group rhinoviruses undergo during uncoating. They additionally emphasize the importance of investigating rhino-viral RNA release in the context of membranes

First insights into the impacts of benthic cyanobacterial mats on fish herbivory functions on a nearshore coral reef

Benthic cyanobacterial mats (BCMs) are becoming increasingly common on coral reefs. In Fiji, blooms generally occur in nearshore areas during warm months but some are starting to prevail through cold months. Many fundamental knowledge gaps about BCM proliferation remain, including their composition and how they influence reef processes. This study examined a seasonal BCM bloom occurring in a 17-year-old no-take inshore reef area in Fiji. Surveys quantified the coverage of various BCM-types and estimated the biomass of key herbivorous fish functional groups. Using remote video observations, we compared fish herbivory (bite rates) on substrate covered primarily by BCMs (> 50%) to substrate lacking BCMs (< 10%) and looked for indications of fish (opportunistically) consuming BCMs. Samples of different BCM-types were analysed by microscopy and next-generation amplicon sequencing (16S rRNA). In total, BCMs covered 51 ± 4% (mean ± s.e.m) of the benthos. Herbivorous fish biomass was relatively high (212 ± 36 kg/ha) with good representation across functional groups. Bite rates were significantly reduced on BCM-dominated substratum, and no fish were unambiguously observed consuming BCMs. Seven different BCM-types were identified, with most containing a complex consortium of cyanobacteria. These results provide insight into BCM composition and impacts on inshore Pacific reefs

CONSCIÊNCIA NA SUBSTITUIÇÃO DO USO DE ANIMAIS NO ENSINO: ASPECTOS HISTÓRICOS, ÉTICOS E DE LEGISLAÇÃO

o especismo, a discriminação de direitos em favor da espéciehumana e em detrimento de outras espécies, é praticado também noambiente acadêmico. O uso indiscriminado de animais não-humanospara o ensino e a pesquisa é uma prática antiga, que foi consolidadadurante a Idade Moderna. Mesmo diante da tecnologia atual, osanimais ainda são usados, de forma prejudicial, para a demonstraçãode conhecimentos. Como reação de um número crescente depesquisadores em todas as áreas do conhecimento, tem havido umaumento da criação, aperfeiçoamento e consolidação de métodossubstitutivos e, por parte dos estudantes, a objeção de consciência.Assim, a substituição do uso prejudicial de animais por métodosmais eficazes é uma realidade possível e positiva no desenvolvimento ético dos humanos pelo refinamento do respeito aos não-humanos, possibilitando o desenvolviment de habilidades necessárias à prática profissional

Sensitivity of phytoplankton, zooplankton and macroinvertebrates to hydrogen peroxide treatments of cyanobacterial blooms

Addition of hydrogen peroxide (H2O2) is a promising method to acutely suppress cyanobacterial blooms in lakes. However, a reliable H2O2 risk assessment to identify potential effects on non-target species is currently hampered by a lack of appropriate ecotoxicity data. The aim of the present study was therefore to quantify the responses of a wide diversity of freshwater phytoplankton, zooplankton and macroinvertebrates to H2O2 treatments of cyanobacterial blooms. To this end, we applied a multifaceted approach. First, we investigated the 24-h toxicity of H2O2 to three cyanobacteria (Planktothrix agardhii, Microcystis aeruginosa, Anabaena sp.) and 23 non-target species (six green algae, eight zooplankton and nine macroinvertebrate taxa), using EC50 values based on photosynthetic yield for phytoplankton and LC50 values based on mortality for the other organisms. The most sensitive species included all three cyanobacterial taxa, but also the rotifer Brachionus calyciflores and the cladocerans Ceriodaphnia dubia and Daphnia pulex. Next, the EC50 and LC50 values obtained from the laboratory toxicity tests were used to construct a species sensitivity distribution (SSD) for H2O2. Finally, the species predicted to be at risk by the SSD were compared with the responses of phytoplankton, zooplankton and macroinvertebrates to two whole-lake treatments with H2O2. The predictions of the laboratory-based SSD matched well with the responses of the different taxa to H2O2 in the lake. The first lake treatment, with a relatively low H2O2 concentration and short residence time, successfully suppressed cyanobacteria without major effects on non-target species. The second lake treatment had a higher H2O2 concentration with a longer residence time, which resulted in partial suppression of cyanobacteria, but also in a major collapse of rotifers and decreased abundance of small cladocerans. Our results thus revealed a trade-off between the successful suppression of cyanobacteria at the expense of adverse effects on part of the zooplankton community. This delicate balance strongly depends on the applied H2O2 dosage and may affect the decision whether to treat a lake or not.</p

Shifts in phytoplankton and zooplankton communities in three cyanobacteria-dominated lakes after treatment with hydrogen peroxide

Cyanobacteria can reach high densities in eutrophic lakes, which may cause problems due to their potential toxin production. Several methods are in use to prevent, control or mitigate harmful cyanobacterial blooms. Treatment of blooms with low concentrations of hydrogen peroxide (H2O2) is a promising emergency method. However, effects of H2O2 on cyanobacteria, eukaryotic phytoplankton and zooplankton have mainly been studied in controlled cultures and mesocosm experiments, while much less is known about the effectiveness and potential side effects of H2O2 treatments on entire lake ecosystems. In this study, we report on three different lakes in the Netherlands that were treated with average H2O2 concentrations ranging from 2 to 5 mg L−1 to suppress cyanobacterial blooms. Effects on phytoplankton and zooplankton communities, on cyanotoxin concentrations, and on nutrient availability in the lakes were assessed. After every H2O2 treatment, cyanobacteria drastically declined, sometimes by more than 99%, although blooms of Dolichospermum sp., Aphanizomenon sp., and Planktothrix rubescens were more strongly suppressed than a Planktothrix agardhii bloom. Eukaryotic phytoplankton were not significantly affected by the H2O2 additions and had an initial advantage over cyanobacteria after the treatment, when ample nutrients and light were available. In all three lakes, a new cyanobacterial bloom developed within several weeks after the first H2O2 treatment, and in two lakes a second H2O2 treatment was therefore applied to again suppress the cyanobacterial population. Rotifers strongly declined after most H2O2 treatments except when the H2O2 concentration was ≤ 2 mg L−1, whereas cladocerans were only mildly affected and copepods were least impacted by the added H2O2. In response to the treatments, the cyanotoxins microcystins and anabaenopeptins were released from the cells into the water column, but disappeared after a few days. We conclude that lake treatments with low concentrations of H2O2 can be a successful tool to suppress harmful cyanobacterial blooms, but may negatively affect some of the zooplankton taxa in lakes. We advise pre-tests prior to the treatment of lakes to define optimal treatment concentrations that kill the majority of the cyanobacteria and to minimize potential side effects on non-target organisms. In some cases, the pre-tests may discourage treatment of the lake.</p

Shifts in phytoplankton and zooplankton communities in three cyanobacteria-dominated lakes after treatment with hydrogen peroxide

Cyanobacteria can reach high densities in eutrophic lakes, which may cause problems due to their potential toxin production. Several methods are in use to prevent, control or mitigate harmful cyanobacterial blooms. Treatment of blooms with low concentrations of hydrogen peroxide (H2O2) is a promising emergency method. However, effects of H2O2 on cyanobacteria, eukaryotic phytoplankton and zooplankton have mainly been studied in controlled cultures and mesocosm experiments, while much less is known about the effectiveness and potential side effects of H2O2 treatments on entire lake ecosystems. In this study, we report on three different lakes in the Netherlands that were treated with average H2O2 concentrations ranging from 2 to 5 mg L−1 to suppress cyanobacterial blooms. Effects on phytoplankton and zooplankton communities, on cyanotoxin concentrations, and on nutrient availability in the lakes were assessed. After every H2O2 treatment, cyanobacteria drastically declined, sometimes by more than 99%, although blooms of Dolichospermum sp., Aphanizomenon sp., and Planktothrix rubescens were more strongly suppressed than a Planktothrix agardhii bloom. Eukaryotic phytoplankton were not significantly affected by the H2O2 additions and had an initial advantage over cyanobacteria after the treatment, when ample nutrients and light were available. In all three lakes, a new cyanobacterial bloom developed within several weeks after the first H2O2 treatment, and in two lakes a second H2O2 treatment was therefore applied to again suppress the cyanobacterial population. Rotifers strongly declined after most H2O2 treatments except when the H2O2 concentration was ≤ 2 mg L−1, whereas cladocerans were only mildly affected and copepods were least impacted by the added H2O2. In response to the treatments, the cyanotoxins microcystins and anabaenopeptins were released from the cells into the water column, but disappeared after a few days. We conclude that lake treatments with low concentrations of H2O2 can be a successful tool to suppress harmful cyanobacterial blooms, but may negatively affect some of the zooplankton taxa in lakes. We advise pre-tests prior to the treatment of lakes to define optimal treatment concentrations that kill the majority of the cyanobacteria and to minimize potential side effects on non-target organisms. In some cases, the pre-tests may discourage treatment of the lake.</p