Plasma membrane localization is required for RasA-mediated polarized morphogenesis and virulence of Aspergillus fumigatus

Ras is a highly conserved GTPase protein that is essential for proper polarized morphogenesis of filamentous fungi. Localization of Ras proteins to the plasma membrane and endomembranes through posttranslational addition of farnesyl and palmitoyl residues is an important mechanism through which cells provide specificity to Ras signal output. Although the Aspergillus fumigatus RasA protein is known to be a major regulator of growth and development, the membrane distribution of RasA during polarized morphogenesis and the role of properly localized Ras signaling in virulence of a pathogenic mold remain unknown. Here we demonstrate that Aspergillus fumigatus RasA localizes primarily to the plasma membrane of actively growing hyphae. We show that treatment with the palmitoylation inhibitor 2-bromopalmitate disrupts normal RasA plasma membrane association and decreases hyphal growth. Targeted mutations of the highly conserved RasA palmitoylation motif also mislocalized RasA from the plasma membrane and led to severe hyphal abnormalities, cell wall structural changes, and reduced virulence in murine invasive aspergillosis. Finally, we provide evidence that proper RasA localization is independent of the Ras palmitoyltransferase homolog, encoded by erfB, but requires the palmitoyltransferase complex subunit, encoded by erfD. Our results demonstrate that plasma membrane-associated RasA is critical for polarized morphogenesis, cell wall stability, and virulence in A. fumigatus.     

Prevalence of malaria and related haemosporidian parasites in two shorebird species with different winter habitat distribution

Parasites can have strong effects on host life-history and behaviour, and result in changes in host population dynamics and community structure. We applied a PCR-based technique and examined prevalence of malaria and related haemosporidian parasites in two arctic breeding shorebird species: the Semipalmated Sandpiper (Calidris pusilla) and the Pectoral Sandpiper (C. melanotos). During the non-breeding season, Semipalmated Sandpipers inhabit coastal marine habitats, whereas Pectoral Sandpipers are found in inland areas. In accordance with the hypothesis that the risk of parasite infection is higher in a species wintering in freshwater areas, we found Plasmodium sp. infection during the breeding season only in Pectoral Sandpipers, whereas Semipalmated Sandpipers were parasite free. However, even in Pectoral Sandpipers sampled in the arctic, prevalence of malaria parasites was very low (<3% of individuals, n = 114). Overall, three different Plasmodium sp. lineages were found, one of which has never been described before.     

Localization of the sulfate/anion exchanger in the rat liver

Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis.     

Genetic and Pathobiologic Characterization of Pandemic H1N1 2009 Influenza Viruses from a Naturally Infected Swine Herd

Since its initial identification in Mexico and the United States, concerns have been raised that the novel H1N1 influenza virus might cause a pandemic of severity comparable to that of the 1918 pandemic. In late April 2009, viruses phylogenetically related to pandemic H1N1 influenza virus were isolated from an outbreak on a Canadian pig farm. This outbreak also had epidemiological links to a suspected human case. Experimental infections carried out in pigs using one of the swine isolates from this outbreak and the human isolate A/Mexico/InDRE4487/2009 showed differences in virus recovery from the lower respiratory tract. Virus was consistently isolated from the lungs of pigs infected with A/Mexico/InDRE4487/2009, while only one pig infected with A/swine/Alberta/OTH-33-8/2008 yielded live virus from the lung, despite comparable amounts of viral RNA and antigen in both groups of pigs. Clinical disease resembled other influenza virus infections in swine, albeit with somewhat prolonged virus antigen detection and delayed viral-RNA clearance from the lungs. There was also a noteworthy amount of genotypic variability among the viruses isolated from the pigs on the farm. This, along with the somewhat irregular pathobiological characteristics observed in experimentally infected animals, suggests that although the virus may be of swine origin, significant viral evolution may still be ongoing.The zoonotic potential of swine influenza viruses is well recognized (18), and pigs have been considered a leading candidate for the role of intermediate host in the generation of reassortant influenza A viruses with pandemic potential. This has been largely based on genomic analysis of influenza A viruses isolated from swine and the fact that α2,3-linked sialic acid (avian-like) and α2,6-linked sialic acid (human-like) receptors are both abundant in the swine respiratory tract (12). Despite this, there is no direct evidence that the reassortment of the 1957 and the 1968 human pandemic viruses occurred in pigs (28). Furthermore, it is very likely that the 1918 pandemic virus was introduced to pigs from humans (8, 31). The origins of influenza A viruses that have been isolated from pigs include those that are wholly human or avian, as well as reassortants containing swine, human, and avian genes (2, 20, 29). Although there have been several instances of swine-to-human transmission, for example, that of triple-reassortant swine influenza (H1) viruses (rH1N1), which appeared after 1998, they did not lead to establishment of sustained transmission in the human population (23).In the early spring of 2009, Mexico and the United States reported clusters of human pneumonia cases caused by a novel H1N1 influenza A virus. This virus subsequently spread across the globe at an unprecedented rate, prompting the WHO to declare a pandemic in June 2009. Phylogenetic analysis has inferred that the virus is likely a reassortant between a North American triple-reassortant swine H1N1 or H1N2 virus and a Eurasian lineage H1N1 swine influenza virus (7, 19). Bayesian molecular-clock analysis of each gene of this novel H1N1 virus (24) concluded that the mean evolutionary rate is typical of that of swine influenza viruses but that the duration of unsampled diversity for each gene segment had means that ranged from 9.24 to 17.15 years, suggesting that the proposed ancestors of this virus may have been circulating undetected for nearly a decade. Inadequate surveillance and characterization of influenza A viruses that circulate in swine have been blamed for this evolutionary gap.On 28 April 2009 the Canadian Food Inspection Agency (CFIA) became involved in a suspected outbreak of swine influenza on a pig farm in Leslieville, Alberta, Canada. The farm was a 220-sow farrow-to-finish operation consisting of approximately 2,200 animals that ranged from newborn piglets to market weight pigs. The animals were not vaccinated against swine influenza, and although there had been prior problems with porcine reproductive and respiratory syndrome virus and Mycoplasma hypopneumoniae, two etiologic agents of the swine respiratory disease complex, the herd had been stable with respect to respiratory disease. Beginning 20 April, approximately 25% of the pregrower and grower pigs in two of the barns exhibited respiratory problems with clinical signs that included an acute onset of coughing, lethargy, and loss of appetite. These clinical signs were preceded by the hiring of a carpenter on 14 April to work on the ventilation system in the same two barns. This individual had been ill for 2 days after his return from Mexico on 12 April (10). Given the evolving situation in Mexico and the United States, the CFIA and Alberta Agriculture and Rural Development decided to place the herd under quarantine and to carry out a full epidemiological and laboratory investigation.Here, we report on the characterization of the first pandemic H1N1 2009 viruses to be isolated from a naturally infected pig herd. Genetic sequence data from several viruses isolated from this outbreak have provided a glimpse into the mutation frequencies associated with replication of the virus in the swine host. Experimental infections of pigs comparing one of these swine isolates with the human isolate A/Mexico/InDRE4487/2009(H1N1) were also carried out and have provided insights into the pathobiological behavior of these viruses in pigs.     

Dehydration improves cryopreservation of coconut (Cocos nucifera L.)

Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatisation and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut cultivars and is useful for the routine cryopreservation of coconut genetic resources.     

Regulation of mucous cell metaplasia in bronchial asthma

Mucous cell metaplasia (MCM), defined by the appearance of mucous cells in airways where mucous cells were not present, is a consistent pathologic characteristic in the peripheral airways of bronchial asthma. Under mild inflammatory conditions MCM occurs as a result of pre-existing airway epithelial cells (AECs) starting to express mucin genes and differentiating into mucous cells. Under extensive inflammatory responses, AECs proliferate, and the development of MCM involves the differentiation of pre-existing and proliferating cells into mucous cells. Epithelial cell numbers per mm basal lamina are increased by approximately 30%. IL-13 is the central cytokine that is responsible for MCM in asthma through GABA-R- and STAT6-mediated mechanisms involving the calcium-activated chloride channel CLCA. IL-13 is also responsible for the proliferation of AECs by causing cells to produce TGFalpha that acts on the epidermal growth factor (EGF) receptor. Normally, resolution of MCM involves two distinct mechanisms. 1) Some of the metaplastic mucous cells stop the synthesis of mucus and dedifferentiate into Clara or serous cells to reconstitute the epithelium. 2) When proliferation of epithelial cells had occurred, approximately 30% of metaplastic cells are eliminated during the resolution process. Thus, a safe approach to reducing IL-13-induced MCM would involve blocking mucous synthesis and storage, blocking secretion of stored mucus, and eliminating hyperplastic mucous cells. Understanding the molecular mechanisms of each of these processes is necessary for developing effective therapies for reducing mucous hypersecretion in asthma and leading to a repaired epithelium.     

Philopatry of winter moult area in migratory Great Reed Warblers Acrocephalus arundinaceus demonstrated by stable isotope profiles

Stable carbon- (δ¹³C), nitrogen- (δ¹⁵N) and hydrogen (δD) isotope profiles in feathers of migratory Great Reed Warblers Acrocephalus arundinaceus recaptured for 2 or more years in 6 successive years were examined to test whether the isotope profiles of individual warblers appeared to be consistent between years. Similar isotopic signatures in successive years suggested that individual birds tended to return and grow their feathers in Afro-tropical wintering habitats that generate similar δ¹³C, δ¹⁵N and δD signatures. Previous studies have shown that Great Reed Warblers exhibit strong natal and breeding philopatry, with most of the surviving birds returning to the breeding site. The present study of feather δ¹³C, δ¹⁵N and δD isotopic values demonstrate the year-to-year fidelity might also include the African moulting sites in this migratory species.