Papillomavirus particles assembled in 293TT cells are infectious in vivo

Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article, we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal. Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected 293TT cells.     

Regulation of Gi by the CB1 cannabinoid receptor C-terminal juxtamembrane region: structural requirements determined by peptide analysis

A CB1 cannabinoid receptor peptide fragment from the C-terminal juxtamembrane region autonomously inhibits adenylyl cyclase activity in a neuroblastoma membrane preparation. The cannabinoid receptor antagonist, SR141716A, failed to block the response. The peptide was able to evoke the response in membranes from Chinese hamster ovary (CHO) cells that do not express the CB1 receptor. These studies are consistent with a direct activation of Gi by the peptide. To test the importance of a BXBXXB sequence, Lys403 was acetylated, resulting in a peptide having similar affinity but reduced efficacy. N-Terminal truncation of Arg401 resulted in a 6-fold loss of affinity, which was not further reduced by sequential truncation of up to the first seven amino acids, four of which are charged. N-Terminal-truncated peptides exhibited maximal activity, suggesting that Gi activation can be conferred by the remaining amino acids. Truncation of the C-terminal Glu417 or substitution of Glu417 by a Leu or of Arg401 by a Norleucine reduced activity at 100 microM. The C-terminal juxtamembrane peptide was constrained to a loop peptide by placement of Cys residues at both terminals and disulfide coupling. This modification reduced the affinity 3-fold but yielded near-maximal efficacy. Blocking the Cys termini resulted in a loss of efficacy. Circular dichroism spectropolarimetry revealed that all C-terminal juxtamembrane peptide analogues exist in a random coil conformation in an aqueous environment. A hydrophobic environment (trifluoroethanol) failed to induce alpha-helix formation in the C-terminal juxtamembrane peptide but did so in less active peptides. The anionic detergent sodium dodecyl sulfate induced alpha-helix formation in all analogues except the loop peptide, where it induces a left-handed PII conformation. It is concluded that alpha-helix formation is not required for Gi activation.     

Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA,the Central Component of the β-Barrel Assembly Machine

The β-barrel assembly machinery (BAM) mediates folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. BAM is a five-protein complex consisting of the β-barrel OMP BamA and lipoproteins BamB, -C, -D, and -E. High resolution structures of all the individual BAM subunits and a BamD-BamC complex have been determined. However, the overall complex architecture remains elusive. BamA is the central component of BAM and consists of a membrane-embedded β-barrel and a periplasmic domain with five polypeptide translocation-associated (POTRA) motifs thought to interact with the accessory lipoproteins. Here we report the crystal structure of a fusion between BamB and a POTRA3–5 fragment of BamA. Extended loops 13 and 17 protruding from one end of the BamB β-propeller contact the face of the POTRA3 β-sheet in BamA. The interface is stabilized by several hydrophobic contacts, a network of hydrogen bonds, and a cation-π interaction between BamA Tyr-255 and BamB Arg-195. Disruption of BamA-BamB binding by BamA Y255A and probing of the interface by disulfide bond cross-linking validate the physiological relevance of the observed interface. Furthermore, the structure is consistent with previously published mutagenesis studies. The periplasmic five-POTRA domain of BamA is flexible in solution due to hinge motions in the POTRA2–3 linker. Modeling BamB in complex with full-length BamA shows BamB binding at the POTRA2–3 hinge, suggesting a role in modulation of BamA flexibility and the conformational changes associated with OMP folding and insertion.     

Inoculation of millet with azospirillum

Summary Millet plants (Pennisetum glaucum) were grown at three levels of nitrogen fertilization with and without an inoculum of live nitrogen-fixing Azospirillum cells. The highest average rate of nitrogen fixation as estimated from acetylene reduction by excised preincubated roots was only 23g N₂ fixed per ha per day and occurred after treatment with low levels of nitrogen amendment. The average rates of acetylene reduction for intact plants at all treatments were also low. The lack of significant nitrogen fixation due to an Azospirillum-millet association in this study was substantiated by plant dry weight analysis, and determination of the nitrogen content of plants, pot leachate, and soil. There was significant correlation between the total nitrogen content of the plants per pot at the termination of the experiment and the amount of nitrogen fertilizer added initially, but there was no effect of inoculum on final total nitrogen content.     

Role of Us9 Phosphorylation in Axonal Sorting and Anterograde Transport of Pseudorabies Virus

Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread in vivo. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.     

Interactions between a decapacitation factor and mouse spermatozoa appear to involve fucose residues and a GPI-anchored receptor

Epididymal mouse spermatozoa have a surface-associated decapacitation factor (DF) that can be removed precociously by centrifugation, resulting in acceleration of capacitation and increased fertilizing ability. Addition of exogenous DF to capacitated suspensions inhibits fertilizing ability and reverses capacitation in acrosome-intact cells. DF appears to regulate a Ca²⁺-ATPase, located primarily in the postacrosomal region. The present investigations of DF↮spermatozoon interaction indicate that DF can be removed from uncapacitated cells by treatment with phosphatidylinositol-specific phospholipase C (PIC), suggesting the involvement of a glycosylphosphatidylinositol (GPI) moiety. However, exogenous DF cannot reassociate with PIC-treated spermatozoa, suggesting that DF may bind to spermatozoa via a GPI-anchored receptor. DF binding appears to involve fucose residues, since depletion of endogenous DF followed by brief exposure to fucose (0.1–10 mM) prevented DF reassociation with cells. Furthermore, 5 mM fucose could displace DF from uncapacitated cells, accelerating capacitation and resulting in a higher proportion of fertilized oocytes, with increased polyspermy, than obtained with untreated controls. FITC-labelled fucosylated BSA bound specifically to the postacrosomal region, binding being inhibited by both excess fucose and crude DF. UEA I, a lectin with specificity for fucose residues, bound to the postacrosomal region of cells preincubated in fucose but not crude DF, and blocked DF binding to DF-depleted cells. These results are consistent with the DF binding, via fucose residues, to a GPI-anchored receptor. Fucose binding sites are in the same region where Ca²⁺-ATPase, the enzyme regulated by DF, has been localized; these results support the hypothesis that DF modulates capacitation by regulating enzyme activity and hence the intracellular Ca²⁺ concentration. Mol. Reprod. Dev. 51:193–202, 1998. © 1998 Wiley-Liss, Inc.     

A role for alphaV integrin subunit in TGF-beta-stimulated osteoclastogenesis

TGF-beta increases bone resorption in vivo and greatly increases osteoclast formation stimulated by receptor activator of NF-kappaB ligand (RANKL) in vitro. TGF-beta does not independently affect the differentiation state of RAW264.7 preosteoclasts, but increases cell attachment to vitronectin. This effect is mediated by increased expression of alphaV integrin subunit mRNA and protein. Concomitant with induction of osteoclast differentiation, RANKL causes relocation of alphaV to focal sites in the cell. This effect is potentiated by TGF-beta. Integrin blockade disrupts both attachment to vitronectin and RANKL-induced osteoclast formation, but culture on vitronectin has little effect. Ectopic expression of alphaV stimulates multinucleation of RAW264.7 cells and increases the number of osteoclasts formed in the presence of RANKL. These data suggest that TGF-beta potentiates RANKL-induced osteoclast formation, in part by increased expression of the alphaV integrin subunit, which may contribute to cell fusion.     

What works in conservation? Using expert assessment of summarised evidence to identify practices that enhance natural pest control in agriculture

This paper documents an exercise to synthesize and assess the best available scientific knowledge on the effectiveness of different farm practices at enhancing natural pest regulation in agriculture. It demonstrates a novel combination of three approaches to evidence synthesis—systematic literature search, collated synopsis and evidence assessment using an expert panel. These approaches follow a logical sequence moving from a large volume of disparate evidence to a simple, easily understandable answer for use in policy or practice. The example of natural pest regulation in agriculture was selected as a case study within two independent science-policy interface projects, one European and one British. A third funder, a private business, supported the final stage to translate the synthesized findings into a useful, simplified output for agronomists. As a whole, the case study showcases how a network of scientific knowledge holders and knowledge users can work together to improve the use of science in policy and practice. The process identified five practices with good evidence of a benefit to natural pest regulation, with the most beneficial being ‘Combine trap and repellent crops in a push–pull system’. It highlights knowledge gaps, or potential research priorities, by showing practices considered important by stakeholders for which there is not enough evidence to make an assessment of effects on natural pest regulation, including ‘Alter the timing of pesticide application.’ Finally, the process identifies several important practices where the volume of evidence of effects on natural pest regulation was too large (>300 experimental studies) to be summarised with the resources available, and for which focused systematic reviews may be the best approach. These very well studied practices include ‘Reduce tillage’ and ‘Plant more than one crop per field’.     

Two-category model of task allocation with application to ant societies

In many network models of interacting units such as cells or insects, the coupling coefficients between units are independent of the state of the units. Here we analyze the temporal behavior of units that can switch between two 'category' states according to rules that involve category-dependent coupling coefficients. The behaviors of the category populations resulting from the asynchronous random updating of units are first classified according to the signs of the coupling coefficients using numerical simulations. They range from isolated fixed points to lines of fixed points and stochastic attractors. These behaviors are then explained analytically using iterated function systems and birth-death jump processes. The main inspiration for our work comes from studies of non-hierarchical task allocation in, e.g., harvester ant colonies where temporal fluctuations in the numbers of ants engaged in various tasks occur as circumstances require and depend on interactions between ants. We identify interaction types that produce quick recovery from perturbations to an asymptotic behavior whose characteristics are function of the coupling coefficients between ants as well as between ants and their environment. We also compute analytically the probability density of the population numbers, and show that perturbations in our model decay twice as fast as in a model with random switching dynamics. A subset of the interaction types between ants yields intrinsic stochastic asymptotic behaviors which could account for some of the experimentally observed fluctuations. Such noisy trajectories are shown to be random walks with state-dependent biases in the 'category population' phase space. With an external stimulus, the parameters of the category-switching rules become time-dependent. Depending on the growth rate of the stimulus in comparison to its population-dependent decay rate, the dynamics may qualitatively differ from the case without stimulus. Our simple two-category model provides a framework for understanding the rich variety of behaviors in network dynamics with state-dependent coupling coefficients, and especially in task allocation processes with many tasks.     

Directly observed therapy (DOT) for individuals with HIV: successes and challenges

Many HIV-infected individuals have not reaped the benefits of combination antiretroviral therapy due to inability either to adhere to medications or to access care. It is now recognized that innovative approaches are needed to increase access and adherence to highly active antiretroviral therapy (HAART), especially among these hard-to-reach populations. Due to the success of directly observed therapy (DOT) for the treatment of Mycobacterium tuberculosis (TB), our group and others have questioned whether DOT can be adapted to deliver HAART to hard-to-reach communities. In this review, we discuss the results of pilot programs that have utilized DOT in multiple different settings and use case studies to explore the diverse issues that can arise when implementing these programs. As we continue to gain more experience with observed therapy, we will be able to better identify the key components for a successful intervention.