Fourier transform infrared (FT-IR) spectroscopy of genomic DNA to discriminate F1 progenies from their paternal lineage of Chinese cabbage (Brassica rapa subsp. pekinensis)

Fourier transform infrared spectroscopy (FT-IR) spectroscopy in combination with multivariate analysis was used to discriminate two different F1 hybrid lines from their parental inbred lines. Genomic DNA was isolated from leaves of three parental lines and two F1 hybrid lines of Brassica campestris subsp. pekinensis. Purified genomic DNA was analyzed by FT-IR spectroscopy in the spectral region from 4,000 to 400 cm^?1. FT-IR spectra confirmed typical spectral differences between the frequency regions of N–H stretching (amide I) and C=O stretching vibrations (amide II) as well as PO₂ ^? ionized asymmetric and symmetric stretching. Principal component analysis was able to discriminate between F1 hybrid progenies depending on their parental lineages, even though they share the same maternal background. Partial least squares discriminant analysis gave a more clear discrimination between the two parental lines and their hybrid progenies. These FT-IR spectral differences might be directly related to subtle changes in the base functional group and backbone structures of genomic DNA. Considering these results, this technique could provide a solid research foundation for FT-IR spectral-based rapid diagnosis, selection, and discrimination of parental lines from their progenies. Furthermore, this technique could be applied to test purity in the hybrid seed industry.     

Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection

The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.Avian H5N1 influenza viruses, currently circulating mainly in southeast Asia, are likely to cause the next influenza pandemic (18, 26, 37). The supply of embryonated eggs for traditional influenza vaccine production may be endangered in this case. Efforts to produce inactivated H5N1 vaccines in permanent cells have resulted in large-scale manufacturing, for instance, in Vero cells (21). This approach, based either on fermentation of H5N1 wild-type (wt) viruses (21) or on viruses attenuated by reverse genetics (9, 31), is the most straightforward strategy for egg-independent, rapid vaccine production.A further approach that may result in more widely available, egg-independent H5 vaccines is the use of recombinant viral vectors expressing protective antigens. Promising protection results were obtained so far with adenovirus-based vectors in mouse models (13, 14). Adenovirus vectors are usually produced in permanent complementing cell lines (11) and have been widely used in clinical trials. Cancellation of a recent trial involving human immunodeficiency virus adenovirus vectors due to suspected enhancement of disease, however, may complicate the future use of these vectors (41). Poxvirus vectors, including recombinant modified vaccinia virus Ankara (MVA) (1, 43), constitute a further class of vectors that have been used to express H5N1 influenza virus antigens (5, 22, 44, 46). Usually, however, the large-scale production of MVA is carried out in primary chicken cells, since these are the most efficient production substrates and are also accepted by regulators. In a pandemic, this production platform may not be available because permanent nontumorigenic avian cell lines are currently not available for production.In this study, we used a permanent cell line, modified Vero cells, to produce nonreplicating vaccinia virus vectors expressing the H5 hemagglutinin (HA), the major influenza virus protective antigen. The defective vaccinia virus (dVV) vectors are safe due to their lack of replication capacity in normal hosts, while they share the superior immunizing properties of poxviral live vaccines (15, 33). Previously, a permanent cell line based on rabbit kidney cells was engineered to express the essential vaccinia virus D4R gene encoding the enzyme uracil-DNA-glycosylase. This cell line allowed the construction of replication-deficient vaccinia virus vectors (15). In this work, a complementing system based on Vero cells was established and used to produce the defective vaccinia virus vector dVV-HA5. The vector was used to immunize mice and was compared to an inactivated whole-virus (whv) vaccine and to replicating control viruses. The dVV-HA5 candidate vaccine induced neutralizing antibodies and full protection, similar to results with an inactivated whv vaccine. Further, it is important to ensure that the immune responses generated by a pandemic influenza vaccine give long-lived, broad, cross-clade protection. While antibody responses to influenza virus provide protective immunity, T-cell responses are also thought to play an important role in clearance of and recovery from infections. Thus, a vaccine which can produce both effective humoral and T-cell responses would be advantageous. A vaccinia virus vector-based pandemic influenza vaccine has the potential to provide this advantage.     

Models for analysing species’ presence/absence data at two time points

Species’ presence/absence at two time points is a very common form of ecological data. It is the simplest type of longitudinal study and has fundamental applications in ecological succession, environmental monitoring, and climate change scenarios. Despite its widespread commonality the use of statistical regression to analyse such data has been wanting. We propose the use of the bivariate odds-ratio model to analyse these data. Seldomly used in ecology, it is argued as being suitable, especially within a constrained ordination framework. In particular, this paper presents the constrained ordination-odds ratio framework as a potentially important key in understanding the underlying processes of niche theory dynamics, e.g., local extinction and colonization probabilities can be described in terms of it. Some of the mathematical and statistical challenges associated with more ambitious extensions are highlighted. As examples, with an underlying Poisson abundance model, a complementary log-log link for the marginal probabilities is shown to be more appropriate. We then develop this model based on the zero-inflated Poisson distribution since excess absences relative to a Poisson distribution is frequent in practice. Two vegetation data sets are used for illustrative purposes.     

Gene Ontology annotation of the rice blast fungus, <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background

Magnaporthe oryzae, the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/MultiDownloads.html. However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of M. oryzae genome assembly.

Methods

A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of M. oryzae and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked.

Results

In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site http://amigo.geneontology.org/cgi-bin/amigo/go.cgi. Additionally, the genome of M. oryzae is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website http://scotland.fgl.ncsu.edu/smeng/GoAnnotationMagnaporthegrisea.html. The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System.

Conclusion

Our analysis provides comprehensive and robust GO annotations of the M. oryzae genome assemblies that will be solid foundations for further functional interrogation of M. oryzae.

     

Parasite annexins--new molecules with potential for drug and vaccine development

In the last few years, annexins have been discovered in several nematodes and other parasites, and distinct differences between the parasite annexins and those of the hosts make them potentially attractive targets for anti-parasite therapeutics. Annexins are ubiquitous proteins found in almost all organisms across all kingdoms.Here, we present an overview of novel annexins from parasitic organisms, and summarize their phylogenetic and biochemical properties, with a view to using them as drug or vaccine targets. Building on structural and biological information that has been accumulated for mammalian and plant annexins, we describe a predicted additional secondary structure element found in many parasite annexins that may confer unique functional properties, and present a specific antigenic epitope for use as a vaccine.     

p21-Activated kinases are required for transformation in a cell-based model of neurofibromatosis type 2

Background

NF2 is an autosomal dominant disease characterized by development of bilateral vestibular schwannomas and other benign tumors in central nervous system. Loss of the NF2 gene product, Merlin, leads to aberrant Schwann cell proliferation, motility, and survival, but the mechanisms by which this tumor suppressor functions remain unclear. One well-defined target of Merlin is the group I family of p21-activated kinases, which are allosterically inhibited by Merlin and which, when activated, stimulate cell cycle progression, motility, and increased survival. Here, we examine the effect of Pak inhibition on cells with diminished Merlin function.

Methodology/Principal Findings

Using a specific peptide inhibitor of group I Paks, we show that loss of Pak activity restores normal cell movement in cells lacking Merlin function. In addition, xenografts of such cells form fewer and smaller tumors than do cells without Pak inhibition. However, in tumors, loss of Pak activity does not reduce Erk or Akt activity, two signaling proteins that are thought to mediate Pak function in growth factor pathways.

Conclusions/Significance

These results suggest that Pak functions in novel signaling pathways in NF2, and may serve as a useful therapeutic target in this disease.     

Survey of bed bugs in infested premises in Malaysia and Singapore

A total of 54 bed bug‐infested sites (hotels, public accommodations, and residential premises) in Malaysia and Singapore was surveyed between July, 2005 and December, 2008. Only one species of bed bug was found, the tropical bed bug Cimex hemipterus (Fabricius). Bed bug infestations were common in hotels and public accommodations when compared to residential premises. The three most common locations of infestation within an infested premise were the bedding (31.1%), the headboard (30.3%), and cracks and crevices surrounding the baseboard, wall, or floor (23.5%). We speculate that the route of movement of bed bugs in hotels and public accommodations is more direct than in residential premises.     

Aerosolized Bacillus anthracis infection in New Zealand white rabbits: natural history and intravenous levofloxacin treatment

The natural history for inhalational Bacillus anthracis (Ames strain) exposure in New Zealand white rabbits was investigated to better identify potential, early biomarkers of anthrax. Twelve SPF Bordetella-free rabbits were exposed to 150 LD(50) aerosolized B. anthracis spores, and clinical signs, body temperature, complete blood count, bacteremia, and presence of protective antigen in the blood (that is, antigenemia) were examined. The development of antigenemia and bacteremia coincided and preceded both pyrexia and inversion of the heterophil:lymphocyte ratio, an indicator of infection. Antigenemia was determined within 1 h by electrochemiluminescence immunoassay, compared with the 24-h traditional culture needed for bacteremia determination. Rabbits appeared clinically normal until shortly before succumbing to anthrax approximately 47 h after challenge or approximately 22 h after antigenemia, which suggests a relatively narrow therapeutic window of opportunity. To evaluate the therapeutic rabbit model, B. anthracis-exposed rabbits were treated (after determination of antigenemia and later confirmed to be bacteremic) intravenously with the fluoroquinolone antibiotic levofloxacin for 5 d at a total daily dose of 25 or 12.5 mg/kg, resulting in nearly 90% and 70% survival, respectively, to the study end (28 d after challenge). The peak level for 12.5 mg/kg was equivalent to that observed for a 500-mg daily levofloxacin dose in humans. These results suggest that intravenous levofloxacin is an effective therapeutic against inhalational anthrax. Taken together, our findings indicate that antigenemia is a viable and early biomarker for B. anthracis infection that can be used as a treatment trigger to allow for timely intervention against this highly pathogenic disease.     

β1 Integrin Establishes Endothelial Cell Polarity and Arteriolar Lumen Formation via a Par3-Dependent Mechanism

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Highlights? β1 integrin deletion results in loss of endothelial polarity and vessel patency ? β1 integrin regulates cell-cell adhesion protein distribution in the endothelium ? Polarity loss is typified by decreased Par3 expression ? Par3 can partially rescue lumen formation in the β1 integrin mutant