Switching species tropism: an effective way to manipulate the feline coronavirus genome

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.     

The Ovicidal,Larvacidal and Adulticidal Properties of 5,5′-Dimethyl-2,2′-Bipyridyl against Drosophila melanogaster

Insecticide resistance has limited the number of available chemical options for insect pest control. Hence there is a need for new chemistries with novel modes of action. Here we investigate the mode of action for an insecticide that has not yet been released for commercial use. The ovicidal, larvacidal and adulticidal effects of 5,5′-dimethyl -2, 2′-dipyridyl (termed Ha44), which is being developed as a treatment for head lice, were evaluated in the Drosophila melanogaster model system. Ha44 demonstrated significant activity against embryos and was capable of arresting development at a number of stages of embryogenesis. The effects of Ha44 on embryos was shown to be reversible following the addition of the metal ions Fe(II) and Fe(III), Cu and Zn. When larvae were exposed to Ha44, lethality was recorded at similar concentrations to those observed for embryos. Using an eYFP reporter system it was shown that Ha44 was able to reduce the levels of both copper and zinc in the digestive tract, confirming the binding of Ha44 to these metals in vivo. Ha44 has further been shown to inhibit a zinc containing metalloproteinase in vitro. Exposure of adult flies to Ha44 resulted in lethality, but at higher concentrations than those observed for embryos and larvae. The median lethal dose in adult flies was shown to be associated with the type of exposure, with an LD-50 of 1.57 mM being recorded following the direct contact of flies with Ha44, while an LD-50 of 12.29 mM was recorded following the ingestion of the compound. The capacity of Ha44 to act on all stages of the life-cycle and potentially via a range of targets suggests that target site resistance is unlikely to evolve.     

Comparable low-level mosaicism in affected and non affected tissue of a complex CDH patient

In this paper we present the detailed clinical and cytogenetic analysis of a prenatally detected complex Congenital Diaphragmatic Hernia (CDH) patient with a mosaic unbalanced translocation (5;12). High-resolution whole genome SNP array confirmed a low-level mosaicism (20%) in uncultured cells, underlining the value of array technology for identification studies. Subsequently, targeted Fluorescence In-Situ Hybridization in postmortem collected tissues demonstrated a similar low-level mosaicism, independently of the affected status of the tissue. Thus, a higher incidence of the genetic aberration in affected organs as lung and diaphragm cannot explain the severe phenotype of this complex CDH patient. Comparison with other described chromosome 5p and 12p anomalies indicated that half of the features presented in our patient (including the diaphragm defect) could be attributed to both chromosomal areas. In contrast, a few features such as the palpebral downslant, the broad nasal bridge, the micrognathia, microcephaly, abnormal dermatoglyphics and IUGR better fitted the 5p associated syndromes only. This study underlines the fact that low-level mosaicism can be associated with severe birth defects including CDH. The contribution of mosaicism to human diseases and specifically to congenital anomalies and spontaneous abortions becomes more and more accepted, although its phenotypic consequences are poorly described phenomena leading to counseling issues. Therefore, thorough follow-up of mosaic aberrations such as presented here is indicated in order to provide genetic counselors a more evidence based prediction of fetal prognosis in the future.     

Ovomucoid intervening sequences specify functional domains and generate protein polymorphism

Two thirds of the natural chicken ovomucoid gene has been sequenced, including all exons and the intron sequences surrounding all fourteen intron/ exon junctions. The junction sequences surrounding four of the introns are redundant; however, the sequences surrounding the other three introns contain no redundancies and thus the splicing sites at either end of these three introns are unambiguous. The splicing in all cases conforms to the GT-AG rule. The ovomucoid gene sequence around intron F can be used to predict the cause of an internal deletion polymorphism in the ovomucoid protein, which is an apparent error in the processing of the ovomucoid pre-mRNA. We also compare the structural organization of the ovomucoid gene with the ovomucoid protein sequence to examine theories of the evolution of ovomucoids as well as the origin of intervening sequences. This analysis suggests that the present ovomucoid gene evolved from a primordial ovomucoid gene by two separate intragenic duplications. Furthermore, sequence analyses suggest that introns were present in the primordial ovomucoid gene before birds and mammals diverged, about 300 million years ago. Finally, the positions of the introns within the ovomucoid gene support the theory that introns separate gene segments that code for functional domains of proteins and provide insight on the manner by which eucaryotic genes were constructed during the process of evolution.     

Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.     

Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA

Background

The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized.

Principal Findings

The binding of glycine betaine to purified OpuA and OpuAC (K_D = 4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher.

Conclusions

Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.     

An integrated approach for automating validation of extracted ion chromatographic peaks

SUMMARY: Accurate determination of extracted ion chromatographic peak areas in isotope-labeled quantitative proteomics is difficult to automate. Manual validation of identified peaks is typically required. We have integrated a peak confidence scoring algorithm into existing tools which are compatible with analysis pipelines based on the standards from the Institute for Systems Biology. This algorithm automatically excludes incorrectly identified peaks, improving the accuracy of the final protein expression ratio calculation. SOURCE AND SUPPLEMENTARY INFORMATION: http://www.chem.uky.edu/research/lynn/Nelson.pdf.     

Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.     

Modeling Neisseria meningitidis B metabolism at different specific growth rates

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. At the Netherlands Vaccine Institute (NVI) a vaccine against serogroup B organisms is currently being developed. This study describes the influence of the growth rate of N. meningitidis on its macro-molecular composition and its metabolic activity and was determined in chemostat cultures. In the applied range of growth rates, no significant changes in RNA content and protein content with growth rate were observed in N. meningitidis. The DNA content in N. meningitidis was somewhat higher at the highest applied growth rate. The phospholipid and lipopolysaccharide content in N. meningitidis changed with growth rate but no specific trends were observed. The cellular fatty acid composition and the amino acid composition did not change significantly with growth rate. Additionally, it was found that the PorA content in outer membrane vesicles was significantly lower at the highest growth rate. The metabolic fluxes at various growth rates were calculated using flux balance analysis. Errors in fluxes were calculated using Monte Carlo Simulation and the reliability of the calculated flux distribution could be indicated, which has not been reported for this type of analysis. The yield of biomass on substrate (Y(x/s)) and the maintenance coefficient (m(s)) were determined as 0.44 (+/-0.04) g g(-1) and 0.04 (+/-0.02) g g(-1) h(-1), respectively. The growth associated energy requirement (Y(x/ATP)) and the non-growth associated ATP requirement for maintenance (m(ATP)) were estimated as 0.13 (+/-0.04) mol mol(-1) and 0.43 (+/-0.14) mol mol(-1) h(-1), respectively. It was found that the split ratio between the Entner-Doudoroff and the pentose phosphate pathway, the sole glucose utilizing pathways in N. meningitidis, had a minor effect on ATP formation rate but a major effect on the fluxes going through for instance the citric-acid cycle. For this reason, we presented flux ranges for underdetermined parts of metabolic network rather than presenting single flux values, which is more commonly done in literature.