Although in western countries intestinal obstruction caused by sigmoid volvulus is rare, its mortality remains significant in patients with a late diagnosis [12]. The aim of this work is to assess which are the results of different surgical timings and procedures performed in the different clinical presentations of this disease. Methods We realized a retrospective case note review of patients treated surgically for a sigmoid volvulus in the Department of General Surgery, St Maria

Hospital, Terni, from January 1996 till January 2009. We included in find more this study a group of 23 patients (15 men and 8 women), which were diagnosed at the Emergency Department with abdominal pain and obstructive symptoms and then admitted into other Departments for treatment. Nine patients were primarily admitted into the surgery unit with intestinal obstruction symptoms, while 14 patients were admitted for a subocclusion (8 patients were admitted

in a medical unit and 6 patients in the surgery division). MAPK inhibitor The patients were divided in 2 groups on the basis of the clinical onset: obstructed patients (9 patients) and subocclusive patients groups (14 patients) according to the following criteria: obstructed patients had abdominal distension with no flatus, tenderness and a clearly positive plain abdominal X-ray, whereas subocclusive patients had no flatus, moderate abdominal distension, and a doubtful plain abdomen X-ray. All patients underwent clinical examination and an abdominal X-ray. We identified patients affected by the comorbidities included into Satariano’s co-morbidity index [13], uncooperative patients with degenerative and cognitive diseases, patients with clinical signs of peritonitis and patients with a diagnostic abdominal X-ray for sigmoid volvulus or intestinal occlusion. We assessed 30-day postoperative mortality relating it to the surgical timing and treatment employed for each group. Results The mean age of patients with obstruction was 76 years (69-85

years). In this group 4 patients O-methylated flavonoid were affected by >2 comorbidities and 5 patients by <2 comorbidities. Three patients were uncooperative and 2 of these were bed-bound. Four patients had clinical signs and symptoms of peritonitis and ileus, showing a diagnostic abdominal X-ray for sigmoid volvulus or intestinal occlusion, while the 5 remaining patients presented clinical and radiological signs of occlusion, but no clinical signs of peritonitis (Table 1). All the patients underwent emergency surgery; we performed a sigmoid resection in the 4 patients with clinical signs and symptoms of peritonitis and in 3 out of the 5 patients showing only clinical and radiological signs of occlusion, while an intestinal derotation with colopexy was performed in the 2 remaining patients.

Samples were dried and treated with 3 M nitric acid overnight at room temperature then quickly boiled. Total manganese content was determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) at North Carolina State University Analytical Service Laboratory. Total manganese and iron was measured Kinase Inhibitor Library mouse in LB medium as above using a 5X concentration of medium. Results Growth of Δfur under anaerobic and aerobic conditions Iron is an essential element for redox reactions in biology. However, it is an important factor in oxygen toxicity due to its involvement in hydroxyl radicals (HO·)

formation via Fenton chemistry [57]. Therefore, we compared the effects of a deletion of fur on growth kinetics under both anaerobic and aerobic conditions. Data in Figure 1 demonstrate that Δfur was not compromised in its growth kinetics under either anaerobic or aerobic conditions. Figure 1 Growth kinetics of Δ fur (black square compared to 14028s (white square). Selleck Z-IETD-FMK Cells were grown in LB-MOPS-X medium as described in Methods; (A) Anaerobic growth; (B) Aerobic growth. Effect of Fur on the anaerobic transcriptome of S. Typhimurium Under anaerobic conditions, the absence of fur resulted in the differential

expression of 298 genes (Additional File 2: Table S2). These genes were organized by Cluster of Orthologous Groups (COGs) and the numbers of genes within each COG are shown in Table 2. The absence of fur resulted in increased expression (i.e., Fur acted as a repressor) of 226 genes. However, the absence of Fur resulted in decreased expression (i.e., Fur acted as an activator) of 72 genes, most likely via an indirect mechanism. Table 2 Number of Differentially Expressed Genes in Δfur Differentially Expressed Genes in Δfur Cluster of Orthologous Groups Number of Genes “”Fur Repressed”" a Number

of Genes “”Fur Activated”" b Total No COG 30 9 39 Energy Production and Conversion 16 18 34 Cell Cycle Control 3 0 3 Amino Acid Metabolism and Transport 7 16 23 Nucleotide Metabolism and Transport 7 4 11 Carbohydrate Metabolism and Transport 9 4 13 Coenzyme Metabolism and Transport 6 0 6 Lipid Metabolism and Transport 5 0 5 Translation 46 0 46 Transcription 9 2 11 Replication, Recombination, and Repair 5 1 6 Cell Wall/Membrane/Envelope Biogenesis 14 3 17 Cell Motility 1 0 1 Post-Translational Modification, old Protein Turnover, Chaperone Functions 10 1 11 Inorganic Ion Transport and Metabolism 20 2 22 Secondary Metabolite Biosynthesis, Transport, and Catabolism 5 4 9 General Functional Prediction Only 15 4 19 Function Unknown 9 2 11 Signal Transduction Mechanisms 5 2 7 Intracellular Trafficking and Secretion 3 0 3 Defense Mechanisms 1 0 1 Total 226 72 298 Categorized According to Cluster of Orthologous Groups (COGs) a Genes with increased expression in the absence of fur b Genes with decreased expression in the absence of fur A Fur information matrix, specific for S.

LGG was chosen as a positive control, because human in vivo studies showed that the beneficial effects of LGG are, in part, attributed to a strong colonization of the colonic mucus layer upon oral administration [41]. This strong adhesion capacity of LGG has recently been attributed to a SpaC pilin, which is located on the top of the pili and exerts a strong mucus-binding activity [42]. After 1.5 h of incubation in the upper compartment of the HMI module, LGG showed an adhesion percentage of 15.7 ± 3.2%, as compared to the original concentration see more dosed to the model. This value

is in line with what described by Van den Abbeele et al. [21], who tested the adhesive properties of LGG in presence of a complex gut microbiota in a M-SHIME. The colonization capacity of mucus by LGG was thus confirmed in the HMI module. Finally, the HMI module containing enterocytes in the lower compartment was challenged for the first time with a complex microbiota originated from the simulated ascending colon of the SHIME. In parallel the enterocytes were also directly exposed to the same complex microbiota. A MTT test showed that the viability of ATM/ATR inhibitor clinical trial Caco-2 cells directly exposed to the complex microbial community decreased by 80% after 2 hours of co-culture. In contrast, when the interaction occurred within an HMI module, the cells’ viability

after 48 h of incubation was not significantly different as compared to a control system in which only sterile SHIME medium was dosed (Figure 2). Although the use of cell cultures, such as Caco-2 cells, is not novel for mechanistic studies [29, 43, 44], the output of these reductionist studies is limited by the fact that they are normally

conducted using pure bacterial cultures, a mix of few bacterial strains or filtered growth media. This is mainly related to the fact that mixed microbial slurries are too cytotoxic (Figure 2), thus limiting the experimental Chlormezanone time (a few hours at most) and the adaptation of the host to the microbial metabolism. On the contrary, the HMI module allows to indirectly expose the Caco-2 cells to the gut microbiota for up to 48 h, the average in vivo exposure time of an enterocyte to the content of the gut lumen when migrating from the crypts to the top of the villi [45]. Figure 2 MTT values (expressed as Optical Density – OD) of Caco-2 cells directly exposed for 2 h to the complex microbial community of the ascending colon of a SHIME reactor (direct contact), exposed to the same microbial community within a HMI module (HMI 1 and 2) or to sterile SHIME medium (control) for 48 h. Values are averages ± standard deviation (n = 2). * = statistically different from the control condition according to a Student’s two-tailed t-test (p < 0.05).

CrossRef 7. Norman AG, France R, Ptak AJ: Atomic ordering and phase separation in MBE GaAs[sub 1−x]Bi[sub x]. J Vac Sci Technol B Microelectron Nanometer Struct Process Meas Phenom 2011, 29:03C121.CrossRef 8. Mascarenhas A: Spontaneous ordering in semiconductor alloys. New York: Kluwer Academic/Plenum Publishers; 2002.CrossRef 9. Bastiman F, Cullis AG, David JPR, Sweeney SJ: Bi incorporation in GaAs(100)-2 × 1 and 4 × 3 reconstructions investigated by RHEED

and STM. J Cryst Growth 2012, 341:19–23.CrossRef Blebbistatin clinical trial 10. Gomyo A, Suzuki T, Iijima S: Observation of Strong Ordering in Ga_xIn_1-xP alloy semiconductors. Phys Rev Lett 1988, 60:2645–2648.CrossRef 11. Mascarenhas A, Kurtz S, Kibbler A, Olson JM: Polarized band-edge photoluminescence and ordering in Ga_0.52In_0.48P. Phys Rev Lett 1989, 63:2108–2111.CrossRef 12. Zhang Y, Mascarenhas A, Smith S, Geisz JF, Olson JM, Hanna M: Effects of spontaneous ordering and alloy statistical fluctuations on exciton linewidth in Ga_xIn_1-xP alloys. Phys Rev B 2000, 61:9910–9912.CrossRef 13. Warren BE: X-ray Diffraction. New York: Dover Publications Inc.; 1990:209–216. 14. Mao D, Taylor PC, Kurtz SR, Wu MC, Harrison WA: Average local order parameter in partially ordered GaInP_2. Phys

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order parameter in epitaxial layers of ZnSnP[sub 2]. Appl Phys ABT888 Lett 2017, 2000:76. 17. Cowley J: Short- and long-range order parameters in disordered solid solutions. Phys Rev 1960, 120:1648.CrossRef 18. Kimoto T, Takeda T, Shida S: A method to determine long-range order parameters from SDHB electron diffraction intensities detected by a CCD camera. Ultramicroscopy 2003, 96:105–116.CrossRef 19. Baxter CS, Broom RF, Stobbs WM: The characterisation of the ordering of MOVPE grown III–V alloys using transmission electron microscopy. Surf Sci 1990, 228:102–107.CrossRef 20. Kret S, Ruterana P, Rosenauer A, Gerthsen D: Extracting quantitative information from high resolution electron microscopy. Phys Status Solidi B Basic Res 2001, 227:247–295.CrossRef 21. Urban K: Determination of long-range order parameter in alloys by means of electron diffraction in the electron microscope. Physica Status Solidi A Appl Res 1985, 87:459–471.CrossRef 22. Li JH, Kulik J, Holý V, Zhong Z, Moss SC, Zhang Y, Ahrenkiel SP, Mascarenhas A, Bai J: X-ray diffraction from CuPt-ordered III-V ternary semiconductor alloy films. Phys Rev B 2001, 63:155310.CrossRef 23. Galindo PL, Kret S, Sanchez AM, Laval J-Y, Yáñez A, Pizarro J, Guerrero E, Ben T, Molina SI: The Peak Pairs algorithm for strain mapping from HRTEM images. Ultramicroscopy 2007, 107:1186–1193.CrossRef 24.

The selleck inhibitor method differs from other complicated methods, such as the electronbeam, followed by etching. Figure 3 XRD spectra (a) and wavelength-dependent

reflectance (b). (a) XRD spectra of AZO film surface and antireflection coatings of the flat-top ZnO nanorods and the tapered ZnO nanorods. (b) Wavelength-dependent reflectance of non-selenized CIGS solar cell before (black line) and after (blue and green lines) deposition of antireflection coating of nanorods. The EQE of the CIGS solar devices was also measured to evaluate the effect of ZnO nanorod coating layer on performance improvement. Figure 4a compares the EQE data for the non-selenization CIGS devices with and without the ZnO nanorod antireflection coating layer. The CIGS cell with ZnO nanorods had excellent quantum efficiency at wavelengths ranging from 450 to 950 nm, owing to XMU-MP-1 cost the low optical reflectance of the ZnO nanorods. The quantum

efficiency of non-selenization CIGS cell with ZnO nanostructure drops off at a high energy of approximately around 320 nm -a lower energy than that without the antireflection coatings. This phenomenon is caused by the fact that the optical band gap energy of ZnO is lower than that of the high band gap material, of AZO layer [22], owing to the Burnstein-Moss bandgap effect. Figure 4b plots the photocurrent versus applied voltage (J-V) curve for the CIGS solar cells with and without the ZnO antireflection coatings under AM1.5 illumination. The CIGS solar cell with tapered ZnO nanorods reaches an efficiency as high as 10% to 11%. The cell conversion efficiency is 9.1% with an open-circuit voltage of 0.55 V, a short current density of 22.7 mA/cm2, and a fill factor (FF) of 72.3%. Based

on the J-V curves, the increase of the short-circuit current is believed to be related to the decrease in reflectance nearly that is caused by the ZnO nanostructure antireflective coating layer. The gain in photocurrent due to the antireflective effect could be given by the previous work [23]. In this study, the comparative advantages that are provided by the ZnO nanostructures on non-selenized CIGS solar cells are indicated by the extra gain in the photocurrent G p (G p ≡ ΔJ sc/J sc), 11%, for the tapered ZnO nanorods. The tapered ZnO nanorod coating ultimately increased the efficiency of non-selenized CIGS solar cells by 9.8% from 9.1% to 10%. There are obvious improvements in photocurrent and efficiency enhancement. These are mainly caused by both the reduction of light reflectance and surface recombination centers by the window layer [24–27]. Figure 4 External quantum efficiency (a) and current-voltage characteristics (b) of solar cells. (a) Solar cell before (black line) and after (blue and green lines) deposition of antireflection coating of nanorods. (b) Bare non-selenized CIGS solar cell and flat-top/tapered ZnO nanorod antireflection-coated non-selenized CIGS solar cells.

HKJ participated in the experimental design with SS and performed most of the experiments. SK and AK helped in some experiments. JBP contributed to new reagents. BAW performed mass spectrometry. PJ and LAH helped in Fedratinib in vitro iTRAQ data analysis. HKJ and SS analyzed the data and wrote the manuscript. All authors read and approved the manuscript.”
“Background

Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces botulinum neurotoxin (BoNT), the most potent toxin known [1–3]. BoNT is classified as a Category A biothreat agent by the Centers for Disease Control and Prevention (CDC) because of its lethality and ease of production, transport and dissemination [4, 5]. In addition, BoNT poses several threats to the public health: first, the possibility of foodborne botulism represents a major potential health hazard that requires continual monitoring by the food industry. Second, infant botulism has been the most common form of human botulism in the United States for more than 20 years and hospitalizes approximately 80-100 U.S. infants annually [6]. Third, cases of wound botulism due to intravenous drug use continue to increase [7, 8]. Botulism toxicity results from one

of seven serologically distinct neurotoxins (types A-G) that cause a severe neuroparalytic disease characterized by descending flaccid paralysis [9]. Rarely, unique strains of C. butyricum and C. baratii may also cause human botulism through MAPK Inhibitor Library nmr production and release of BoNT/E and F, respectively [10, 11]. The toxin acts by binding C1GALT1 to peripheral cholinergic nerve endings and inhibiting release of acetylcholine at the neuromuscular

junction. A part of the toxin is a zinc-dependent protease that cleaves target substrate proteins (SNAREs), located either on the plasma membrane or the synaptic vesicle, thereby preventing their binding, fusion and release of neurotransmitter. BoNTs cleave specific amino acids on the target proteins of the SNARE complex. BoNT/A and BoNT/E act on SNAP-25, while BoNT/C targets syntaxin as well as SNAP-25. The remaining toxin types (BoNT/B, BoNT/D, BoNT/E and BoNT/F) all act on synaptobrevin, but at different cleavage sites [12–15]. The potential severity and lethality of the disease warrants sensitive and specific detection and serotyping of toxin and its typing to enable correct administration of serotype-specific antitoxin in a timely manner. Although treatment with Human Botulism Immune Globulin (BabyBIG®) or equine antitoxin is based on clinical findings and should be instituted as rapidly as possible [5, 16, 17], definitive microbiological diagnosis may take several days or even longer. This extended time to diagnosis occurs because detection of the bacterium and its toxin relies on toxicity assessment in mice (the mouse protection bioassay) and lengthy culture assays, which, while sensitive and specific, may be time-consuming and difficult [18, 19].

It can be expected that one-step bait fishing is effective for interactions with slow kinetics—here termed static interactions—whereas it will miss interactions with fast kinetics, which we call dynamic interactions. However, if the affinity is sufficiently high, dynamic interactions should be detectable by two-step bait fishing. On the other hand, two-step bait fishing will selleck screening library probably miss static interactions, because the exogenously added bait might not be able to displace its already bound endogenous counterpart. Detection of interactions by both one-step

and two-step bait fishing can occur if either the interaction is of low dynamics resulting in enough stability for detection EPZ004777 cost by one-step bait fishing but allowing enough exchange for prey binding to the exogenously added bait in two-step bait fishing, or if the interaction is static but prey protein with free bait binding sites is present in wild type cells and thus accessible to the exogenously added bait in two-step bait fishing. As a further difference, in two-step bait fishing the prey proteins are purified from genetically unmodified cells, which excludes effects of chromosomal integration of the tagging vector at the locus of the bait protein upon the expression of interaction partners. This might be of particular importance as

interacting proteins are often located adjacent to each other in the genome or even in one operon [62]. Since the methods detect different Amrubicin subsets of interactions, we applied both of them to all proteins under investigation. A similar strategy,

the combination of MAP (mixing after purification)-SILAC and PAM (purification after mixing)-SILAC was developed by Wang and Huang [63] and demonstrated to outperform standard SILAC experiments for the identification of protein interactions with a broad range of kinetics. Interaction analysis of the Hbt. salinarum taxis signal transduction system Initially, the interactions of the ten known Hbt.salinarum Che proteins were analyzed. Afterwards six additional proteins that were found to be interaction partners were used as baits to confirm the detected interactions and to extend the interaction network (Additional file 5). Overall, the experiments resulted in 5505 reliable protein identifications (ProteinProphet [64]; probability > 0.95), detecting 597 unique proteins (Additional file 3). Of the identifications made, 267 were classified as interactions. Applying the spoke model [65] to derive binary interactions from the copurification data resulted in a final set of 201 unique interactions. The resulting interaction network is depicted in Figure 3. For the sake of clarity, only interactions discussed in the text are included. The complete network is available from Additional file 6.

J Alloys Compd 2007, 438:258–262. 10.1016/j.jallcom.2006.08.030CrossRef 25. Li Y, Li Y, Zhu M, Yang T, Huang J, Jin H, Hu Y: Structure and magnetic properties of Cr-doped ZnO nanoparticles prepared under GSK2245840 research buy high magnetic field. Solid State Commun 2010, 150:751–754. 10.1016/j.ssc.2010.01.027CrossRef 26. Wesselinowa J, Apostolov A: A possibility to obtain room temperature ferromagnetism by transition metal doping of ZnO nanoparticles. J Appl Phys 2010, 107:053917–053917–053915.CrossRef 27. Yousefi R, Zak AK, Jamali-Sheini F: The effect of group-I elements on the structural and optical properties of ZnO nanoparticles. Ceram Int 2013,

39:1371–1377. 10.1016/j.ceramint.2012.07.076CrossRef 28. Khorsand Zak A, Abd Majid WH, Mahmoudian MR, Darroudi M, Yousefi R: Starch-stabilized synthesis of ZnO nanopowders at low temperature and optical selleck properties study. Adv Powder Technol 2013, 24:618–624. 10.1016/j.apt.2012.11.008CrossRef 29. Farag AAM, Yahia IS: Structural, absorption and optical dispersion characteristics of rhodamine B thin films prepared by drop casting technique. Opt Commun 2010, 283:4310–4317. 10.1016/j.optcom.2010.06.081CrossRef 30. Wang D-W, Zhao S-L, Xu Z, Kong C, Gong W: The improvement of near-ultraviolet

electroluminescence of ZnO nanorods/MEH-PPV heterostructure by using a ZnS buffer layer. Org Electron 2011, 12:92–97. 10.1016/j.orgel.2010.09.018CrossRef 31. Khorsand Zak A, Razali R, Abd Majid WH, Darroudi M: Synthesis and characterization of a narrow size distribution of zinc oxide nanoparticles. Int J Nanomedicine 2011, 6:1399–1403.CrossRef 32. Zak AK, Majid WHA: Effect of solvent on structure and optical properties of PZT nanoparticles prepared by sol–gel method, in infrared region. Ceram Int 2011, 37:753–758. 10.1016/j.ceramint.2010.10.020CrossRef 33. Deng X, Sun J, Yu S, Xi J, Zhu W, Qiu X: Steam reforming of ethanol for hydrogen production

over NiO/ZnO/ZrO 2 catalysts. Int J Hydrog Energy 2008, 33:1008–1013. Competing interests The authors declare that they do not have competing interests. Authors’ contributions AKZ carried out the sample preparation, XRD, and UV section. MD carried out the TEM imaging and Auger spectroscopy selleck antibody inhibitor part. AMH was the project leader and contributed in analyzing the data. All authors read and approved the final manuscript.”
“Background The layered transitional quasi-two-dimensional (Q2D) semiconductor oxides MO3 (M = Mo, W), have recently attracted significant interest because they demonstrate quantum confinement effects at the few-layer limit [1, 2]. Among them, tungsten trioxide (WO3) is an n-type semiconductor in an indirect bandgap of 2.6 to 2.9 eV [3] with excellent electrochromic and gasochromic properties [4]. It has electron Hall mobility of ~12 cm2V-1 s-1 at room temperature and responsive to the blue end of the visible spectrum (λ < 470 nm) [5].

The UspE protein is a tandem-like protein consisting of two Usp domains. The UspE domain1 is more related to the UspA sub-family, whereas the domain2 is closer related to the UspFG sub-family. The intracellular copy number of UspA, UspC, UspD, and UspE increases upon stress conditions such as starvation, moderate heat stress, oxidative stress, and osmotic stress [23]. UspG is induced under

osmotic stress and has recently been shown to undergo autophosphorylation and autoadenylation [24]. However, the exact functions of these small proteins are unclear. The degree of similarity of the Usp domain within KdpD (Fig. 1) varies among all known KdpD sequences. To elucidate the role of the Usp domain in KdpD for signaling, we used a “”domain swapping”" approach, wherein the E. coli KdpD-Usp domain was replaced with homologous

Stem Cells inhibitor domains or the six E. coli Usp proteins. These KdpD chimeras were characterized in vivo as well as in vitro. Results “”Domain swapping”" CX-5461 datasheet of the Usp domain within KdpD The N-terminal region of the cytoplasmic input domain containing the KdpD domain (pfam02702) is highly conserved [25], whereas the C-terminal region containing the Usp-domain (cd01987) (I253-P365) is less conserved (Fig. 1). The KdpD-Usp domain of other bacteria, for example Agrobacterium tumefaciens (KdpD/R249-D372), Streptomyces coelicolor (KdpD/R233-I354), Salmonella enterica serotype Typhimurium (KdpD/I253-P365), and Pseudomonas aeruginosa (KdpD/R248-R358) are characterized by different degrees of identity Protein kinase N1 and similarity. The highest degree of sequence identity has the KdpD-Usp domain of S. enterica serotype Typhimurium compared to the corresponding E. coli domain (86% identity, 89% similarity). The other KdpD-Usp domains are less conserved (A. tumefaciens: 30% identity, 45% similarity; P. aeruginosa: 28% identity, 43% similarity; S. coelicolor: 25% identity, 42% similarity). The KdpD-Usp domain belongs to the UspA subfamily. Despite the lack of amino acid sequence

identity, proteins of this (sub)family (UspA, UspC and UspD) are predicted to have a homologous tertiary structure which consists of four to five central β-sheets surrounded by four a-helices [19, 22]. To examine the specifics of the KdpD-Usp domain and its importance in KdpD signaling, we replaced amino acids L221-V358 of E. coli KdpD with the homologous KdpD-Usp domains of A. tumefaciens (L218-I371), S. enterica serotype Typhimurium (L221-V358), S. coelicolor (L202-V355), and P. aeruginosa (L218-Q361) as described in Methods, and designated the chimeras Agrocoli-KdpD, Salmocoli-KdpD, Streptocoli-KdpD, and Pseudocoli-KdpD (Fig. 2) [26]. Furthermore, we exchanged the KdpD-Usp domain of E. coli with the six soluble Usp protein sequences of E. coli, yielding the chimeras KdpD-UspA, KdpD-UspC, KdpD-UspD, KdpD-UspE, KdpD-UspF, and KdpD-UspG (Fig. 2).

2 mol/l NaOH solution, and washed again. Then 0.3 μmol/l pyrosequencing primer was annealed to the purified single-stranded PCR product

and the pyrosequencing was performed on a PyroMark ID system (Qiagen) following the manufacturer’s instructions. The nucleotide dispensation order was GTATCAGACATGAC for analysis of exon 19 and CTGCGTGTCA for analysis of exon 21. Results Pyrosequencing assay of exon 19 deletions In order to test the pyrosequencing method for the analysis of exon 19 deletions, we used DNA from the NCI-H1650 cell line as positive control and DNA extracted from human peripheral blood lymphocytes (PBL) Captisol in vivo as wild-type control. We choose a particular pyrosequencing program with the oligonucleotide dispensation order (GTATCAGACATGAC) because it permits to distinguish wild type and mutated alleles

(table 2) generating for each sample a specific pyrogram (Figure 1A and 1B and Figure 2). These pyrograms correspond to a mix of wild type and mutated alleles. We quantitatively evaluated the exon 19 deletion (c.2235-2249del; TPCA-1 p.Glu746-Ala750del) by determining the ratio between the peak areas of the two adenines dispensed in positions 6 (A6) and 8 (A8). We tested the reproducibility of the technique by analyzing each DNA in 20 consecutive and independent Interleukin-3 receptor runs. We found an A6/A8 ratio of 1.06 ± 0.04 for the wild type sample and 4.59 ± 0.33 for the sample with the deletion. The relative standard deviation (RSD) was respectively 3.9% and 7.2%. Thus, a sample could be considered as mutated if A6/A8 was superior to 1.2 (corresponding to [the mean + 3 standard deviations] of the wild type sample). To demonstrate the assay sensitivity, we also quantified the A6/A8 ratio in various mixtures (10/0, 9/1, 8/2, 7/3, 6/4, 5/5, 4/6, 3/7, 2/8, 1/9 and 0/10) of DNA from the NCI-H1650 cell line with DNA from peripheral blood lymphocytes

(Figure 1C). Each mixture was analyzed 5 times in the same run and we found an A6/A8 ratio varying from 5.27 ± 0.38 (mixture 10/0) to 1.11 ± 0.05 (mixture 0/10). We determined that all the mixtures containing at least 20% of NCI-H1650 DNA have an A6/A8 ratio superior to 1.2 and could be considered as mutated. Table 2 Sequencing of wild type and mutated alleles with a particular program of pyrosquencing nucleotide dispensation during pyrosequencing G T A T C A G A C A T G A C   WT   T A T C AA GG AA     TT   AA   allelic c.2235-2249del   T A T C AA AA     C A T     C sequence of c.2236-2250del   T A T C AA G A C A T     C   c.2237-2251del   T A T C AA GG   C A T     C   c.