Experimental Observation of Proton Bunch Modulation in a Plasma at Varying Plasma Densities.

We give direct experimental evidence for the observation of the full transverse self-modulation of a long, relativistic proton bunch propagating through a dense plasma. The bunch exits the plasma with a periodic density modulation resulting from radial wakefield effects. We show that the modulation is seeded by a relativistic ionization front created using an intense laser pulse copropagating with the proton bunch. The modulation extends over the length of the proton bunch following the seed point. By varying the plasma density over one order of magnitude, we show that the modulation frequency scales with the expected dependence on the plasma density, i.e., it is equal to the plasma frequency, as expected from theory.

Experimental Observation of Plasma Wakefield Growth Driven by the Seeded Self-Modulation of a Proton Bunch.

We measure the effects of transverse wakefields driven by a relativistic proton bunch in plasma with densities of 2.1×10^{14} and 7.7×10^{14} electrons/cm^{3}. We show that these wakefields periodically defocus the proton bunch itself, consistently with the development of the seeded self-modulation process. We show that the defocusing increases both along the bunch and along the plasma by using time resolved and time-integrated measurements of the proton bunch transverse distribution. We evaluate the transverse wakefield amplitudes and show that they exceed their seed value (<15 MV/m) and reach over 300 MV/m. All these results confirm the development of the seeded self-modulation process, a necessary condition for external injection of low energy and acceleration of electrons to multi-GeV energy levels.

Acceleration of electrons in the plasma wakefield of a proton bunch.

High-energy particle accelerators have been crucial in providing a deeper understanding of fundamental particles and the forces that govern their interactions. To increase the energy of the particles or to reduce the size of the accelerator, new acceleration schemes need to be developed. Plasma wakefield acceleration1-5, in which the electrons in a plasma are excited, leading to strong electric fields (so called 'wakefields'), is one such promising acceleration technique. Experiments have shown that an intense laser pulse6-9 or electron bunch10,11 traversing a plasma can drive electric fields of tens of gigavolts per metre and above-well beyond those achieved in conventional radio-frequency accelerators (about 0.1 gigavolt per metre). However, the low stored energy of laser pulses and electron bunches means that multiple acceleration stages are needed to reach very high particle energies5,12. The use of proton bunches is compelling because they have the potential to drive wakefields and to accelerate electrons to high energy in a single acceleration stage13. Long, thin proton bunches can be used because they undergo a process called self-modulation14-16, a particle-plasma interaction that splits the bunch longitudinally into a series of high-density microbunches, which then act resonantly to create large wakefields. The Advanced Wakefield (AWAKE) experiment at CERN17-19 uses high-intensity proton bunches-in which each proton has an energy of 400 gigaelectronvolts, resulting in a total bunch energy of 19 kilojoules-to drive a wakefield in a ten-metre-long plasma. Electron bunches are then injected into this wakefield. Here we present measurements of electrons accelerated up to two gigaelectronvolts at the AWAKE experiment, in a demonstration of proton-driven plasma wakefield acceleration. Measurements were conducted under various plasma conditions and the acceleration was found to be consistent and reliable. The potential for this scheme to produce very high-energy electron bunches in a single accelerating stage20 means that our results are an important step towards the development of future high-energy particle accelerators21,22.

The effectiveness of social resource intervention to promote adherence to HIV medication in a multidisciplinary care setting in Kenya.

Adherence to HIV medication has a dramatic impact on morbidity, mortality and health in people living with HIV. Recent studies have demonstrated good adherence to HIV medication among people in sub-Saharan Africa, but few have investigated factors influencing adherence. The goal of this study was to evaluate the effectiveness of social intervention strategies to enhance adherence to HIV medication. A cross-sectional design study was used to obtain data through self-administered questionnaires from 354 individuals who were prescribed HIV medication at nine satellite centres under the auspice of the Nazareth Hospital in Kenya. Binomial logistics were used to test the relationships between social support and its dimensions with adherence to HIV medication. Composite social support was predictive of adherence to HIV medication (P < 0.05). Among the four dimensions of support, material and emotional support were the strongest predictors.

Avian predators are less abundant during periodical cicada emergences, but why?

Despite a substantial resource pulse, numerous avian insectivores known to depredate periodical cicadas (Magicicada spp.) are detected less commonly during emergence years than in either the previous or following years. We used data on periodical cicada calls collected by volunteers conducting North American Breeding Bird Surveys within the range of cicada Brood X to test three hypotheses for this observation: lower detection rates could be caused by bird calls being obscured by cicada calls ("detectability" hypothesis), by birds avoiding areas with cicadas ("repel" hypothesis), or because bird abundances are generally lower during emergence years for some reason unrelated to the current emergence event ("true decline" hypothesis). We tested these hypotheses by comparing bird detections at stations coincident with calling cicadas vs. those without calling cicadas in the year prior to and during cicada emergences. At four distinct levels (stop, route, range, and season), parallel declines of birds in groups exposed and not exposed to cicada calls supported the true decline hypothesis. We discuss several potential mechanisms for this pattern, including the possibility that it is a consequence of the ecological and evolutionary interactions between predators of this extraordinary group of insects.

Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungus belonging to the Phylum Chytridiomycota, Class Chytridiomycetes, Order Chytridiales, and is the highly infectious aetiological agent responsible for a potentially fatal disease, chytridiomycosis, which is currently decimating many of the world's amphibian populations. The fungus infects 2 amphibian orders (Anura and Caudata), 14 families and at least 200 species and is responsible for at least 1 species extinction. Whilst the origin of the agent and routes of transmission are being debated, it has been recognised that successful management of the disease will require effective sampling regimes and detection assays. We have developed a range of unique sampling protocols together with diagnostic assays for the detection of B. dendrobatidis in both living and deceased tadpoles and adults. Here, we formally present our data and discuss them in respect to assay sensitivity, specificity, repeatability and reproducibility. We suggest that compliance with the recommended protocols will avoid the generation of spurious results, thereby providing the international scientific and regulatory community with a set of validated procedures which will assist in the successful management of chytridiomycosis in the future.

Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay.

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide, associated with declines in amphibian populations. Diagnosis of chytridiomycosis to date has largely relied upon histological and immunohistochemical examination of toe clips. This technique is invasive and insensitive particularly at early stages of infection when treatment may be possible. We have developed a real-time PCR Taqman assay that can accurately detect and quantify one zoospore in a diagnostic sample. This assay will assist the early detection of B. dendrobatidis in both captive and wild populations, with a high degree of sensitivity and specificity, thus facilitating treatment and protection of endangered populations, monitoring of pristine environments and preventing further global spread via amphibian trade.

Effect of season and temperature on mortality in amphibians due to chytridiomycosis.

OBJECTIVE: To investigate the distribution and incidence of chytridiomycosis in eastern Australian frogs and to examine the effects of temperature on this disease. DESIGN: A pathological survey and a transmission experiment were conducted. PROCEDURE: Diagnostic pathology examinations were performed on free-living and captive, ill and dead amphibians collected opportunistically from eastern Australia between October 1993 and December 2000. We conducted a transmission experiment in the laboratory to investigate the effects of temperature: eight great barred frogs (Mixophyes fasciolatus) exposed to zoospores of Batrachochytrium dendrobatidis and six unexposed frogs were housed individually in each of three rooms held at 17 degrees C, 23 degrees C and 27 degrees C. RESULTS: Chytridiomycosis was the cause of death or morbidity for 133 (55.2%) of 241 free-living amphibians and for 66 (58.4%) of 113 captive amphibians. This disease occurred in 34 amphibian species, was widespread around the eastern seaboard of Australia and affected amphibians in a variety of habitats at high and low altitudes on or between the Great Dividing Range and the coast. The incidence of chytridiomycosis was higher in winter, with 53% of wild frogs from Queensland and New South Wales dying in July and August. Other diseases were much less common and were detected mostly in spring and summer. In experimental infections, lower temperatures enhanced the pathogenicity of B. dendrobatidis in M. fasciolatus. All 16 frogs exposed to B. dendrobatidis at 17 degrees C and 23 degrees C died, whereas 4 of 8 frogs exposed at 27 degrees C survived. However, the time until death for the frogs that died at 27 degrees C was shorter than at the lower temperatures. Infections in survivors were eliminated by 98 days. CONCLUSION: Chytridiomycosis is a major cause of mortality in free-living and captive amphibians in Australia and mortality rate increases at lower temperatures.

Cryo-archiving of Batrachochytrium dendrobatidis and other chytridiomycetes.

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide. It has been associated with catastrophic declines of frog populations including those in pristine habitats in Queensland, Australia. To facilitate genetic and disease studies of this fungus and related species, it is essential to have a reliable long-term storage method to maintain genetic integrity of isolates. We have adapted well-established techniques used for the long-term storage of tissue-culture cell lines to the preservation of B. dendrobatidis and other chytridiomycetes. This simple method has allowed us to recover these fungi from storage at -80 degrees C and in liquid nitrogen over an extended period. With this technique it is now possible to preserve saprobic and parasitic isolates from a variety of environmental and disease situations for comparative genetic and biological studies.

Light directed gene transfer by photochemical internalisation.

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.

In vivo processing of nonanchored Yapsin 1 (Yap3p).

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3) was overexpressed in yeast and its processing through the secretory pathway was followed by pulse-labeling and immunoprecipitation studies. In the soluble cell extract, three forms of yapsin 1-87, 74, and 18 kDa-were found. Identification of these forms of yapsin 1 using different antisera suggests that the 87-kDa form is pro-yapsin 1, which is processed into two subunits, alpha (18 kDa) and beta (74 kDa), by cleavage at a loop region not found in traditional aspartic proteases. By use of a temperature-sensitive mutant strain, sec18, the generation of the two subunits was found to occur in the endoplasmic reticulum. An active site-mutated yapsin 1 was not processed into the two subunits, suggesting that this process occurs in an autocatalytic manner.

Role of endosomes in gene transfection mediated by photochemical internalisation (PCI).

BACKGROUND: Most non-viral gene therapy vectors deliver transgenes into cells through the endocytic pathway. Lack of escape from endocytic vesicles in many cases constitutes a major barrier for delivery of the functional gene. We have developed a new technology named photochemical internalisation (PCI) to achieve light-inducible cytosolic delivery of the transgene. The technology is based on a photochemical treatment employing photosensitisers localised in endocytic vesicles. In this work mechanisms involved in PCI-mediated transfection (photochemical transfection) were studied. METHODS: Human melanoma or colon carcinoma cells were pre-incubated with the photosensitiser aluminium phthalocyanine disulfonate (AlPcS2a) followed by treatment with plasmid encoding enhanced green fluorescent protein (EGFP) complexed with poly-L-lysine, N-(1-(2,3-dioleoxyloxy)propyl)-N,N,N,-trimethylammonium-methyl-sulfate (DOTAP) or polyethylenimine (PEI) and light exposure. The expression of the EGFP-gene was scored by fluorescence microscopy and flow cytometry. RESULTS: The photochemical treatment using light doses corresponding to D50 substantially improves the efficiency of transfection mediated by poly-L-lysine and PEI, but not by DOTAP. The treatment does not enhance the delivery of the plasmid complex across the plasma membrane, since the amount of internalised plasmid is similar for irradiated and non-irradiated cells. Light-inducible transfection occurs only under temperature conditions allowing endocytic uptake and is not improved by chloroquine or ammonium chloride, but is inhibited by bafilomycin A1 (agents that increase vesicular pH and interfere with the endocytic transport). CONCLUSIONS: Photochemical transfection occurs through endocytosis, followed by cytosolic release of the transfecting DNA from photochemically permeabilised endocytic vesicles. Release of plasmid from early endosomes seems to be of importance in photochemical transfection, although a role of later endocytic vesicles can, however, not be ruled out.

Identification and characterization of Saccharomyces cerevisiae yapsin 3, a new member of the yapsin family of aspartic proteases encoded by the YPS3 gene.

A new aspartic protease from Saccharomyces cerevisiae, with a high degree of similarity with yapsin 1 and yapsin 2 and a specificity for basic residue cleavage sites of prohormones, has been cloned. This enzyme was named yapsin 3. Expression of a C-terminally truncated non-membrane anchored yapsin 3 in yeast yielded a heterogeneous protein between 135-200 kDa which, upon treatment with endoglycosidase H, migrated as a 60 kDa form. Amino-acid analysis of the N-terminus of expressed yapsin 3 revealed two different N-terminal residues, serine-48 and phenylalanine-54, which followed a dibasic and a monobasic residue respectively. Cleavage of several prohormones by non-anchored yapsin 3 revealed a specificity distinct from that of yapsin 1.

Cleavage efficiency of the novel aspartic protease yapsin 1 (Yap3p) enhanced for substrates with arginine residues flanking the P1 site: correlation with electronegative active-site pockets predicted by molecular modeling.

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.

Activation and processing of non-anchored yapsin 1 (Yap3p).

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel sub-class of aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding approximately 1 microg of yapsin 1/g of wet yeast. N-terminal amino acid analysis of the purified protein confirmed that the propeptide was absent and that the mature enzyme began at Ala68. The mature enzyme was shown to be composed of approximately equimolar amounts of two subunits, designated alpha and beta, that were associated to each other by a disulfide bond. C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at approximately 4.0. When expressed without its pro-region, it was localized intracellularly and lacked activity, indicating that the pro-region was required for the correct folding of the enzyme. The activation of proyapsin 1 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.