Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

PATCHY: simulating and visualizing the effects of stomatal patchiness on photosynthetic CO2 exchange studies

Abstract. In this study, computer modelling has been used to simulate and analyse the effects which non-homogeneous stomatal conductance (patchiness) might have on the appearance of the macroscopic relationship between photosynthetic CO₂ assimilation (A) and intercellular CO₂ (Q) in leaves. The problem was formalized using the assumptions that (1) the biochemical model of Farquhar, von Caemmerer & Berry applies [ Planta , 149 , 78–90 (1980)]; (2) that the parameters for the model are fixed for the period required to determine the relationship; (3) that the distribution of conductances in any leaf area is normal (but restricted to positive values); and (4) that the leaf is perfectly heterobaric. The model is interactive, allowing the user to explore–well beyond the conditions for which data are presented here–the effects of carboxylation capacity, photosynthetic electron transport rate and photorespiration over a range of possible conductances and degrees of patchiness. Regardless of the parameters used in the model, the results fail to predict the change in appearance of the A -C₁ curve which has been attributed to patchiness in other reports: even when conductance varies with a standard deviation of twice its mean value, the effects on the curve are minor. The need for reconsideration of the methods currently used to interpret gas exchange studies is indicated.     

Marked seasonal variation in the wild mouse gut microbiota

Recent studies have provided an unprecedented view of the microbial communities colonizing captive mice; yet the host and environmental factors that shape the rodent gut microbiota in their natural habitat remain largely unexplored. Here, we present results from a 2-year 16 S ribosomal RNA gene sequencing-based survey of wild wood mice (Apodemus sylvaticus) in two nearby woodlands. Similar to other mammals, wild mice were colonized by 10 bacterial phyla and dominated by the Firmicutes, Bacteroidetes and Proteobacteria. Within the Firmicutes, the Lactobacillus genus was most abundant. Putative bacterial pathogens were widespread and often abundant members of the wild mouse gut microbiota. Among a suite of extrinsic (environmental) and intrinsic (host-related) factors examined, seasonal changes dominated in driving qualitative and quantitative differences in the gut microbiota. In both years examined, we observed a strong seasonal shift in gut microbial community structure, potentially due to the transition from an insect- to a seed-based diet. This involved decreased levels of Lactobacillus, and increased levels of Alistipes (Bacteroidetes phylum) and Helicobacter. We also detected more subtle but statistically significant associations between the gut microbiota and biogeography, sex, reproductive status and co-colonization with enteric nematodes. These results suggest that environmental factors have a major role in shaping temporal variations in microbial community structure within natural populations.     

The rise to dominance of the angiosperm kingdom: dispersal,habitat widening and evolution during the Late Cretaceous of Europe

Coiffard, C. & Gomez, B. 2009: The rise to dominance of the angiosperm kingdom: dispersal, habitat widening and evolution during the Late Cretaceous of Europe. Lethaia, Vol. 43, pp. 164–169. The earliest fossil records of angiosperms in Europe occur in the Barremian and consist of freshwater wetland plants. From the Barremian onwards, angiosperms show a stepwise widening of their ecological range with the result that they inhabited most environments by the Cenomanian. Nevertheless, most angiosperms had still restricted habitats, while a few angiosperm trees were confined to disturbed environments, such as channel margins. A Wagner’s Parsimony Method analysis performed on a fossil plant and locality database from the Turonian to the Campanian of Europe indicates continued decrease in richness of ferns and gymnosperms compared with angiosperms, turnover between conifer and palm trees in freshwater‐related swamps at about the Cenomanian/Turonian boundary, and spreading of angiosperm trees through the floodplains. The ecological range of angiosperm trees was increased, being recorded in channel margins from the Cenomanian and spreading over floodplains (e.g. Platanaceae) and swamps (e.g. Arecaceae) by the Campanian. These new ecological ranges and successions went with innovative architectures, such as dicot trees and palm trees. Most living core angiosperm families had their earliest representatives in the Late Cretaceous, which should be considered as the dawn of modern angiosperm forests. □Core angiosperms, Europe, Late Cretaceous, palms, Wagner’s Parsimony Method.     

Cell Potentials and Turgor Pressures in Epidermal Cells of Tradescantia and Commelina

Cell membrane potentials have been measured both in epidermalstrips and intact leaf sections of Tradescantia virginiana andCommelina communis, and in epidermal cells over green and overalbino mesophyll cells of T. albiflora var. albovittata. Membranepotentials (_cell) in strips were considerably lower than thosein intact sections and were insensitive to light and to theabsence or presence of calcium. Their response to external cationlevels was indifferent to ionic species. However, in intactleaf sections incubated with calcium present, membrane potentialsresponded to K⁺ levels but not to Na⁺. were more negative thancells in epidermal strips, and responded to changes in illumination. Long-term recordings of _cell and vacuolar K⁺ levels in T. virginianaduring stomatal closure suggest that the fluctuations of _cellwere unrelated to K⁺ movement (which we could not detect) andthus probably to stomatal movement as well. Turgor pressures measured in epidermal cells of intact leafsections of T. virginiana were found to be of the same magnitudeas those previously reported for epidermal strips. It is concludedthat epidermal cells maintain their solute contents during strippingwithout the involvement of an electrophysiological transportsystem. With the possible exception of lateral subsidiary cells,there was no evidence suggesting that ordinary epidermal cellsare capable of osmotic adjustment even when additional KCI wassupplied in the osmoticum. Absolute turgor levels in intactleaf sections kept at constant external KCI were unrelated tosteady state _cell.     

Biochemical and ecological characterization of two peroxidase isoenzymes from the mangrove, Rhizophora mangle

This study examines phenolic peroxidase (POX) in Rhizophora mangle L. leaves in order to assess its role in phenolic manipulation and H₂O₂ scavenging. Sun-exposed and understorey leaves experiencing varying degrees of nutrient stress were analysed from an oligotrophic cay off the coast of Belize. POX activity was unaffected by growth environment, but increased throughout leaf development and persisted through senescence and after abscission. Histochemical analyses indicated POX activity throughout leaf tissues, especially in the apoplast. Phenolics were similarly broadly distributed. Two isoenzymes of POX were partially characterized with pIs of 4.1 and 6.3 and masses of 65.5 and 54.3 kDa, respectively. The larger, more acidic isoenzyme showed especially high heat stability, showing no reduced activity after 24 h at 60 °C. Rhizophora mangle POX oxidized quercetin preferentially, and, to a lesser extent, coniferyl alcohol, caffeic acid, chlorogenic acid, and p-coumaric acid. It did not oxidize ascorbate, but ascorbate could act as a secondary electron donor in the presence of a phenolic substrate and H₂O₂. However, because quercetin and other aglycones were not present in R. mangle leaves, and because POX showed no activity with the most abundant leaf flavonoid, rutin, it was concluded that detoxification of H₂O₂ is secondary to the other roles of POX in manipulation of phenolics.     

Comparison of marker types and map assumptions using Markov chain Monte Carlo-based linkage analysis of COGA data

We performed multipoint linkage analysis of the electrophysiological trait ECB21 on chromosome 4 in the full pedigrees provided by the Collaborative Study on the Genetics of Alcoholism (COGA). Three Markov chain Monte Carlo (MCMC)-based approaches were applied to the provided and re-estimated genetic maps and to five different marker panels consisting of microsatellite (STRP) and/or SNP markers at various densities. We found evidence of linkage near the GABRB1 STRP using all methods, maps, and marker panels. Difficulties encountered with SNP panels included convergence problems and demanding computations.