Coronavirus replication complex formation utilizes components of cellular autophagy

The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell. However, the mechanism by which these DMVs form has not been determined. Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated. The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12. MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition. MHV replication is impaired in autophagy knockout, APG5-/-, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5-/-cells. In MHV-infected APG5-/-cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen. The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication. Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes.     

Detection of Enterobacter sakazakii in Dried Infant Milk Formula by Cationic-Magnetic-Bead Capture

Enterobacter sakazakii has been associated with life-threatening infections in premature low-birth-weight infants. Contaminated infant milk formula (IMF) has been implicated in cases of E. sakazakii meningitis. Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations would positively contribute towards risk reduction across the infant formula food chain. Here we report on the development of a simple method, combining charged separation and growth on selective agar, to detect E. sakazakii in IMF. This protocol can reliably detect 1 to 5 CFU of E. sakazakii in 500 g of IMF in less than 24 h.     

Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5-15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.     

Implementing plant hydraulic architecture within the LPJ Dynamic Global Vegetation Model

Aim To implement plant hydraulic architecture within the Lund–Potsdam–Jena Dynamic Global Vegetation Model (LPJ–DGVM), and to test the model against a set of observational data. If the model can reproduce major patterns in vegetation and ecosystem processes, we consider this to be an important linkage between plant physiology and larger‐scale ecosystem dynamics. Location The location is global, geographically distributed. Methods A literature review was carried out to derive model formulations and parameter values for representing the hydraulic characteristics of major global plant functional types (PFTs) in a DGVM. After implementing the corresponding formulations within the LPJ–DGVM, present‐day model output was compared to observational data. Results The model reproduced observed broad‐scale patterns in potential natural vegetation, but it failed to distinguish accurately between different types of grassland and savanna vegetation, possibly related to inadequate model representations of water fluxes in the soil and wildfire effects. Compared to a version of the model using an empirical formulation for calculating plant water supply without considering plant hydraulic architecture, the new formulation improved simulated patterns of vegetation in particular for dry shrublands. Global‐scale simulation results for runoff and actual evapotranspiration (AET) corresponded well to available data. The model also successfully reproduced the magnitude and seasonal cycle of AET for most EUROFLUX forests, while modelled variation in NPP across a large number of sites spanning several biomes showed a strong correlation with estimates from field measurements. Main conclusions The model was generally confirmed by comparison to observational data. The novel model representation of water flow within plants makes it possible to resolve mechanistically the effects of hydraulic differences between plant functional groups on vegetation structure, water cycling, and competition. This may be an advantage when predicting ecosystem responses to nonextant climates, in particular in areas dominated by dry shrubland vegetation.     

Linking landscape history and dispersal traits in grassland plant communities

Dispersal limitation and long-term persistence are known to delay plant species’ responses to habitat fragmentation, but it is still unclear to what extent landscape history may explain the distribution of dispersal traits in present-day plant communities. We used quantitative data on long-distance seed dispersal potential by wind and grazing cattle (epi- and endozoochory), and on persistence (adult plant longevity and seed bank persistence) to quantify the linkages between dispersal and persistence traits in grassland plant communities and current and past landscape configurations. The long-distance dispersal potential of present-day communities was positively associated with the amounts of grassland in the historical (1835, 1938) landscape, and with a long continuity of grazing management—but was not associated with the properties of the current landscape. The study emphasises the role of history as a determinant of the dispersal potential of present-day grassland plant communities. The importance of long-distance dispersal processes has declined in the increasingly fragmented modern landscape, and long-term persistent species are expected to play a more dominant role in grassland communities in the future. However, even within highly fragmented landscapes, long-distance dispersed species may persist locally—delaying the repayment of the extinction debt.     

A spatial analytical approach for selecting reintroduction sites for burbot in English rivers

1. Availability of suitable habitat is a prerequisite for species reintroduction success, and to ensure population persistence, investigations of a species’ habitat utilisation throughout its life history should be conducted as part of a feasibility study. 2. Habitat utilisation models for burbot, Lota lota, developed using data from field studies conducted in France and Germany and information from the literature were used to assess the feasibility of reintroducing burbot into rivers of its former native range in eastern England. 3. Per cent tree roots, aquatic vegetation and flow types were important predictors of adult burbot abundance. Furthermore, the habitat utilisation models were supplemented with information from the literature, which suggested that off‐channel habitat such as wetlands and backwaters is important for spawning and nursery stages. 4. An assessment of the habitat availability in the rivers of the burbot’s former native range using variables related to spawning and nursery and adult life stages showed that although adult habitat was widely distributed, the availability of spawning and nursery habitat was less abundant, potentially limiting successful reestablishment. 5. Potential suitable habitat was concentrated in the central and southern areas of the species’ former English distribution. Overall, rivers of the burbot’s former range potentially afford suitable habitat to sustain a reintroduced population. However, sites should be preferentially selected on the basis of having appropriate spawning and nursery areas.     

Twinning propensity and offspring in utero growth covary in rural African women

In humans and other mammals, some females are more likely to experience twin pregnancies than others, but the reasons behind such individual variation are poorly understood. One hypothesis invokes variation in the dynamics of the insulin-like growth factor (IGF) system, which also regulates foetal growth. Using data from a rural African population living in a highly seasonal environment, we test a novel prediction generated by this hypothesis, that maternal twinning status predicts offspring birthweight. We found that among singleton offspring who experience a favourable in utero environment (born January-June), births before and after twins are, respectively, associated with a 134.07 g and 226.41 g increase in birthweight compared with those born to non-twinning mothers. These results were not mediated by maternal anthropometry. This is consistent with a role for the IGF system in individual variation in twinning propensity, a possibility with implications for understanding mechanisms of life-history variation in humans and other vertebrates.     

Iron incorporation and post-malaria anaemia

Background

Iron supplementation is employed to treat post-malarial anaemia in environments where iron deficiency is common. Malaria induces an intense inflammatory reaction that stalls reticulo-endothelial macrophagal iron recycling from haemolysed red blood cells and inhibits oral iron absorption, but the magnitude and duration of these effects are unclear.

Methodology/Principal Findings

We examined the red blood cell incorporation of oral administered stable isotopes of iron and compared incorporation between age matched 18 to 36 months old children with either anaemia post-malaria (n = 37) or presumed iron deficiency anaemia alone (n = 36). All children were supplemented for 30 days with 2 mg/kg elemental iron as liquid iron sulphate and administered ⁵⁷Fe and ⁵⁸Fe on days 1 and 15 of supplementation respectively. ⁵⁷Fe and⁵⁸Fe incorporation were significantly reduced (8% vs. 28%: p<0.001 and 14% vs. 26%: p = 0.045) in the malaria vs. non-malaria groups. There was a significantly greater haemoglobin response in the malaria group at both day 15 (p = 0.001) and 30 (p<0.000) with a regression analysis estimated greater change in haemoglobin of 7.2 g/l (s.e. 2.0) and 10.1 g/l (s.e. 2.5) respectively.

Conclusion/Significance

Post-malaria anaemia is associated with a better haemoglobin recovery despite a significant depressant effect on oral iron incorporation which may indicate that early erythropoetic iron need is met by iron recycling rather than oral iron. Supplemental iron administration is of questionable utility within 2 weeks of clinical malaria in children with mild or moderate anaemia.