Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.     

Applying quality by design (QbD) concept for fabrication of chitosan coated nanoliposomes

In the present investigation, a quality by design (QbD) strategy was successfully applied to the fabrication of chitosan-coated nanoliposomes (CH-NLPs) encapsulating a hydrophilic drug. The effects of the processing variables on the particle size, encapsulation efficiency (%EE) and coating efficiency (%CE) of CH-NLPs (prepared using a modified ethanol injection method) were investigated. The concentrations of lipid, cholesterol, drug and chitosan; stirring speed, sonication time; organic:aqueous phase ratio; and temperature were identified as the key factors after risk analysis for conducting a screening design study. A separate study was designed to investigate the robustness of the predicted design space. The particle size, %EE and %CE of the optimized CH-NLPs were 111.3?nm, 33.4% and 35.2%, respectively. The observed responses were in accordance with the predicted response, which confirms the suitability and robustness of the design space for CH-NLP formulation. In conclusion, optimization of the selected key variables will help minimize the problems related to size, %EE and %CE that are generally encountered when scaling up processes for NLP formulations. The robustness of the design space will help minimize both intra-batch and inter-batch variations, which are quite common in the pharmaceutical industry.     

Ion-beam and gamma-ray irradiations induce thermotolerant mutants in the entomopathogenic fungus Metarhizium anisopliae s.l.

Entomopathogenic fungi, such as Metarhizium anisopliae (Metch.) Sorokin, are important agents for the biological control of insect pests. However, these fungi are not compatible with high temperatures. In this study, mutagenesis using ion beams or gamma rays was used to generate five potentially thermotolerant mutants from two wild-type isolates of M. anisopliae (two using ion beams and three using gamma rays). The mutant isolates had a higher upper thermal limit for vegetative growth compared to the wild types (by 2–3°C) and enhanced tolerance to wet–heat stress of 45°C for conidial germination. At 25°C and 30°C, most mutants were as virulent to maize weevil adults as the wild type, however, one mutant produced using ion beams almost lost virulence entirely. These results indicate that ion beams and gamma rays are useful tools for improving biological characteristics, such as thermotolerance, in entomopathogenic fungi, but that mutants must be carefully evaluated for unpredictable negative side effects.     

Environmental drivers of ectomycorrhizal communities in Europe's temperate oak forests

Ectomycorrhizal fungi are major ecological players in temperate forests, but they are rarely used in measures of forest condition because large‐scale, high‐resolution, standardized and replicated belowground data are scarce. We carried out an analysis of ectomycorrhizas at 22 intensively monitored long‐term oak plots, across nine European countries, covering complex natural and anthropogenic environmental gradients. We found that at large scales, mycorrhizal richness and evenness declined with decreasing soil pH and root density, and with increasing atmospheric nitrogen deposition. Shifts in mycorrhizas with different functional traits were detected; mycorrhizas with structures specialized for long‐distance transport related differently to most environmental variables than those without. The dominant oak‐specialist Lactarius quietus, with limited soil exploration abilities, responds positively to increasing nitrogen inputs and decreasing pH. In contrast, Tricholoma, Cortinarius and Piloderma species, with medium‐distance soil exploration abilities, show a consistently negative response. We also determined nitrogen critical loads for moderate (9.5–13.5 kg N/ha/year) and drastic (17 kg N/ha/year) changes in belowground mycorrhizal root communities in temperate oak forests. Overall, we generated the first baseline data for ectomycorrhizal fungi in the oak forests sampled, identified nitrogen pollution as one of their major drivers at large scales and revealed fungi that individually and/or in combination with others can be used as belowground indicators of environmental characteristics.     

Mechanical limits to maximum weapon size in a giant rhinoceros beetle

The horns of giant rhinoceros beetles are a classic example of the elaborate morphologies that can result from sexual selection. Theory predicts that sexual traits will evolve to be increasingly exaggerated until survival costs balance the reproductive benefits of further trait elaboration. In Trypoxylus dichotomus, long horns confer a competitive advantage to males, yet previous studies have found that they do not incur survival costs. It is therefore unlikely that horn size is limited by the theoretical cost–benefit equilibrium. However, males sometimes fight vigorously enough to break their horns, so mechanical limits may set an upper bound on horn size. Here, I tested this mechanical limit hypothesis by measuring safety factors across the full range of horn sizes. Safety factors were calculated as the ratio between the force required to break a horn and the maximum force exerted on a horn during a typical fight. I found that safety factors decrease with increasing horn length, indicating that the risk of breakage is indeed highest for the longest horns. Structural failure of oversized horns may therefore oppose the continued exaggeration of horn length driven by male–male competition and set a mechanical limit on the maximum size of rhinoceros beetle horns.     

临床危急值与药品不良反应的关联性研究分析

目的:通过监测临床危急值提高药品不良反应(ADR)的上报质量。方法:分析临床药物治疗与出现临床危急值之间的联系,确定可疑的ADR病例。结果:调查中共发现23例疑似ADR报告,占调查期间总上报ADR例数的24.73%;主要ADR表现为血小板减少(13例),其次是尿素升高(5例);给药途径主要为静脉滴注(涉及21例),涉及品种以抗菌药物为主。结论:通过加强对临床危急值的监测,可以提高ADR的报告的质量,减少ADR的漏报率。     

Microhabitats reduce animal's exposure to climate extremes

Extreme weather events, such as unusually hot or dry conditions, can cause death by exceeding physiological limits, and so cause loss of population. Survival will depend on whether or not susceptible organisms can find refuges that buffer extreme conditions. Microhabitats offer different microclimates to those found within the wider ecosystem, but do these microhabitats effectively buffer extreme climate events relative to the physiological requirements of the animals that frequent them? We collected temperature data from four common microhabitats (soil, tree holes, epiphytes, and vegetation) located from the ground to canopy in primary rainforests in the Philippines. Ambient temperatures were monitored from outside of each microhabitat and from the upper forest canopy, which represent our macrohabitat controls. We measured the critical thermal maxima (CT_max) of frog and lizard species, which are thermally sensitive and inhabit our microhabitats. Microhabitats reduced mean temperature by 1–2 °C and reduced the duration of extreme temperature exposure by 14–31 times. Microhabitat temperatures were below the CT_max of inhabitant frogs and lizards, whereas macrohabitats consistently contained lethal temperatures. Microhabitat temperatures increased by 0.11–0.66 °C for every 1 °C increase in macrohabitat temperature, and this nonuniformity in temperature change influenced our forecasts of vulnerability for animal communities under climate change. Assuming uniform increases of 6 °C, microhabitats decreased the vulnerability of communities by up to 32‐fold, whereas under nonuniform increases of 0.66 to 3.96 °C, microhabitats decreased the vulnerability of communities by up to 108‐fold. Microhabitats have extraordinary potential to buffer climate and likely reduce mortality during extreme climate events. These results suggest that predicted changes in distribution due to mortality and habitat shifts that are derived from macroclimatic samples and that assume uniform changes in microclimates relative to macroclimates may be overly pessimistic. Nevertheless, even nonuniform temperature increases within buffered microhabitats would still threaten frogs and lizards.     

Effects of acclimation on the thermal tolerance of the brown planthopper Nilaparvata lugens (Stål)

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