Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich “NR box” motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists. A structural study of the androgen receptor shows how it interacts with coactivator proteins. The results also provide a possible framework for the development of new receptor antagonists The androgen receptor (AR) is the cellular mediator of the actions of the hormone 5-α dihydrotestosterone (DHT). Androgen binding to AR leads to activation of genes involved in the development and maintenance of the male reproductive system and other tissues such as bone and muscle. However, it is the pivotal role of AR in the development and progression of prostate cancer that has led to increasing interest in this nuclear receptor. Presently, hormone-dependent prostate cancer is treated with a combination of strategies that reduce circulating levels of androgens, such as the administration of antiandrogens that compete for the androgen-binding pocket in the core of the C-terminal ligand-binding domain (LBD). The benefits of these treatments are typically transient, with later tumor growth associated with increases in expression levels of AR or its cofactors, or mutations that render AR resistant to antiandrogens . AlternaThe precise details of how AR binds the dozens of coregulator proteins reported to associate with different regions of AR in vivo remain poorly understood . Many nuAR, however, utilizes multiple mechanisms to activate gene transcription. Generally, AR activity is dependent on contributions from multiple transactivation functions that lie within the N-terminal domain (NTD) collectively called AF-1. Although the AR AF-2 can bind to a restricted set of LxxLL motifs  and is rPresently, how the AR AF-2 surface can accommodate residues with bulky aromatic side chains and distinguish FxxLF motifs from LxxLL motifs is not known. To understand the structural basis of this unusual coactivator recognition preference, we characterized the full repertoire of interacting sequences using phage display to define amino acids preferred at the AR coactivator binding interface. Crystal structures of the AR LBD in complex with several phage display–derived peptides reveal the structural basis of FxxLF motif specificity and an induced fit of the receptor that allows accommodation of other related hydrophobic motifs. Comparisons of the structures suggest strategies for the design of AR coactivator antagonists.10 randomized peptides against DHT-bound AR LBD. Selections identified sequences containing hydrophobic motifs that were primarily aromatic in character, consistent with another recent study (Phage display has been used to study coactivator recognition specificity and to identify coactivator motif sequence variants preferred by the estrogen receptor (ER), thyroid hormone receptor (TR) β, and most recently AR . Using pnt study    . SurprisP212121 with one molecule per asymmetric unit and unit cell dimensions similar to those observed in previous AR LBD crystal structures  reveals that the AF-2 cleft reorganizes to accommodate the bulky peptide side chains see B and 4. The preference of AR for motifs with aromatic groups over leucine-rich motifs was assessed with a crystal structure of the AR LBD in complex with the LxxLL peptide. The structure reveals similarities between the binding modes of the LxxLL and FxxLF motifs to AR, and other LxxLL motifs to other nuclear receptors. The LxxLL motif adopts a helical conformation, and interactions of the motif with the AF-2 cleft are predominantly hydrophobic, with the three leucine residues of the motif contributing most of the interactions. However, significant differences can be seen between the binding mode of the LxxLL motif to AR and that of p160-derived LxxLL motifs to other nuclear receptors. First, flanking residues were largely disordered, with only two N-terminal flanking residues and one C-terminal residue visible in electron density maps see  and 3B. Side chains of residues flanking the first leucine of the motif make additional hydrophobic interactions with the AR surface see B. Trp+2 To understand how the AR AF-2 accommodates tryptophan residues, structures of AR in complex with peptides containing tryptophan substitutions at the +1 or +5 position, or both, were determined . SurprisFinally, effects of substitutions at the +4 position were assessed in structures of AR in complex with peptides containing FxxFF and FxxYF motifs . SurprisBy contrast, the conformation of FxxYF was the closest to FxxLF see A. Other Together, the structures described above permit an assessment of the way that individual subsites of the AR AF-2 cleft accommodate hydrophobic groups. The indole rings of tryptophan and the phenyl rings of phenylalanine fit into their pockets analogously with the +1 and +5 residues bound facedown and edgewise, respectively, into the AF-2 cleft. On the other hand, the position of the +4 residue is variable, with binding in this shallow pocket largely dictated by the position of the peptide backbone caused by the bound conformations of the +1 and +5 residues see C. Small The binding mode detected in the +1 pocket is the most conserved of the three hydrophobic subsites see C. The beBinding of tryptophans in the +5 pocket is slightly more variable see C. Trp+5 The crystal structures reported here reveal how AR binds coactivator motifs with bulky aromatic hydrophobic groups and permit construction of a profile of the AR coregulator interface see . In someThe same characteristics that make the AR AF-2 ideal for binding of longer, aromatic side chains also make it less well suited for binding of shorter, branched side chains. Although changes in the position of Met734 widen the groove towards the +5 subsite to permit binding of leucine residues, the gross features of the groove remain largely the same see B. As a rUnlike the conserved interaction modes of aromatic residues with the +1 and +5 sites, binding interactions at the +4 site are variable and characterized by nonspecific interactions. This finding agrees with the relatively high conservation of residues at the +1 and +5 positions of AR-interacting motifs and suggests that these residues drive peptide interaction with the LBD, whereas the +4 site is less critical. Indeed, the +4 pocket is shallow, surface exposed, and relatively featureless, explaining the assortment of residues selected at the +4 position. It is likely that any hydrophobic residue that does not clash with surrounding residues would be suitable at this subsite.While peptide motif recognition is governed by hydrophobic interactions, polar interactions from backbone atoms and residues outside the core motif also contribute. With the exception of FxxFF, motifs containing phenylalanines at the +1 and +5 positions present canonical main chain interactions with both charge clamp residues, Lys720 and Glu897. This finding stands in contrast to predictions of previous studies , which cOn the other hand, Glu897 interacts with the FxxLF peptide backbone, but is disengaged from the LxxLL peptide backbone. One possible explanation for the apparent requirement for Glu897 in LxxLL binding is that it might interact with residues outside of the core motif. The corresponding glutamate of GR, Glu 755, forms hydrogen bonds with the −3 asparagine of TIF2 NR box 3 , and GluSequence alignment of NR coactivator sequences shows that positively charged residues are favored N-terminal to the core hydrophobic motif while negatively charged residues are favored C-terminal to the motif . Our phaFinally, the AR AF-2 surface is an attractive target for pharmaceutical design. Selective peptide inhibitors that bind the AF-2 surface of liganded ERα, ERβ, and TRβ have been developed , and simE. coli strain BL21 (DE3) STAR in the presence of 10 μM DHT. Induction was carried out with 30 μM IPTG at 17 °C for 16–18 h. E. coli cells were lysed in buffer (10 mM Tris, [pH 8.0], 150 mM NaCl, 10% glycerol, 1 mM TCEP, 0.2 mM PMSF) supplemented with 0.5 μg/ml lysozyme, 5 U/ml benzonase, 0.5% CHAPS, and 10 μM DHT. All buffers for further purification steps contained 1 μM DHT. Soluble cell lysate was adsorbed to Glutathione Sepharose 4 Fast Flow resin (Amersham Biosciences), washed with buffer containing 0.1% n-octyl β-glucoside, and eluted with 15 mM glutathione. After cleavage of the GST moiety with thrombin, final purification of the AR LBD was carried out using a HiTrap SP cation exchange column (Amersham Biosciences). Eluted AR LBD was dialyzed overnight at 4 °C against buffer containing 50 mM HEPES (pH 7.2), 10% glycerol, 0.2 mM TCEP, 20 μM DHT, 150 mM Li2SO4, and 0.1% n-octyl β-glucoside, then concentrated to greater than 4 mg/ml for crystallization.Expression and purification of the AR LBD for crystallization were performed essentially as described . The cDNE. coli biotin ligase (Avidity) into BL21 (DE3) STAR cells. Protein expression was carried out as above but with induction supplemented with 50 μM biotin to ensure quantitative biotinylation of AR LBD.Purification of AR LBD for use in phage affinity selection was carried out as above without the final dialysis and concentration steps. The expression construct contained the AR LBD as an inframe fusion with GST in a modified pGEX-2T vector containing both a flexible region and an AviTag sequence (Avidity, Denver, Colorado, United States) allowing in vivo biotinylation. The GST–AR LBD fusion expression plasmid was cotransformed with a plasmid-encoding 10 different random or biased amino acid sequences were added to the wells containing immobilized AR LBD and incubated for 3 h at 4 °C. After washing, bound phage were eluted using pH 2 glycine. Enrichment of phage displaying target-specific peptides was monitored after each round of affinity selection using an anti-M13 antibody conjugated to horseradish peroxidase in an ELISA–type assay.Phage affinity selections were performed essentially as described . BiotinySynthetic peptides corresponding to the deduced amino acid sequences from receptor-specific phage were tested for their ability to interact with purified AR LBD using a FRET–based assay format. Peptides were synthesized according to the deduced amino acid sequence displayed on phage with an additional C-terminal amino acid sequence consisting of SGSGK to allow the attachment of a biotin tag . Flourophor conjugates were prepared by incubating either biotinylated peptides with streptavidin-cryptate , or biotinylated AR LBD with streptavidin-XL665 (Cis Bio). Interaction between peptide and AR LBD was monitored by the ratio of energy transfer by excitation at 320 nm and emission at 625 nm and 665 nm.Affinities of peptides to the AR LBD were determined with a Biacore (Piscataway, New Jersey, United States) 2000 instrument. A peptide derived from silencing mediator for RXR and TR 2 (SMRT2) served as a negative control. 1 mM peptide stock solutions in DMSO were diluted into HBS-P buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 0.005% Surfactant P20) to generate 10 μM working solutions. HBS-P buffer was flowed through the cells to achieve a stable baseline prior to immobilization of the biotinylated peptides. To achieve the binding of approximately 250 RU of peptides to individual cells, working solutions of peptides were diluted to 100 nM in HBS-P buffer. Unbound streptavidin sites were blocked by injection of a 1 mM biotin solution at a rate of 10 μl/min.Purified AR LBD was diluted into HBS-P buffer to a concentration of 10 μM and injected into all four Flowcells using the Kinject protocol at a flow rate of 10 μl/min (contact time 360 s, dissociation time 360 s). Following the dissociation phase, the surface of the chip was regenerated to remove residual AR LBD by QuickInject of buffer containing 10 mM HEPES and 50% ethylene glycol (pH 11). Following the establishment of a stable baseline, the same procedure was repeated using a series of AR LBD dilutions (5 μM, 1 μM, and 300 nM) in an iterative manner. Analysis of the data was performed using BIAevaluation 3.0 software (Biacore). The SMRT2 signals were subtracted as background from the three remaining peptide signals. Data were best fit using the two-state conformational change model , 2002.Purified, concentrated AR LBD was combined with 3x to 6x molar excess of peptide and incubated 1 h at room temperature before crystallization trials. Complexes were crystallized using the hanging drop vapor diffusion method. Protein–peptide solution was combined in a 1:1 ratio with a well solution consisting of 0.6–0.8 M sodium citrate and 100 mM Tris or HEPES buffer (pH 7–8). Crystals typically appeared after 1–2 d, with maximal size attained within 2 wk. For data collection, crystals were swiped into a cryo-protectant solution consisting of well solution plus 10% glycerol before flash freezing in liquid nitrogen. The addition of ethylene glycol to a well concentration of 10%–20% was later found to both improve crystal quality and enable the freezing of crystals directly out of the drop.A-weighted 2Fo − Fc, Fo − Fc, and simulated annealing composite omit maps. Superposition of structures was performed with LSQMAN , beamline 8.3.1, with either a ADSC Quantum 315 or Quantum 210 CCD detector. Data were processed using Denzo and Scalepack . Moleculh LSQMAN . Buried h LSQMAN . Coordinhttp://www.ebi.ac.uk/swissprot) accession numbers for the gene products discussed in this paper are AR (P10275), ARA54 (Q9UBS8), ARA55 (Q9Y2V5), ARA70 (Q13772), ER (P03372, Q92731), glucocorticoid receptor-interacting protein 1 NR box 3 (Q61026 ), GR (P04150), NR box 3 of TIF2 (Q15596), and TR β (P10828).The Swiss-Prot (http://www.rcsb.org/pdb) accession numbers for the structures used in this paper are FxxFF (1T73), FxxLF (1T7R), FxxLW (1T79), FxxYF (1T7M), LxxLL (1T7F), unbound (1T7T), WxxLF (1T74), and WxxVW (1T76).The Protein Data Bank (


One of the most time-consuming tasks after performing a gene expression experiment is the biological interpretation of the results by identifying physiologically important associations between the differentially expressed genes. A large part of the relevant functional evidence can be represented in the form of graphs, e.g. metabolic and signaling pathways, protein interaction maps, shared GeneOntology annotations, or literature co-citation relations. Such graphs are easily constructed from available genome annotation data. The problem of biological interpretation can then be described as identifying the subgraphs showing the most significant patterns of gene expression. We applied a graph-based extension of our iterative Group Analysis (iGA) approach to obtain a statistically rigorous identification of the subgraphs of interest in any evidence graph.We validated the Graph-based iterative Group Analysis (GiGA) by applying it to the classic yeast diauxic shift experiment of DeRisi et al., using GeneOntology and metabolic network information. GiGA reliably identified and summarized all the biological processes discussed in the original publication. Visualization of the detected subgraphs allowed the convenient exploration of the results. The method also identified several processes that were not presented in the original paper but are of obvious relevance to the yeast starvation response.GiGA provides a fast and flexible delimitation of the most interesting areas in a microarray experiment, and leads to a considerable speed-up and improvement of the interpretation process. Microarray experiments can provide a comprehensive picture of gene expression levels in biological samples. In a typical application they compare expression of several thousand genes under two different conditions , using a small number of replicate experiments. Various techniques have been developed to rank genes according to their expression changes, e.g. based on the t-statistic  or the sThe main challenge to the biologist is contained in the next step of the analysis. It consists in identifying the biologically relevant expression changes, the "big picture" of the experiment. As microarray experiments tend to generate unexpected observations in areas outside the specialized expertise of the experimentalist, this can be quite difficult and time-consuming. A principled mechanism to identify the significant higher-level features of the experimental results would therefore be very useful.The biological interpretation process consists to a large extent of finding evidence connecting certain genes that are differentially expressed. This evidence can consist, e.g., of joint participation in some physiological process, physical interaction at the protein level, reported co-expression in earlier microarray experiments, a shared functional annotation, etc. This kind of evidence can intuitively be represented as a graph, and this feature is regularly used to visualize biological data, in the form of metabolic or signaling pathways or protein interaction maps. The task can then be described as the identification of subgraphs that as a whole show a statistically significant expression change. This would allow the biologist to focus her analysis on the most promising areas, without prior bias, while at the same time presenting the relevant evidence underlying each association for critical evaluation.We have recently developed an approach, iterative Group Analysis (iGA) that identifies significantly changed functional classes of genes in a microarray experiment . In contHere we extend this approach to the analysis of "evidence graphs", which offers much larger flexibility of the annotations that can be used and allows substantially improved visualization. Evidence graphs can be represented as bigraphs with two types of nodes, one for genes and one for the associated "evidence" Fig. . For evam) and, if present, all adjacent nodes of ranks equal or smaller than m. Hence, at each step of the extension process the newly extended subgraph is not adjacent to any outside node with a rank lower than m  or unchanged based on an arbitrary selected significance cut-off and thus discards most of the relative-change information (gene ranks) used by GiGA . Therefore, this method is difficult to apply to very noisy or unreplicated experiments where a reliable delimitation of the "changed" genes becomes impossible.The recently released commercial Pathway Analysis software from Ingenuity Systems To validate the GiGA approach, we used the yeast diauxic shift experiment by DeRisi et al. . In thisIn their original publication, DeRisi et al. highlight the following changes during starvation: Rechanneling of metabolites into the tricarboxylic acid (TCA) and glyoxylate cycle, increase in aerobic respiration (cytochrome c oxidase and reductase), gluconeogenesis, and carbohydrate storage . In contrast, 95% of ribosomal proteins, as well as tRNA synthetases and translation elongation/initiation factors were strongly down-regulated. Twenty hours after the initial inoculation of the sample about 20% of all genes showed at least a two-fold change in expression.Table This connectivity between genes and functional classes is provided by GiGA. Table The performance of GiGA (as well as iGA) is best appreciated when compared to the results of an extensive expert interpretation of the same data. Table It is important to be aware that each of the highlighted subgraphs has to be carefully evaluated for its biological relevance. On the one hand, the sheer number of possible subgraphs in an evidence network creates a major multiple-testing problem, which means that some of the detected associations may be due to chance. Random permutations of the expression data – which can be generated by the GiGA software – can give an idea of the expected false-discovery rate. On the other hand, functional annotations are at present notoriously unreliable and spurious edges may affect the details of the results. Also, not all genes within a detected subgraph will necessarily show a strong expression change, because sometimes less strongly affected genes may connect those genes that do change. Such a relation is for example expected for many transcription factors and their targets . Nonethem1 and m2 whenever their mass difference (Δm = |m1 - m2|) can be explained by a common biochemical transformation (e.g. dehydrogenation: Δm = 2* mass(hydrogen)). The set of relevant transformations can easily be collected from any biochemistry textbook. In addition, one can introduce edges for condensation reactions between observed masses, i.e. if m1 + m2 = m3 + mass(H2O) then edges between m1 and m3, and m2 and m3 are added to the evidence network. We are currently developing the application of GiGA to this kind of data in the Sir Henry Wellcome Functional Genomics Facility at the University of Glasgow ; data not shown).The GiGA method is not restricted to use with exhaustively annotated genomes. It can work on a wide variety of "evidence" to build the necessary network, including hypothetical predicted functions or associations. It is even possible to apply GiGA to metabolomics results, which are characterized by the absence of any significant amount of annotation – usually only exact molecular masses and their differential abundance are known. In that case, an evidence network can be built from the measured masses themselves, linking compounds The present analysis of a biologically well-understood test case demonstrates the reliable performance of GiGA. The method automatically identifies all relevant physiological processes, puts them into context, summarizes them in an intuitive format, and associates them with the underlying evidence Fig.  and 3. I. GeneOntology annotations  were obtained from Affymetrix . The enzyme substrate networks were built based on information contained in the annotation of the yeast proteome in the SwissProt database . The GiGA algorithm has been implemented as a Perl script and compiled as a Windows command line executable. These files are available  as Yeast gene expression data for the diauxic shift experiment were obtained from the Stanford Microarray Database RB devised and implemented the GiGA technique and drafted the manuscript. AA and PH supervised the project. All authors read and approved the final manuscript.GiGA program. For use from the Windows command line.Click here for fileGiGA source code.Click here for fileGiGA manual. Describes the use of GiGA applied to the example data .Click here for fileGene expression data. Sorted list of genes, based on expression during the yeast diauxic shift.Click here for fileEvidence network. List of gene pairs connecting genes whenever their gene products are enzymes that share a common substrate. Based on annotation derived from SwissProt.Click here for fileGenenames file. Contains descriptive names of the yeast genes contained in Additional file 4.Click here for fileExample output in text format. List of significantly affected subgraphs detected in the experimental data  using GiGA with default settings.Click here for file.Example output in graph-description language format. Contains the same results as Additional file 7, but in a format that can be visualized and explored using graph-layout software, e.g. aiSee, which is freely available for academics at Click here for file


The early stages consecutive to infection of sheep (e.g. primo-infection) by Bovine leukemia virus mutants are largely unknown. In order to better understand the mechanisms associated with this period, we aimed at analyzing simultaneously three parameters: B-lymphocytosis, cell proliferation and viral replication.Sheep were experimentally infected either with a wild type BLV provirus or with selected mutants among which: a virus harboring an optimalized LTR promoter with consensus cyclic AMP-responsive elements, two deletants of the R3 or the G4 accessory genes and a fusion-deficient transmembrane recombinant. Seroconversion, as revealed by the onset of an anti-viral antibody response, was detected at 3 to 11 weeks after inoculation. At seroconversion, all sheep exhibited a marked increase in the numbers of circulating B lymphocytes expressing the CD5 and CD11b cluster of differentiation markers and, interestingly, this phenomenon occurred independently of the type of virus. The net increase of the absolute number of B cells was at least partially due to accelerated proliferation as revealed, after intravenous injection of bromodeoxyuridine, by the higher proportion of circulating BrdU+ B lymphocytes. BLV proviral DNA was detected by polymerase chain reaction in the leucocytes of all sheep, as expected. However, at seroconversion, the proviral loads were lower in sheep infected by the attenuated proviruses despite similar levels of B cell lymphocytosis.We conclude that the proviral loads are not directly linked to the extent of B cell proliferation observed during primo-infection of BLV-infected sheep. We propose a model of opportunistic replication of the virus supported by a general activation process of B lymphocytes. Bovine leukemia virus (BLV) is an oncogenic retrovirus closely related to the primate T-cell leukemia viruses . These vA major advantage of the BLV system is the possibility to study viral genetic determinants in relation with infectivity and pathogenicity in vivo. A strategy, which we previously described, is based on the use of a cloned BLV provirus whose sequence can be mutagenized in vitro. Well characterized mutants can subsequently be injected into sheep and compared to the wild type virus (WT) . This ex(i) the R3 and G4 accessory genes are required for efficient viral spread in vivo, although their deletion or mutation does not hamper infectivity (mutants CRX3 and IG4 described by ,19,20).(ii) restoring a CRE consensus  in the triplicate motif of the imperfectly conserved Tax-responsive sequence (TxRE: AGACGTCA, TGACGGCA, TGACCTCA) increases LTR promoter activity, as expected, but restricts the proviral loads in vivo, suggesting that repression of expression is required for immune escape .(iii) formation of multinucleated syncytia by envelope dependent cell fusion in vitro is paradoxically not required for infectivity or efficient viral spread in vivo .Importantly, only the A60V envelope mutant behaves as wild type in terms of infectivity and pathogenesis, in contrast to the others (CRX3, IG4 and CRE) therefore referred to as attenuated. The two categories of viruses, either wild type (WT and A60V) or attenuated (CRX3, IG4 and CRE) thus permit to characterize and compare the processes occurring during primo-infection.+ (triangles) and CD8+ (crosses) T cell populations remained remarkably constant over extended periods of time, as expected  and mutants deleted in the R3 and G4 genes (CRX3 and IG4 proviruses) are impaired in their ability to propagate efficiently within their host ,19,21. T3/ml of blood to 4,858 103/ml, respectively, at days 21 and day 28; see squares), while the absolute counts of CD4+ (triangles) and CD8+ (crosses) T cells remained relatively constant (as did the γδ T cell population, data not shown). Furthermore, maximal B cell accumulation corresponded to the day of seroconversion characterized by the onset of an anti-BLV humoral response , the latter behaving as wild type in terms of pathogenesis and infectivity in vivo. Interestingly, B cell lymphocytosis occurred in all sheep independently of the type of provirus Figure  was indeExpression of the CD5 and CD11b cluster of differentiation markers has been associated with BLV-infection, although B lymphocytes negative for these receptors are also less efficient targets for the virus ,24. TherIn terms of absolute cell counts , it appeared that B+CD5+ or B+CD11b+ lymphocytes accumulated at the seroconversion day in wild type infected sheep 4535 and 4536  . Figure Since BLV mutants also induce a transient lymphocytosis, a kinetics of BrdU incorporation was performed in 4 additional sheep infected with proviruses IG4, CRX3, CRE and GP30  yielded a 1 kb fragment as expected. Under similar conditions, a weaker signal was generated by amplification of genomic DNA isolated from animals infected by mutant proviruses  or so-called attenuated mutants (IG4, CRX3 and CRE). Experimental infection of sheep with these two types of viruses led to a surprising observation, namely, a similar extent of transient lymphocytosis independently of the proviral loads . AnswerTrypanosoma evansi and Pasteurella haemolytica [With the aim to correlate lymphocytosis with a defined B cell sub-population, we have demonstrated that two surface molecules, CD5 and CD11b, whose expression has been previously associated with late stages of BLV infection -15,23,24molytica ,36. In tDifferences in the infectious potential of the attenuated viruses are the consequence of mutations in accessory genes (R3 and G4) or in the LTR promoter (CRE) ,21. AlthIn conclusion, we have characterized here the initial steps consecutive to BLV infection of sheep. We show that this period is characterized by a transient accumulation of CD5+ / CD11b+ B lymphocytes resulting, at least in part, from increased proliferation. Furthermore, the extent of B cell lymphocytosis is not directly linked to the proviral loads reached by the wild type and mutant viruses. On basis of a comparative leukemia approach, these results could be informative for the related human T-lymphotropic viruses.Roche Diagnostics) in 1 ml of HBS (20 mM HEPES-150 mM NaCl, [pH 7.4]) and injected intradermally into the back of each sheep. Plasmids containing the mutant proviruses pBLVIG4  techniques [Twelve sheep of one year old were kept under controlled conditions at the Veterinary and Agrochemical Research Centre (Machelen, Belgium). Two animals (n° 4533 and 4534) were used as uninfected controls whereas sheep n° 4535 and 4536 were experimentally infected with a BLV wild type cloned provirus (strain 344) . Brieflyg frame; , pBLVCRXg frame; ) and pBLg frame; ) were inchniques .VMRD Inc. Cells were then labeled with a rat anti-mouse IgG1 phycoerythrin (PE)-antibody (Becton Dickinson Immunocytometry Systems) or with a goat anti-mouse IgG2b fluorescein isothiocyanate (FITC)-conjugate . Finally, PBMCs were analyzed by flow cytometry on a Becton Dickinson FACScan flow cytometer. Ten thousand events were collected for each sample and data were analyzed with the Cellquest software (Becton Dickinson Immunocytometry Systems).Peripheral blood mononuclear cells (PBMCs) were isolated by Percoll gradient centrifugation and their viability was estimated by trypan blue dye exclusion ). PBMCs Sigma Aldrich) resuspended in physiologic serum (NaCl 0.9%). To evaluate BrdU-incorporation, blood was collected at three and seven days after each BrdU injection. The red blood cells were lysed with 1× FACS Lysing Solution (Becton Dickinson Immunocytometry Systems), the leucocytes were washed twice with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich) and incubated in the presence of biotinylated 1H4 monoclonal antibody for 30 min at 4°C. Next, the cells were labeled with streptavidin-phycoerythrin (Becton Dickinson Immunocytometry Systems) and incubated with 1× FACS Permeabilizing Solution (Becton Dickinson Immunocytometry Systems). Finally, leucocytes were stained with anti-BrdU FITC antibody in the presence of DNase (Becton Dickinson Immunocytometry Systems) and analyzed by flow cytometry.Each week during two months, sheep were injected intravenously with 500 mg of 5-bromo-2'-deoxyuridine (Wizard®Genomic DNA Purification Kit (Promega). An aliquot of 300 μl of blood were mixed with 900 μl of Cell Lysis Solution and incubated for 10 minutes at room temperature. After two washes with the same buffer, the cells were resuspended in 300 μl of Nuclei Lysis Solution and incubated for one hour at 37°C. Then, the samples were digested during 15 minutes at 37°C in the presence of 1.5 μl of RNase Solution. Proteins were precipitated by adding 100 μl of Protein Precipitation Solution to the nuclear lysates. After centrifugation at 13,000 g, the supernatant was mixed with an equal volume of isopropanol, centrifuged and ethanol precipitated. Five hundred nanograms of the purified DNAs were amplified in the presence of 200 μM of deoxynucleotides, 2.5 U of Taq DNA polymerase, and 200 ng of primers. The primers used (PCRTA 5'-CTCTTCGGGATCCATTACCTGA-3' and PCRTC 5'-CCTGCATGATCTTTCATACAAAT-3') encompass the region from position 7999 to 6990 of the BLV tax gene [gapdh gene. The samples were denatured for 5 min at 94°C, and amplified by 25 cycles of PCR (30 s at 94°C, 30 s at 57°C, and 1 min at 72°C). After a final elongation step of 10 min at 72°C, 20 μl of the amplification products were resolved on a 1% agarose gel, transferred to a Hybond N+ membrane (Amersham Pharmacia Biosciences), and hybridized either with a BLV tax (a 1-kb ClaI insert from plasmid pGEM7zfLOR1) or with a gapdh probe labeled with α-32P dCTP. Quantification of 32P signal was performed using a PhosphorImager .DNA isolations were performed directly on blood using the tax gene . In parapol gene and the 18S ribosomal DNA sequences essentially as described in reference [Real-time PCR was performed using 6FAM-labeled MGB probes specific for the BLV eference .BLV: Bovine Leukemia Virus; BrdU: 5-bromo-2'-deoxyuridine; CRE: Cyclic-AMP Response Element; PBMCs: Peripheral Blood Mononuclear Cells; WT: Wild Type.The author(s) declare that they have no competing interests.CD carried out the most experimental work and drafted the manuscript. MS and FM performed the sample collections and the determination of the proviral loads. PK was responsible for the sheep studies. RK participated to experimental design and interpretation of data. LW conceived the study, its design and coordination. All authors read and approved the final manuscript


Diatoms are unicellular algae with ornate silica shells. Their dazzling ability to build tiny structures could inspire applications in the semiconductor industry, drug delivery, and engineering Thalassiosira pseudonana (http://genome.jgi-psf.org/diatom/).Diatoms, unicellular algae with ornate silica shells, have fascinated amateur and professional biologists ever since the invention of the microscope. But these days, diatoms and their exquisite shells are also attracting the attention of nanotechnologists who hope that diatoms will teach them how to make minute structures currently beyond the capabilities of materials scientists. And now these nanotechnologists, together with ecologists interested in the global carbon cycle—in which diatoms play a central role—have a genomic blueprint to help them in their studies: the annotated genome sequence of Diatoms, microalgae that are found in all aquatic and moist environments, first appeared more than 180 million years ago. Since then, diatom diversity has literally exploded; no one is sure how many living species there are—probably about 100,000—or why there are so many different types. Plant molecular biologist Chris Bowler  explains that molecular phylogeny and morphological studies suggest that diatoms originated ‘probably as the result of a eukaryote being invaded or engulfed by a photosynthetic eukaryote, most probably a red alga’.The basic structure of all diatoms is similar: a single cell, often with a large vacuole, contained within a silica shell or frustule made of two overlapping halves or valves joined by girdle bands, which are also made of silica. The girdle bands form the rims of the two valves and allow unidirectional growth of the diatom during vegetative division. ‘The shell is rather like a Camembert cheese box or a petri dish’, explains marine ecologist Christian Hamm (Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany).There are only two main types of diatom: centric diatoms, which often have a circular symmetry, and pennate diatoms, which are usually bilaterally symmetrical. Nevertheless, diatom shells come in a dazzling array of forms and sizes ; Box 1. Richard Gordon, Professor of Radiology at the University of Manitoba in Winnipeg, Canada, somewhat accidentally laid the foundations of ‘diatom nanotechnology’ in 1988 when he was invited to give a lecture at an engineering conference. ‘I'm not an engineer’, explains Gordon, ‘but I knew engineers were interested in what was then called microfabrication so I told them about diatoms because they are so good at making small things’. Gordon, a keen diatom hobbyist, explained to his audience how diatoms could make a three-dimensional micro- or nanoscale structure for them without them lifting a finger. By contrast, says Gordon, ‘nanotechnology techniques then and now are tedious, involving painstakingly building three-dimensional structures up layer by layer’.Such tedious techniques are currently used in the semiconductor industry. At present, explains Michael Sussman, Director of the Biotechnology Center at the University of Wisconsin-Madison (Madison, Wisconsin, United States), ‘features are etched onto circuit boards using light. However, the wavelength of light limits the smallest size that can be achieved, and for the next generation of faster computers, engineers need to get denser features onto computer chips than is possible with light etching’. Diatoms, says Sussman, ‘are natural-born lithographers in the nanometre range. If we could work out how diatoms lay down micro lines of silica, then we may be able to simulate it’. The proteins that diatoms use to direct silica deposition could be very useful to the semiconductor industry, says Sussman.There are other ways in which diatoms could help us clumsy humans build nanoscale ‘widgets’. Molecular biologist Mark Hildebrand  is a member of a collaborative project trying to develop genetically engineered micro/nanodevices . Already, engineers are using diatoms to help them build extremely sensitive sensors based on microfluidic devices, he explains. Hildebrand is also interested in the optical properties of diatoms. ‘Information processing technology is moving from electronically to optically based hardware, which allows more information to be carried and stored. Optical systems need materials with regularly repeating structures with features below the micrometre size range. These are very difficult to make by standard manufacturing techniques, but diatoms make structures like this all the time’.It might also be possible to use diatom shells as delivery vehicles for drugs, suggests chemical engineer Tony Rogers, an assistant professor at Michigan Technological University (Houghton, Michigan, United States). ‘They have a uniform nanoscale pore structure and are chemically inert and biocompatible’. Rogers envisages loading diatoms with a drug that would then leach out into the blood stream at a rate dependent on the diatom species used. By incorporating ferromagnetic particles within the diatom structure, it might be possible to use a magnet to guide the drug to the right organ, he suggests.Diatom structures are not just of interest to people interested in tiny objects. As Hamm comments, ‘in diatoms, Nature has solved many of the problems that engineers want to solve. For example, diatoms are particularly good at making lightweight but strong structures. Because it is possible to scale static structures like shells, diatoms can teach us how to make lightweight constructions for the aerospace and car industry’.Some of the potential applications of diatoms can be investigated right now, using naturally occurring diatoms. In addition, subtle but important changes can be induced in diatoms by varying the amount of silica in their environment or changing the water flow. Gordon also envisages a device he calls a compustat, which would be used to select diatoms for a specific purpose. Diatoms taken from the sea, for example, would be individually examined using a computer-controlled microscope. ‘We would tell the computer what characteristics we were looking for, and it would go through the culture, zapping those diatoms furthest from the ideal with a laser beam. The culture would then be allowed to grow up again and the process repeated until we got the sort of diatoms we wanted’, says Gordon.Gordon has not built a compustat yet—it may not work, he says, because we don't know how far we can push diatoms by forced evolution. And even if the compustat does work, to make the most of the nanotechnological potential of diatoms, we need to know exactly how diatoms make their shells. At present, all we know is that silicon transporters and a group of long-chain, polyamine-containing proteins called silaffins, which act as nucleation points for silica deposition, are involved. This is where the diatom sequencing project at the United States Department of the Environment's Joint Genome Initiative (JGI) at Walnut Creek, California, comes in.T. pseudonana, a marine centric diatom ‘We believe that knowing this genome will help us to figure out how to mimic the processes that diatoms use to construct their very precise structures, and that we can then learn how to create similarly precise structures ourselves’. Also, he adds, diatoms are extremely important on an ecological level.Daniel Rokhsar, Department Head for Computational Genomics at JGI, explains why his institute undertook the sequencing and computer annotation of the genome of T. pseudonana, explains further. Diatoms are responsible for between 25% and 40% of all the primary productivity of the oceans, she says. ‘They also keep the biological pump going. By fixing carbon dioxide and then sinking, diatoms draw carbon dioxide out of the atmosphere and take it into the deeper waters of the ocean, where it is retained for longer than it would be if the diatoms stayed near the surface’.Oceanographer Ginger Armbrust (University of Washington, Seattle, Washington, United States), Principal Investigator on the sequencing project for T. pseudonana, she continues, was chosen as the first diatom to sequence in part because it has a small genome, but mainly because it represents a cosmopolitan genus of diatoms and its physiology has been well studied. Once the primary sequence of the genome had been determined, molecular biologists, oceanographers, and ecologists from around the world gathered at JGI for a ‘genome jamboree’. ‘The first of these was in October 2002, a massive brainstorming session at which we all dug around in the genome for our favourite genes and tried to get a feel for what was there’, explains Bowler. ‘It was really refreshing to get the insights of oceanographers and ecologists into what this genome was telling us’.T. pseudonana genome can tell them about the difference between photosynthesis on land and in the sea. They also want to investigate how these organisms adapt to their environment. ‘Now that we have the genome’, says Armbrust, ‘we can investigate how gene expression varies at different places in the water column, for example. This will be the first time a eukaryotic genome has been interpreted in this ecological sort of way’.Among other things, Armbrust and her collaborators are interested in finding out what the T. pseudonana genome is that we can figure out quite a bit from it about how this diatom deals with organic materials, but it is hard to figure out what it is doing with silicon’, admits Rokhsar. ‘The only way we can really figure out what a gene is doing is by comparing it with known genes in other organisms, but because diatoms are so unique in their use of silicon, we don't have that option. We literally just have the parts list’.‘One of the striking things about the T. pseudonana genes is important in silicon metabolism, Sussman is using microarrays to investigate how silicon concentrations affect gene expression patterns in the diatom. ‘There may be a few hundred genes whose expression changes in response to silicon stress’, he predicts, ‘and we can then focus on the role that these genes play in silicon metabolism’. In another approach, Hildebrand is purifying the proteins present in diatom shells. ‘Once we have isolated these proteins, we can get a little bit of protein sequence, and from there go back to the genome to pull the gene out’, he explains.To get a hook on which of the 10,000 or so T. pseudonana. Unfortunately, this can't currently be done. ‘The only diatom we can genetically manipulate is Phaeodactylum tricornutum, a pennate diatom’, explains Bowler. P. tricornutum, he says, is the ‘lab rat’ of the diatom world but is much less important ecologically than T. pseudonana  by oceanographer David Thomas. ‘What we saw down the microscopes just blew our socks off’, says Parker-Eaton. ‘We could see how the plankton moved, the forms, the incredible different layers within the diatoms. What we particularly liked about diatoms was their complexity—they are totally unlike any other life form’.Navicula sp. The main body is sycamore, the ‘blobs’ are resin, and the object is coloured with inks. The piece is held together by magnets but splits in half to reveal two silver inserts at its centre. More examples of these artists' work can be seen at http://www.louiseandsarah.com.Parker-Eaton and Hibbert translate what they see down the microscope into objects made of wood and silver, usually small enough to hold in the hand.


Macaca nemestrina rhadinovirus-2 (MneRV2). These primers showed little similarity to the corresponding sequences of the macaque RV1 rhadinoviruses, retroperitoneal fibromatosis herpesvirus Macaca nemestrina (RFHVMn) and Macaca mulatta (RFHVMm). To determine viral loads per cell, an additional TaqMan QPCR assay was developed to detect the single copy cellular oncostatin M gene.Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) have been identified in macaques and other Old World non-human primates. We have developed a real-time quantitative PCR (QPCR) assay using a TaqMan probe to differentially detect and quantitate members of the rhadinovirus-2 (RV2) lineage. PCR primers were derived from sequences within ORF 60 and the adjacent ORF 59/60 intergenic region which were highly conserved between the macaque RV2 rhadinoviruses, rhesus rhadinovirus (RRV) and 6 cells were detected in PBMC from randomly sampled macaques from the Washington National Primate Research Center. Screening tissue from other primate species, including another macaque, Macaca fascicularis, and a baboon, Papio cynocephalus, revealed the presence of novel rhadinoviruses, MfaRV2 and PcyRV2, respectively. Sequence comparison and phylogenetic analysis confirmed their inclusion within the RV2 lineage of KSHV-like rhadinoviruses.We show that the RV2 QPCR assay is linear from less than 2 to more than 300,000 copies using MneRV2 DNA, and is non-reactive with RFHVMn DNA up to 1 billion DNA templates per reaction. RV2 loads ranging from 6 to 2,300 viral genome equivalent copies per 10We describe a QPCR assay which provides a quick and sensitive method for quantitating rhadinoviruses belonging to the RV2 lineage of KSHV-like rhadinoviruses found in a variety of macaque species commonly used for biomedical research. While this assay broadly detects different RV2 rhadinovirus species, it is unreactive with RV1 rhadinovirus species which commonly co-infect the same primate hosts. We also show that this QPCR assay can be used to identify novel RV2 rhadinoviruses in different primate species. Rhadinovirus genus of the gammaherpesviruses are lymphotrophic and are associated with a variety of lymphoproliferative diseases. Herpesvirus saimiri (HVS), the prototype rhadinovirus isolated from the South American squirrel monkey, causes fulminant T-cell lymphomas in closely related host species [Members of the  species . Kaposi' species . Other r species ,4.M. mulatta) in the New England National Primate Research Center (NENPRC) [M. nemestrina RV2 (MneRV2) [We and others have demonstrated the existence of two distinct lineages of KSHV-like rhadinoviruses in Old World non-human primates ,6. The r(NENPRC)  and pig-(MneRV2) ,10,11. W(MneRV2) ,13, phyl(MneRV2) . Althoug(MneRV2) -17.While complete genomic sequences have been obtained for two closely related strains of the RV2 lineage rhadinovirus of rhesus macaques, RRV strain H26-95 from the NENPRC  and RRV M. nemestrina, which has been previously shown to contain a co-infection of both MneRV-2 and RFHVMn rhadinoviruses [The ORF 59 and ORF 60 genes show high levels of homology between the related rhadinoviruses, KSHV and RRV, with 52% and 70% identity at the amino acid level, respectively . Using tMultiple alignment of the RRV and MneRV2 nucleotide sequences revealed large regions of identical sequences within both the ORF 59 and ORF 60 coding regions and the ORF 59/60 intergenic region. As shown in Figure M. nemestrina OSM gene (data not shown). Multiple alignment of the human, monkey and macaque OSM sequences revealed a region within exon 3 which was highly conserved. Using Primer Express software, a set of primers  and a probe (OSM-FAM) were identified  . We had ied Fig.  which coThe RV2 and OSM QPCR assays were optimized using DNA obtained from the spleen of a rhesus macaque, MmuA01111, which we have previously determined to contain RRV DNA in a background of macaque genomic DNA . Initial5 copies of MneRV2, with a slope of -3.320 (100% efficiency) and r2 = 0.997. For the OSM assay, MmuA01111 genomic DNA was assayed in duplicate using 4-fold dilutions, with the amount of DNA tested ranging from 0.06 ng  up to 1 μg . The assay was linear across this range with a slope of -3.322 (100% efficiency) and r2 = 0.999  was assayed in duplicate using 4-fold dilutions. As seen in Figure 999 Fig. .4 RRV copies per μg genomic DNA (MmuA01111 DNA undiluted) with a slope of -3.318 (100% efficiency) and r2 = 0.988  to more than 1.7 × 106 copies of the KSHV genome and a sample containing 109 copies of a PCR product of the ORF59/60 junctional region from RFHVMn were used as templates in the RV2 assay. The RV-2 QPCR assay was negative for these templates under the standard reaction conditions.To ensure that the RV2 assay does not detect RV1 viruses, the assay was performed using DNA from the human and macaque RV1 rhadinoviruses. A DNA sample from the KSHV infected BCBL-1 cell line  containiM. mulatta and M. nemestrina, RRV and MneRV2, respectively, we tested to see if this assay could be used to identify a novel RV2 rhadinovirus in M. fascicularis. DNA was obtained from spleen tissue of Mfa95044, an M. fascicularis from the Tissue Distribution Program at the WaNPRC. Approximately 250 ng of spleen DNA produced a positive result in the RV2 QPCR assay with an average cycle threshold (CT) of 31.9 cycles. In order to prove that the assay detected a novel rhadinovirus, CODEHOP primers were used in a PCR amplification reaction with the Mfa95044 spleen DNA to obtain the ORF59/60 intergenic region of this rhadinovirus as described in the Materials and Methods. An 832 bp PCR product was obtained and sequenced. A comparison of this sequence with the corresponding region from RRV and MneRV2 showed 94% and 86% nucleotide identity, respectively. The nucleotide identity with the corresponding region in RFHV and KSHV was only 59% and 60%, respectively. Phylogenetic analysis showed a close clustering of the M. fascicularis sequence with the RRV sequence and a more distant relationship with the MneRV2 sequence, confirming its origin from an RV2 rhadinovirus of M. fascicularis, herein termed MfaRV2 , herein termed PcyRV2. Phylogenetic analysis demonstrated that while PcyRV2 clustered within the RV2 rhadinovirus lineage, it branched off separately from the macaque RV2 rhadinoviruses as expected for a baboon rhadinovirus  [Previously, an RV2 rhadinovirus, PapRV2, was detected in a baboon ( anubis) , and a p anubis)  to ampliT values . The cumulative fluorescence curve for the MneRV2 and RRV samples were superimposable with slopes typical of those seen in the assays performed in Figures M. fascicularis and baboon templates produced fluorescence curves with significantly decreased slopes, indicating lower amplification efficiencies. The efficiencies of these PCR reactions were calculated to be approximately 81% (r2 = 0.900) for the MfaRV2 and 72% (r2 = 0.929) for the PcyRV2, however, the low levels of virus in these samples made it difficult to accurately determine the efficiencies, as indicated by the correlation coefficients.In order to compare the ability of the RV2 QPCR assay to detect different rhadinovirus templates, test samples containing roughly equivalent viral copy numbers in a background of genomic DNA were prepared. DNA from purified MneRV2, DNA from MmuA01111 spleen which contains RRV, and DNA from Mfa95044 spleen which contains MfaRV2 were diluted in DNA from a virus negative macaque to have approximately the same virus load as that found in the baboon lymphocyte DNA containing PcyRV2. As shown in Figure The novel ORF 59/60 intergenic regions of MfaRV2 and PcyRV2 were aligned with the corresponding sequences of RRV, MneRV2, RFHVMn, and KSHV. Also aligned was a partial sequence of the ORF 59/60 region obtained from RFHVMm . As shown in Figure 6 cells  and RV2 (71 bp), viral loads can even be determined in formalin-fixed paraffin embedded tissue in which significant degradation of the DNA has occurred. Due to the similarities in sequence of the human, macaque and African green monkey OSM genes, the OSM QPCR assay may be suitable for quantitation of DNA in tissue from a number of other Old World primate species.M. nemestrina, 1 of 7 M. fascicularis and 1 of 2 macaques whose species is not known. In these macaques, the viral copy number was determined to range from 6–2300 per 106 cells. Although the copy number in the single positive M. fascicularis was calculated to be 250 viruses per 106 cells, this would be a low estimate due to the 81% efficiency of the amplification of that template, as discussed above. Our results for RV2 rhadinoviruses in the macaque species tested at the WaNPRC were similar to those determined for RRV in rhesus macaques at the Tulane National Primate Research Center [6 cells. In the other 28 animals, the RRV load was below the level of detection. While RRV was detected more frequently in SIV-infected macaques in this study, the virus load was similar to that seen in healthy macaques.We have screened DNA from a number of random PBMC samples from macaques at the WaNPRC for the presence of an RV2 rhadinovirus. We detected RV2 rhadinovirus DNA in 6 of 30 macaques; 4 of 20 h Center . In the Macaca fascicularis) and the lymphocytes of a baboon . The standard RV2 assay had an amplification efficiency less than 100% with the M. fascicularis and P. cynocephalus templates which cautions against its use for accurate quantitation of the MfaRV2 and PcyRV2 rhadinoviruses. The primer and probe binding regions of these two rhadinoviruses showed nucleotide mismatches which correlate with the decrease amplification efficiency of the assay.The Tulane RRV assay had a similar sensitivity to our RV2 assay, with a lower limit of one RRV genome per 10,000 cell equivalents however, it was designed to specifically target only RRV while our RV2 assay is capable of detecting RRV, MneRV2 and other macaque and baboon rhadinoviruses. In this report, we have used the RV2 assay to detect novel RV2 rhadinovirus homologs in both the spleen of a crab-eating macaque  442N were provided by R. Shibata while at the National Institutes of Health, Bethesda, MD. This pig-tailed macaque had been experimentally infected with a pathogenic SHIV strain [Macaca mulatta (Mmu) YN91-224, an SIV-infected rhesus macaque diagnosed with RF, was kindly provided by H. McClure, Yerkes National Primate Research Center. Fresh frozen spleen tissue samples were also obtained from Macaca mulatta (Mmu) A01111 at the WaNPRC, a rhesus macaque that had been experimentally infected with SIV which we have shown to be co-infected with the RV1 and RV2 macaque rhadinoviruses, RFHVMm and RRV, respectively (unpublished observations). Fresh frozen spleen tissue from a Macaca fascicularis (Mfa) 95044 and lymphocytes from a baboon  (Pcy78404) were kindly provided by H. Bielefeldt-Ohmann and C.-C. Tsai, respectively, from the WaNPRC. DNA from the PBMC of thirty random healthy colony macaques was also obtained from the virus screening program at the WaNPRC.Fresh frozen spleen tissue samples from V strain . We haveV strain . Fresh fThe KSHV-infected pleural effusion lymphoma cell line, BCBL-1, was obtained from D. Ganem (Howard Hughes Institute – UCSF), and was carried in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, streptomycin, glutamine, and β-mercaptoethanol. Rhesus primary fetal fibroblasts (RPFF) were kindly provided by Dr. Michael Axthelm (ONPRC).M. nemestrina, MneJ97167, at the WaNPRC. The MneRV2 was used to infect cultures of RPFF and viral particles were harvested from culture supernatent by high speed centrifugation. Viral DNA used as positive controls in the PCR assays was obtained by disruption of the viral particles using phenol/chloroform and ethanol precipitation.An isolate of MneRV2, was obtained from an DNA was extracted from frozen tissues using standard proteinase K-phenol/chloroform extractions and concentrated by ethanol precipitation.The protein sequences of the ORF 59 and ORF 60 genes from KSHV and RRV were aligned using ClustalW. The consensus-degenerate hybrid oligonucleotide primer (CODEHOP) technique ,21 was uTo obtain the ORF 59/60 junctional regions between the RDEL motif of ORF 60 and the PQFV motif of ORF 59 of MneRV2, PcyRV2, RFHVMn, and RFHVMm, DNA was obtained from different sources and used in PCR amplification with different CODEHOP PCR primers. Reactions were performed in 1 μM forward and reverse primers, 200 μM each dNTP, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 2.5 units Platinum Taq polymerase (Invitrogen) using a 55–70°C annealing temperature gradient (BioRad Icycler). For MneRV2, PCR amplification was performed on Mne442N spleen DNA using primers RDELa and PQFVb. For PcyRV2, PCR amplification was performed on lymphocyte DNA from baboon Pcy78404, using SRDEa and QFVRb. In both cases an ~830 bp PCR fragment was obtained and sequenced. To obtain the sequence of RFHVMn which had a low copy number, it was necessary to amplify the RDEL-PQFV region in two fragments. A CODEHOP primer NFFEa . Phylogenetic tree output was produced using TreeView.The RV2 assay was designed to amplify a 71-bp amplicon from the ORF 59/60 junctional region of macaque viruses belonging to the RV2 rhadinovirus lineage using consensus primers "RV2a" (forward primer 5'-TCTGAATATGTCACATCCGTTCATA-3') and "RV2b" (reverse primer 5'-GGCCCGGAAAATGAGTAACA-3') with a TaqMan probe "RV2" 5'-(6-FAM)-TGATCTGTAGTCCCCATGTGTCC-(BHQ-1)-3' (Table T) determined using the Bio-Rad software. The viral load was calculated as a cellular genome copy equivalent by using the formula:Reactions (50 μl) contained approximately 250–1000 ng of template DNA, 1 μM forward and reverse primers, 100 nM probe, 200 μM each dNTP, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 2.5 units Platinum Taq polymerase (Invitrogen). Magnesium chloride concentrations were 4.0 mM for the RV2 assay and 2.0 mM for the OSM assay. After activation of the polymerase by incubation for 1 minute at 95°C, amplification was performed on a Bio-Rad iCycler equipped with an optical module for 45 cycles of 95°C for 30 s, 62°C for 30 s and 72°C for 30 s. The copy number for each assay was calculated from the cycle threshold  = Viral copy number/diploid OSM copy numberSamples were assayed in duplicate and the means were determined. Standard deviations were calculated using the sum of the errors of the viral and OSM copy number determinations.T, cycle threshold; KSHV/HHV8, Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8; Mfa, Macaca fascicularis; MfaRV2, Macaca fascicularis rhadinovirus-2; Mm/Mmu, Macaca mulatta; Mn/Mne, Macaca nemestrina; MneRV2, Macaca nemestrina rhadinovirus-2; ORF, open-reading frame; OSM, oncostatin M; Pcy, Papio cynocephalus; PcyRV2, Papio cynocephalus rhadinovirus-2; PCR, polymerase chain reaction; QPCR, quantitative PCR; RFHV, retroperitoneal fibromatosis herpesvirus; RRV, rhesus rhadinovirus; RV1, rhadinovirus-1; RV2, rhadinovirus-2;AGM, African green monkey; CODEHOP, consensus-degenerate hybrid oligonucleotide primer; CThe author(s) declare that they have no competing interests.Design and conception of the study (AGB, TMR); development of the methods for amplification of the ORF59/60 regions (AGB, TMR); Development of the QPCR assays and quantitative analysis (AGB, AMB); Virus isolation and preparation (MET); Sequence analysis, alignment and phylogeny (AGB, AMB, TMR); Manuscript preparation (AGB, AMB, MET, TMR). All authors read and approved the final manuscript.


Feeding normal rats with high dietary levels of saturated fat leads to pathological conditions, which are quite similar to syndrome X in humans. These conditions such as hypertriglyceridemia, hypercholesterolemia, obesity, and hyperglycemia might induce hypertension through various mechanisms. Metabolic syndrome and the resulting NIDDM represent a major clinical challenge because implementation of treatment strategies is difficult. Vascular abnormalities probably contribute to the etiology of many diabetic complications including nephropathy, neuropathy, retinopathy, and cardiomyopathy. It has been shown that in Streptozotocin induced diabetic animals there is an increase in maximal responses to 5-Hydroxytryptamine and Angiotensin II. The purpose of this study was to evaluate High fat diet fed rats for the development of hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and hyperglycemia and to assess their vascular responses to 5-Hydroxytryptamine and Angiotensin II.Male Sprague Dawley rats were used for this study and were divided into two equal groups. One of the groups was fed with normal pellet diet and they served as the control group, whereas the other group was on a high fat diet for 4 weeks. Body weight, plasma triglycerides, plasma cholesterol, and plasma glucose were measured every week. Intraperitoneal glucose tolerance test was performed after 4 weeks of feeding. At the end of fourth week of high fat diet feeding, thoracic aortae were removed, and cut into helical strips for vascular reactivity studies. Dose-response curves of 5-Hydroxytryptamine and Angiotensin II were obtained.2, with 5-Hydroxytryptamine and Angiotensin II in both groups but Emax was increased.There was no significant difference in pDThese results suggest that hypertension in high fat diet rats is associated with increased in vitro vascular reactivity to 5-HT and Ang II. Syndrome X comprises a plethora of conditions such as obesity, dyslipidemia, impaired glucose tolerance, insulin resistance and hypertension . It placThe aim of this study was to elucidate the contractile responses to 5-HT and Ang II in HFD fed rat thoracic aorta which will provide an avenue for further exploratory studies. We have selected HFD fed rat model for our study because it is a useful model of the putative effects of excess fat intake in humans and it represents the major sub type of diabetes mellitus, non insulin dependent diabetes mellitus (NIDDM).HFD fed rats showed significant increase in body weight as compared to NPD fed rats Table . In addimax . The order of potency of agonists in both groups was Ang II>5-HT>KCl.Cumulative concentration response curves of 5-HT and Ang II for both HFD fed rat thoracic aortae showed an increase in Emax Fig  and 3 wiObesity is a major risk factor for several metabolic diseases, frequently clustering to form the metabolic syndrome or syndrome X . Obese pHyperglycemia is observed in insulin resistance where glucose utilization is reduced. We saw significant elevations in blood glucose levels. Intraperitoneal glucose tolerance tests confirm severe glucose intolerance. Oversupply of dietary lipids causes insulin resistance in rats . Randle Insulin resistance with compensatory hyperinsulinemia is a prominent feature of metabolic syndrome. The most common reason for the development of hyperinsulinemia in insulin resistance is obesity. It stands as one of the major cardiovascular risk factors in patients with obesity. The present study on HFD rats demonstrated higher plasma insulin levels than control values. This marked hyperinsulinemia could be due to a combination of increased β-cell mass and decreased insulin clearance, as well as failure of insulin to suppress hepatic gluconeogenesis .Elevated cholesterol is also observed in insulin resistant individuals. For this reason we measured plasma cholesterol levels which were found to be more than normal values. Previous studies have reported a down regulation of LDL receptors and associated decrease in LDL clearance, increased total cholesterol levels .According to National Cholesterol Education Program's Adult Treatment Panel III (Third report) easily measured clinical findings for syndrome X includes increased abdominal circumference, elevated triglycerides, low high-density lipoprotein-cholesterol, and elevated fasting blood glucose and/or elevated blood pressure. Three of these five are required for diagnosis. Our study demonstrated three of the clinical parameters indicating conditions of syndrome X in HFD fed rats . InsulinIn order to elaborate the pathways that connect syndrome X to hypertension we have studied the contractile responses to 5-HT and Ang II in both HFD and NPD fed rat thoracic aortae. Previously we have demonstrated increased contractile responses with synthetic alpha adrenoceptor agonist, phenylephrine, in HFD fed rat thoracic aorta . This en2 value. Endothelial denudation obviates any related mechanisms such as impairment of NO release, increased destruction of EDRF and substrate availability for the production of EDRF. Hence, the probable reasons for these enhanced 5-HT and Ang II responses may be due to receptor mediated or non-receptor mediated pathways.The present vascular studies demonstrated that the magnitude of responses to 5-HT and Ang II was significantly enhanced in HFD fed animals without change in pD2A upregulation as observed in spontaneously hypertensive rats or due to serotonin acting through alpha adrenoceptors [The role of non-receptor mediated contraction can be ruled out for there was no change in contractile response to KCl. Vascular studies have also shown functional evidence that hypertension developed in HFD fed rats may be associated with enhanced vasoreactivity to various vasoconstrictor agents. Further the enhanced responses to 5-HT in HFD fed rats could be due to increased PKC as previously reported with STZ and alloxan (ALL) induced diabetic animals . Increasoceptors .Increased Ang II responses in HFD fed rats are may be due to upregulation of Ang II receptors as observed in hyperinsulinemia or via amplified secondary messenger systems . A propoIn summary, the present study has shown that HFD feeding in rats produces conditions similar to syndrome X. Increased vasocontractile responses observed in the model are not only mediated via alpha adrenoceptors but also due to 5-HT and Ang II , India), 160–200 g, were kept in controlled environmental conditions with room temperature 22 ± 2°C, humidity 55 ± 5% and 12-h light/dark cycles. All the animals had free access to food and water. The rats were divided into two dietary groups and fed with standard rat normal pellet diet (NPD)  and HFD . Composition of HFD is described in Table Blood samples from the retro orbital plexus of anaesthetized rats (Pentobarbitone 45 mg/kg, i.p.,) were collected into the heparinized tubes and immediately centrifuged at 5000 rpm for the separation of plasma. Plasma was stored at -20°C until assayed. The plasma was used for the estimation of glucose , triglycerides, and cholesterol  by commercial kits. Plasma insulin was determined by radioimmuno assay using rat insulin as standard (Linco research, St. Charles, MO, USA)Glucose tolerance tests were carried out after four weeks of feeding of both NPD and HFD. After an overnight fast, blood samples were collected from the retro orbital plexus. Glucose levels were measured at time zero (0 min) and glucose was injected into the rats (2 g/Kg/4 ml, i.p.,). Additional blood samples were taken at 15, 30, 60 and 120 min. following the glucose load. Plasma glucose levels were measured by the glucose oxidase reaction (GOD/POD) using commercial kit . Area under the curve (AUC) was calculated for both NPD and HFD fed rats.3, 2.6 CaCl2 2H2O 1.2 NaH2PO4, 1.2 MgCl2 6H2O, 5.5 glucose). With the help of a steel rod, aortic endothelium was deliberately denuded. The aorta was cut into helical strips 3 mm wide, 20 mm long and then placed in a well-oxygenated (95% O2-5% CO2) bath of 10-ml KHS with one end connected to a tissue holder and the other to an isotonic transducer . The tissue was equilibrated for 60 min under a resting tension of 1.0 g. At the beginning of each experiment, aortic strips were primed with depolarizing concentration (90 mM) potassium chloride (KCl). After the equilibration period, contractile responses to various concentrations of 5-HT (10 nM-30 μM) and Ang II (1 nM-300 nM) were recorded.After 4 weeks of feeding, rats were sacrificed by cervical dislocation. The section of the aorta from between the aortic arch and the diaphragm was removed from the euthanized rats and placed in oxygenated, modified Krebs-Henseleit solution  and the concentration necessary to produce 50% of its maximal response (EC50) were determined. The EC50 values were converted to the negative logarithms and expressed as pD2. Results were shown as mean ± SEM; n refers to the number of animals from which vessels were taken. Agonist potencies and maximal effects were compared by student's t test by using statistical software (GraphPad Prism 3.01). Values were considered significantly different at p < 0.05.Contraction responses are expressed as percentage. For each contractile agent, both the maximal contraction (EThe following drugs were used in this study: 5-HT (RBI, USA), Ang II (Bachem, Switzerland), All drugs were dissolved in KHS. Drugs were added to the organ chambers in volumes not greater than 0.2 ml.HFD – High fat dietNPD – Normal pellet dietNIDDM – Non insulin dependent diabetes mellitusSTZ – Streptozotocin5-HT – 5-HydroxytryptamineAng II – Angiotensin IIKHS – Krebs-Henseleit solutionGOD/POD – Glucose oxidase/peroxidaseAUC – area under the curvemax – Maximal responseE50 – Concentration required producing 50% of maximal responseEC2 – -log EC50pDSG carried out the all experimentations. PR has conceived the study and participated in its design and coordination. Authors read and approved the final manuscript.


Spirituality has become a subject of interest in health care as it is was recognized to have the potential to prevent, heal or cope with illness. There is less doubt that values and goals are important contributors to life satisfaction, physical and psychological health, and that goals are what gives meaning and purpose to people's lives. However, there is as yet but limited understanding of how patients themselves view the impact of spirituality on their health and well-being, and whether they are convinced that their illness may have "meaning" to them. To raise these questions and to more precisely survey the basic attitudes of patients with severe diseases towards spirituality/religiosity (SpR) and their adjustment to their illness, we developed the SpREUK questionnaire.In order to re-validate our previously described SpREUK instrument, reliability and factor analysis of the new inventory (Version 1.1) were performed according to the standard procedures. The test sample contained 257 German subjects (53.3 ± 13.4 years) with cancer (51%), multiple sclerosis (24%), other chronic diseases (16%) and patients with acute diseases (7%).As some items of the SpREUK construct require a positive attitude towards SpR, these items (item pool 2) were separated from the others (item pool 1). The reliability of the 15-item the construct derived from the item pool 1 respectively the 14-item construct which refers to the item pool 2 both had a good quality . Factor analysis of item pool 1 resulted in a 3-factor solution  which explains 53.8% of variance. Factor analysis of item pool 2 pointed to a 2-factor solution  which explains 58.8% of variance. Generally, women had significantly higher SpREUK scores than male patients. Univariate variance analyses revealed significant associations between the sub-scales and SpR attitude and the educational level.The current re-evaluation of the SpREUK 1.1 questionnaire indicates that it is a reliable, valid measure of distinct topics of SpR that may be especially useful of assessing the role of SpR in health related research. The instrument appears to be a good choice for assessing a patients interest in spiritual concerns which is not biased for or against a particular religious commitment. Moreover it addresses the topic of "positive reinterpretation of disease" which seems to be of outstanding importance for patients with life-changing diseases. Spirituality has become a subject of interest in health care, and an increasing number of studies, commentaries and reviews examine the connection between religiosity/spirituality and health, its potential to prevent, heal or cope with diseases -10. MoreHowever, although religiosity and spirituality were interchangeable words, these constructs may not be identical. It is well established to divide Religiosity into three sub-constructs: Intrinsic, Extrinsic, and Quest Religiosity -20, whilSpiritual Well-Being Scale [Daily Spiritual Experience Scale [Santa Clara Strength of Religious Faith Questionnaire [Functional Assessment of Chronic Illness Therapy – Spiritual Well-Being (FACIT-Sp) scale has a much more open design [The measurability and operability of spirituality and religiosity remains a problem and thus several questionnaires address this topic. Most of them measure beliefs of specific religious groups, and ask about the relationship with God  and their adjustment to their illness, we developed the SpREUK questionnaire ,29,36-39In this article we report the re-validation of the SpREUK 1.1 questionnaire , an instrument designed to examine attitudes of patients with life-threatening and chronic diseases towards spirituality/religiosity.All individuals were informed of the purpose of the study, were assured of confidentiality, and gave informed consent to participate. The patients were recruited consecutively in the cancer service, the multiple sclerosis service, and two internal medical units of the Communal Hospital in Herdecke (Germany). All subjects completed the questionnaire by themselves. Demographic information is provided in Table The sample contained 257 subjects of whom 70% were women. The mean age was 53.3 ± 13.4 years. The majority had a Christian nomination (80%), 17% had no religious orientation, and 3% other nominations. Cancer was diagnosed in 51%, multiple sclerosis in 24%, and other chronic diseases in 16% (i.e. Hepatitis C, liver cirrhosis, inflammatory bowel disease, severe hypertension etc.); 7% of the individuals were patients with acute diseases . Patients in final stages of their disease were not enrolled.The items of the SpREUK 1.0 were developed with the patients' input  and experts' statements (physicians, priest and chaplains working with patients) ,36, rathAccording to a previously conducted reliability and factor analysis ,37 the SIn order to more precisely differentiate the three topics guidance, control and message of disease in scale 2 of the version 1.0, for the current version of the questionnaire, six new items were added . All items were scored on a 5-point scale from disagreement to agreement . The SpREUK scores are referred to a 100% level (4 "applied very much" = 100%).Reliability and factor analysis of the new inventory were performed according to the standard procedures. Next, to combine several items with similar content, we relied on the technique of factor analysis which examines the correlations among a set of variables, and to achieve a set of more general "factors." Factor analyses were repeated rotating different numbers of items in order to arrive at the solution which demonstrated both the best simple structure and the most coherence.Differences in the SpREUK scores were tested using the Kruskal-Wallis-Test for asymptomatic significance. We judged p < 0.05 significant, and 0,05 < p < 0.10 as a trend.To tested the impact of several variables on the SpREUK sub-scales, we performed analysis of univariate variance (ANOVA). As in several cases Levene's test for equality of variances was significant, and we judged p < 0.01 as significant.All statistical analyses were performed with SPSS for Windows 10.0.In order to eliminate items from the item pool that were not contributing to the questionnaire reliability, the reliability of the scale and distinct sub-scales was evaluated with internal consistency coefficients, which reflect the degree to which all items on a particular scale measure a single  concept.Our item pool consisted of the previously established set of items ,36,37 anReliability analysis revealed that 6 items from the new item pool 1 had a poor corrected item-total correlation and thus were eliminated : F1.2 , F1.3 , F2.1 , F2.2 . F2.3 , and F3.1 . One item (F3.6 "The "true being" ("inner core") can not be affected by illness") was omitted because of a weak reliability (0.2997) and – even more important – it points to a distinct "field of meaning" that would need more items in the questionnaire, and thus will be used as marker item until the construct will be revised for this topic.As shown in Table Thus, the internal consistency of the 29-item SpREUK 1.1 construct was sufficiently high. The level of difficulty  is 0.6205 for item pool 1 resp.  0.6014 for item pool 2. With the exception of item F3.7 , all values are in the acceptable range from 0.2 to 0.8.To combine several items with similar content, we relied on the technique of factor analysis which examines the correlations among a set of variables, and to achieve a set of more general "factors." Factor analyses were repeated rotating different numbers of items in order to arrive at the solution which demonstrated both the best simple structure and the most coherence.With a Kaiser-Mayer-Olkin value of 0.850 (item pool 1) resp. 0.939 (item pool 2), which measures the degree of common variance, the 15 resp. 14-item-pool seems to be suitable. Barlett's test for non-sphericity was highly significant (p < 0,001).Primary factor analysis of item pool 1 pointed to a 5-factor solution. However, due to a low item number in the tentative subscales 2–5 (with 2 or 3 items each), we favoured the more appropriate 3-factor solution which explains 53.8% of variance  reveal that items F1.8 ("looking for purpose and meaning in life") load good on sub-scale 2 (0.414). Analysis of the side-loadings of item pool 2 revealed that several items load also on the other sub-scale. Moreover, sub-scale 4 showed a strong but negative inter-correlation with sub-scale 5 , the lowest for sub-scale 1 ("Search for meaningful support"). Means and standard deviations for study variables are provided in Table Women had significantly higher SpREUK scores than male patients. With respect to age, the lowest SpREUK scores were found in the group of < 30 years of age. With increasing age, the trust in a higher supporting presence  and the beneficial effects of resp. support through SpR increased.With respect to the marriage status, widowed patients obviously has to rely on external guidance  but not the patients living with a partner not married with. Widowed and divorced patients find support in external relations through their SpR engagement , while – in contrast to married patients which may find hold in their partnership – especially divorced patients are in search for meaningful support .Search for meaningful support and positive interpretation of diseases were depending on the educational level, as patients with lower educational level had significantly lower scores than those with a higher level. A higher educational level was associated with higher scores in the sub-scales 4 and 5 which deals with the beneficial effects of SpR.Illness itself (but not the duration of disease) has a significant impact on the SpREUK scores, as MS patients had the lowest scores in all 5 sub-scales. The SpREUK scores of cancer patients revealed slight differences when compared to patients with other chronic diseases.With the exception of sub-scale 2, patients without confessional affiliations had the lowest scores for all sub-scales, indicating that the "message of disease" was not depending on a denomination. Surprisingly, the few patients with other than a Christian orientation had the highest scores for sub-scales 1, 3, 4, 5.(R+S+); 35% as religious, but not spiritual (R+S-); 23% as neither religious nor spiritual (R-S-); 10% claimed that they were spiritual, but not religious (R-S+). Thus, the numbers of patients with denominational affiliation and self-reported spiritual/religious attitudes is somewhat similar. A spiritual attitude (R+S+ and R-S+) was associated with "search for meaningful support" and "positive interpretation of disease", while a religious attitude (R+S+ and R+S-) was associated with the highest scores for the "trust in external guidance" sub-scale 3.Since nominational affiliation is not necessarily identical with religiosity or spirituality, we asked whether the patients would describe themselves as religious or spiritual ,34,35. TThe living area and the duration of diseases had no significant impact on the SpREUK scores.As shown in Table In detail Table , prayingNext we tested the impact of several variables on the SpREUK sub-scales, such as sex and marital status, educational level and confession, age and SpR attitude, and disease and duration of disease. Using the method of univariate analyses of variance we identified several sources of variability Table :SpR attitude is an important covariate for the "Search for meaningful support", "Positive interpretation of disease", "Trust in external guidance", and both "Support through SpR" sub-scales.• The educational level is an important covariate for "Search for meaningful support", "Trust in external guidance" and to a minor content for "Support in relations with the External life through SpR" – but not for the "Positive interpretation of disease".• The Age is an important covariate only for "Trust in external guidance".• Confession is an important covariate for "Trust in external guidance".• Disease itself has an impact on the "Search for meaningful support• Data from the current analysis demonstrate the reliability and validity of the SpREUK construct. Moreover, the sub-scales 1 and A (= 4) and B (= 5) of the preliminary version 1.0 were confirmed in the new version 1.1. In order to more precisely differentiate the three topics guidance, control and message of disease from the SpREUK version 1.0, six new items were added. Due to this fact, some items from the original item pool decreased the reliability of the construct and thus, two items from the sub-scale 1 had to be deleted  of disease" has a good correlation with an existentialistic practice, which seems to be of outstanding importance for patients with life-changing diseases. It may be desirable to use such a measure that allows to assess attitudes which are independent of any religion or specific belief.A third strength is that the validation was performed in a sample with at least two different types of life-changing diseases (cancer and MS, and other chronic diseases) and a healthy control group.Beyond conceptual boundaries, our instrument differentiates the self-addressed "religious" and "spiritual" attitudes of the patients with life-threatening diseases and heeds their search for support and meaning, and integrates the topic of "meaning in illness". We cannot exclude the possibility that these topics are not relevant for healthy individuals.In future studies we have to correlate our scales with other relevant instruments which measure aspects of SpR. Nevertheless, evaluation of the SpREUK questionnaire indicates that it is a reliable, valid measure of distinct topics of SpR that may be especially useful of assessing the role of non-religious spirituality in health related research. The focus of a larger study is to enrol patients from the highly secular Eastern Europe, and to run longitudinal studies with cancer, multiple sclerosis patients, but also cardiac failure and spinal cord damage.The SpREUK with its additional SpREUK-P manual to measure a patient's engagement in distinct forms of SpR practice is currently available in English and German language.AB conceived the study, designed and developed the questionnaire, performed statistical analysis and drafted the manuscript. TO participated to conceive and design the study, performed additional statistical analysis and helped to draft the manuscript. PFM conceived the study and participated in the design and development of the questionnaire. All authors read and approved the final manuscript.