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Licensed live attenuated virus vaccines capable of expressing transgenes from other pathogens have the potential to reduce the number of childhood immunizations by eliciting robust immunity to multiple pathogens simultaneously. Recombinant attenuated measles virus (rMV) derived from the Edmonston Zagreb vaccine strain was engineered to express simian immunodeficiency virus (SIV) Gag protein for the purpose of evaluating the immunogenicity of rMV as a vaccine vector in rhesus macaques. rMV-Gag immunization alone elicited robust measles-specific humoral and cellular responses, but failed to elicit transgene (Gag)-specific immune responses, following aerosol or intratracheal/intramuscular delivery. However, when administered as a priming vaccine to a heterologous boost with recombinant adenovirus serotype 5 expressing the same transgene, rMV-Gag significantly enhanced Gag-specific T lymphocyte responses following rAd5 immunization. Gag-specific humoral responses were not enhanced, however, which may be due to either the transgene or the vector. Cellular response priming by rMV against the transgene was highly effective even when using a suboptimal dose of rAd5 for the boost. These data demonstrate feasibility of using rMV as a priming component of heterologous prime-boost vaccine regimens for pathogens requiring strong cellular responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To validate the Dutch translation of the Fear of Progression Questionnaire-Short Form (FoP-Q-SF) for patients with systemic sclerosis (SSc). Although concerns about the future are often expressed by patients with SSc, there is no valid quantitative measure available to assess the extent to which patients with SSc are troubled by those concerns., Methods: Measurement properties of the FoP-Q-SF were assessed using a cross-sectional design that included 215 patients with SSc. Patients completed the FoP-Q-SF as well as questionnaires on physical and psychological functioning. Psychometric properties of the FoP-Q-SF were assessed using the Consensus-Based Standards for the Selection of Health Status Measurement Instruments checklist., Results: The mean ± SD FoP-Q-SF score in patients with SSc was 30.05 ± 8.97. There were no indications of floor or ceiling effects. Confirmatory factor analysis supported the single-factor structure of the questionnaire (χ(2) [52] = 96.84, P < 0.001, root mean square error of approximation = 0.064, chi-square/df ratio = 1.86). Cronbach's alpha was 0.86 for the questionnaire. Most of our a priori hypotheses (11 of 12) were confirmed, supporting the construct validity of the questionnaire., Conclusion: A valid measure is now available to assess fear of disease progression in patients with SSc, which is significant since fear of progression is one of the most important stressors in this patient population., (Copyright © 2012 by the American College of Rheumatology.)

Isolated microspores and pollen suspension of Brassica napus "Topas" cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a "comet-tail" form. These GC "tail" MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a "whole-mount" immunodetection technique and confocal laser-scanning microscopy was essential.

Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26-Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years.

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A 'mild' heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3-8 celled.

The use of adenoviruses (Ad) as vaccine vectors against a variety of pathogens has demonstrated their capacity to elicit strong antibody and cell-mediated immune responses. Adenovirus serotype C vectors, such as Ad serotype 5 (Ad5), expressing Ebolavirus (EBOV) glycoprotein (GP), protect completely after a single inoculation at a dose of 10(10) viral particles. However, the clinical application of a vaccine based on Ad5 vectors may be hampered, since impairment of Ad5 vaccine efficacy has been demonstrated for humans and nonhuman primates with high levels of preexisting immunity to the vector. Ad26 and Ad35 segregate genetically from Ad5 and exhibit lower seroprevalence in humans, making them attractive vaccine vector alternatives. In the series of studies presented, we show that Ad26 and Ad35 vectors generate robust antigen-specific cell-mediated and humoral immune responses against EBOV GP and that Ad5 immune status does not affect the generation of GP-specific immune responses by these vaccines. We demonstrate partial protection against EBOV by a single-shot Ad26 vaccine and complete protection when this vaccine is boosted by Ad35 1 month later. Increases in efficacy are paralleled by substantial increases in T- and B-cell responses to EBOV GP. These results suggest that Ad26 and Ad35 vectors warrant further development as candidate vaccines for EBOV.

The heavy fermion compound Ce3Pd20Si6 is one of the rare examples of a cubic system with a readily accessible quantum critical point. Ce atoms at the two different sites 4a and 8c of the crystal structure have recently been shown to have different crystal field ground states (Γ7 and Γ8, respectively) and are assumed to be responsible for the two different low-temperature phase transitions at T(L) and T(U), which have been tentatively attributed to antiferromagnetic and antiferroquadrupolar order, respectively. Here we present electrical resistivity measurements in a wide temperature range (50 mK-300 K) on two new representatives of the La substitution series Ce(3 - x)La(x)Pd20Si6, x=1/3 and 2/3. Put in the context of previously published data our results indicate that La preferentially occupies the 4a site and that Ce ions at the 8c site have a sizably larger Kondo temperature. Low-temperature resistivity measurements in applied magnetic fields suggest that (disorder smeared) quantum critical points exist in the x=1/3 and 2/3 samples at fields below 1 T.

The immunogenicity and durability of genetic vaccines are influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown limited comparative efficacy as a stand-alone platform in primates, due possibly to suboptimal gene expression or cell targeting. Here, regimens using DNA plasmids modified for optimal antigen expression and recombinant adenovirus (rAd) vectors, all encoding the glycoprotein (GP) gene from Angola Marburg virus (MARV), were compared for their ability to provide immune protection against lethal MARV Angola infection. Heterologous DNA-GP/rAd5-GP prime-boost and single-modality rAd5-GP, as well as the DNA-GP-only vaccine, prevented death in all vaccinated subjects after challenge with a lethal dose of MARV Angola. The DNA/DNA vaccine induced humoral responses comparable to those induced by a single inoculation with rAd5-GP, as well as CD4(+) and CD8(+) cellular immune responses, with skewing toward CD4(+) T-cell activity against MARV GP. Vaccine regimens containing rAd-GP, alone or as a boost, exhibited cellular responses with CD8(+) T-cell dominance. Across vaccine groups, CD8(+) T-cell subset dominance comprising cells exhibiting a tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) double-positive functional phenotype was associated with an absence or low frequency of clinical symptoms, suggesting that both the magnitude and functional phenotype of CD8(+) T cells may determine vaccine efficacy against infection by MARV Angola.

The canonical view of heavy fermion quantum criticality assumes a single quantum critical point separating the paramagnet from the antiferromagnet. However, recent experiments on Yb-based heavy fermion compounds suggest the presence of non-Fermi liquid behavior over a finite zero-temperature region. Using detailed susceptibility and transport measurements we show that the classic quantum critical system, Ge-substituted YbRh(2)Si(2), also displays such behavior. We advance arguments that this is not due to a disorder-smeared quantum critical point, but represents a new class of metallic phase.

Single crystals of the type-I clathrate Ba(8)Ni(3.5)Ge(42.1)square(0.4) (space group Pm3n, no. 223, a = 10.798(2) A, l = 30 mm, slashed circle = 8 mm) were grown from the melt using the Bridgman technique. Their composition, determined by microprobe analysis, reveals a distinctly lower Ni content than previously reported for the lower limit (x = 5.4) of the homogeneity range of the clathrate-I phase Ba(8)Ni(x)Ge(46-x). From single crystal X-ray diffraction data we introduce a crystal structure model that takes point defects (vacancies) square in the Ge network into account. It reveals that both Ni and square accumulate at a single site (6c) and that, as a consequence, the Ge network distorts considerably. Ba(8)Ni(3.5)Ge(42.1)square(0.4) shows metal-like behaviour (drho/dT > 0) albeit with high resistivity at room temperature (rho(300 K) approximately 1 mOmega cm). Together with the low charge carrier concentration of 2.3 e(-)/unit cell at 300 K this is typical of a degenerate semiconductor. The lattice thermal conductivity is distinctly smaller than that of Ba(8)Ge(43)square(3), where the vacancies partially order, and smaller than those of Ba-Ni-Ge type-I clathrates without vacancies, suggesting that disordered vacancies efficiently scatter heat-transporting phonons. We provide evidence that the maximum value of the thermoelectric figure of merit reached in Ba(8)Ni(3.5)Ge(42.1)square(0.4), ZT(680 K) congruent with 0.21, can be further improved by adjusting the charge carrier concentration.

A vaccine that elicits both specific antibodies and IFN-gamma-producing T cells is required to protect against pre-erythrocytic malaria. Among the most promising approaches to induce such complex immunity are heterologous prime-boost vaccination regimens, in particular ones containing live viral vector. We have demonstrated previously that adenovectors serotype 35 (Ads35) encoding the circumsporozoite (CS) antigen or liver-stage antigen-1 (LSA-1) are highly effective in improving the T-cell responses induced by immunizations with protein-based vaccines in a heterologous prime-boost schedule. Here we evaluated the potential of a heterologous prime-boost vaccination that combines the Ad35.CS vector with the serologically distinct adenovector Ad5.CS, in rhesus macaques, after establishing the potency in mice. We show that the heterologous Ad35.CS/Ad5.CS prime-boost regimen elicits both antibody responses and robust IFN-gamma-producing CD8(+) T-cell responses against the CS antigen. Analysis of the quality of the antibody responses in rhesus macaques, using indirect immunofluorescence assay (IFA) with Plasmodium falciparum-coated slides, demonstrated that this heterologous prime-boost regimen elicits a high titer of antibodies that are able to bind to P. falciparum sporozoites. Level of the IFA response was superior to the response measured with sera of an adult human population living in endemic malaria region. In conclusion, the combination of Ad35.CS, a vaccine based on a rare serotype adenovirus, with Ad5.CS or possibly another adenovector of a distinct serotype, induces a complex immune response that is required for protection against malaria, and is thus a highly promising approach for pediatric vaccination.

Recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus (Ad) serotype 5-mediated liver transduction in vivo. FX binds to the adenovirus hexon hypervariable regions (HVRs). Here, we perform a systematic analysis of FX binding to Ad5 HVRs 5 and 7, identifying domains and amino acids critical for this interaction. We constructed a model of the Ad5-FX interaction using crystallographic and cryo-electron microscopic data to identify contact points. Exchanging Ad5 HVR5 or HVR7 from Ad5 to Ad26 (which does not bind FX) diminished FX binding as analyzed by surface plasmon resonance, gene delivery in vitro, and liver transduction in vivo. Exchanging Ad5-HVR5 for Ad26-HVR5 produced deficient virus maturation. Importantly, defined mutagenesis of just 2 amino acids in Ad5-HVR5 circumvented this and was sufficient to block liver gene transfer. In addition, mutation of 4 amino acids in Ad5-HVR7 or a single mutation at position 451 also blocked FX-mediated effects in vitro and in vivo. We therefore define the regions and amino acids on the Ad5 hexon that bind with high affinity to FX thereby better defining adenovirus infectivity pathways. These vectors may be useful for gene therapy applications where evasion of liver transduction is a prerequisite.

Reverse genetics, the generation of influenza viruses from cDNA, presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 cells, among the most extensively characterized and documented cells, support growth of all influenza viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated on PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.

The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated cellular binding and transduction of Ad5-based vectors in vitro. Neutralizing activity correlated well with the ability to inhibit Ad5-mediated liver transduction, suggesting that prescreening patient sera in this manner accurately predicts the efficacy of Ad5-based gene therapies. Neutralization in vitro can be partially bypassed by pseudotyping with Ad45 fiber protein, indicating that a proportion of neutralizing antibodies are directed against the Ad5 fiber.

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

The inaccessibility of the zygote and proembryos of angiosperms within the surrounding maternal and filial tissues has hampered studies on early plant embryogenesis. Somatic and gametophytic embryo cultures are often used as alternative systems for molecular and biochemical studies on early embryogenesis, but are not widely used in developmental studies due to differences in the early cell division patterns with seed embryos. A new Brassica napus microspore embryo culture system, wherein embryogenesis highly mimics zygotic embryo development, is reported here. In this new system, the donor microspore first divides transversely to form a filamentous structure, from which the distal cell forms the embryo proper, while the lower part resembles the suspensor. In conventional microspore embryogenesis, the microspore divides randomly to form an embryonic mass that after a while establishes a protoderm and subsequently shows delayed histodifferentiation. In contrast, the embryo proper of filament-bearing microspore-derived embryos undergoes the same ordered pattern of cell division and early histodifferentiation as in the zygotic embryo. This observation suggests an important role for the suspensor in early zygotic embryo patterning and histodifferentiation. This is the first in vitro system wherein single differentiated cells in culture can efficiently regenerate embryos that are morphologically comparable to zygotic embryos. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during the early stages of plant embryogenesis.

Glucosinolates and their breakdown products have been recognized for their effects on plant defense, human health, flavor and taste of cruciferous vegetables. Despite this importance, little is known about the regulation of the biosynthesis and degradation in Brassica rapa. Here, the identification of quantitative trait loci (QTL) for glucosinolate accumulation in B. rapa leaves in two novel segregating double haploid (DH) populations is reported: DH38, derived from a cross between yellow sarson R500 and pak choi variety HK Naibaicai; and DH30, from a cross between yellow sarson R500 and Kairyou Hakata, a Japanese vegetable turnip variety. An integrated map of 1068 cM with 10 linkage groups, assigned to the international agreed nomenclature, is developed based on the two individual DH maps with the common parent using amplified fragment length polymorphism (AFLP) and single sequence repeat (SSR) markers. Eight different glucosinolate compounds were detected in parents and F(1)s of the DH populations and found to segregate quantitatively in the DH populations. QTL analysis identified 16 loci controlling aliphatic glucosinolate accumulation, three loci controlling total indolic glucosinolate concentration and three loci regulating aromatic glucosinolate concentrations. Both comparative genomic analyses based on Arabidopsis-Brassica rapa synteny and mapping of candidate orthologous genes in B. rapa allowed the selection of genes involved in the glucosinolate biosynthesis pathway that may account for the identified QTL.

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants.

The presence of replication-competent adenovirus (RCA) is a safety concern for biologics based on recombinant adenoviruses and RCA testing is therefore mandatory for release of clinical material. RCA, which arises from homologous recombination between Ad5 vectors and HEK-293 cells, can be eliminated by the use of PER.C6 cells in combination with a matched vector. However, little is known on RCA formation with vectors based on adenovirus serotypes other than Ad5 and reliable RCA assays to test them are generally lacking. Here we report on the development and qualification of a sensitive RCA assay for Ad35, a promising alternative to Ad5 vectors. The assay is able to detect 1 RCA in 3x10(10) vector particles with 95% confidence, thus meeting current FDA requirements, and can discriminate between RCA and other rare CPE-causing entities, including helper dependent E1 positive particles (HDEP). Using this assay, the first batches of Ad35 vectors produced in PER.C6 cells were analysed and found to be free of RCA and HDEP. Based on the statistical model used, we anticipate that our approach to RCA assay development can be broadly applicable to other adenoviral vectors.

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4 degrees C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9 degrees C and thereafter at 28 degrees C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 muM zeatin and 5 muM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.

Background: Measurement of thoracic excursion (TEM) during maximal ventilation is part of the physiotherapy assessment in children with cystic fibrosis (CF)., Objectives: The purpose of our study was to examine the reliability of TEM and its relation with pulmonary function., Methods: Thoracic excursions were measured using a measuring tape. Thirty children participated in an intra-observer and inter-observer reliability study. Reliability was determined by calculating the typical error in repeated measurement, limits of agreement and correlation coefficients. Cross-sectional data from the annual check-ups were used to measure the relation between TEM and pulmonary function., Results: In the intra-observer reliability study the typical error was 0.31 cm, the limits of agreement were +/-0.86 cm. Pearson's r and ICC were 0.96 and 0.95, respectively. In the inter-observer reliability study these values were 0.56 cm, +/-1.55 cm, 0.85 and 0.85, respectively. TEM correlated significantly with height (0.31, P<0.001), FVC (0.44, P<0.001), FEV1 (0.41, P<0.001) and TLC (0.19, P<0.05), and there was a significant inverse correlation with RV%TLC (-0.45, P<0.001)., Conclusions: TEM is a reliable assessment tool. Thoracic excursion is significantly, although moderately correlated with pulmonary function.

A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (DeltaE1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV(+)) individuals is as low as that among HIV(-) individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.

In seed plants, successful fertilization requires correct regulation of pollen tube growth. At germination and during growth, the pollen tube interacts with tissues from the pistil while the pollen tube extends via tip growth. Despite the fact that much research has been devoted to the mechanisms regulating pollen tube growth, many aspects are currently unknown. Previously, we have isolated a pollen-specific gene from tobacco--NTP303--that probably functions during pollen tube growth. NTP303 is part of a family of five members. Its expression is regulated both at the transcriptional and at the translational level. While NTP303 transcripts accumulate to high levels between early bi-cellular and mature pollen stages, NTP303 protein is hardly detectable until germination and pollen tube growth. In order to elucidate the role and function of NTP303 in the pollen tube, we studied the effect of NTP303 gene silencing on pollen function. Therefore, we have transformed tobacco plants with NTP303 co-suppression and anti-sense gene constructs. In these plants, the kanamycin resistance trait--which was linked to the NTP303-silencing gene--was not transmitted through the male gametophyte. This indicated that lowering the transcript level of NTP303 and/or its family members interferes with pollen function. Because we could not find a readily distinguishable phenotype in pollen from the hemizygous anti-sense and co-suppression plants, we rescued the defective pollen to produce doubled haploid plants that were homozygous for the NTP303 anti-sense gene. We found that in pollen from these plants the transcript levels of all NTP303 family members were reduced. Although pollen and pollen tubes from these plants appeared completely normal in vitro, the pollen tubes showed slower growth rates in vivo and arrested in the style before they reached the ovary, so that fertilization failed. These data demonstrate that NTP303 and its family members are essential for normal pollen tube growth and indicate several possible functions.

A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napus hypocotyl transformation protocol and a promoterless gus::nptII tagging construct, a series of 20 kanamycin-resistant tagged lines was produced. Most of the regenerated plants showed hardly any GUS activity in leaf, stem and root tissues. However, expression was readily restored in callus tissue induced on in vitro leaf segments. Genomic sequences upstream of the gus::nptII insertions were isolated via plasmid rescue. Three clones originating from single copy T-DNA lines were selected for further evaluation. The rescued plasmids were cloned as linear fragments in binary vectors and re-transformed to Brassica napus hypocotyl and Solanum tuberosum stem segments. The new sequences maintained their promoter activity, demonstrated by transient and stable GUS activity after transformation. Furthermore, the promoters provided sufficient expression of the nptII gene to yield transgenic plants when using kanamycin as selective agent. Database searching (BLASTN) revealed that the promoters have significant homology with three Arabidopsis BAC clones, one Arabidopsis cDNA and one Brassica napus cDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters.

Purpose: To adapt the American PEDI into a Dutch version, and to establish the content validity of this pediatric functional status instrument., Methods: The adaptation process was based on current scientific guidelines in the field of cross-cultural research. Thirty-one allied health professionals completed a validity questionnaire for the content validity study., Results: The topic 'bicycling' was added to the questionnaire, and adaptations of the text were made without losing the content of the original PEDI. At least 81% of the respondents rated that the most important facets in examining functional status in childhood were represented in the Dutch PEDI. Eighty-seven per cent and 71% of the respondents rated that the PEDI is feasible for discriminative purposes and evaluative purposes, respectively., Conclusion: The expert panel confirmed the functional content and feasibility of the Dutch PEDI for pediatric rehabilitation outcome measurement.

Ever since the initial discovery of the molecules and genes involved in disease resistance in plants, attempts have been made to engineer durable disease resistance in economically important crop plants. Unfortunately, many of these attempts have failed, owing to the complexity of disease-resistance signalling and the sheer diversity of infection mechanisms that different pathogens use. Although disease-resistant transgenic plants or seeds are not yet available commercially, future product development seems likely as our current level of understanding of pathogenesis and plant defence improves.

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications.

Temperature controls the developmental fate of isolated Brassica napus microspores in vitro. Culture at 32.5°C leads to sporophytic development and the formation of embryos. Here we show that culture at 17.5°C leads to gametophytic development, and the formation of pollen-like structures at high frequencies (up to 80% after 7 days in culture). Early stages of both developmental pathways are observed in culture at 25.0°C, and embryos are produced at low frequencies (0.7%) at that temperature. Culturing B. napus microspores at 32.5°C versus 17.5°C brings the switch from gametophytic to sporophytic development under simple experimental control and provides a convenient tool for investigating the cellular and molecular mechanisms controlling this developmental switch.

Suspension cultures were initiated from leaf explant-derived callus of cucumber,Cucumis sativus cv. Hokus, and maintained under two different conditions; (I) continuously in medium with 5 μM 2,4-D + 5 μM BA, and (II) alternately three cultures in medium containing 5 μM NAA + 5 μM BA and one culture in 5 μM 2,4-D + 5 μM BA. After plating on solid medium with 0.5 μM KIN + 0.1 μM IAA, suspension aggregates from long-term culture in medium with 2,4-D developed into callus, and subsequently formed somatic embryos. These embryos, however, hardly developed into plants. They showed growth arrest and several structural abnormalities. In contrast, organogenesis took place when suspension aggregates from NAA containing medium were plated on solid medium with 0.5 μM KIN + 0.1 μM IAA. Numerous adventitious buds were regenerated, which quite normally developed into plants. Sucrose at low concentration of 1% improved plant formation. On the average thirty complete plants were obtained from each ml of suspension. It is discussed why adventitious buds develop into plants so well, whereas somatic embryos are prone to growth arrest and abnormal development.