In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.     

Modulation of the Acetylcholine System in the Superior Cervical Ganglion of Rat: Effects of GABA and Hypoglossal Nerve Implantation After In Vivo GABA Treatment

gamma-Aminobutyric acid (GABA) was applied to the superior cervical ganglion (SCG) of CFY rats in vitro and in vivo, with or without implantation of a hypoglossal nerve, to evaluate the effects of these experimental interventions on the acetylcholine (ACh) system, which mainly serves the synaptic transmission of the preganglionic input. Long-lasting GABA microinfusion into the SCG in vivo apparently resulted in a "functional denervation." This treatment reduced the acetylcholinesterase (AChE; EC 3.1.1.7) activity by 30% (p less than 0.01) and transiently increased the number of nicotinic acetylcholine receptors, but had no significant effect on the choline acetyltransferase (acetyl-coenzyme A:choline-O-acetyltransferase; EC 2.3.1.6) activity, the ACh level, or the number of muscarinic acetylcholine receptors. The relative amounts of the different molecular forms of AChE did not change under these conditions. In vivo GABA application to the SCG with a hypoglossal nerve implanted in the presence of intact preganglionic afferent synapses exerted a significant modulatory effect on the AChE activity and its molecular forms. The "hyperinnervation" of the ganglia led to increases in the AChE activity (to 142.5%, p less than 0.01) and the 16S molecular form (to 200%, p less than 0.01). It is concluded that in vivo GABA microinfusion and GABA treatment in the presence of additional cholinergic synapses has a modulatory effect on the elements of the ACh system in the SCG of CFY rats.     

Land use changes could modify future negative effects of climate change on old‐growth forest indicator species

Aim

Climate change is expected to have major impacts on terrestrial biodiversity at all ecosystem levels, including reductions in species‐level distribution and abundance. We aim to test the extent to which land use management, such as setting‐aside forest from production, could reduce climate‐induced biodiversity impacts for specialist species over large geographical gradients.

Location

Sweden.

Methods

We applied ensembles of species distribution models based on citizen science data for six species of red‐listed old‐forest indicator fungi confined to spruce dead wood. We tested the effect on species habitat suitabilities of alternative climate change scenarios and varying amounts of forest set‐aside from production over the coming century.

Results

With 3.6% of forest area set‐aside from production and assuming no climate change, overall habitat suitabilities for all six species were projected to increase in response to maturing spruce in set‐aside forest. However, overall habitat suitabilities for all six species were projected to decline under climate change scenario RCP4.5 (intermediate–low emissions), with even greater declines projected under RCP 8.5 (high emissions). Increasing the amount of forest set‐aside to 16% resulted in significant increases in overall habitat suitability, with one species showing an increase. A further increase to 32% forest set‐aside resulted in considerably more positive trends, with three of six species increasing.

Main conclusions

There is interspecific variation in the importance of future macroclimate and resource availability on species occurrence. However, large‐scale conservation measures, such as increasing resource availability through setting aside forest from production, could reduce future negative effects from climate change, and early investment in conservation is likely to reduce the future negative impacts of climate change on specialist species.     

Investigating fairness in global supply chains: applying an extension of the living wage to the Western European clothing supply chain

Purpose

This paper explores the issue of fairness in global supply chains. Taking the Western European clothing supply chain as a case study, we demonstrate how applying a normative indicator in Social Life Cycle Assessment (SLCA) can contribute academic and practical insights into debates on fairness. To do so, we develop a new indicator that addresses some of the limitations of the living wage for SLCA.

Methods

We extend the standard form of living wage available for developing countries to include income tax and social security contributions. We call this extension ‘living labour compensation’. Using publically available data, we estimate net living wages, gross living wages, and living labour compensation rates for Brazil, Russia, India, and China (BRIC) in 2005. We then integrate living labour compensation rates into an input-output framework, which we use to compare living labour compensation and actual labour compensation in the BRIC countries in the Western European clothing supply chain in 2005.

Results and discussion

We find that in 2005, actual labour compensation in the Western European clothing supply chain was around half of the living labour compensation level, with the greatest difference being in the Agricultural sector. Therefore, we argue that BRIC pay in the Western European clothing supply chain was unfair. Furthermore, our living labour compensation estimates for BRIC in 2005 are ~ 35% higher than standard living wage estimates. Indeed, adding income taxes and employee social security contributions alone increases the living wage by ~ 10%. Consequently, we argue there is a risk that investigations based on living wages are not using a representative measure of fairness from the employee’s perspective and are substantially underestimating the cost of living wages from an employer’s perspective. Finally, we discuss implications for retailers and living wage advocacy groups.

Conclusions

Living labour compensation extends the living wage, maintaining its strengths and addressing key weaknesses. It can be estimated for multiple countries from publically available data and can be applied in an input-output framework. Therefore, it is able to provide a normative assessment of fairness in complex global supply chains. Applying it to the Western European clothing supply chain, we were able to show that pay for workers in Brazil, Russia, India, and China is unfair, and draw substantive conclusions for practice.

     

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans‐cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis‐Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages.     

Lidocaine block of cardiac sodium channels

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.