Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters.

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and fine specificity of human antibody response against non-polymorphic domains.

BACKGROUND: Two long synthetic peptides representing the dimorphic and constant C-terminal domains of the two allelic families of Plasmodium falciparum merozoite surface proteins 2 are considered promising malaria vaccine candidates. The aim of the current study is to characterize the immune response (epitope mapping) in naturally exposed individuals and relate immune responses to the risk of clinical malaria. METHODS: To optimize their construction, the fine specificity of human serum antibodies from donors of different age, sex and living in four distinct endemic regions was determined in ELISA by using overlapping 20 mer peptides covering the two domains. Immune purified antibodies were used in Western blot and immunofluorescence assay to recognize native parasite derivate proteins. RESULTS: Immunodominant epitopes were characterized, and their distribution was similar irrespective of geographic origin, age group and gender. Acquisition of a 3D7 family and constant region-specific immune response and antibody avidity maturation occur early in life while a longer period is needed for the corresponding FC27 family response. In addition, the antibody response to individual epitopes within the 3D7 family-specific region contributes to protection from malaria infection with different statistical weight. It is also illustrated that affinity-purified antibodies against the dimorphic or constant regions recognized homologous and heterologous parasites in immunofluorescence and homologous and heterologous MSP2 and other polypeptides in Western blot. CONCLUSION: Data from this current study may contribute to a development of MSP2 vaccine candidates based on conserved and dimorphic regions thus bypassing the complexity of vaccine development related to the polymorphism of full-length MSP2.

The attractiveness of nations in global competition : an empirical assessment of the effects of country attractiveness on the success of strategy for hosting international sports events, 1990-2012

This dissertation aims to investigate empirical evidence on the importance and influence of attractiveness of nations in global competition. The notion of country attractiveness, which has been widely developed in the research areas of international business, tourism and migration, is a multi-dimensional construct to measure a country's characteristics with regard to its market or destination that attract international investors, tourists and migrants. This analytical concept provides an account of the mechanism as to how potential stakeholders evaluate more attractive countries based on certain criteria. Thus, in the field of international sport-event bidding, do international sport event owners also have specific country attractiveness for their sport event hosts? The dissertation attempts to address this research question by statistically assessing the effects of country attractiveness on the success of strategy for hosting international sports events. Based on theories of signaling and soft power, country attractiveness is defined and measured as the three dimensions of sustainable development: economic, social, and environmental attractiveness. This thesis proceeds to examine the concept of sport-event-hosting strategy and explore multi-level factors affecting the success in international sport-event bidding. By exploring past history of the Olympic Movement from theoretical perspectives, the thesis proposes and tests the hypotheses that economic, social and environmental attractiveness of a country may be correlated with its bid wins or the success of sport-event-hosting strategy. Quantitative analytical methods with various robustness checks are employed with using collected data on bidding results of major events in Olympic sports during the period from 1990 to 2012. The analysis results reveal that event owners of international Olympic sports are likely to prefer countries that have higher economic, social, and environmental attractiveness. The empirical assessment of this thesis suggests that high country attractiveness can be an essential element of prerequisites for a city/country to secure in order to bid with an increased chance of success.

Anchoring of an immunogenic Plasmodium falciparum circumsporozoite protein on the surface of Dictyostelium discoideum.

The circumsporozoite protein (CSP), a major antigen of Plasmodium falciparum, was expressed in the slime mold Dictyostelium discoideum. Fusion of the parasite protein to a leader peptide derived from Dictyostelium contact site A was essential for expression. The natural parasite surface antigen, however, was not detected at the slime mold cell surface as expected but retained intracellularly. Removal of the last 23 amino acids resulted in secretion of CSP, suggesting that the C-terminal segment of the CSP, rather than an ectoplasmic domain, was responsible for retention. Cell surface expression was obtained when the CSP C-terminal segment was replaced by the D. discoideum contact site A glycosyl phosphatidylinositol anchor signal sequence. Mice were immunized with Dictyostelium cells harboring CSP at their surface. The raised antibodies recognized two different regions of the CSP. Anti-sporozoite titers of these sera were equivalent to anti-peptide titers detected by enzyme-linked immunosorbent assay. Thus, cell surface targeting of antigens can be obtained in Dictyostelium, generating sporozoite-like cells having potentials for vaccination, diagnostic tests, or basic studies involving parasite cell surface proteins.

Bentho-planktonic evidence from the Austrian Alps for a decline in sea-surface carbonate production at the end of the Triassic

A high-resolution micropalaeontological study, combined with geochemical and sedimentological analyses was performed on the Tiefengraben, Schlossgraben and Eiberg sections (Austrian Alps) in order to characterize sea-surface carbonate production during the end-Triassic crisis. At the end-Rhaetian, the dominant calcareous nannofossil Prinsiosphaera triassica shows a decrease in abundance and size and this is correlated with a increase in delta O-18 and a gradual decline in delta C-13(carb) values. Simultaneously, benthic foraminiferal assemblages show a decrease in diversity and abundance of calcareous taxa and a dominance of infaunal agglutinated taxa. The smaller size of calcareous nannofossils disturbed the vertical export balance of the biological carbon pump towards the sea-bottom, resulting in changes in feeding strategies within the benthic foraminiferal assemblages from deposit feeders to detritus feeders and bacterial scavengers. These micropalaeontological data combined with geochemical proxies suggest that changes in seawater chemistry and/or cooling episodes might have occurred in the latest Triassic, leading to a marked decrease of carbonate production. This in turn culminated in the quasi-absence of calcareous nannofossils and benthic foraminifers in the latest Triassic. The aftermath (latest Triassic earliest Jurassic) was characterised by abundance peaks of ``disaster'' epifaunal agglutinated foraminifera Trochammina on the sea-floor. Central Atlantic Magmatic Province (CAMP) paroxysmal activity, superimposed on a major worldwide regressive phase, is assumed to be responsible for a deterioration in marine palaeoenvironments. CAMP sulfuric emissions might have been the trigger for cooling episodes and seawater acidification leading to disturbance of the surface carbonate production at the very end-Triassic.

Identification of a novel 45-kDa cell surface molecule involved in activation of the human Jurkat T cell line.

This report describes a surface molecule, Tp45, which appears to be involved in interleukin 2 production and Ca2+ mobilization by Jurkat cells. The Tp45 molecule was identified by a monoclonal antibody, MX13, on the surface of either T3/TCR+ or T3/TCR- human T cell lines. Biochemical data showed that mAb MX13 precipitated a single polypeptide chain of 45 kDa both under reduced and nonreduced conditions from lysates of 125I-surface-labeled cells. Sequential immunodepletion experiments using lysates of 125I-labeled T3/TCR+ cells showed that Tp45 was distinct from the alpha chain of the TCR complex. However, incubation of such cells with either anti-T3 or anti-TCR monoclonal antibody induced complete modulation of both the T3/TCR complex and Tp45. Conversely, complete modulation of both Tp45 and the T3/TCR complex was observed after incubation with anti-Tp45 antibody. Functional studies showed that anti-Tp45 antibody induced high levels of interleukin 2 production in Jurkat cells. In addition, incubation of these cells with the antibody resulted in Ca2+ mobilization from internal stores. Anti-Tp45 antibody reacted with 3-19% peripheral blood (E-rosette-positive) T cells in individual donors. The magnitude of the proliferative response elicited by anti-Tp45 antibody for peripheral blood T cells was lower than that induced by an anti-T3 antibody. This observation is compatible with the idea that only a subpopulation of T cells is reactive with anti-Tp45. Multicolor flow cytometry analysis showed that the Tp45+ cells belong preferentially to the T8 subset.

Detection of human growth hormone doping in urine: out of competition tests are necessary.

The misuse of human growth hormone (hGH) in sport is deemed to be unethical and dangerous because of various adverse effects. Thus, it has been added to the International Olympic Committee list of banned substances. Until now, the very low concentration of hGH in the urine made its measurement difficult using classical methodology. Indeed, for routine diagnosis, only plasma measurements were available. However, unlike blood samples, urine is generally provided in abundant quantities and is, at present, the only body fluid allowed to be analysed in sport doping controls. A recently developed enzyme-linked immunosorbent assay (Norditest) makes it now possible, without any extraction, to measure urinary hGH (u-hGH) in a dynamic range of 2-50 ng hGH/l. In our protocol, untreated and treated non-athlete volunteers were followed. Some of them received therapeutical doses of recombinant hGH (Norditropin) for one week either intramuscularly (three increasing doses) or subcutaneously (12 i.u. every day). The u-hGH excretion after treatment showed dramatic increases of 50-100 times the basal values and returned to almost the mean normal level after 24 h. u-hGH was also measured in samples provided by the anti-doping controls at major and minor competitions. Depending on the type of efforts made during the competition, the hGH concentration in urine was dramatically increased. Insulin-like growth factor binding proteins and beta 2-microglobulins in urine and/or in blood could be necessary for the correct investigation of any hGH doping test procedure.

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

The genetic consequences of hatchery-induced sperm competition in a salmonid

Supportive breeding is an important tool in conservation management, but its long-term genetic consequences are not well understood. Among the factors that could affect the genetics of the offspring is sperm competition as a consequence of mixed-milt fertilizations - which is still a common practice in many hatcheries. Here, we measured and combined the relevant factors to predict the genetic consequences of various kinds of hatchery-induced sperm competition. We drew a random sample of male Coregonus zugensis (an Alpine whitefish) from a hatchery program and quantified their in vitro sperm potency by integrating sperm velocity during the first minute after activation, and their in vitro milt potency by multiplying sperm potency with milt volume and sperm cell density. We found that not controlling for sperm density and/or milt volume would, at a constant population size, decrease the variance effective number of male breeders N-em by around 40-50%. This loss would decrease with increasing population growth rates. Partial multifactorial breeding and the separate rearing of in total 799 batches of eggs revealed that neither sperm nor milt potency was significantly linked to egg survival. Sperm and milt potency was also not significantly correlated to other potential quality measures such as breeding tubercles or condition factor. However, sperm potency was correlated to male age and milt potency to male growth rate. Our findings suggest that hatchery-induced sperm competition not only increases the loss of genetic variation but may also induce artificial selection, depending on the fertilization protocol. By not equalizing milt volume in multi-male fertilization hatchery managers lose relatively more genetic variation and give fast-growing males a reproductive advantage, while equalizing milt volume reduces the loss of genetic variation and favors younger males who may have fast sperm to compensate for their subdominance at the spawning place. (c) 2007 Elsevier Ltd. All rights reserved.

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells.

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.

Using 3D surface datasets to understand landslide evolution: From analogue models to real case study

Early detection of landslide surface deformation with 3D remote sensing techniques, as TLS, has become a great challenge during last decade. To improve our understanding of landslide deformation, a series of analogue simulation have been carried out on non-rigid bodies coupled with 3D digitizer. All these experiments have been carried out under controlled conditions, as water level and slope angle inclination. We were able to follow 3D surface deformation suffered by complex landslide bodies from precursory deformation still larger failures. These experiments were the basis for the development of a new algorithm for the quantification of surface deformation using automatic tracking method on discrete points of the slope surface. To validate the algorithm, comparisons were made between manually obtained results and algorithm surface displacement results. Outputs will help in understanding 3D deformation during pre-failure stages and failure mechanisms, which are fundamental aspects for future implementation of 3D remote sensing techniques in early warning systems.

Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates.

Merozoite surface protein 2 (MSP2) is a promising vaccine candidate against Plasmodium falciparum blood stages. A recombinant 3D7 form of MSP2 was a subunit of Combination B, a blood stage vaccine tested in the field in Papua New Guinea. A selective effect in favour of the allelic family not represented by the vaccine argued for a MSP2 vaccine consisting of both dimorphic variants. An alternative approach to recombinant manufacture of vaccines is the production of long synthetic peptides (LSP). LSP exceeding a length of well over 100 amino acids can now be routinely synthesized. Synthetic production of vaccine antigens cuts the often time-consuming steps of protein expression and purification short. This considerably reduces the time for a candidate to reach the phase of clinical trials. Here we present the evaluation of two long synthetic peptides representing both allelic families of MSP2 as potential vaccine candidates. The constructs were well recognized by human immune sera from different locations and different age groups. Furthermore, peptide-specific antibodies in human immune sera were associated with protection from clinical malaria. The synthetic fragments share major antigenic properties with native MSP2. Immunization of mice with these antigens yielded high titre antibody responses and monoclonal antibodies recognized parasite-derived MSP2. Our results justify taking these candidate poly-peptides into further vaccine development.