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The study of radiation biology on laser-based accelerators is most interesting due to the unique irradiation conditions they can produce, in terms of peak current and duration of the irradiation. In this paper we present the implementation of a beam transport system to transport and shape the proton beam generated by laser-target interaction for in vitro irradiation of biological samples. A set of four permanent magnet quadrupoles is used to transport and focus the beam, efficiently shaping the spectrum and providing a large and relatively uniform irradiation surface. Real time, absolutely calibrated, dosimetry is installed on the beam line, to enable shot-to-shot control of dose deposition in the irradiated volume. Preliminary results of cell sample irradiation are presented to validate the robustness of the full system.

We present a novel, to the best of our knowledge, Hartmann wave front sensor for extreme ultraviolet (EUV) spectral range with a numerical aperture (NA) of 0.15. The sensor has been calibrated using an EUV radiation source based on gas high harmonic generation. The calibration, together with simulation results, shows an accuracy beyond λ /39 root mean square (rms) at λ =32 n m . The sensor is suitable for wave front measurement in the 10 nm to 45 nm spectral regime. This compact wave front sensor is high-vacuum compatible and designed for in situ operations, allowing wide applications for up-to-date EUV sources or high-NA EUV optics.

Radiotherapy is a cornerstone of cancer management. The improvement of spatial dose distribution in the tumor volume by minimizing the dose deposited in the healthy tissues have been a major concern during the last decades. Temporal aspects of dose deposition are yet to be investigated. Laser-plasma-based particle accelerators are able to emit pulsed-proton beams at extremely high peak dose rates (~10 9  Gy/s) during several nanoseconds. The impact of such dose rates on resistant glioblastoma cell lines, SF763 and U87-MG, was compared to conventionally accelerated protons and X-rays. No difference was observed in DNA double-strand breaks generation and cells killing. The variation of the repetition rate of the proton bunches produced an oscillation of the radio-induced cell susceptibility in human colon carcinoma HCT116 cells, which appeared to be related to the presence of the PARP1 protein and an efficient parylation process. Interestingly, when laser-driven proton bunches were applied at 0.5 Hz, survival of the radioresistant HCT116 p53 -/- cells equaled that of its radiosensitive counterpart, HCT116 WT, which was also similar to cells treated with the PARP1 inhibitor Olaparib. Altogether, these results suggest that the application modality of ultrashort bunches of particles could provide a great therapeutic potential in radiotherapy.

Somatostatin (SST) a neuropeptide involved in the central modulation of several physiological functions, is co-distributed in the same hypothalamic areas as kisspeptin (KP), the most potent secretagogue of the gonadotropin-releasing hormone (GnRH) secretion known to date. As SST infused intracerebroventricularly (icv) evoked a potent inhibition of GnRH release, we explored neuroanatomical relationships between KP and SST populations in male and female rats. For that, intact males and ovariectomised oestradiol-replaced females were killed and their brains processed in order to simultaneously detect KP, SST and synapsin, a marker for synapses. We observed numerous appositions of KP on SST neurones both in female and male arcuate nucleus (ARC) and ventromedial hypothalamus. A large association between SST terminals and KP neurones at the level of the pre-optic area (POA) was also observed in female rats and in a more limited frame in males. Finally, most KP neurones from the ARC showed SST appositions in both sexes. To determine whether SST could affect KP cell activity, we assessed whether SST receptors (SSTR) were present on KP neurones in the ARC. We also looked for the presence of SSTR1 and SSTR2A in the brain of male rats. Brains were processed through a sequential double immunocytochemistry in order to detect KP and SSTR1 or KP and SSTR2A. We observed overlapping distributions of immunoreactive neurones for SSTR1 and KP and counted approximately one third of KP neurones with SSTR1. In contrast, neurones labelled for SSTR2A or KP were often juxtaposed in the ARC and the occurrence of double-labelled neurones was sporadic (<5%). These results suggest that SST action on KP neurones would pass mainly through SSTR1 at the level of the ARC. This article is protected by copyright. All rights reserved., (This article is protected by copyright. All rights reserved.)

Development of efficient soft x-ray laser plasma amplifiers adapted to seeded operation, requires a better control over amplifier transverse spatial extent, brilliance control and gain lifetime. Here it is shown that pumping the plasma amplifier with one long and two short pump pulses (1L2S) provides advantages in terms of control for the specified parameters in the case of Ni-like Ag x-ray laser. Also, significant tunability of the gain lifetime in the 1L2S pumping scheme for Ne-like Ti x-ray laser is observed. Direct harmonics seeding and chirped harmonics seeding amplification approaches may benefit from the control of the gain lifetime, in terms of better use of the pump energy and as a way to reduce the amplified spontaneous emission in x-ray lasers.

Harmonic seeded operation of a neon-like titanium plasma-based soft x-ray laser is described. The plasma amplifier is pumped with a variation of the grazing incidence technique involving a fast and localized ionization step. We discuss its effect on gain dynamics by measuring the amplifying factor as a function of the delay between pump pulse and harmonic seed. Two different regimes are pointed out, following the pumping scheme used. For one of them, a delay in the gain generation compared with the pumping laser pulse is observed.

We have investigated a new scheme for laser plasma transient collisional soft x-ray lasers based on the use of an additional laser to produce the preplasma. Soft x-ray emission measurements made for different solid targets are presented and discussed. A significant enhancement of the SXRL emission as compared to double-pulse single-beam grazing incidence (DGRIP) using the same pump laser is reported for 13.9- and 32.6-nm SXRL wavelengths.

An alternative, novel multiple pulse generation scheme was implemented directly after the optical compressor output of an x-ray pump laser. The new method uses a polarization sensitive thin film beam splitter and a half-wavelength wave plate for tuning the energy ratio in the multiple short pulses. Based on this method, an extensive study was made of the running parameters for a grazing incidence pumped silver x-ray laser (XRL) pumped with a long pulse of 145 mJ in 6 ns at 532 nm and up to 1.45 J in few picoseconds at 810 nm. Fivefold enhancement in the emission of the silver XRL was demonstrated using the new pump method.

The accurate characterization of thermal electron transport and the determination of heating by suprathermal electrons in laser driven solid targets are both issues of great importance to the current experiments being performed at the National Ignition Facility, which aims to achieve thermonuclear fusion ignition using lasers. Ionization, induced by electronic heat conduction, can cause the opacity of a material to drop significantly once bound-free photoionization is no longer energetically possible. We show that this drop in opacity enables measurements of the transmission of extreme ultraviolet (EUV) laser pulses at 13.9 nm to act as a signature of the heating of thin (50 nm) iron layers with a 50-nm thick parylene-N (CH) overlay irradiated by 35-fs pulses at irradiance 3×10(16) Wcm(-2). Comparing EUV transmission measurements at different times after irradiation to fluid code simulations shows that the target is instantaneously heated by hot electrons (with approximately 10% of the laser energy), followed by thermal conduction with a flux limiter of ≈0.05.

NF-κB transcription factors are crucial for many cellular processes. NF-κB is activated by viral infections to induce expression of antiviral cytokines. Here, we identified a novel member of the human NF-κB family, denoted RelAp43, the nucleotide sequence of which contains several exons as well as an intron of the RelA gene. RelAp43 is expressed in all cell lines and tissues tested and exhibits all the properties of a NF-κB protein. Although its sequence does not include a transactivation domain, identifying it as a class I member of the NF-κB family, it is able to potentiate RelA-mediated transactivation and stabilize dimers comprising p50. Furthermore, RelAp43 stimulates the expression of HIAP1, IRF1, and IFN-β - three genes involved in cell immunity against viral infection. It is also targeted by the matrix protein of lyssaviruses, the agents of rabies, resulting in an inhibition of the NF-κB pathway. Taken together, our data provide the description of a novel functional member of the NF-κB family, which plays a key role in the induction of anti-viral innate immune response.

Rabies remains one of the most ancient and deadly of human infectious diseases. This viral zoonosis is transmitted principally by the saliva of infected dogs, inducing a form of encephalomyelitis that is almost invariably fatal. Since the first implementation, by Louis Pasteur in 1885, of an efficient preventive post-exposure treatment, more effective protocols and safer products have been developed, providing almost 100% protection if administered early enough. However, this disease still represents a major, but neglected public health problem, with an estimated 55,000 human deaths due to rabies reported each year, mostly in Africa and Asia. Once the first clinical signs appear, there is no effective treatment. A ray of hope emerged in 2004, with the report of a patient recovering from rabies after aggressive, innovative treatment. However, this case was not clearly reproduced and the identification of targets for antiviral treatment in cases of rabies infection remains a major challenge. In this context, this review presents the state-of-the art in the prevention and curative treatment of human rabies. We begin by describing the viral etiological agent and the disease it causes, to provide an essential background to rabies. An overview of the post-exposure prophylaxis of rabies in humans is then given, from its initial implementation to possible future developments. Finally, an analysis of the various antiviral compounds tested in rabies in vitro, in animal models or in humans is presented, focusing in particular on potential new strategies.

The family Rhabdoviridae contains important pathogens of humans, livestocks or even crops. The matrix protein of rhabdovirus is a constituent of the virion, forming a layer between the viral envelope and the nucleocapsid. It is crucial for the bullet-like morphology of the virion. There is a strong structural convergence between the lyssavirus and vesiculovirus matrix proteins. These matrix proteins are able to self assemble to form non-covalent polymers, acting as a scaffold for the other components of the virion. The matrix protein of rhabdovirus also has non-structural functions within the viral cycle: it is involved in the expression and replication of the viral genome, and in the virion budding. Moreover, it is a key mediator of the interactions between the virus and the infected cell, even involved in the death of the cell. To conclude, the repertoire of the matrix protein of rhabdovirus functionalities keeps on expanding.

The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication., (Copyright 2010 Elsevier B.V. All rights reserved.)

The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein.

Mokola virus (MOKV) is a nonsegmented, negative-sense RNA virus that belongs to the Lyssavirus genus and Rhabdoviridae family. MOKV phosphoprotein P is an essential component of the replication and transcription complex and acts as a cofactor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. Here we present a structure for this domain of MOKV P, obtained by expression of full-length P in Escherichia coli, which was subsequently truncated during crystallization. The structure has a high degree of homology with P of rabies virus, another member of Lyssavirus genus, and to a lesser degree with P of vesicular stomatitis virus (VSV), a member of the related Vesiculovirus genus. In addition, analysis of the crystal packing of this domain reveals a potential binding site for the nucleoprotein N. Using both site-directed mutagenesis and yeast two-hybrid experiments to measure P-N interaction, we have determined the relative roles of key amino acids involved in this interaction to map the region of P that binds N. This analysis also reveals a structural relationship between the N-RNA binding domain of the P proteins of the Rhabdoviridae and the Paramyxoviridae.

We identified 2 cases of European bat lyssavirus subtype 1 transmission to domestic carnivores (cats) in France. Bat-to-cat transmission is suspected. Low amounts of virus antigen in cat brain made diagnosis difficult.

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.

Lyssaviruses are RNA viruses with single-strand, negative-sense genomes responsible for rabies-like diseases in mammals. To date, genomic and evolutionary studies have most often utilized partial genome sequences, particularly of the nucleoprotein and glycoprotein genes, with little consideration of genome-scale evolution. Herein, we report the first genomic and evolutionary analysis using complete genome sequences of all recognised lyssavirus genotypes, including 14 new complete genomes of field isolates from 6 genotypes and one genotype that is completely sequenced for the first time. In doing so we significantly increase the extent of genome sequence data available for these important viruses. Our analysis of these genome sequence data reveals that all lyssaviruses have the same genomic organization. A phylogenetic analysis reveals strong geographical structuring, with the greatest genetic diversity in Africa, and an independent origin for the two known genotypes that infect European bats. We also suggest that multiple genotypes may exist within the diversity of viruses currently classified as 'Lagos Bat'. In sum, we show that rigorous phylogenetic techniques based on full length genome sequence provide the best discriminatory power for genotype classification within the lyssaviruses.

The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 A resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 56.9-57.2, c = 187.9-188.6 A, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-beta-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.

Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. VP7 and NSP4 are two glycosylated proteins. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In the meantime, viral particles were blocked as enveloped particles in the ER. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event.

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.