PURIFICATION AND EFFECTIVENESS OF VACCINES AND ANTIVIRAL COMPOUNDS

Viral infections account for over 13 million deaths per year. Antiviral drugs and vaccines are the most effective method to treat viral diseases. Antiviral compounds have revolutionized the treatment of AIDS, and reduced the mortality rate. However, this disease still causes a large number of deaths in developing countries that lack these types of drugs. Vaccination is the most effective method to treat viral disease; vaccines prevent around 2.5 million deaths per year. Vaccines are not able to offer full coverage due to high operational costs in the manufacturing processes. Although vaccines have saved millions of lives, conventional vaccines often offer reactogenic effects. New technologies have been created to eliminate the undesired side effects. However, new vaccines are less immunogenic and adjuvants such as vaccine delivery vehicles are required. This work focuses on the discovery of new natural antivirals that can reduce the high cost and side effects of synthetic drugs. We discovered that two osmolytes, trimethylamine N-oxide (TMAO) and glycine reduce the infectivity of a model virus, porcine parvovirus (PPV), by 4 LRV (99.99%), likely by disruption of capsid assembly. These osmolytes have the potential to be used as drugs, since they showed antiviral activity after 20 h. We have also focused on improving current vaccine manufacturing processes that will allow fast, effective and economical vaccines to be produced worldwide. We propose virus flocculation in osmolytes followed by microfiltration as an economical alternative for vaccine manufacturing. Osmolytes are able to specifically flocculate hydrophobic virus particles by depleting a hydration layer around the particles and subsequently cause virus aggregation. The osmolyte mannitol was able to flocculate virus particles, and demonstrate a high virus removal, 81% for PPV and 98.1% for Sindbis virus (SVHR). Virus flocculation with mannitol, followed by microfiltration could be used as a platform process for virus purification. Finally, we perform biocompatibility studies on soft-templated mesoporous carbon materials with the aim of using these materials as vaccine delivery vehicles. We discovered that these materials are biocompatible, and the degree of biocompatibility is within the range of other biomaterials currently employed in biomedical applications.

Nest use is influenced by the positions of nests and drinkers in aviaries.

The influence of the nest location and the placement of nipple drinkers on nest use by laying hens in a commercial aviary was assessed. Twenty pens in a laying hen house were equipped with the same commercial aviary system, but the pens differed in the nest location and the placement of nipple drinkers. Nests were placed along the walls in 10 pens, and nipple drinkers were installed in front of the nests in 5 of these pens. The other 10 pens were equipped with nests placed on a tier within the aviary (integrated nests). Nipple drinkers were installed in front of the nests in 5 of these pens. A total of 225 Lohmann Selected Leghorns were housed per pen. The hens were offered 4 nests per pen: 2 facing the service corridor of the laying hen house and 2 facing the outdoor area. The numbers of nest eggs and mislaid eggs were counted daily per pen. At 25, 36, and 43 wk of age, the nest platforms were videotaped and the behavior of laying hens in front of the nests was analyzed. The nest location affected the stationary and locomotive behaviors in front of the nests. Hens in front of the integrated nests and the nests with drinkers displayed more stationary behaviors than hens in front of wall-placed nests or nests without drinkers. No difference in the number of nest eggs could be detected, but the integration of the nests inside the aviary led to a more even distribution of hens while nest searching. In the pens with wall-placed nests, significantly more hens laid eggs in the nests at the wall near the service corridor than at the wall near the outdoor area. Due to this imbalance, crowding in front of the preferred nests occurred and pushing and agonistic interactions on the nest platforms were significantly more frequent. Placement of nipple drinkers in front of nests had no effect on the number of eggs laid in those nests.

Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ(54)-σ(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

Simulation of the Impact of Dams and Fishing Weirs on Reproductive Potential of Silver-Phase American Eels in the Kennebec River Basin, Maine

I modeled the cumulative impact of hydroelectric projects with and without commercial fishing weirs and water-control dams on the production, survival to the sea, and potential fecundity of migrating female silver-phase American eels, Anguilla rostrata in the Kennebec River basin, Maine, This river basin has 22 hydroelectric projects, 73 water-control dams, and 15 commercial fishing weir sites. The modeled area included an 8,324 km(2) segment of the drainage area between Merrymeeting Bay and the upper limit of American eel distribution in the basin. One set of input,, (assumed or real values) concerned population structure (Le., population density and sex ratio changes throughout the basin, female length-class distribution, and drainage area between dams), Another set concerned factors influencing survival and potential fecundity of migrating American eels (i.e., pathway sequences through projects, survival rate per project by length-class. and length-fecundity relationship). Under baseline conditions about 402,400 simulated silver female American eels would be produced annually reductions in their numbers due to dams and weirs would reduce the realized fecundity (i.e., the number of eggs produced by all females that survived the migration). Without weirs or water-control dams, about 63% of the simulated silverphase American eels survived their freshwater spawning migration run to the sea when the survival rate at each hydroelectric dam was 9017, 40% survived at 80% survival per dam, and 18% survived at 60% survival per dam. Removing the lowermost hydroelectric dam on the Kennebec River increased survival by 6.0-7.6% for the basin. The efficient commercial weirs reduced survival to the sea to 69-76%( of what it would have been without weirs', regardless of survival rates at hydroelectric dams. Water-control dams had little impact on production in this basin because most were located in the upper reaches of tributaries. Sensitivity analysis led to the conclusion that small changes in population density and female length distribution had greater effects on survival and realized fecundity than similar changes in turbine survival rate. The latter became more important as turbine survival rate decreased. Therefore, it might be more fruitful to determine population distribution in basins of interest than to determine mortality rate at each hydroelectric project.

Role of Methyl Salicylate on Oviposition Deterrence in Arabidopsis thaliana

Plants attacked by herbivores have evolved different strategies that fend off their enemies. Insect eggs deposited on leaves have been shown to inhibit further oviposition through visual or chemical cues. In some plant species, the volatile methyl salicylate (MeSA) repels gravid insects but whether it plays the same role in the model species Arabidopsis thaliana is currently unknown. Here we showed that Pieris brassicae butterflies laid fewer eggs on Arabidopsis plants that were next to a MeSA dispenser or on plants with constitutively high MeSA emission than on control plants. Surprisingly, the MeSA biosynthesis mutant bsmt1-1 treated with egg extract was still repellent to butterflies when compared to untreated bsmt1-1. Moreover, the expression of BSMT1 was not enhanced by egg extract treatment but was induced by herbivory. Altogether, these results provide evidence that the deterring activity of eggs on gravid butterflies is independent of MeSA emission in Arabidopsis, and that MeSA might rather serve as a deterrent in plants challenged by feeding larvae.

Effects of variation in nest curtain design on pre-laying behaviour of domestic hens

Laying hens in loose-housing systems select a nest daily in which to lay their eggs among many identical looking nests, they often prefer corner nests. We investigated whether heterogeneity in nest curtain appearance – via colours and symbols – would influence nest selection and result in an even distribution of eggs among nests. We studied pre-laying behaviour in groups of 30 LSL hens across two consecutive trials with eight groups per trial. Half of the groups had access to six identical rollaway group-nests, while the others had access to six nests of the same type differing in outer appearance. Three colours (red, green, yellow) and three black symbols (cross, circle, rectangle) were used to create three different nest curtain designs per pen. Nest position and the side of entrance to the pens were changed at 28 and 30 weeks of age, respectively, whereby the order of changes was counterbalanced across trials. Nest positions were numbered 1–6, with nest position 1 representing the nest closest to the pen entrance. Eggs were counted per nest daily from week of age 18 to 33. Nest visits were recorded individually with an RFID system for the first 5 h of light throughout weeks 24–33. Hens with access to nests differing in curtain appearance entered fewer nests daily than hens with identical nests throughout the study but both groups entered more nests with increasing age. We found no other evidence that curtain appearance affected nest choice and hens were inconsistent in their daily nest selection. A high proportion of eggs were laid in corner nests especially during the first three weeks of lay. The number of visits per egg depended upon nest position and age: it increased with age and was higher after the nest position change than before in nest position 1, whereas it stayed stable over time in nest position 6. At 24 weeks of age, gregarious nest visits (hens visiting an occupied nest when there was at least one unoccupied nest) and solitary nest visits (hens visiting an unoccupied nest when there was at least one occupied nest) accounted for a similar amount of nest visits, however, after the door switch, gregarious nest visits made up more than half of all nest visits, while the number of solitary nest visits had decreased. The visual cues were too subtle or inadequate for hens to develop individual preferences while nest position, entrance side, age and nest occupancy affected the quantity and type of nest visits.

Human complement component C3 -binding proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that uses the host mononuclear phagocyte as a niche for survival and replication during infection. Complement component C3 has previously been shown to enhance the binding of M. tuberculosis to mononuclear phagocytes. Using a C3 ligand affinity blot protocol, we identified a 30 kDa C3-binding protein in M. tuberculosis as heparin-binding hemagglutinin (HbhA). HbhA was found to be a hydrophobic protein that localized to the cell membrane/cell wall fraction of M. tuberculosis, and this protein has previously been shown by others to be located on the surface of M. tuberculosis. The C3-binding activity of HbhA was localized to the C-terminus of the protein, which consists of lysine-alanine repeats. Full-length recombinant HbhA coated onto latex beads was shown to mediate the adherence of the beads to murine macrophage-like cells in both a C3-dependent and a C3-independent manner. An in-frame 576 by deletion in the hbhA gene was created in a virulent strain of M. tuberculosis using a PCR technique known as gene splicing by overlap extension (SOEing). Using the ΔhbhA mutant, HbhA was found not to be necessary for growth of M. tuberculosis in laboratory media or in macrophage-like cells, nor is HbhA required for adherence of M. tuberculosis to macrophage-like cells. HbhA is, however, required for infectivity of M. tuberculosis in mice. Mice infected with the ΔhbhA mutant show decreased growth in the lungs, liver, and spleen compared to mice infected with the wild-type strain. Using the ΔhbhA mutant strain, we were able to purify and identify a second 30-kDa C3-binding protein, HupB. These data demonstrate that HbhA is required for the in vivo but not the in vitro survival of M. tuberculosis and that HbhA is not necessary for the adherence of M. tuberculosis to the macrophage-like cells used in these studies. The expression of two proteins that bind human C3 may aid in the efficient binding of M. tuberculosis to complement receptors for uptake into mononuclear cells, or may influence other aspects of the host-parasite interaction. ^

Plasmid -encoded virulence determinants of Borrelia burgdorferi

Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease, infects both mammals and ticks. Its genome, sequenced in 1997, consists of one linear chromosome and over 20 linear and circular plasmids. Continuous passage of organisms in culture causes them to lose certain plasmids and also results in loss of infectivity in mammals. In this work, 19 B. burgdorferi clonal isolates were examined for infectivity in mice and for plasmid content utilizing polymerase chain reaction (PCR). Two plasmids, a 28 kilobase (kb) linear plasmid (Ip28-1) and a 25 kb linear plasmid (Ip25) were found to be required for full infectivity. Previous studies had demonstrated that Ip28-1 contains the vls locus, which is involved in antigenic variation and immune evasion. Gene BBE22 on Ip25 is predicted to encode the nicotinamidase PncA, an enzyme that converts nicotinamide to nicotinic acid as part of a pathway for NAD synthesis. To examine the potential role of BBE22 in infectivity, a shuttle vector containing BBE22 (pBBE22) was constructed and used to transform B. burgdorferi clone 5A13, which contains all plasmids except lp25. Transformation with pBBE22 restored infectivity of clone 5A13 in mice, whereas 5A13 transformed with the shuttle vector alone was not infectious. To determine whether BBE22 acts as a nicotinamidase in vivo, a Salmonella typhimurium pncA− nadB− transposon mutant was transformed with pBBE22 or with pQE30:BBE22, which contained BBE22 in an E. coli expression vector. Both constructs complemented the Salmonella mutant, permitting growth in minimal media plus nicotinamide. Salmonella cells over-expressing BBE22 also exhibited nicotinamidase activity, as determined by ammonia production in the presence of nicotinamide. Site-directed mutagenesis of BBE22 at the predicted active site (resulting in a Cys120Ala substitution) abrogated the ability to restore infectivity to B. burgdorferi 5A13 and to complement the pncA mutation in S. typhimurium. These studies indicate that BBE22 is a nicotinamidase required for NAD synthesis and survival of B. burgdorferi in mammals. This is also the first demonstration of ‘molecular Koch's postulates’ in B. burgdorferi, i.e. that a specific gene is essential for infectivity of the Lyme disease spirochete. ^

Egg parasitism of Piezodorus guildinii and Nezara viridula (Hemiptera: Pentatomidae) in soybean, alfalfa and red clover

Piezodorus guildinii Westwood and Nezara viridula (L.) (Hemiptera: Pentatomidae) are important soybean pests. P. guildinii causes more injury and is less susceptible to insecticides compared to N. viridula. N. viridula egg parasitoids are well studied; however, little is known about parasitoids of P. guildinii. Alfalfa, soybean and red clover were sampled during several seasons to characterize the abundance of both stink bugs, to determine their egg parasitoids, and to estimate parasitoids impact. In the field, Telenomus podisi (Ashmead),Trissolcus urichi (Crawford) and Trissolcus basalis (Wollaston) (Hymenoptera: Platygastridae) emerged from P. guildinii, while only T. basalis (Wollaston) (Hymenoptera: Platygastridae) emerged from N. viridula. The proportions of parasitized eggs (i. e., the parasitoid impact) and egg masses, as well as the number of parasitized eggs/total number of eggs of the parasitized egg masses, were similar for alfalfa and soybean. Parasitism was not observed in red pclover. Parasitoid impact was lower during the dry growing seasons. Although P. guildinii field parasitism by T. urichi was less significant, laboratory experiments from the bibliography indicate that this wasp species performs well on this host. Trissolcus urichi would be an important biological control agent against P. guildinii, principally when the stink bug is more abundant.

(Table 1) Enantiomer fractions of egg yolk and plasma samples from Svalbard glaucous gulls (Larus hyperboreus)

Glaucous gulls (Larus hyperboreus) and their eggs from Svalbard (Norwegian Arctic) have been used as biomonitors of contaminants in the marine environment. In this study, the enantiomer fractions (EFs) of chiral chlordanes and atropisomeric polychlorinated biphenyl (PCB) congeners were determined in the blood plasma of adult male and female glaucous gulls from three breeding colonies in Svalbard. Plasma EFs were similar in magnitude and direction to EFs previously reported in glaucous gulls from other arctic food webs, suggesting overall similarities in the biochemical processes influencing the EFs of bioaccumulated organochlorine (OC) contaminants within the food webs at those locations. Additionally, EFs in yolk of eggs collected concurrently from within the same nesting colonies varied with location, laying date, and OC concentrations, and may be influenced by changes in the local feeding ecology between those colonies. No differences were found between the EFs for any analyte in female gulls compared to those found in egg yolk, indicating that processes involved in the maternal transfer of chlordanes and PCBs to eggs do not modulate the stereochemical ratio between enantiomers. Therefore, the use of eggs as a valuable and noninvasive means of OC biomonitoring may also extend to enantiomer compositions in glaucous gulls, and perhaps also in other seabird species from arctic regions.

(Table 1) Stage of the lemming cycle and nesting parameters of the greater snow geese between 1996-2005, Bylot Island

Resource pulses are common in various ecosystems and often have large impacts on ecosystem functioning. Many animals hoard food during resource pulses, yet how this behaviour affects pulse diffusion through trophic levels is poorly known because of a lack of individual-based studies. Our objective was to examine how the hoarding behaviour of arctic foxes (Alopex lagopus) preying on a seasonal pulsed resource (goose eggs) was affected by annual and seasonal changes in resource availability. We monitored foraging behaviour of foxes in a greater snow goose (Chen caerulescens atlanticus) colony during 8 nesting seasons that covered 2 lemming cycles. The number of goose eggs taken and cached per hour by foxes declined 6-fold from laying to hatching, while the proportion of eggs cached remained constant. In contrast, the proportion of eggs cached by foxes fluctuated in response to the annual lemming cycle independently of the seasonal pulse of goose eggs. Foxes cached the majority of eggs taken (> 90%) when lemming abundance was high or moderate but only 40% during the low phase of the cycle. This likely occurred because foxes consumed a greater proportion of goose eggs to fulfill their energy requirement at low lemming abundance. Our study clearly illustrates a behavioural mechanism that extends the energetic benefits of a resource pulse. The hoarding behaviour of the main predator enhances the allochthonous nutrients input brought by migrating birds from the south into the arctic terrestrial ecosystem. This could increase average predator density and promote indirect interactions among prey.

Investigation of knot nests on Ellesmere Island

Knots arrive on Ellesmere Island in late May or early June. At Hazen Camp small flocks were present on 3 June 1966, but the main influx occurred 5 June when many flocks were seen ranging in size from 6 to 60 individuals. The sexes appeared to arrive together, but the manner of pair-formation was not determined. By 7 June pairs were distributed over the tundra with large feeding flocks forming at snowfree wet marshy areas. Most nests were on Dryas-hummocked slopes and tundra, either dry or moist, with some on clay plains and summits in a mixed Dryas and Salix vegetation. A census area of 240 ha supported at least 3 breeding pairs, and possibly 5; the total number of pairs breeding in the Hazen Camp study area was estimated to be about 25 (1.09 pairs/km**2). Egg-laying (4 nests) extended from 15 to 28 June, with 3 of the 4 sets completed between 20 and 23 June. Both sexes incubated, one of the pair more regularly than the other. The song-flight display of the male was performed most frequently during egglaying and incubation. The incubation period of the last egg in one clutch was established as being between 21.5 and 22.4 days. Four nests hatched between 12 and 20 July, and the hatching period of the entire clutch was less than 24 hours. Four of 7 nests (57 %) survived and egg survival (53 %) was low. Families left the nesting area so on after hatching, concentrating at ponds where food was readily available for the young. Both adults attended the young during the pre-fledging period, but the females apparently departed before the young had hedged. Males left once the young could fly and the adult fall migration was complete by early August. Most 01 the young departed belore mid-August. Fall migration is complete by late August or early September. The breeding season appears to be timed to peak load supply for the young. Adult Chironomidae emergence was highest between 3 and 17 July, the period during which most successful nests hatched. The increasing scarcity of adult insects for the young after mid-July was offset by family movements over the tundra and the early departure of half the adult population. Food also seemed to influence the distribution of breeding pairs aver the tundra, restricting them to the general vicinity of marshes, streams, and ponds where food is most available when the young hatch. Territoriality in the Knot appears to be closely associated with the protection of the nest against predators and has at least a local effect in regulating the number of breeding pairs. Plant material was important in the diet of adult Knots throughout the summer and the primary food from the time of arrival until mid-June. After mid-June the percentage of animal matter increased as dipterous insects became available (especially adult Chironomidae), but plant materials continued to constitute a large part of the diet, usually more than 50 %. The food of the young before fledging consisted principally of adult chironomids.

Contribution of woody habitat islands to the conservation of birds and their potential ecosystem services in an extensive Colombian rangeland

The conservation of birds and their habitats is essential to maintain well-functioning ecosystems including human-dominated habitats. In simplified or homogenized landscapes, patches of natural and semi-natural habitat are essential for the survival of plant and animal populations. We compared species composition and diversity of trees and birds between gallery forests, tree islands and hedges in a Colombian savanna landscape to assess how fragmented woody plant communities affect forest bird communities and how differences in habitat characteristics influenced bird species traits and their potential ecosystem function. Bird and tree diversity was higher in forests than in tree islands and hedges. Soil depth influenced woody species distribution, and canopy cover and tree height determined bird species distribution, resulting in plant and bird communities that mainly differed between forest and non-forest habitat. Bird and tree species and traits widely co-varied. Bird species in tree islands and hedges were on average smaller, less specialized to habitat and more tolerant to disturbance than in forest, but dietary differences did not emerge. Despite being less complex and diverse than forests, hedges and tree islands significantly contribute to the conservation of forest biodiversity in the savanna matrix. Forest fragments remain essential for the conservation of forest specialists, but hedges and tree islands facilitate spillover of more tolerant forest birds and their ecological functions such as seed dispersal from forest to the savanna matrix.

Effects of CO2-driven ocean acidification on early life stages of marine medaka (Oryzias melastigma)

The potential effects of elevated CO2 level and reduced carbonate saturation state in marine environment on fishes and other non-calcified organisms are still poorly known. In present study, we investigated the effects of ocean acidification on embryogenesis and organogenesis of newly hatched larvae of marine medaka (Oryzias melastigma) after 21 d exposure of eggs to different artificially acidified seawater (pH 7.6 and 7.2, respectively), and compared with those in control group (pH 8.2). Results showed that CO2-driven seawater acidification (pH 7.6 and 7.2) had no detectable effect on hatching time, hatching rate, and heart rate of embryos. However, the deformity rate of larvae in pH 7.2 treatment was significantly higher than that in control treatment. The left and right sagitta areas did not differ significantly from each other in each treatment. However, the mean sagitta area of larvae in pH 7.6 treatment was significantly smaller than that in the control (p = 0.024). These results suggest that although marine medaka might be more tolerant of elevated CO2 than some other fishes, the effect of elevated CO2 level on the calcification of otolith is likely to be the most susceptibly physiological process of pH regulation in early life stage of marine medaka.

Lenght measurements and carbon biomass of individual zooplankton from METEOR cruise M87/1 in April 2012

Zooplankton samples were taken in five depth strata using a Multinet type Midi, with 50 µm nets. The samples were taken during the second leg only, three times at station 1, two times at station 2 and once at station 3. Zooplankton were identified to species / genus and life-stage, and at least 300 individuals were counted per sample. 10 individuals of each stage / species were measured and the numbers of eggs counted.