MBA4 was grown in minimal medium containing acetate (squares) or MCA (circles). Uptakes of 50 μM of [2-14C]acetate were assayed in the presence of 0, 5, 10, 25, and 50 μM of CCCP for a period of 1 min. Data shown are the means of three independent experiments, and the error bars represent the standard deviations. A limitation of SGC-CBP30 mouse employing CCCP is that

it cannot discriminate selleck inhibitor between proton-coupled symport and Na+-coupled symport [17, 20]. As it is difficult to remove sodium from the buffers completely and radioactive MCA and acetate were provided in the form of a sodium salt, the effect of pH on acetate- and MCA- uptake was examined with an aim to find out the possible involvement of proton(s). In acetate uptake of acetate-grown cells, the uptake rate decreased steadily as pH increased from 4 to 8 (Figure 5, squares). In acetate uptake of MCA-grown cells, the uptake rate increased slightly as pH increased from 4 to 5 and then dropped gradually as pH increases (Figure 5, circles). The uptake rates were much lower than that of acetate-grown cells in similar assay conditions. In MCA uptake of MCA-grown cells, the uptake rate increased slightly as pH increased from 4 to 6 and dropped swiftly from pH 7 to 8 (Figure 5, triangles). These results showed that acetate- and MCA- transport systems have

different sensitivities to pH. Nonetheless, the involvement of proton(s) in acetate transport is noticeable. Figure 5 Effect of pH on acetate- and MCA- uptake. MBA4 was grown in minimal Saracatinib mouse medium containing acetate or MCA, harvested and resuspended in potassium phosphate buffers of various pH values. Uptakes of 50 μM of [2-14C] labelled acetate or MCA were assayed for a period of 1 min. Squares represent acetate uptake of acetate-grown cells, circles represent acetate uptake of MCA-grown cells, and triangles represent MCA uptake of MCA-grown cells. Data shown are the means of three independent experiments, and the error bars represent the standard deviations. Discussion In this study, we demonstrated Teicoplanin the presence of distinct acetate- and MCA-

transport system in MBA4. This is supported by: (i) the observation that the inducible substrates for acetate- and MCA- uptake activity were different; (ii) the two transport systems have different competing solutes and (iii) a difference in dependency on pH for the two systems. The failure of pyruvate-grown cells to take up acetate suggested that the acetate-transport system in MBA4 was inducible. Both acetate and MCA were able to induce acetate-uptake activity although to a different level. Acetate permease MctC of Corynebacterium glutamicum is also inducible. MctC exhibits a high affinity for acetate and propionate and low affinity for pyruvate. In this case, the expression was higher in pyruvate- than in acetate-grown cells. As a result, both pyruvate- and acetate-grown cells showed comparable acetate-uptake activities [18].

), nor did they host basidiomycetes whereas

only very few nursery Tozasertib supplier plants had been contaminated with Eutypa lata (1.4 %). While most adult plants contracted esca-associated fungal species, the majority of nursery plants hosted fungi that were more typically associated with young vine decline (Figs. 3, 4), i.e. various species of Cylindrocarpon (incidence: 57.5 %, cumulated relative abundance: 8 %), a genus that was completely absent from adult plants. The Cell Cycle inhibitor genus Cadophora had a much higher incidence (57.5 %) in nursery plants than in adult plants (asymptomatic: 1.7 %, esca-symptomatic: 1.5 %). Consequently nursery plants hosted presumed fungal pathogens with a high incidence, but there was a clear shift in the involved fungal genera and species during plant maturation (Figs. 3, 4). The fungal community associated with the wood of adult V. vinifera plants LB-100 purchase was highly similar in both

symptomatic and asymptomatic plants, but very different from nursery plants Apart from the generally assumed pathogens, other species of the fungal community could be involved in the expression of esca-disease. When comparing the systematic structure of the fungal communities associated with the different plant types (Fig. 5, inferred from Table 1), the most frequently isolated OTUs belonged to the Dothideomycetes and the Sordariomycetes, with a dominance of Dothideomycetes in adults plants (54.9-56.9 % of the fungal isolates). Both classes were equally represented in nursery plants (40.4 % of the isolates are Sordariomycetes and 38.31 % are Dothideomycetes) [Fig. 5a]. Taken together, both classes represented more than 73 % of the isolates in all plant categories. The two other dominant classes in all plant categories were Eurotiomycetes (asymptomatic: 13.8 %,

esca-symptomatic: 13.6 %, nursery: 5 %) and Leotiomycetes (asymptomatic: 6.6 %, esca-symptomatic: 5.1 %, nursery: 10.3 %) but with a dominance of the former in adults plants and of the latter in nursery plants. Fungal isolates of the five remaining classes represented less than 6 % of the fungal community of each of the plant types. The comparison of the systematic placement of our fungal isolates revealed a clear shift from nursery plants to adult grapevine plants: Dothideomycetes and Eurotiomycetes increased in frequency at the expense of Leotiomycetes and Sordariomycetes. These frequency shifts were observed for both http://www.selleck.co.jp/products/Pomalidomide(CC-4047).html esca-symptomatic and asymptomatic plants. Fig. 5 Systematic structure of the fungal communities respectively associated with the different plant types. a. Distribution of the fungal isolates in the different classes; b. Distribution of the fungal isolates in the different orders. Plant types: 1. asymptomatic, 2. esca-symptomatic, 3. nursery The fungal communities hosted by the adult plants, symptomatic or not, were also very similar based on the distribution of the isolates in the different fungal orders (Fig. 5b). If Pleosporales were the most diverse in all plant types (asymptomatic: 27.

aureus. The amount of 260 and 280 nm absorbing material in S. aureus cell supernatants treated with AKBA (relative to the total released upon complete cell lysis) was 12 and 15% at 90 min while it was 15 and 19% at 120 min respectively (Figure 4), which was significantly higher than the untreated control (P < 0.05). Figure 3 Uptake of propidium iodide in cell of S. aureus PND-1186 cost ATCC 29213. Cells of S. aureus were treated with AKBA at 64 μg/ml for 60 and 120 min. Control group included cells

untreated with AKBA. AKBA treated cells significantly increases the fluorescence compared with untreated control (P < 0.05). Data represent the mean and standard deviations (±SD) of two different experiments performed in triplicate. *, P < 0.05 (Student's t test). Figure 4 Effect of AKBA on the leakage of 260 and 280 nm absorbing materials in S. aureus ATCC cells. Control group (treated with lytic enzymes and considered as 100% leakage) and treated with AKBA at 64 μg/ml for 90 and 120 min. No compound added served as untreated control. Values are means (±SD) from three independent determinations. *, P < 0.05 (Student's t test), AKBA treated group compared to untreated control group. Discussion and conclusion The gum exudate or the resin obtained from the bark of Boswellia serrata has been widely used by the practitioners

www.selleckchem.com/products/AZD0530.html of the Indian systems of medicine for various medical conditions such as arthritis, asthma, ulcers, and skin diseases; currently it is being extensively used in various formulations for the treatment of inflammation related disorders [13–15]. The major chemical components of gum resin can be divided into three groups: volatile oils or lower terpenoids, higher

terpenoids, and carbohydrates. The higher terpenoids comprises of β-boswellic acids as the main triterpenic acid along with 11-keto-β-boswellic acids and their acetates [23]. The in vitro antibacterial activity results of four medroxyprogesterone boswellic acid compounds revealed AKBA to be the most potent antibacterial compound against Gram-positive pathogens, but it showed no significant antibacterial activity (MIC >128 μg/ml) against the Gram negative bacteria. AKBA exerted bacteriostatic antibacterial activity against S. aureus ATCC 29213 (Figure 1) and exhibited a good PAE of 4.8 h at 2 × MIC concentration. Staphylococci cause a large percentage of catheter associated infections, and like many other pathogens, rather than living as free planktonic cells within the host they tend to form a multilayered community of sessile bacterial cells known as a biofilm on medical implants or damaged tissue [7, 24, 12]. Biofilm infections are Selleckchem Birinapant difficult to treat due to their inherent antibiotic resistance [7, 12, 25]. AKBA effectively inhibited the staphylococcal biofilm and also reduced the preformed biofilm of these bacterial pathogens (P < 0.01).

6 mPa.s) is equal to the dynamic viscosity of octadecene at 303 K. The PL peak position of Si NPs is equal to 1.702 eV in octadecene at 303 K and is equal to 1.68 eV in squalane at 368 K. Therefore, there is a difference of 22 meV between the two PL peak positions which is very close to the shift given by the Varshni expression

on bulk Si (17.5 meV) in the same temperature range (from 303 down to 368 K). Hence, when corrected from the viscosity effect, the red shift that we observed (around −0.3 meV/K) with temperature is close to the one reported by different groups. Conclusion Si NPs JNK-IN-8 prepared by electrochemical etching of bulk Si have been functionalized with alkyl chains (octadecene) for dispersion in NPLs like lubricants for mechanical bearings. Their potential application as fluorescent nanosensors for temperature measurement in lubricated contact with optical access has been evaluated. The important variation of the fluorescence emission energy with temperature (−0.9 meV/K) allows simple temperature measurement in squalane. Nevertheless, we have shown that this variation is mainly due to energy

exchange between Si NPs promoted by viscosity reduction when the temperature is increased. For static condition in the fluid, this indirect temperature sensing via viscosity change is convenient, but in dynamic conditions of Pictilisib ic50 the mechanical contact, a more intrinsic measurement like PL lifetime [21] is needed. Authors’ information HH has obtained his Master’s degree in Physics and Materials in June 2011 at University of Poitiers (France).

Idoxuridine In October 2011, he started his current Ph.D. project at Lyon Institute of Nanotechnologies. His main scientific interest focuses on synthesis, chemical functionalization, and optical characterization of silicon-based semiconductor nanostructures. SAA received his Master’s degree in Chemistry from Kiev National Taras Shevchenko University in 1998 and then his Ph.D. degree in Chemistry at the same university in 2003 for his work on the ‘Immobilization of organic acids on silica gel surface, thermochemical and catalytic properties of materials obtained’. Currently, SAA is working as an associate professor in the Chemistry Faculty of the same university. Since 2004, SAA has close scientific collaboration with INSA Lyon (France); he LY333531 solubility dmso participated in European projects such as INTAS, IRSES, and LST. Fields of his research interests are as follows: surface chemistry of nanostructured materials (semiconductors, inorganic oxides), surface functionalization and characterization, and application of nanostructures in LDI mass spectrometry, sensors, and catalysis. GG received his Master’s degree in Solid State Physics from Claude Bernard University in Lyon (France) in 1970 and then his Ph.D.

A sum score ranging from 5 to 30 was calculated—a high score indicates a high level of exhaustion; standard deviation 5.9. Psychometric properties which were shown to be excellent were published for the Swedish version in Hanson et al. (2008). Depressive symptoms were measured with a brief subscale from the Hopkins Symptom Checklist (SCL-90). The scale measures one-week prevalence and includes six items covering the depressive core symptoms ‘feeling

blue’; ‘feeling no interest in things’; ‘feeling lethargy or low in energy’; ‘worrying too Paclitaxel cell line much about things’; ‘blaming yourself for things’; ‘feeling everything is an effort’. Response options cover five steps from ‘not at all’ to ‘a great deal’. A sum score of depressive symptoms ranging from 0 to 24 was calculated with a high score BVD-523 molecular weight indicating a high probability of clinical depression; standard deviation 5.1. Scale characteristics for this short Swedish version which were shown to be excellent have been published by Magnusson Hanson et al. (2009). Statistical methods In the first step product moment correlations

were calculated between all explanatory study variables using accumulated scores for both cultural activity and the work-related variables. Since the study variables were normally or close to normally distributed, multiple linear regressions were performed in the next step using cultural activity as explanatory and health variables as outcome. find more Each wave was analysed separately. The regressions were made in three successive versions: 1. with adjustment for age, gender and nlog (income) from the actual study year and education from TGF-beta inhibitor self-reported data in 2006.   2. In addition to 1. also with adjustment

for listening/non-listening manager.   3. In addition to 2. also with adjustment for psychological demands and decision latitude at work.   Since income has had a more important role as a confounding factor in the association between cultural activities and health in previous research (Bygren et al. 1996) and since decision latitude and education are strongly correlated, we decided to use income and decision latitude but not education as a proxy for socioeconomic status in the multiple regressions. A final step was two series of prospective analyses, one from 2006 to 2008 and one from 2008 to 2010, in the form of multiple linear regressions. In these analyses age, gender and logarithmically transformed income as well as the work-related variables listening/non-listening manager, psychological demands and decision latitude and finally cultural activities at work and psychological state (emotional exhaustion and depressive symptoms, respectively) at start of follow-up (2006 and 2008, respectively) were used as predictors and subsequent psychological states (emotional exhaustion and depressive feeling 2008 and 2010, respectively) as outcomes.

coli AZD1152 clinical trial real-time PCR (R2 = 0.94) and for C. jejuni real-time PCR (R2 = 0.86). Among the PCR-culture positive samples for the experimentally infected pig, 72.5% of the samples had a difference in cell

number of less than 1 log, 25% of less than 2 logs, and 2.5% of less than 2.5 logs for C. coli real-time PCR assay. For C. jejuni real-time PCR assay, the results obtained by real-time PCR matched equally the results obtained by culture: 67% of the samples had a difference in cell number of less than 1 log, 29% of less than 2 logs, and 4% of less https://www.selleckchem.com/products/Bortezomib.html than 3 logs. Figure 4 Correlation between real-time PCR and microaerobic culture for faecal samples of Campylobacter experimentally infected pigs. Scatter plot showing the differences and correlations between the real-time PCR and the microaerobic culture method for the faecal samples of pigs experimentally infected with Campylobacter for the detection of (a) C. coli and (b) C. jejuni. Data for Campylobacter-positive samples versus Campylobacter-negative samples by both methods fall close 3MA to the line equivalence: a- Campylobacter-positive ( n = 40) and Campylobacter-negative

( n = 25) samples respectively with a coefficient of correlation of 0.90 (R2 = 0.90). b- Campylobacter-positive ( n = 24) and Campylobacter-negative ( n = 25) samples respectively with a coefficient of correlation of 0.93 (R2 = 0.93). Analysis of field samples of naturally contaminated pigs No C. jejuni was identified among the faecal, feed, and environmental samples from the different pig herds by conventional PCR or by our C. jejuni real-time PCR assay. Conversely, all the Campylobacter tested were identified as C. coli by both methods. The specificity and the sensitivity for the C. coli real-time PCR assay with the different field samples are reported in Table 4. Table 4 Comparison of Campylobacter

coli real-time PCR and microaerobic culture in (4.1) faecal, (4.2) feed, and (4.3) environmental samples of naturally contaminated pigs       Microaerobic culture         + – Total 4.1 Campylobacter coli detection in faecal samples   + 125 1 126 Amino acid   Real-time PCR – 3 17 20     Total 128 18 146 4.2 Campylobacter coli detection in feed samples   + 21 1 22   Real-time PCR – 2 26 28     Total 23 27 50 4.3 Campylobacter coli detection in environmental samples   + 34 2 36   Real-time PCR – 3 47 50     Total 37 49 86 4.1 Sensitivity Se = 97.7%, Specificity Sp = 94.4%, Kappa K = 0.96 4.2 Sensitivity Se = 91.3%, Specificity Sp = 96.2%, Kappa K = 0.89 4.3 Sensitivity Se = 91.9%, Specificity Sp = 95.9%, Kappa K = 0.89 For the different field samples tested, the quantification results obtained by C. coli real-time PCR matched equally the results obtained by bacterial culture: 58% of the samples had a difference in cell number of less than 1 log, 37% of less than 2 logs, and 5% of less than 3 logs.

The available literature on RTW and sick leave has been focused mainly on the determinants find more of the return to work of employees on short-term sick leave, while largely ignoring the importance of the determinants of long-term sick leave. Literature shows that there is no international

consensus about the definition of long-term sick leave and short-term sick leave. In the present study, we define long-term sick leave as sickness absence during at least 1.5 years. A systematic review showed that most studies on sick leave are based on sickness absence periods of 6 weeks or less, and there is much less literature about sick leave periods longer than 6 weeks (RG7112 nmr Dekkers-Sánchez et al. 2008). The importance of early work resumption for employees on sick leave has been highlighted by several previous studies (e.g. Bernacki et al. 2000; Tveito et al. 2004). The literature suggests that the impact of factors related to sick leave and absence from work can vary through the different stages of illness (Krause et al. 2001; Burton et al. 2003). The initial onset of absence from work is almost always due to medical reasons. Sufficient evidence suggests that both medical and non-medical factors play a role in the maintenance of sick leave (Dekkers-Sánchez et al. 2008). This diversity of factors could explain why the resumption of work is increasingly difficult as the time absent from work increases

(WHO Prostatic acid phosphatase 2003). Despite the importance of long-term sickness absence, previous research has shown that there is a lack of scientific knowledge on C646 supplier the factors associated with long-term sick leave (Dekkers-Sánchez et al. 2008). Literature shows that the causes of long-term sick leave and complex may involve medical, psychosocial, financial, organisational and work-related factors (Alexanderson

2004). Therefore, a proper workability assessment should take into account all factors that seem responsible for the maintenance of the sickness absence. After 2 years of sick leave, these complex conditions require a multifactorial analysis, including the medical situation, work situation and personal situation of the claimant. This implies that the assessment of workability should include not only the medical factors, but also the non-medical factors responsible for a decreased ability to perform work. With better knowledge about the factors associated with sickness absence, IPs can make useful recommendations to achieve RTW, which is in concordance with the Dutch legislation, aiming at improving RTW outcomes. Despite the important role of physicians in the RTW process, little is known about the views of physicians on the factors that should be addressed in the evaluation of the work ability of employees on long-term sick leave. Therefore, enhancing the knowledge of physicians regarding these relevant factors is warranted.

This is in contrast to the results for P. falciparum cultured in the presence of Neocuproine throughout the culture period (48 h to 96 h) (Figure  4). Pretreatment of uninfected RBCs with two copper chelators, Neocuproine (for Cu1+) and Cuprizone (for Cu2+), individually or in combination, caused partial growth arrest of the parasite, and the effect was independent of the concentrations tested (Figure  6b). To avoid a possible effect of

intrinsic copper ions in the surrounding culture medium, CP-690550 molecular weight GFSRPMI, tests were also performed in CDRPMI, and showed similar results (Figure  6c). These results implied that chelation of Cu1+ ions of the parasite by Neocuproine may be reversible, or that Cu ions (Cu1+ and Cu2+) may be replenished by RBCs, because removal of Cu ions from RBCs caused growth arrest (Figure  6b,c). Figure 6 Growth of P. falciparum co-cultured with PfRBCs and RBCs that were pretreated

separately with the chelators. Synchronized PfRBCs at the ring stage and RBCs were treated with CP673451 graded concentrations of Neocuproine and/or Cuprizone for 0.5 h or 2.5 h at room temperature. After washing, both treated RBCs and PfRBCs were mixed (pretreated PfRBCs plus non-treated RBCs (a) or non-treated PfRBCs plus pretreated RBCs (b, c)) at a ratio of more than 10 times RBCs to PfRBCs, and cultured in GFSRPMI (b) or CDRPMI (a, c) for 95 h. RBCs were pretreated for 2.5 h (b, c); (*) indicates a significant difference versus no treatment with Neocuproine and/or Cuprizone.

(N + C) indicates the PF-02341066 cost mixture of Neocuproine and Cuprizone (1:1). Arrested development of the parasite Amisulpride with CDM-16alone, and profoundly down-regulated expression of copper-binding proteins The CDMs formulated for the development of P. falciparum contain specific NEFAs and phospholipids with specific fatty acid moieties. The effectiveness of the different NEFAs in sustaining the development of P. falciparum varies markedly, depending on their type, total amount, and combination, and the result ranges from complete development to growth arrest at the ring stage. The most effective combination of NEFAs has been found to be C18:1 and C16:0 [4, 5]. P. falciparum was cultured asynchronously with different concentrations and ratios of two NEFAs (C18:1 and C16:0), individually or in combination, in the presence of phospholipids. The mixtures of NEFAs, but not individual C16:0 or C18:1, sustained parasite growth (Figure  7). The NEFAs required pairing at different ratios: the maximum effect was obtained with 100 μM C18:1 plus 60 μM C16:0. This culture medium represents CDRPMI, and the growth rate was comparable to that in GFSRPMI. These experiments also showed that profound growth arrest of the parasite occurred in CDM enriched with either C16:0 or C18:1 (Figure  7). Figure 7 Growth of asynchronous P. falciparum cultured for 95 h in the presence of NEFAs. The two NEFAs, C18:1 and C16:0, were added to CDM, alone or in combination, at various concentrations and ratios.

The SRA–DNA interaction may serve as an MCC950 research buy anchor to keep UHRF1

at a hemi-methylated CpG site where it recruits the DNMT1 for DNA methylation maintenance [9, 11]. Thus, UHRF1 plays a fundamental role in the inheritance EPZ5676 cost of the DNA epigenetic marks from the mother cell to the daughter cells. It also appears that preventing the transmission of these marks via knock-down of UHRF1 leads to an activation of pro-apoptotic pathways [9, 12–16]. In agreement with this hypothesis, UHRF1 down-regulation has been shown to inhibit cell growth and induces apoptosis of colorectal cancer through p16INK4A up-regulation [17]. Some bioactive plants components have been shown to have cancer inhibition activities by reducing DNA hypermethylation of key cancer-causing genes

through their DNA methyltransferase (DNMT) inhibition properties [18]. In this context, recently we found that the epigallocatechin-3-gallate (EGCG), a natural anti-cancer drug induces G1 cell arrest and apoptosis in Jurkat cells by down-regulating UHRF1 and DNMT1 expression, with subsequent up-regulation of p16 INK4A gene [19]. L. guyonianum has been used in traditional medicines to treat gastric infections. It has also been employed as an anti-bacterial drug in the treatment of bronchitis [20]. L. feei has been similarly used in the treatment of bronchitis and stomach infections [21]. Previous investigations revealed that methanol extract from L. feei leaves contained potential anti-fungal Selleck Rabusertib constituents that could be PIK3C2G employed against Candida albicans and anti-bacterial constituents useful against E. coli[22]. More recently, our laboratory demonstrated that L. guyonianum aqueous gall extract was able to induce splenocyte proliferation and to stimulate macrophage activation [23]. Chemical investigation of Limoniastrum genus has been reported in literature. Indeed, bioguided fractionation of leaves

extract from Limoniastrum feei led to the isolation of several polyphenolic constituents such as Gallic acid, Epigallocatechin gallate, Quercetin and Myricetin [24]. A subsequent article noted that ethyl acetate extract of L. guyonianum contained gallocatechin, epigallocatechin, and epigallocatechin-3-O-gallate [25]. Several groups have reported that epigallocatechin gallate exhibited antitumor effects that were discovered from various cancer cell lines, animal models and clinical studies [26]. For example, in vivo studies showed that epigallocatechin gallate administration decreased H1299 xenograft tumor growth [27]. Furthermore, myricetin treatment significantly inhibited the tumor growth on T24 bladder cancer xenografts model [28]. In the same way, it was demonstrated that gallic acid plays a critical role as an anticancer agent in vivo by decreasing MNNG/HOS xenograft tumor growth in Balb/C mice [29].

After drying, each sample was finely ground in a mortar, sieved, homogenized and stored at −20°C until DNA extraction was performed. Soil DNA extraction A DNA extraction procedure was specifically developed

for all the four types of soil analysed in this study. Three Geneticin molecular weight replicates (5 g each) were prepared for each soil sample, re-suspended in 6–7 ml of CTAB lysis buffer (2% CTAB, 2% Polyvinylpyrrolidon, VE-822 nmr 2 M NaCl, 20 mM EDTA, 100 mM Tris–HCl, pH 8) and processed according the detailed protocol described in Additional file 2. Brown crude DNA solutions (about 3 ml in volume) from each reaction were obtained following this extraction phase and 1 ml aliquots were then purified using the Nucleospin Plant II kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions with slight modifications (see Additional file 2). Total DNAs were finally

eluted in 65 μl of elution buffer (5 mM Tris/HCl, pH 8.5). The amount of DNA in each extract was quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). The quality of the total DNAs was evaluated with optical density (OD) 260/280 nm and 260/230 nm ratios. Extractions with OD ratios less than 1.4 and DNA quantity less than 25 ng μl–1 were repeated. In addition soil DNA extracts were PCR-amplified with primer pair ITS1-ITS4 [39] to confirm the absence of DNA polymerase inhibitors. Extracts with positive ITS1-ITS4 amplification products (from 500 bp to 1000 bp) were considered suitable for Tideglusib in vitro quantitative Erastin solubility dmso PCR (qPCR) assays. Purified DNAs were stored at −80°C until processed. Primer and probe selection ITS1-5.8 S-ITS2 rDNA sequences of T. magnatum and other truffle

species were retrieved from GenBank database (http://​www.​ncbi.​nlm.​nih.​gov/​; date of accession: June, 2008) and aligned with Multalign [40] to identify species-specific domains for primer and probe selection. Oligonucleotide design was carried out with Primer3 software (http://​frodo.​wi.​mit.​edu/​primer3/​) [41] with the following parameters: amplicon size 90–110, primer size 18–22 bp (opt. 20 bp), melting temperature 58-62°C (opt. 60°C), GC content 40-60% (opt. 50%), Max Self Complementarity = 5. Secondary structures and dimer formation were verified using Oligo Analyzer 1.0.3 software (Freeware, Teemu Kuulasmaa, Finland) and specificity was firstly evaluated in silico using BLASTN algorithm (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi). A primer pair and the respective probe was selected for both the ITS1 and the ITS2 region (Table 2) and their specificity was then confirmed with qualitative PCR against genomic DNA of different mycorrhizal, saprobic and pathogenic fungi (Table 3). The specificity of the oligonucleotides selected as probes was tested in PCR reactions using their opposite primers (TmgITS1rev with TmgITS1prob and TmgITS2for with TmgITS2prob).