Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

Localization to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division

The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus (group III coronavirus) (J. A. Hiscox et al., J. Virol. 75:506-512, 2001), indicating that nucleolar localization of the N protein is a common feature of the coronavirus family and is possibly of functional significance. Nucleolar localization signals were identified in the domain III region of the N protein from all three coronavirus groups, and this suggested that transport of N protein to the nucleus might be an active process. In addition, our results suggest that the N protein might function to disrupt cell division. Thus, we observed that approximately 30% of cells transfected with the N protein appeared to be undergoing cell division. The most likely explanation for this is that the N protein induced a cell cycle delay or arrest, most likely in the G2/M phase. In a fraction of transfected cells expressing coronavirus N proteins, we observed multinucleate cells and dividing cells with nucleoli (which are only present during interphase). These findings are consistent with the possible inhibition of cytokinesis in these cells.

Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats

The interaction between the gut microbiota and their mammalian host is known to have far-reaching consequences with respect to metabolism and health. We investigated the effects of eight days of oral antibiotic exposure (penicillin and streptomycin sulfate) on gut microbial composition and host metabolic phenotype in male Han-Wistar rats (n = 6) compared to matched controls. Early recolonization was assessed in a third group exposed to antibiotics for four days followed by four days recovery (n = 6). Fluorescence in situ hybridization analysis of the intestinal contents collected at eight days showed a significant reduction in all bacterial groups measured (control, 1010.7 cells/g feces; antibiotic-treated, 108.4). Bacterial suppression reduced the excretion of mammalian-microbial urinary cometabolites including hippurate, phenylpropionic acid, phenylacetylglycine and indoxyl-sulfate whereas taurine, glycine, citrate, 2-oxoglutarate, and fumarate excretion was elevated. While total bacterial counts remained notably lower in the recolonized animals (109.1 cells/g faeces) compared to the controls, two cage-dependent subgroups emerged with Lactobacillus/Enterococcus probe counts dominant in one subgroup. This dichotomous profile manifested in the metabolic phenotypes with subgroup differences in tricarboxylic acid cycle metabolites and indoxyl-sulfate excretion. Fecal short chain fatty acids were diminished in all treated animals. Antibiotic treatment induced a profound effect on the microbiome structure, which was reflected in the metabotype. Moreover, the recolonization process was sensitive to the microenvironment, which may impact on understanding downstream consequences of antibiotic consumption in human populations.

Evolution of network structure in polymer-stabilised liquid crystals

Experiments were performed to investigate the evolution of structure and morphology of the network in polymer-stabilised liquid crystals. In situ optical microscopy revealed that the morphology was significantly altered by extraction of the LC host, while scanning electron microscopy showed that the network morphology was also dependent on the polymerisation conditions and closely related to the depletion of monomer, as monitored by high performance liquid chromatography. Transmission electron microscopy allowed observation of internal structure, resolving microstructure on the order of 0. 1 μm.

A transmission electron microscopy study of polymer-stabilised liquid crystal structure

A method has been established for observing the internal structure of the network component of polymer-stabilised liquid crystals. In situ photopolymerisation of a mesogenic diacrylate monomer using ultraviolet light leads to a sparse network (∼1 wt%) within a nematic host. Following polymerisation, the host was removed through dissolution in heptane, revealing the network. In order to observe a cross-section through the network, it was embedded in a resin and then sectioned using an ultramicrotome. However, imaging of the network was not possible due to poor contrast. To improve this, several reagents were used for network staining, but only one was successful: bromine. The use of a Melinex-resin composite for sectioning was also found to be advantageous. Imaging of the network using transmission electron microscopy revealed solid “droplets” of width 0.07–0.20 μm, possessing an open, yet homogeneous structure, with no evidence for any large-scale internal structures.

Long-distance dispersal and its influence on adaptation to host resistance in a heterogeneous landscape

Small propagules like pollen or fungal spores may be dispersed by the wind over distances of hundreds or thousands of kilometres,even though the median dispersal may be only a few metres. Such long-distance dispersal is a stochastic event which may be exceptionally important in shaping a population. It has been found repeatedly in field studies that subpopulations of wind-dispersed fungal pathogens virulent on cultivars with newly introduced, effective resistance genes are dominated by one or very few genotypes. The role of propagule dispersal distributions with distinct behaviour at long distances in generating this characteristic population structure was studied by computer simulation of dispersal of clonal organisms in a heterogeneous environment with fields of unselective and selective hosts. Power-law distributions generated founder events in which new, virulent genotypes rapidly colonized fields of resistant crop varieties and subsequently dominated the pathogen population on both selective and unselective varieties, in agreement with data on rust and powdery mildew fungi. An exponential dispersal function, with extremely rare dispersal over long distances, resulted in slower colonization of resistant varieties by virulent pathogens or even no colonization if the distance between susceptible source and resistant target fields was sufficiently large. The founder events resulting from long-distance dispersal were highly stochastic and exact quantitative prediction of genotype frequencies will therefore always be difficult.

Probiotics, prebiotics, and the host microbiome: the science of translation

Recent advances in our understanding of the community structure and function of the human microbiome have implications for the potential role of probiotics and prebiotics in promoting human health. A group of experts recently met to review the latest advances in microbiota/microbiome research and discuss the implications for development of probiotics and prebiotics, primarily as they relate to effects mediated via the intestine. The goals of the meeting were to share recent advances in research on the microbiota, microbiome, probiotics, and prebiotics, and to discuss these findings in the contexts of regulatory barriers, evolving healthcare environments, and potential effects on a variety of health topics, including the development of obesity and diabetes; the long-term consequences of exposure to antibiotics early in life to the gastrointestinal (GI) microbiota; lactose intolerance; and the relationship between the GI microbiota and the central nervous system, with implications for depression, cognition, satiety, and mental health for people living in developed and developing countries. This report provides an overview of these discussions.

Complexity and anisotropy in host morphology make populations less susceptible to epidemic outbreaks

One of the challenges in epidemiology is to account for the complex morphological structure of hosts such as plant roots, crop fields, farms, cells, animal habitats and social networks, when the transmission of infection occurs between contiguous hosts. Morphological complexity brings an inherent heterogeneity in populations and affects the dynamics of pathogen spread in such systems. We have analysed the influence of realistically complex host morphology on the threshold for invasion and epidemic outbreak in an SIR (susceptible-infected-recovered) epidemiological model. We show that disorder expressed in the host morphology and anisotropy reduces the probability of epidemic outbreak and thus makes the system more resistant to epidemic outbreaks. We obtain general analytical estimates for minimally safe bounds for an invasion threshold and then illustrate their validity by considering an example of host data for branching hosts (salamander retinal ganglion cells). Several spatial arrangements of hosts with different degrees of heterogeneity have been considered in order to separately analyse the role of shape complexity and anisotropy in the host population. The estimates for invasion threshold are linked to morphological characteristics of the hosts that can be used for determining the threshold for invasion in practical applications.

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

Bis(3-endo-camphoryl)phosphinic acid, a non-racemic helical supramolecular host with aquapores

Bis(3-endo-camphoryl)phosphinic acid (1) was prepared by the reaction of the lithium enolate of D-(+)-camphor and phosphorous trichloride followed by an oxidative work up. Compound 1 crystallizes from wet toluene as monohydrate 1·H2O, which was investigated by X-ray crystallography. Molecules of 1 are associated by strong hydrogen bonds giving rise to the formation of a supramolecular helix. The interior channel of the helix is filled by a one-dimensional (1D) string of water molecules that are also associated by hydrogen bonding. The 1D string adopts a twisted zigzag conformation. Although the hydrogen bond networks are not cross-linked both the screw of the helix and the twist of the 1D string of water molecules are left-handed (M) and controlled by the chiral camphoryl residues situated on the exterior of the helix. The overall supramolecular structure is strongly reminiscent of aquaporin-1, a significant membrane-channel protein responsible for the transport of water into the cells.

Structure and properties of self-assembled narural rubber/multi-walled carbon nanotube composites

Natural rubber (NR)/multi-walled carbon nanotube (MWCNTs) composites were prepared bycombining self-assembly and latex compounding techniques. The acid-treated MWCNTs (H2SO4: HNO3=3:1,volume ratio) were self-assembled with poly (diallyldimethylammonium chloride) (PDDA) through electrostaticadhesion. In the second assembling, NR/MWCNTs composites were developed by mixing MWCNTs/PDDAsolution with NR latex. The results show that MWCNTs are homogenously distributed throughout the NRmatrix as single tube and present a great interfacial adhesion with NR phase when MWCNTs contents areless than 3 wt%. Moreover, the addition of the MWCNTs brings about the remarkable enhancement in tensilestrength and crosslink density compared with the NR host, and the data peak at 2 wt% MWCNTs loadings.When more MWCNTs are loaded, aggregations of MWCNTs are gradually generated, and the tensile strengthand crosslink both decrease to a certain extent.

Population biology of swift (Apus apus) ectoparasites in relation to host reproductive success

1We censused ectoparasite populations of adult and nestling swifts over the course of the host's breeding season. Nearly all of the birds were infested with chewing lice and two-thirds of the nests were infested with louse flies. Feather mites were observed but not quantified.2Lice and louse flies both showed aggregated distributions among hosts. Louse eggs, hatched lice and adult louse flies had negative binomial distributions, whereas the aggregated distribution of louse fly pupae was not adequately described by negative binomial or Poisson models.3Transmission of lice from parents to offspring was documented. A comparison of the age structure of lice on parents and offspring indicated that most transmission was by nymphal lice.4Host reproductive success and survival appeared to be independent of the number of lice or louse flies. Neither parasite correlated with the number, body mass, or date of fledging of young birds, nor with the overwinter survival of adults. We caution, however, that experimental manipulations of parasite load are required for a definitive test of the impact of ectoparasites on evolutionary fitness components.

Ultra-structure and histochemistry of digestive cells of Podisus nigrispinus (Hemiptera: Pentatomidae) fed with prey reared on bt-cotton

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ileum of the Cephalotes ants: A specialized structure to harbor symbionts microorganisms

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mistletoes Play Different Roles in a Modular Host-Parasite Network

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)