50, P = 0.001) (Figure  1) and the percent CX-5461 chemical structure change in fat mass (r = 0.44, P < 0.001) and significantly and negatively related to fluid intake (r = -0.54, P < 0.05) (Figure  1) and percent change in plasma urea (r = -0.53, P < 0.05). Men’s’ absolute ranking in the race was not related to changes in plasma [Na+], or percent

changes in urine specific gravity (P > 0.05). Changes in body mass were significantly and negatively related to the number of achieved kilometers during the 24 hours also in women (r = -0.80, P < 0.001). Their absolute ranking during the race was significantly and positively related to the change in body mass (r = 0.70, P < 0.05), the percent change in body mass (r = 0.77, P < 0.05) (Figure  1), and significantly and negatively related find more to fluid intake (r = -0.73, P < 0.05) (Figure  1) during the race. Women’ absolute ranking in the race was not related to percent change in fat mass, or percent change in urine specific gravity (P > 0.05). Changes in body composition with regard to anthropometric, urine and blood measurements The correlation matrix of post-race body mass, change in body mass, percent change in body mass, post-race fat mass, percent GSK126 ic50 change in fat mass, percent change in extracellular fluid and percent change in plasma urea for men is shown

in Table  4. The correlation matrix of change in body mass, percent change in body mass and percent change in fat mass for women is presented in Table  5. Table 4 Correlation matrix of PR BM, ΔBM, %ΔBM, PR FM, %ΔFM, %ΔECF and %Δ plasma urea

for men (n = 37) PR BM 0.20 0.33* 0.63** 0.17 0.35* -0.10 ΔBM 0.99** 0.19 0.30 0.88** -0.44 %ΔBM 0.53* 0.33* 0.83** -0.50* PR FM 0.45** Cobimetinib 0.29 -0.53* %ΔFM -0.05 -0.31 %ΔEXW -0.52* %ΔPU PR BM – post-race body mass, ΔBM – change in body mass, %ΔBM – percent change in body mass, PR FM – post-race body mass, %ΔFM – percent change in fat mass, %ΔECF – percent change in extracellular fluid, %Δ plasma urea – percent change in plasma urea. Output file contain both the Pearson’s r values and the scatter plot, one star (*) above the Pearson value represents significance level P < 0.05, two stars (**) P < 0.001. Table 5 The correlation matrix of ΔBM, %ΔBM and %ΔFM for women (n = 12) ΔBM 0.99** 0.35 %ΔBM 0.36 %ΔFM ΔBM – change in body mass, %ΔBM – percent change in body mass, %ΔFM – percent change in fat mass. Output file contain both the Spearman’s rank correlation coefficient and the scatter plot, one star (*) above the Spearman value represents significance level P < 0.05, two stars (**) P < 0.01. In male ultra-MTBers (n = 37) body mass decreased significantly during the race by 2.0 ± 1.6 kg, equal to 2.6 ± 2.1% (P < 0.001) (Table  2, also Figure  2). Fat mass decreased significantly by 1.4 ± 1.2 kg (P < 0.001), percent body fat decreased significantly by 1.4 ± 1.4% (P < 0.001), whereas skeletal muscle mass decreased non-significantly by 0.6 ± 2.7% (P > 0.05) (Table  2, also Figure  2).

It can be seen that less stretching force was needed for the present study compared to those of Hsieh and Liu [1]. Figure 8 Representative of DNA recoiling at different times (Δ t  = 5 s) for 1× TBE. Figure 9 Graphs showing (a) relaxation time vs viscosity and (b) μ vs . Figure 10 Comparisons with those of previous studies. Finally, data for mean stretch ratio were correlated in a power law from of Wi as x/L c = 0.17 Wi0.265, as indicated ASK inhibitor in Figure 11a. Teixeira et al.’s [14] and Smith et al.’s [15] results were also included in Figure 11a. Again, the present results show a large stretch with a definite Wi. Another correlation of mean stretch ratio as a function

of Pe is shown in Figure 11b. A straight line relation was found in the form of x/L c = 5.37 × 10−5 Pe + 0.18, and the initial stretch length was obtained as Pe equals zero in this study. Figure 11 Graphs showing (a) mean stretch ratio vs Wi

and (b) mean stretch ratio vs Pe. Conclusions DNA click here molecule dynamics in curved (semi-circle, 0° ≤ θ ≤ 180°) microchannels with different radii for five different buffer solutions of 1× Tris-acetate-EDTA (TAE), 1× Tris-borate-EDTA (TBE), 1× Tris-EDTA (TE), 1× Tris-phosphate-EDTA (TPE), and 1× Tris-buffered saline (TBS) with a variety of viscosity such as 40, 60, and 80 cP were extensively studied for 10−4 ≤ Re ≤10−3 and 5 ≤ Wi ≤12. The major findings drawn are as follows: 1. Radius effect was significantly noted with maximum stretch ratio occurring at the center of the semi-circle (θ = 90°) with a radius of 500 μm.   2. The oscillatory/recovery nature of the present stretching behavior was found.   3. The buffer solution type seems to have no significant influence on the stretch ratio, with no viscosity effect.   4. The correlation of x/L c was developed for parameters of Wi and Pe, respectively, with different functional relationships.   Authors’

information SSH is a professor at the Department of Mechanical and mTOR activation Electro Mechanical Engineering, National Sun Yat-Sen University, Kaohsiung, Taiwan, Republic of China. FHW is a student working towards a master’s degree at the Department of Mechanical and Electro Mechanical Engineering, National Sun Yat-Sen University, Kaohsiung, MycoClean Mycoplasma Removal Kit Taiwan, Republic of China. MJT is a student working towards a master’s degree at the Department of Mechanical and Electro Mechanical Engineering, National Sun Yat-Sen University, Kaohsiung, Taiwan, Republic of China. Acknowledgements This work was supported by the National Science Council (NSC) of Taiwan under contract number NSC 101-2221-E-110-043-MY3. References 1. Randall GC, Schultz KM, Doyle PS: Methods to electrophoretically stretch DNA: microcontractions, gels, and hybrid gel-microcontraction devices. Lab Chip 2006, 6:516–525.CrossRef 2.

1) of the genus Hypocrea/Trichoderma. For ITS sequences search GenBank under the respective taxon or strain numbers. Taxon

Name in part I Strain Accession rpb2 Accession tef1 Hypocrea albolutescens H. sp. 1 CBS 119286 FJ860517 FJ860609 H. atlantica H. sp. 11 C.P.K. 1896 FJ860545   H. atlantica H. sp. 11 CBS 120632   FJ860649 H. auranteffusa H. sp. 2 CBS 119284 FJ860520 FJ860613 H. austriaca H. sp. 3 CBS 122494 FJ860525 FJ860619 H. bavarica H. sp. 4 C.P.K. 2021 FJ860526 FJ860620 H. calamagrostidis H. sp. 5 CBS 121133 FJ860528 FJ860622 H. margaretensis Autophagy Compound Library clinical trial H. sp. 6 C.P.K. 3127 FJ860529 FJ860625 H. junci H. sp. 9 CBS 120926 FJ860540 FJ860641 H. luteffusa H. sp. 10 CBS 120537 FJ860543 FJ860645 H. luteocrystallina H. sp. 8 CBS 123828 FJ860544 FJ860646 H. neorufoides H. sp. 12 C.P.K. 1900 FJ860553   H. neorufoides H. sp. 12 CBS 119506   FJ860657 H. pachypallida H. sp. 13 CBS 120533 FJ860559   H. pachypallida H. sp. 13 CBS 122126   FJ860662 H. phellinicola H. sp. 14 CBS 119283 FJ860569 FJ860672 H. rhododendri H. sp. 15 CBS 119288 FJ860578 FJ860685 H. sambuci H. sp. 16 WU 29467 FJ860585 FJ860693 H. silvae-virgineae H. sp. 7 CBS 120922 FJ860587 FJ860696 H. subeffusa H. PCI-34051 order sp. 17 CBS 120929 FJ860597 FJ860707 H. valdunensis H. sp. 18 CBS 120923 FJ860605 FJ860717 Results and discussion Overview and phylogeny of the European Hypocreas

Of the 75 species of Hypocrea/Trichoderma so far recognised as forming teleomorphs in Europe 56 species have hyaline ascospores. These species are here described in detail and illustrated by colour plates, including cultures and anamorphs. The number of species described in this volume includes 16 new holomorphs, two new teleomorphs and nine anamorphs of species previously described as teleomorphs. Phylogenetic placement and relationships of all species are shown on the strict consensus tree (Fig. 1) based on a combined analysis of sequences of RNA polymerase STK38 II subunit b (rpb2) and translation elongation factor 1 alpha (tef1) exon of the genus comprising 135 species. The tree is the same as presented by Jaklitsch (2009), but names are inserted for the species

cited there only with a number. See Jaklitsch (2009) for a discussion of the tree topology. Sectional and clade names are used in a phylogenetic sense. This means that they are not necessarily congruent with the Trichoderma sections defined by Bissett (1991a) and that they are used synonymously for both Hypocrea and Trichoderma. Fig. 1 Strict consensus tree of LY3023414 datasheet length 5952 resulting from a maximum parsimony (MP) analysis of 1529 characters of the combined rpb2 – tef1 exon alignment of 135 species of Hypocrea/Trichoderma. Broad black lines represent nodes with MP bootstrap values (BS) = 70–100 and Bayesian posterior probabilities (PP) = 95–100, broad grey lines nodes with BS < 70 and PP = 95–100; asterisks (*) mark nodes with BS > 70 and PP < 95.

Ott et al. found that a reduction in FDG uptake of more than 35% for metabolic responders predicted

a favorable response in gastric cancer patients FG-4592 manufacturer two weeks after initiation of chemotherapy [11], while metabolic non-responders or FDG non-avid Vorinostat chemical structure tumors received an unfavorable prognosis. Cancer cells theoretically require a greater amount of glucose consumption than healthy tissue because of increased cell division [12, 13] or anaerobic respiration in tumors [14]. Many cancers increase glucose transport through glucose transporter 1 (GLUT1) and glucose phosphorylation by hexokinase (HK) [15–17]. A correlation between FDG uptake and GLUT1 expression has been found in gastric cancer patients [1, 3, 7, 8], but

these studies were conducted by non-quantitative immunohistochemistry analysis, such as negative or positive staining that can vary by evaluator. We therefore evaluated the expression of glucose metabolism-related proteins through quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and compared the results to maximum SUV of FDG-PET. In addition, we also analyzed the expression of proliferating cell nuclear antigen (PCNA) as a valid marker of proliferation [18] and hypoxia-inducible factor 1 alpha (HIF1α) as a marker of hypoxia [19] to elucidate either of these mechanisms, i.e., tumor proliferation or tumor hypoxia, contribute to FDG uptake. We then discuss the significance and HTS assay difficulties involved with the clinical application of FDG-PET in gastric cancer due to FDG uptake mechanisms. Materials and methods Patients This retrospective study involved 50 patients (29 male and 21 female; mean age ± standard error of measurement [SEM], 65.8 ± 1.4 years) with gastric cancer who underwent same FDG-PET system before gastrectomy in Kagawa University from July 2005 to March 2010. Tumor specimens were snap-frozen at the time of surgery, and stored at −80°C. Participants were divided into 25 cases of intestinal tumors and 25 cases of non-intestinal tumors based on histopathological diagnoses. When focal FDG

uptake was not found in the stomach, SUV was calculated from a lesion determined by histology results after gastrectomy. The International Union Against Cancer Janus kinase (JAK) staging system was used to determine clinicopathological parameters associated with FDG uptake. The protocol was approved by the institutional review board of our institution, and all patients provided written informed consent. FDG-PET imaging FDG-PET images were acquired with a PET scanner (ECAT EXACT HR+, Siemens/CTI, Knoxville, TN, USA). Patients fasted at least five hours before FDG injection. Images were reviewed on a Sun Microsystems workstation (Siemens/CTI) along transverse, coronal, and sagittal planes with maximum intensity projection images.

Ann Surg 2003, 238:349–355. discussion 355–347PubMed 52. Brandt CP, McHenry CR, Jacobs

DG, Piotrowski JJ, Priebe PP: Polypropylene mesh closure after emergency laparotomy: morbidity and outcome. Surgery 1995, 118:736–740. discussion 740–731PubMedCrossRef 53. Tons C, Schachtrupp A, Rau M, Mumme T, Schumpelick V: Abdominal compartment syndrome: prevention and treatment. Der Chirurg; Zeitschrift fur alle Gebiete der operativen Medizen 2000, 71:918–926.PubMedCrossRef 54. Sugrue M, Jones F, Janjua KJ, Deane SA, Bristow P, Hillman K: Temporary abdominal closure: a prospective evaluation selleck products of its effects on renal and respiratory physiology. J Trauma 1998, 45:914–921.PubMedCrossRef 55. Weinberg JA, George RL, Griffin RL, Stewart AH, Reiff DA, Kerby JD, Melton SM, Rue LW 3rd: Closing the open abdomen: improved success with Wittmann Patch staged abdominal {Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|buy Anti-diabetic Compound Library|Anti-diabetic Compound Library ic50|Anti-diabetic Compound Library price|Anti-diabetic Compound Library cost|Anti-diabetic Compound Library solubility dmso|Anti-diabetic Compound Library purchase|Anti-diabetic Compound Library manufacturer|Anti-diabetic Compound Library research buy|Anti-diabetic Compound Library order|Anti-diabetic Compound Library mouse|Anti-diabetic Compound Library chemical structure|Anti-diabetic Compound Library mw|Anti-diabetic Compound Library molecular weight|Anti-diabetic Compound Library datasheet|Anti-diabetic Compound Library supplier|Anti-diabetic Compound Library in vitro|Anti-diabetic Compound Library cell line|Anti-diabetic Compound Library concentration|Anti-diabetic Compound Library nmr|Anti-diabetic Compound Library in vivo|Anti-diabetic Compound Library clinical trial|Anti-diabetic Compound Library cell assay|Anti-diabetic Compound Library screening|Anti-diabetic Compound Library high throughput|buy Antidiabetic Compound Library|Antidiabetic Compound Library ic50|Antidiabetic Compound Library price|Antidiabetic Compound Library cost|Antidiabetic Compound Library solubility dmso|Antidiabetic Compound Library purchase|Antidiabetic Compound Library manufacturer|Antidiabetic Compound Library research buy|Antidiabetic Compound Library order|Antidiabetic Compound Library chemical structure|Antidiabetic Compound Library datasheet|Antidiabetic Compound Library supplier|Antidiabetic Compound Library in vitro|Antidiabetic Compound Library cell line|Antidiabetic Compound Library concentration|Antidiabetic Compound Library clinical trial|Antidiabetic Compound Library cell assay|Antidiabetic Compound Library screening|Antidiabetic Compound Library high throughput|Anti-diabetic Compound high throughput screening| closure.

J Trauma 2008, 65:345–348.PubMedCrossRef 56. Tieu BH, Cho SD, Luem N, Riha G, Mayberry J, Schreiber MA: The use of the Wittmann Patch facilitates a high rate of fascial closure in severely injured trauma patients and critically ill emergency surgery patients. J Trauma 2008, 65:865–870.PubMedCrossRef 57. Wittmann DH: Staged abdominal repair: Development and current practice of an advanced operative technique for diffuse suppurative peritonitis. Acta Chirurgica Austriaca 2000, 32:171–178. 58. Hadeed JG, Staman GW, Sariol HS, Kumar S, Ross SE: Delayed primary closure in www.selleckchem.com/products/bv-6.html damage control laparotomy: The value of the Wittmann patch. Am Surg 2007, 73:10–12.PubMed 59. Schnuriger B, Inaba K, Wu T, Eberle BM, Belzberg H, Demetriades D: Crystalloids after primary colon resection and anastomosis

at initial trauma laparotomy: Excessive volumes are associated with anastomotic leakage. J Trauma 2011, 70:603–610.PubMedCrossRef 60. Cotton BA, Guy JS, Morris JA Jr, Abumrad NN: The cellular, metabolic, and systemic consequences of aggressive fluid resuscitation strategies. Shock 2006, Baricitinib 26:115–121.PubMedCrossRef 61. Cotton BA, Gunter OL, Isbell J, Au BK, Robertson AM, Morris JA Jr, St Jacques P: Damage control hematology: the impact of a trauma exsanguination protocol on survival and blood product utilization. J Trauma 2008, 64:1177–1182. discussion 1182–1173PubMedCrossRef 62. Holcomb JB, Jenkins D, Rhee P, Johannigman J, Mahoney P, Mehta S, Cox ED, Gehrke MJ, Beilman GJ, Schreiber M, et al.: Damage control resuscitation: directly addressing the early coagulopathy of trauma. J Trauma 2007, 62:307–310.PubMedCrossRef 63. Holcomb JB, Wade CE, Michalek JE, Chisholm GB, Zarzabal LA, Schreiber MA, Gonzalez EA, Pomper GJ, Perkins JG, Spinella PC, et al.: Increased plasma and platelet to red blood cell ratios improves outcome in 466 massively transfused civilian trauma patients. Ann Surg 2008, 248:447–458.PubMed 64.

Total DNA enriched in bacterial endosymbionts was extracted from viscera of 20–30 adult female insects in sterile conditions and mechanically homogenized. In order to reduce insect DNA contamination, the samples were subjected to consecutive centrifugations at 1150 g and 1300 g for 10 minutes, and genomic DNA was obtained from the supernatant following a CTAB (Cetyltrimethylammonium LGK-974 in vitro bromide) extraction method [48]. Genome sequencing and assembly The purified genomic DNA was shotgun sequenced using 454/Roche GS-FLX Titanium technology at the Genomics and Health area of the Public Health Research Center (CSISP, Generalitat Valenciana). One half-plate

single-ends, and one-fourth plate paired-ends (3 kb of fragment size) sequencing experiments were performed, yielding a total of 1.3 million reads. Sequences of eukaryotic origin were eliminated after a taxonomic assignation process by Galaxy [49]. Filtered reads were automatically assembled by MIRA [50] and the resulting

contigs were manually edited with the Gap4 program from the Staden package software [51]. The remaining gaps in the genome of M. endobia str. PCVAL were closed by ABI sequencing of PCR products obtained with designed primers, at the sequencing facility of the Universitat de València. Potential oriC on both genomes were sought with the OriginX program [52]. Total DNA samples obtained from the P. citri populations from Murcia and Almassora were used to further analyze the rplQ region PXD101 research buy from

the T. princeps genome. The region comprised between genes rpoA and aroK was amplified and sequenced using the primers rpoA-F (5′-TGCCAGGCCTAGTGCTAAACATCA-3′) and aroK-R (5′-TGTCGCCAGGACTGCTATCAATGT-3′). Gene annotation and functional analysis ARAGORN [53], tRNAscan Racecadotril [54], and Rfam [55] sowftware packages were used for RNA genes prediction. Coding genes were annotated by BASys (Bacterial Annotation System, [56], RAST [57] and refined by BLAST searches [58]. Finally, functional domain studies in Pfam database [59] were performed when coding-genes functionality assessment was required. Artemis [60] and MEGA5 [61] programs were used for genome statistics calculation and codon usage analysis. Metabolic capabilities were analyzed with Blast2Go [62] and KAAS [63] programs. Functional information from the BioCyc [64], KEEG [65] and BRENDA [66] databases were also used in this context. Genome alignments were performed using MAFFT [67]. Annotated ORFs were considered as functional genes following two non-exclusionary criteria: the conservation of at least 80% of the sequence length of the closest orthologs found by BLAST in non-redundant databases, and/or the maintenance of the essential functional domains detected by Pfam [59]. Accession numbers The genome sequence of M. endobia strain PCVAL has been deposited at the GenBank (accession number CP003881).

1980). The idea behind this model

is that individuals are active problem solvers who make sense of a threat to their health by developing their own cognitive representation of the threat, which, in turn, determines how they then respond to it (Petrie and Weinman 2006). GSK2126458 The concept of “illness perceptions” has been a focus of many research studies evaluating and predicting patient outcomes in the past decades and has been adapted and advocated by many authors as shown by several reviews (Hagger and Orbell 2003; Coutu et al. 2008; Fadyl and McPherson 2008). Initially, Leventhal et al. (1980) distinguished five domains considered to be important when assessing these illness representations or perceptions, including (1) the identity of the illness

based on the diagnosis or symptoms associated with it; (2) the timeline of the illness (3) the short- and long-term consequences; (4) the factors contributing to the illness and (5) ways to control or cure the illness. Although illness representations were initially assessed using interviews, the drawbacks of this method led to the development of measures such as the Implicit INK 128 nmr Model of Illness Questionnaire (Turk et al. 1986), the Illness Cognition Questionnaire (Evers et al. 2001) and the Illness Perception Questionnaire (IPQ) (Weinman et al. 1996) or subsequent modifications such as the revised IPQ (IPQ-R) (Moss-Morris et al. 2002) or the brief version of the IPQ (IPQ-B) (Broadbent et al. 2006). These quantitative measures all use the five domains identified by Leventhal, although the revised IPQ (IPQ-R) also further developed the model by including new dimensions, i.e., ‘emotional’ and ‘coherence’ representations. Factors closely linked to several illness representation dimensions have also been used in several

other one-dimensional or multi-dimensional questionnaires measuring psychosocial dimensions (Coutu et al. 2008). These include questionnaires on catastrophizing (Sullivan et al. 1995), self-efficacy, or attitudes or experiences of pain (Gibson and Strong 1996; Jensen et al. from 1987; Edwards et al. 1992), but do not aim to describe all dimensions considered to be important in the link between representations, coping behavior and outcomes as described in the common sense model. Illness perceptions directly influence the individual’s emotional response to the disease or complaint and their coping behavior as has been shown in studies on treatment adherence, which could be, for example, a physician’s recommendation regarding return to work. The common sense model assumes a causal link between illness representations, the coping strategies patients adopt in response to their illness and the health outcomes of patients. The IPQ and subsequent revisions are based on assessing just the first stage of the common sense model of self-regulation, i.e., interpretation of the cognitive or emotional representation of the health threat.

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interrogans 2 09131462 Human serum 55/ml L. borgpetersenii

1 09117472 Human serum 60/ml L. borgpetersenii 1 09233024 Human serum 200/ml L. interrogans 1 08121411 Human serum 320/ml L. interrogans 4 09100462 Human serum 320/ml L. interrogans 5 09031188 Human serum 920/ml L. interrogans 1 08095345 Human serum (fatal case) 1100/ml L. interrogans 1 09043326 Human serum 1100/ml L. interrogans 5 09210289 Human serum 1100/ml L. interrogans 5 09145359 Human serum 1600/ml L. interrogans 1 09044463 Human serum (fatal case) 5800/ml L. interrogans 5 09243410 Human serum (fatal case) 6300/ml L. interrogans 1 Deer 16 Deer kidney < 50/mg L. borgpetersenii 2 Deer 39 Deer kidney Tucidinostat < 50/mg L. interrogans 1 Deer 3 Deer kidney selleckchem 50/mg L. interrogans 4 Deer 10 Deer kidney 80/mg L. borgpetersenii 2 Deer 13 Deer kidney 82/mg L. interrogans 1 Deer 9 Deer kidney 88/mg L. borgpetersenii 2 Deer 14 Deer kidney 300/mg L. borgpetersenii 2 Deer 15 Deer kidney 675/mg L. borgpetersenii 2 Deer 21 Deer kidney 625/mg L. borgpetersenii 2 Deer 2 Deer kidney 1100/mg L. interrogans 4 Deer 27 Deer kidney 3700/mg L. interrogans 4 GenBank accession numbers of the sequences obtained from these specimens are provided in additional file 1 Table S2. DNA extraction For human samples, total DNA from serum (200 μl) was extracted using an automatic method on an EasyMAG apparatus (Biomerieux). For bacterial cultures and animal samples, total DNA from a culture

pellet, or kidney (ca. 25 mg) was extracted using mafosfamide the QIAamp DNA minikit (Qiagen) following the manufacturer’s

instructions. PCR analysis The real time PCR routinely used for leptospirosis diagnosis targets the lfb1 gene as described by Mérien et al. [15] and was run on a LightCycler LC 2.0 using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science, New Zealand). For the MLST study, we used the typing scheme described by Thaipadungpanit et al. that uses the sequence polymorphism of pntA, sucA, pfkB, tpiA, mreA, glmU and fadD [20]. Amplifications were performed in a 25 μl total volume containing 1-10 ng genomic DNA, 5 pmol of each primer, 200 μM dNTP with 1.25 mM MgCl2. Two different DNA polymerases were used for DNA amplification: either 1 unit of Red Hot Taq DNA Polymerase, Thermo Scientific (ABgene) or 1.25 units of FastStart High Fidelity PCR System (Roche Applied Science), in their corresponding 1× buffer. A GeneAmp PCR system 9700 (Applied Biosystem) was used to perform PCR with an initial denaturation step at 94°C for 2 minutes, followed by 35 cycles of 94°C for 20 seconds, variable annealing temperature for 30 seconds, 72°C for 50 seconds for Red Hot Taq DNA Polymerase and 40 cycles of 94°C for 30 seconds, variable annealing temperatures for 30 seconds, 72°C for 50 seconds for FastStart High Fidelity DNA Polymerase, then 72°C for 7 minutes. PCR product size, primer sequences and annealing temperatures are shown in Table 3.