抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

BackgroundCoronavirus disease 2019 (Covid-19) is associated with diffuse lung damage. Glucocorticoids may modulate inflammation-mediated lung injury and thereby reduce progression to respiratory failure and death.MethodsIn this controlled, open-label trial comparing a range of possible treatments in patients who were hospitalized with Covid-19, we randomly assigned patients to receive oral or intravenous dexamethasone (at a dose of 6 mg once daily) for up to 10 days or to receive usual care alone. The primary outcome was 28-day mortality. Here, we report the final results of this assessment.ResultsA total of 2104 patients were assigned to receive dexamethasone and 4321 to receive usual care. Overall, 482 patients (22.9%) in the dexamethasone group and 1110 patients (25.7%) in the usual care group died within 28 days after randomization (age-adjusted rate ratio, 0.83; 95% confidence interval [CI], 0.75 to 0.93; P<0.001). The proportional and absolute between-group differences in mortality varied considerably according to the level of respiratory support that the patients were receiving at the time of randomization. In the dexamethasone group, the incidence of death was lower than that in the usual care group among patients receiving invasive mechanical ventilation (29.3% vs. 41.4%; rate ratio, 0.64; 95% CI, 0.51 to 0.81) and among those receiving oxygen without invasive mechanical ventilation (23.3% vs. 26.2%; rate ratio, 0.82; 95% CI, 0.72 to 0.94) but not among those who were receiving no respiratory support at randomization (17.8% vs. 14.0%; rate ratio, 1.19; 95% CI, 0.92 to 1.55).ConclusionsIn patients hospitalized with Covid-19, the use of dexamethasone resulted in lower 28-day mortality among those who were receiving either invasive mechanical ventilation or oxygen alone at randomization but not among those receiving no respiratory support. (Funded by the Medical Research Council and National Institute for Health Research and others; RECOVERY ClinicalTrials.gov number, NCT04381936. opens in new tab; ISRCTN number, 50189673. opens in new tab.)

/pdf/dexamethasone-in-hospitalized-patients-with-covid-19-2p8rrjo50m.pdf

Dexamethasone in Hospitalized Patients with Covid-19

Evolution and ecology of influenza A viruses.

Background: The RNA-dependent RNA polymerase of Influenza A virus is a determinant of viral pathogenicity and host range that is responsible for transcribing and replicating the negative sense segmented viral genome (vRNA). Transcription produces capped and polyadenylated mRNAs whereas genome replication involves the synthesis of an alternative plus-sense transcript (cRNA) with unmodified termini that is copied back to vRNA. Viral mRNA transcription predominates at early stages of viral infection, while later, negative sense genome replication is favoured. However, the "switch" that regulates the transition from transcription to replication is poorly understood. Results: We show that temperature strongly affects the balance between plus and minus-sense RNA synthesis with high temperature causing a large decrease in vRNA accumulation, a moderate decrease in cRNA levels but (depending on genome segment) either increased or unchanged levels of mRNA. We found no evidence implicating cellular heat shock protein activity in this effect despite the known association of hsp70 and hsp90 with viral polymerase components. Temperature-shift experiments indicated that polymerase synthesised at 41°C maintained transcriptional activity even though genome replication failed. Reduced polymerase association with viral RNA was seen in vivo and in confirmation of this, in vitro binding assays showed that temperature increased the rate of dissociation of polymerase from both positive and negative sense promoters. However, the interaction of polymerase with the cRNA promoter was particularly heat labile, showing rapid dissociation even at 37°C. This suggested that vRNA synthesis fails at elevated temperatures because the polymerase does not bind the promoter. In support of this hypothesis, a mutant cRNA promoter with vRNA-like sequence elements supported vRNA synthesis at higher temperatures than the wild-type promoter. Conclusion: The differential stability of negative and positive sense polymerase-promoter complexes explains why high temperature favours transcription over replication and has implications for the control of viral RNA synthesis at physiological temperatures. Furthermore, given the different body temperatures of birds and man, these finding suggest molecular hypotheses for how polymerase function may affect host range.

/pdf/temperature-sensitive-influenza-a-virus-genome-replication-2jznmbswsj.pdf

Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes

A novel functional domain of an alpha-like DNA polymerase. The binding site on the herpes simplex virus polymerase for the viral UL42 protein.

The swine-origin H1N1 influenza A virus emerged in early 2009 and caused the first influenza pandemic in 41 years. The virus has spread efficiently to both the Northern and the Southern Hemispheres and has been associated with over 16,000 deaths. Given the virus’s recent zoonotic origin, there is concern that the virus could acquire signature mutations associated with the enhanced pathogenicity of previous pandemic viruses or H5N1 viruses with pandemic potential. We tested the hypothesis that mutations in the polymerase PB2 gene at residues 627 and 701 would enhance virulence but found that influenza viruses containing these mutations in the context of the pandemic virus polymerase complex are attenuated in cell culture and mice.

Paul Digard

Papers

Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes

A novel functional domain of an alpha-like DNA polymerase. The binding site on the herpes simplex virus polymerase for the viral UL42 protein.

The PB2-E627K Mutation Attenuates Viruses Containing the 2009 H1N1 Influenza Pandemic Polymerase

Options for the control of influenza VI

Characterisation of influenza A viruses with mutations in segment 5 packaging signals