Accuracy of an optical active-marker system to track the relative motion of rigid bodies

The measurement of relative motion between two moving bones is commonly accomplished for in vitro studies by attaching to each bone a series of either passive or active markers in a fixed orientation to create a rigid body (RB). This work determined the accuracy of motion between two RBs using an Optotrak optical motion capture system with active infrared LEDs. The stationary noise in the system was quantified by recording the apparent change in position with the RBs stationary and found to be 0.04 degrees and 0.03 mm. Incremental 10 degrees rotations and 10-mm translations were made using a more precise tool than the Optotrak. Increasing camera distance decreased the precision or increased the range of values observed for a set motion and increased the error in rotation or bias between the measured and actual rotation. The relative positions of the RBs with respect to the camera-viewing plane had a minimal effect on the kinematics and, therefore, for a given distance in the volume less than or close to the precalibrated camera distance, any motion was similarly reliable. For a typical operating set-up, a 10 degrees rotation showed a bias of 0.05 degrees and a 95% repeatability limit of 0.67 degrees. A 10-mm translation showed a bias of 0.03 mm and a 95% repeatability limit of 0.29 mm. To achieve a high level of accuracy it is important to keep the distance between the cameras and the markers near the distance the cameras are focused to during calibration.     

Biocidal activity of three wood essential oils against Ixodes scapularis (Acari: Ixodidae), Xenopsylla cheopis (Siphonaptera: Pulicidae), and Aedes aegypti (Diptera: Culicidae)

The biocidal activity of three steam distilled wood essential oils-incense cedar, Calocedrus decurrens (Torr.) Florin; Port-Orford-cedar, Chamaecyparis lawsoniana (A. Murr.) Parl.; and western juniper, Juniperus occidentalis (Hook)--were evaluated against adult Aedes aegypti (L.) (Diptera: Culicidae) and Xenopsylla cheopis (Rothchild) (Siphonaptera: Pulicidae) and nymphal Ixodes scapularis Say (Acari: Ixodidae). In vitro laboratory bioassays were conducted to establish baseline dose-mortality data through 24 h. Incense cedar heartwood was the most toxic to all three vector species followed in order of activity by western juniper and Port-Orford-cedar based on LC50 and LC90 values. Ae. aegypti were substantially more susceptible to the oils than either I. scapularis or X. cheopis.     

NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.     

A vectored measles virus induces hepatitis B surface antigen antibodies while protecting macaques against measles virus challenge

Hepatitis B virus (HBV) acute and chronic infections remain a major worldwide health problem. Towards developing an anti-HBV vaccine with single-dose scheme potential, we engineered infectious measles virus (MV) genomic cDNAs with a vaccine strain background and expression vector properties. Hepatitis B surface antigen (HBsAg) expression cassettes were inserted into this cDNA and three MVs expressing HBsAg at different levels generated. All vectored MVs, which secrete HBsAg as subviral particles, elicited humoral responses in MV-susceptible genetically modified mice. However, small differences in HBsAg expression elicited vastly different HBsAg antibody levels. The two vectors inducing the highest HBsAg antibody levels were inoculated into rhesus monkeys (Macaca mulatta). After challenge with a pathogenic MV strain (Davis87), control naive monkeys showed a classic measles rash and high viral loads. In contrast, all monkeys immunized with vaccine or a control nonvectored recombinant vaccine or HBsAg-expressing vectored MV remained healthy, with low or undetectable viral loads. After a single vaccine dose, only the vector expressing HBsAg at the highest levels elicited protective levels of HBsAg antibodies in two of four animals. These observations reveal an expression threshold for efficient induction of HBsAg humoral immune responses. This threshold is lower in mice than in macaques. Implications for the development of divalent vaccines based on live attenuated viruses are discussed.     

Purification of host cell enzymes involved in adeno-associated virus DNA replication

Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase delta, but not polymerase epsilon or alpha, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase delta complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase delta, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase delta, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.     

Disruption of diacylglycerol kinase delta (DGKD) associated with seizures in humans and mice

We report a female patient with a de novo balanced translocation, 46,X,t(X;2)(p11.2;q37)dn, who exhibits seizures, capillary abnormality, developmental delay, infantile hypotonia, and obesity. The 2q37 breakpoint observed in association with the seizure phenotype is of particular interest, because it lies near loci implicated in epilepsy in humans and mice. Fluorescence in situ hybridization mapping of the translocation breakpoints showed that no known genes are disrupted at Xp11.2, whereas diacylglycerol kinase delta (DGKD) is disrupted at 2q37. Expression studies in Drosophila and mouse suggest that DGKD is involved in central nervous system development and function. Electroencephalographic assessment of Dgkd mutant mice revealed abnormal epileptic discharges and electrographic seizures in three of six homozygotes. These findings implicate DGKD disruption by the t(X;2)(p11.2;q37)dn in the observed phenotype and support a more general role for DGKD in the etiology of seizures.     

Genome partitioning of genetic variation for height from 11,214 sibling pairs

Height has been used for more than a century as a model by which to understand quantitative genetic variation in humans. We report that the entire genome appears to contribute to its additive genetic variance. We used genotypes and phenotypes of 11,214 sibling pairs from three countries to partition additive genetic variance across the genome. Using genome scans to estimate the proportion of the genomes of each chromosome from siblings that were identical by descent, we estimated the heritability of height contributed by each of the 22 autosomes and the X chromosome. We show that additive genetic variance is spread across multiple chromosomes and that at least six chromosomes (i.e., 3, 4, 8, 15, 17, and 18) are responsible for the observed variation. Indeed, the data are not inconsistent with a uniform spread of trait loci throughout the genome. Our estimate of the variance explained by a chromosome is correlated with the number of times suggestive or significant linkage with height has been reported for that chromosome. Variance due to dominance was not significant but was difficult to assess because of the high sampling correlation between additive and dominance components. Results were consistent with the absence of any large between-chromosome epistatic effects. Notwithstanding the proposed architecture of complex traits that involves widespread gene-gene and gene-environment interactions, our results suggest that variation in height in humans can be explained by many loci distributed over all autosomes, with an additive mode of gene action.     

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.     

Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence

Trehalose fulfils a wide variety of functions in cells, acting as a stress protectant, storage carbohydrate and compatible solute. Recent evidence, however, indicates that trehalose metabolism may exert important regulatory roles in the development of multicellular eukaryotes. Here, we show that in the plant pathogenic fungus Magnaporthe grisea trehalose-6-phosphate (T6P) synthase (Tps1) is responsible for regulating the pentose phosphate pathway, intracellular levels of NADPH and fungal virulence. Tps1 integrates glucose-6-phosphate (G6P) metabolism with nitrogen source utilisation, and thereby regulates the activity of nitrate reductase. Activity of Tps1 requires an associated regulator protein Tps3, which is also necessary for pathogenicity. Tps1 controls expression of the nitrogen metabolite repressor gene, NMR1, and is required for expression of virulence-associated genes. Functional analysis of Tps1 indicates that its regulatory functions are associated with binding of G6P, but independent of Tps1 catalytic activity. Taken together, these results demonstrate that Tps1 is a central regulator for integration of carbon and nitrogen metabolism, and plays a pivotal role in the establishment of plant disease.