jgi-psf.org, http://broad.mit.edu, http://vmd.vbi.vt.edu). Second, the noncanonical abiotic/biotic reaction pathway reported in thermal-tolerant bacteria requires hydrothermal environments to form DPD (Nichols et al., 2009). Such conditions are not encountered by these oomycete ‘water molds.’ Lastly, in the pentose-phosphate pathway, DPD is formed spontaneously by converting pentose phosphates to d-ribulose-5-phosphate using isomerases (RPI). On searching oomycete genome databases, we found that pentose Z-VAD-FMK ic50 phosphates are common metabolic products, and all four published genome sequences of Phytophthora species contain conserved sequences for RPI, suggesting that zoosporic oomycetes may

form DPD through the central intermediate ribose-5-phosphate. Silencing the RPI gene and testing mutant AI-2 production may provide direct evidence to test this presumption. However, it is possible that other unknown pathways are responsible for the production of AI-2. Although it is not clear whether oomycetes use AI-2 to encode information for communication within the population to coordinate behaviors such as aggregation and plant infection, AI-2 production by Pythiaceae species raises the possibility that zoosporic pathogens may use AI-2 as a common signal to communicate with bacteria. Communication with bacteria may be beneficial to these pathogens as shown

by their ability to survive in soil with a wide range of bacteria and their tolerance to frequent culture contamination by bacteria. Selleck Inhibitor Library It will be interesting to know whether this cross-kingdom relationship is bridged by AI-2. In fact, triggering the luminescence of V. harveyi by ZFF (Fig. 1) has verified that oomycetes can communicate with bacteria and affect their quorum sensing through this molecule. This process may provide oomycetes an advantage in fitness and

possibly virulence. Bacteria and bacterial metabolites have been shown to stimulate Phytophthora reproduction (Zentmyer, 1965) and contribute to Phytophthora colonization on plants (Yang et al., 2001). Etofibrate We gratefully acknowledge supplies of isolates of Phytophthora and Pythium from Drs Brett Tyler, Michael Benson, and Gary Moorman, and expression strains for AI-2 production from Drs Kenneth Cornell, Michael Riscoe, Mark Hilgers, and Martha Ludwig. We thank Dr Brett Tyler for assistance with oomycete bioinformatics, and Patricia Richardson for reading this manuscript. This work is supported in part by grants to C.H. from USDA-CSREES (2005-51101-02337) and to Z.S.Z. from NIAID/NIH (1R01AI058146). This is publication number 939 from the Barnett Institute. “
“Clostridium difficile, a Gram-positive spore-forming anaerobe, causes infections in humans ranging from mild diarrhoeal to potentially life-threatening pseudomembranous colitis. The availability of genomic information for a range of C.

Host penetration by biotrophic mycoparasites is believed to be mediated by both mechanical and enzymatic mechanisms; strict regulation of chitinase and chitosanase lytic enzymes is a reported characteristic Selleck Nutlin3a of biotrophs (Manocha, 1987). In contrast to the F. graminearum 3-ADON chemotype, 15-ADON co-cultured with S. mycoparasitica formed irregular mycelia, leading to the morphological hyphae alteration or formation hyphal ‘rosettes’ at the contact zone. Similarly, deformation of mycelia and hyphae has been observed in F. oxysporum pathogens challenged with antagonistic bacteria (Chaurasia et al., 2005). To date, no biotrophic mycoparasitic fungi have been reported

to suppress F. graminearum growth or to prevent mycotoxin accumulation in kernels, food and feed.

Further studies are underway to show the direct effect of mycoparasite on mycotoxin accumulation and to use S. mycoparasitica as a potential biocontrol agent for managing F. graminearum toxigenic chemotypes. Finally, this is the first report of the ability of S. mycoparasitica to parasitize and hinder the growth of F. graminearum 3- and 15-ADON hosts, as well as to decrease trichothecene gene accumulation. Specific differences in S. mycoparasitica interaction with 3- and 15-ADON chemotypes are the subject of ongoing research. This research was financially Protein kinase N1 supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant, and the Saskatchewan Agriculture Development Fund (ADF) to V.V. and a Departmental Devolved INK 128 mw Scholarship to Y.K.G. “
“A Phoma sp. was isolated and characterized as endophytic and as a pathogen of Larrea tridentata (creosote bush) growing in the desert region of southern Utah, USA. This fungus produces a unique mixture of volatile organic compounds (VOCs), including

a series of sesquiterpenoids, some alcohols and several reduced naphthalene derivatives. Trans-caryophyllene, a product in the fungal VOCs, was also noted in the VOCs of this pungent plant. The gases of Phoma sp. possess antifungal properties and is markedly similar to that of a methanolic extract of the host plant. Some of the test organisms with the greatest sensitivity to the Phoma sp. VOCs were Verticillium, Ceratocystis, Cercospora and Sclerotinia while those being the least sensitive were Trichoderma, Colletotrichum and Aspergillus. We discuss the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship with implications for utilization as an energy source. Cresote bush, Larrea tridentata, is a prominent plant in the Mojave, Sonoran and Chihuahuan deserts of North America.

As the prevalence of HIV infection in adults in Catalonia is 0.6% [31], HIV-positive patients were

overrepresented among those with confirmed influenza A H1N1 infection. The increased rate of diagnosis of influenza A H1N1 infection in HIV-positive adults relative to that in HIV-negative individuals might suggest that HIV-positive patients are more vulnerable to influenza A H1N1 infection than the general adult population, but the overall findings of our study, indicating that influenza A H1N1 infection in HIV-positive adults had a similar or even Omipalisib more benign presentation and prognosis than that in the general adult population, argue against that conclusion. Alternatively, this increased rate of diagnosis might have been a consequence of a higher proportion of HIV-positive patients relative to HIV-negative controls having a diagnosis of influenza A H1N1 infection confirmed. Because the health care of HIV-positive patients is already linked to the hospital, they are more likely than HIV-negative patients to go to hospital whenever they feel unwell, and this may be especially true for those without any underlying comorbidity or those with comorbidities not cared for at the hospital. This reasoning would explain not only the higher-than-expected representation of HIV-positive

patients among those adults ERK high throughput screening with confirmed influenza A H1N1 infection, but also the shorter time interval between the onset of symptoms and the diagnosis

of influenza A H1N1 infection in HIV-positive patients. Because the highest influenza A H1N1 rates have been reported in children and younger adults [13], we should have expected younger HIV-infected adults to be the individuals mainly affected. However, HIV-positive adults with confirmed influenza A H1N1 infection had representative features of the HIV-infected adult population receiving care at our institution, O-methylated flavonoid suggesting that influenza A H1N1 does not preferentially target a specific age group of HIV-infected adults. The clinical presentation was similar in HIV-positive and HIV-negative patients, except for gastrointestinal symptoms, which were more common in HIV-positive patients. It has been suggested that gastrointestinal symptoms occur more frequently in influenza A H1N1 infection than in seasonal influenza infection, especially in adults [32]. Gastrointestinal symptoms are a common problem in HIV-positive persons [33], and this might have contributed to the higher frequency of digestive symptoms seen in HIV-positive individuals with influenza A H1N1 infection. In agreement with current expectations for HIV-positive adults on effective antiretroviral therapy [25], most HIV-positive patients with confirmed influenza A H1N1 infection in our cohort showed good virological control.

Then, from week 48 to week 96, if viral load was maintained PARP inhibitor at < 50 copies/mL, patients could be switched to darunavir 800/100 mg once a day (qd). Randomization was centralized and stratified by HIV-1 RNA level (< vs. ≥ 100 000 copies/mL) prior to the first antiretroviral treatment. Seventeen Agence Nationale de Recherche sur le SIDA et les hépatites virales (ANRS) clinical sites participated in

the body composition substudy; participation was based on the availability of dual-energy X-ray absorptiometry (DEXA). Anthropometric measurements were obtained at baseline and at weeks 48 and 96. Total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and glucose were measured on patients in a fasting state at baseline and every 24 weeks. Body composition was measured by a whole-body DEXA scan using Hologic Inc. (Waltham, MA, USA) and Lunar (GE Healthcare, Madison, WI, USA) devices at baseline and at weeks 48 and 96. All DEXA scans were performed

according to standardized protocols, using the same device for each patient, and according to the manufacturer’s recommendations [23-25]. Data were centrally analysed in a blinded manner by a single investigator. A bone evaluation was performed, including bone Selleck CAL101 mineral density (BMD) measurements in the lumbar spine and femoral neck, and parathyroid hormone (PTH), serum 25-hydroxyvitamin D, calcium and phosphate levels were assessed only at week 96 in a subset of patients. DEXA scans were subjected to quality controls to verify the absence of drift. The T-scores were calculated for each body site using the appropriate reference curve for each Immune system device.

Osteoporosis was defined as a T-score ≤–2.5, and osteopenia as a T-score of >–2.5 and ≤–1, according to World Health Organization (WHO) definitions [26]. Although these categories were created to classify postmenopausal women, we applied this definition to all patients whatever their age or gender. Adverse clinical and laboratory events were assessed by site investigators and scored according to the ANRS adverse-event grading scale. An independent Data and Safety Monitoring Board (DSMB) reviewed interim efficacy and safety. The primary objective of this body composition substudy was to compare the two randomized treatment groups for changes in limb and trunk fat measured by DEXA. Changes in limb and trunk fat were assessed both as absolute quantitative values (kg) and as percentage changes relative to baseline. The sample size was chosen to detect a treatment difference of 0.5 kg in limb fat, with a common standard deviation of 1.0. Using a Wilcoxon rank-sum test, a sample size of 75 people per arm has an 83% power to detect at least a 0.5 kg difference between the two groups at the 5% significance level.

Secondary structures of TDH and TRH were predicted from CD data using the cdpro program package (Sreerama & Woody, 2000). The cdpro suite contains modified versions of three methods: selcon3, continll, and cdsstr. All methods are based on comparison of the far-UV CD spectrum of the protein undergoing testing with CD spectra of reference proteins with a known three-dimensional structure. Using three methods and one set of reference proteins, we obtained the predicted secondary structures. We performed analytical ultracentrifugation experiments using an Optima XL-1 analytical

ultracentrifuge (Beckman Coulter, Fullerton, CA) with a Beckman An-50 Ti rotor. Sedimentation equilibrium experiments were carried Selleckchem BMS-354825 out in cells with a six-channel www.selleckchem.com/products/bmn-673.html centerpiece and quartz windows. The sample concentrations used were 0.15, 0.31, and 0.59 mg mL−1 dissolved

in 10 mM phosphate buffer (pH 7.4) and 100 mM NaCl. We set the absorbance wavelength at 280 nm. Data were obtained at 2600 g (6000 rpm) and 5900 g (9000 rpm) at 20 °C. A total equilibration time of 22 h was used for each speed, with a scan taken at 18 h to ensure that equilibrium had been reached. We calculated the partial specific volume of the protein, solvent density, and solvent viscosity from standard tables using the program sednterp (version 1.09). Data analysis was performed by global analysis Amisulpride of datasets obtained at different loading concentrations and rotor speeds using ultraspin software (MRC Center for Protein Engineering, Cambridge, UK; http://www.mrc-cpe.cam.ac.uk/ultraspin).

The homology model of TRH was built by the program modeller (Marti-Renom et al., 2000) using the crystal structure of TDH (PDB: 3A57). Sample preparation was performed as described previously (Fukui et al., 2005; Hamada et al., 2007). We diluted samples containing 20 μg mL−1 TRH with 10 mM sodium phosphate (pH 7.4). For negative staining, 4 μL of the solution was applied to a copper grid supporting a thin continuous carbon film, left for 1 min, and then stained with three drops of 2% uranyl acetate. Images were recorded by a BioScan CCD camera (Gatan) with a pixel size of 3.1 Å, using a JEM1010 electron microscope (Jeol, Tokyo, Japan). We incubated protein samples (0.2 mg mL−1) with 10 μM ThT in 50 mM glycine–NaOH (pH 8.5) according to a previous report (Fukui et al., 2005). Fluorescence of ThT was measured at 485 nm with an excitation wavelength of 450 nm using an FP-777 (Jasco) spectrofluorometer. The kinetic of fibril formation was described previously (Hamada & Dobson, 2002; Fukui et al., 2005). Each kinetic traces was fitted to the stretched exponential function F=F∞+ΔF exp[(−kt)n].

Because A. hydrophila is also a component of the normal intestinal flora of healthy fish, virulence mechanisms are not well understood. Considering that fish models used for the examination of A. hydrophila genes associated with virulence have not been well defined, we established an infection model using the free-living, ciliate protozoa Tetrahymena thermophila. The expression of A. hydrophila virulence genes following infection of T. thermophila was assessed by reverse transcription-PCR and demonstrated that the aerolysin (aerA) Selleck ICG-001 and Ahe2 serine protease (ahe2) genes (not present in the avirulent A. hydrophila NJ-4 strain) in the

virulent J-1 strain were upregulated 4-h postinfection. Furthermore, the presence of intact A. hydrophila J-1 within T. thermophila suggested

Dinaciclib datasheet that these bacteria could interfere with phagocytosis, resulting in the death of the infected protozoan 48-h postinfection. Conversely, A. hydrophila NJ-4-infected T. thermophila survived the infection. This study established a novel T. thermophila infection model that will provide a novel means of examining virulence mechanisms of A. hydrophila. Aeromonas hydrophila has been receiving increasing attention recently both as an opportunistic and as a primary pathogen of both humans and aquatic and terrestrial animals (Bi et al., 2007). Aeromonas hydrophila pathogenesis is mediated by various cell bound and secreted virulence factors including aerolysin (Singh et al., 2009), cytotoxic enterotoxin (Chopra et al., 2000), extracellular serine protease Branched chain aminotransferase (Cascon et al., 2001), elastase (Cascon & Yugueos,

2000) and S-layer (Murray et al., 1988), which can play a role in affecting disease severity. However, the precise pathogenesis mechanism is not known. The pathogenesis resulting from A. hydrophila infections might be not exclusively virulence factor mediated and can also be affected by host species resistance mechanisms. In order to develop more effective anti-infective therapies, it is important to study the pathogenesis mechanism at the cellular and molecular levels using adequate host organisms. Although fish are excellent models for assessing the lethal dose 50% of A. hydrophila (Rodriguez et al., 2008) or for examining host immune responses (Rodriguez et al., 2009), they are not ideal for dissecting host–pathogen interactions at the molecular level (Pradel & Ewbank, 2004). Many model organisms have been used to study bacterial pathogenesis. For instance, the nematode Caenorhabditis elegans and the insect Drosophila melanogaster or even unicellular Dictyostelium discoideum amoebae have proven to be useful hosts to measure bacteria virulence (Kurz & Ewbank, 2007). Previously, the amoeba D.

These data confirmed that the identified pqqABCDEF operon was essential, at minimum, for several steps of the PQQ biosynthetic pathway in P. ananatis. However, it cannot be excluded that some additional genes from other loci of the P. ananatis SC17(0) chromosome participated in PQQ synthesis as well. To test this possibility, the cloned pqq operon was transferred from P. ananatis SC17(0) into E. coli. Ponatinib chemical structure In E. coli, the primary pathway for glucose consumption is the phosphoenolpyruvate/carbohydrate phosphotransferase system (PTS) (for a review, see Deutscher et al., 2006). The GDH-mediated pathway

in this organism does not work because of the absence of the PQQ biosynthesis route. In E. coli, mGDH is synthesized only in apoenzyme form; however, the holoenzyme can be formed in the presence of exogenous PQQ. We expected to observe a similar effect after integration of the P. ananatis putative pqq operon into the E. coli chromosome. To support this hypothesis,

one copy of the pqq operon was introduced into the double mutant strain, with inactivated PTS and the mannose permease selleck kinase inhibitor system. The strain used as a recipient, named MG1655-2Δ, is unable to grow on the glucose minimal medium because of the absence of effective glucose uptake. Synthesis of PQQ in the MG1655-2Δ-pqq strain, which has pqq operon integrated at the φ80attB site, could lead to direct oxidation of glucose to gluconic acid by PQQ-mGDH. The growth properties of MG1655-2Δ-pqq were compared with those

of the wild-type strain and to MG1655-2Δ, grown with the addition of exogenous PQQ, on the minimal medium with glucose as the sole carbon source. As shown in Fig. 1, integration of the pqq operon resulted in the restoration of MG1655-2Δ-pqq growth on glucose minimal medium. However, MG1655-2Δ-pqq showed a prolonged lag time unlike the wild-type strain or MG1655-2Δ growing in the ID-8 presence of PQQ in the medium (+PQQ). In addition, MG1655-2Δ-pqq grew at a slower rate than MG1655-2Δ under +PQQ conditions; however, it had a higher final OD. Comparison of the growth properties of MG1655-2Δ and MG1655-2Δ-pqq suggests that the introduction of the pqq operon allowed the formation of an active GDH, resulting in the production of gluconic acid from glucose and its further utilization. We attempted to determine whether E. coli strains containing the pqq operon are able to accumulate PQQ in the culture medium. In our experiments, we could detect about 0.25 μg L−1 of PQQ in the assay system. However, no PQQ was observed during MG1655-2Δ-pqq growth on the minimal medium with gluconate as the sole carbon source. It is possible that pqq genes cloned with their native regulatory regions from P. ananatis are poorly expressed in E. coli. Conversely, the P. ananatis SC17(0) strain with the native pqq operon accumulates up to 9 mg L−1 of PQQ.

Empty vector (pDB1568) was used as negative control and plasmids containing iscS or nifS from A. vinelandii as positive controls. No growth was observed on nonsupplemented medium after 72 h at 37 °C, PFT�� mw although control strains grew as expected (Fig. 3a). These results indicate the E. faecalis SUF machinery is not able to complement the ISC system of Proteobacteria, even in E. coli, which is slightly evolutionarily different from A. vinelandii in terms of the presence of SUF machinery in the latter. Several Proteobacteria representatives possess the SUF. genes together with the

housekeeping ISC machinery. However, E. faecalis possess the only SUF system with high homology with the corresponding E. coli SUF genes, with the addition of sufU, similar to E. coli iscU. Genetic experiments were performed to assess the possibility that the cloned E. faecalis SUF genes can complement E. coli mutants lacking one or more of the components of the SUF system. SUF mutants of E. coli have no apparent growth phenotype. However, combination of an SUF mutation (or mutations)

with an iscS mutation is lethal unless a plasmid is present in trans that provides either iscS or the missing SUF function(s) (Trotter et al., 2009). To guarantee BKM120 solubility dmso the complementation of the iscS mutant, the complementing element needs to fill the gaps caused by the absence of iscS. This is what seems to occur in vivo when the E. coli sufABCDSE system produces viable strains of E. coli ISC mutants (Takahashi & Tokumoto, 2002). This system plays roles related not only to [Fe–S] cluster formation, but also to nicotinic acid and thiamine biosynthesis. Escherichia coli strains JW1670-1 (ΔsufS), GSO97 (ΔsufSE), and GSO92 (ΔsufABCDSE) were used as recipient strains for phage P1 transduction

experiments in which the donor strain (EESC42) contained ΔiscS∷kan and a tightly linked Tn10, which these confers tetracycline resistance. In each transduction, tetracycline resistance was selected and kanamycin resistance scored as described by Outten et al. (2004). The appearance of viable kanR transductants would indicate complementation of either iscS or SUF function(s) by the resident plasmid. As negative and positive control plasmids, the empty vectors pDB1568 and pDB943 (which encodes iscS from A. vinelandii) were used. Azotobacter vinelandii IscS was able to complement all double mutants, whereas the only complementation observed using the test strains was with strain GSO92 (ΔsufABCDSE), containing pEFSE121 (which encodes sufCDSUB). Tetracycline-resistant transductants were obtained that displayed resistance to kanamycin and ampicillin, and grew on glucose minimal medium (containing arabinose) after 48 h of incubation (Fig. 3b).

The surprise signal of the unsigned PE also yielded a highly focal activation in the midbrain anatomically consistent with the substantia nigra (SN)/ventral tegmental area and activity

in the anterior insula (Fig. 4A). We did not observe a significant correlation with blood oxygenation level-dependent (BOLD) responses in the amygdala for signed PEs in a complementary analysis (inspected at a threshold of P < 0.05, family-wise error corrected). In a second step, activity in a different amygdala subregion was found to be negatively correlated with the associability at the time of CS onset (Fig. 4B and Table 3B), whereas no positive correlation could be observed in the amygdala (even at a liberal threshold of P < 0.01, uncorrected). As the negative associability Ixazomib supplier indicates the reliability of prior predictions, the observed negative correlations suggest that activity in the amygdala increased whenever outcome predictions became more reliable and decreased when

outcome predictions were poor. According to the anatomical atlas as well as the probabilistic maps (Table 4), the observed amygdala activation can be assigned to the BLA. However, it should be noted that, although the probabilistic maps and the anatomical atlas yielded the same amygdala subregions in the present study, the location of amygdala nuclei can differ between both methods. To further approve the functional dissociation of the CM and BLA, we directly compared the mean activity with unsigned PE and negative associability signals in those areas [associability Protease Inhibitor Library nmr beta values were inversed for the purpose of this analysis to indicate the strength (and not the direction) of the correlation]. More specifically, we extracted the betas for both signals from all voxels falling into the CM and BLA, respectively. The CM was approximated by a combination of the bilateral superficial and the centromedial amygdala masks and the BLA was defined by bilateral basolateral amygdala masks using the maximum

probability maps to define regions of interest Histone demethylase (Eickhoff et al., 2005). A 2 × 2 repeated-measures anova with factors region (CM, BLA) and signal (unsigned PE, negative associability) on the mean beta coefficients from individual subjects revealed a significant region-by-signal interaction (F1,20 = 12.39, P < 0.01) indicating that the two subdivisions of the amygdala are differentially engaged in representing the unsigned PE and negative associability (Fig. 5B). In addition, subsequent t-tests showed that the unsigned PE correlated significantly more strongly with activity in the CM than in the BLA (t20 = 2.54, P < 0.05), whereas the negative associability function revealed a larger correlation with BOLD responses in the BLA as compared with the CM (t20 = 2.76, P < 0.05).

l-methionine, l-leucine, l-isoleucine and l-threonine were found to be catalysed by the investigated enzymes, producing l-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family ABT-888 for which free l-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed. Hydroxylation of

free canonical l-amino acids is an interesting and growing field of biotechnology and molecular biology research. Introduction of a functional hydroxyl group into amino acid molecules makes possible synthesis of fine chemicals (Blaskovich et al., 1998). In addition, the hydroxylated amino acid may itself be biologically active with pharmacological significance (Jette et al., 2009) and/or may be involved in bacterial metabolic regulation (Ogawa et al., 2011). In bacteria, the hydroxylation of free l-amino acids

SP600125 is usually catalysed by specific Fe(II)/α-ketoglutarate-dependent dioxygenases (Hausinger, 2004). Because both l-amino acids and α-ketoglutarate are involved in cell metabolism, metabolic engineering of Escherichia coli could be used for the microbiological production of target hydroxylated l-amino acids (Shibasaki SPTLC1 et al., 2000; Smirnov et al., 2010; Ogawa et al., 2011).[ Correction added after online publication 17 April 2012: Kim et al., 2010 and Smirnov et al., 2010 references swapped throughout ]. Thus, identification of novel l-amino acid dioxygenases or l-amino acid hydroxylation activities may facilitate industrial bioprocesses

to produce novel pharmaceuticals and synthons for organic chemistry. In addition, understanding the hydroxylation of free l-amino acids could facilitate the discovery of novel biosynthetic processes and regulatory mechanisms in bacteria. Recently, we described the cloning and characterization of l-isoleucine-4-hydroxylase (IDO) from Bacillus thuringiensis (Kodera et al., 2009; Smirnov et al., 2010; Hibi et al., 2011; Ogawa et al., 2011). IDO hydroxylated several hydrophobic aliphatic l-amino acids, including l-leucine, and generated l-methionine sulfoxide from l-methionine. In this work, we used IDO homologues from several bacteria to examine the substrate specificities of novel dioxygenases in regard to other canonical l-amino acids and to determine kinetic constants for l-isoleucine, l-leucine and l-methionine. To construct the pET-HT-IDO, pET-HT-PAA, pET-HT-MFL and pET-HT-GOX plasmids, the following DNA fragments were amplified: (1) a 776-bp ‘IDO’ fragment, using the primers svs 335 (5′-TATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCAAAATGAGTGGCTTTAGCATAGAAGA-3′) and svs 336 (5′-CAGCCGGATCCTTATTTTGTCTCCTTATAA-3′) and the pEL-IDO plasmid (Smirnov et al.