These results show there is no real consensus of proteins identif

These results show there is no real consensus of proteins identified between the LPI™ FlowCell method and more established methods such as 2D GE and 2D-LC-MS/MS (Additional file 2). Instead these methods complement each other and therefore when designing experiments to identify outer membrane proteins it is important to try a range of approaches to maximise the coverage of OMPs detected. Finally, when collating the results from both digests performed in this study, different classes of membrane proteins with varying functions were also identified. A total of 69 proteins were

identified as being outer membrane proteins of which 54 were identified with two or more peptide find more hits (Additional file 1). Using the database UniProtKB http://​www.​uniprot.​org some of the functions of the outer membrane proteins were deduced. These included the transporters BtuB which

is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other biologically significant proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell check details wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. To further verify the functions of the outer membrane proteins identified in the present study, manual mining of the data, which involved searching through literature containing information on the proteins of interest, was also undertaken. This approach shed further light on outer membrane proteins identified

that were not apparent using UniProtKB, a shortcoming of using a single approach to verify the functions of proteins [23]. These included membrane-bound lytic murein transglycosylase (MltB and MltC) which is important for cell growth [24], conjugal transfer surface exclusion protein (TraT) which is responsible for resistance to bacterial killing by serum [25] and RcsF protein which is part crotamiton of the Rcs phosphorelay signalling pathway responding to GDC-0449 purchase peptidoglycan damage by regulating colanic acid capsular exopolysaccharide synthesis, and has also been seen to enhance bacterial survival in the presence of antibiotics [26]. Conclusions The present study aimed to elucidate the expression of outer membrane proteins in Salmonella Typhimurium using LPI™ FlowCells. The membrane preparations largely excluded most of the cytosolic proteins that co-purifies with it when using currently available fractionation procedures and therefore achieved a wider coverage of the membrane subproteome than had been reported.

The up-Ri fragment was cloned into the SphI/SpeI site of the pTZ5

The up-Ri fragment was cloned into the SphI/SpeI site of the pTZ57-down-Ri plasmid. The plasmid was cut with BamHI/BglII and the fragment was cloned into BamHI of vector ksgt between the gpd promoter and TrpC terminator, resulting in plasmid OptRi. The OptRi plasmid

was transformed into C. gloeosporioides together with the gGFP vector, which confers resistance to hygromycin. Fungal transformation Fungal transformation was performed by electroporation of germinated spores as previously described [20]. Hygromycin-resistant colonies were collected and the presence of either Popt-gfp or OptRi plasmid was verified by PCR. Transgenic isolates obtained with Anlotinib the Popt-gfp plasmid were compared and detailed www.selleckchem.com/products/nct-501.html analyses were performed with isolate Popt-gfp6. For OptRi, isolates containing the silencing cassette were propagated and the expression levels of CgOPT1 were compared. Detailed analyses were carried out with isolates Ori51 and Ori83, which gave similar results in all cases. Sporulation assay Fungi were cultured on CD or EMS plates. For media with IAA, the calculated amount of IAA was dissolved in ethanol and applied on a Whatman filter paper, the ethanol was air-dried and then the filter was click here placed

between two layers of agar medium. Plates were prepared 1 day before inoculation to allow diffusion of IAA into the medium. Control plates were prepared in a similar fashion with filters containing an equivalent volume of air-dried ethanol. Each plate was inoculated with a 3-mm2 mycelium cube that was excised from a 5-day-old culture. After 5 days, the spores were washed from the plates and counted. Selleck Rucaparib Three plates were used as replicates in each experiment and all experiments were repeated several times. Data are the mean results of three experiments. Plant inoculation Inoculation experiments were performed with 12-day-old Aeschynomene virginica plants as described previously [26]. Plants were sprayed to runoff with spore suspension

containing 0.05% (v/v) Tween 20. Control plants were sprayed with similar volumes of 0.05% Tween 20. Six plants per treatment were used as replicates in each experiment and all experiments were repeated several times. Symptoms were recorded and fresh weight determined 6 days post-inoculation. Microscopy Fluorescent and light microscopy were performed with a Zeiss Axioskop 2 epifluorescent microscope, or with an Olympus SZX 12 fluorescent stereoscope equipped with an eGFP filter. Confocal microscopy was performed with a Zeiss CLSM 510 laser-scanning confocal microscope. Computational analysis CgOPT1 homologous sequences were identified by BlastpX [27] analyses at the NCBI database http://​www.​ncbi.​nlm.​nih.​gov/​. For details of species and retrieved sequences see Additional file 1 and Additional file 2. Multiple alignments were performed by the ClustalW program [28]. Phylogenetic analyses were conducted with the PHYLIP package [29], available online at http://​mobyle.​pasteur.​fr.

The

The click here spray voltage was 3 kV, the tube lens offset −132 V and the skimmer offset 5 V. The ion transfer capillary temperature and vaporizer temperature were 250 and 300°C respectively. All the amines

give a m/z signals that correspond to the structure [M-H]-. Each amine was injected at 1 to 10 μg.mL-1 for mass characterization. Collision-induced dissociation (CID) was performed from 20 to 30 eV under 1.5 mTor of argon. PCR amplification L. plantarum identification was performed by 16S ribosomal RNA gene sequencing and multiplex PCR using recA gene-derived primers [43]. Chromosomal DNA from L. plantarum was extracted using the Wizard Genomic Kit (Promega, Charbonnières les Bains, France). Amplification and sequencing of the 16S gene was performed using the High Fidelity Taq polymerase (Roche, Meylan, France) and the universal primers BSF8 and

BSR1541 [54]. Amplification conditions were 94°C for 2 min, 10 cycles of 94°C for 15 s, 52°C for 30 s, and 72°C for 1 min 30 s, followed by 20 cycles with an additional time of 5 s for each elongation reaction and a final extension at 72°C for 10 min. Multiplex PCR protocol developed by Torriani et al. [43] was performed with Go Taq polymerase (Promega, Charbonnières JNK-IN-8 nmr les Bains, France) and was modified for dNTP concentration (0.2 mM inside of 12 μM) and for annealing time (20 s inside of 10 s). The L. plantarum IR BL0076 tyrDC and tyrP genes were amplified by PCR using High Fidelity Taq polymerase (Roche, Meylan, France) and primers tyrSa

and nhaCa based on the tyrS and nhaC sequences which flanked tyrDC and tyrP genes of L. brevis NS77 [GenBank : EU195891]. Amplification was performed in a final volume of 50 μL, with 5 μL of Expand High Fidelity buffer (Roche, Meylan, France), 1 μL of 10 mM dNTP mix (Fermentas, Villebon sur Yvette, SPTLC1 France), 1 μL of each primer at 20 μM, 2.6 U of Expand High Fidelity enzyme mix (Roche, Meylan, France), and 1 μL of extract DNA. The amplification program was applied in a Bio-Rad thermocycler following the manufacturer’s instructions for long fragments. PCR fragments were purified using the GenElute PCR purification kit (Sigma, Saint Quentin Fallavier, France) and sent to Benckman Coulter Genomics (United kingdom) for sequencing. Total RNA extraction and RT-PCR L. plantarum RNA was extracted after various periods of growth in media 1 and 2 (when the cultures reached at OD600nm = 1.1, 1.6 and 1.8). Aliquots of 25 mL of culture were harvested, and the cells pelleted and washed with 10 mL of Tris HCl 10 mM pH 8. The cells were then broken in 1 mL of Tri-Reagent (Sigma, Saint Quentin Fallavier, France) in a screw cap tube containing 200 mg of beads (100 μm) in a BIX 1294 Precellys 24 ultrasound device (Ozyme, Saint Quentin en Yvelines, France) programmed as follows: 6500, 3 × 30 s, twice. Cell fragments were pelleted by centrifugation (12,000 × g, 10 min, 4°C) and the supernatant was transferred to an Eppendorf tube and 200 μL of chloroform was added.

Poster No 101 Drastic Decreased Expression of Activating Recepto

Poster No. 101 Drastic Decreased Expression of Activating Receptors on NK Cells in Human Lung Tumor Microenvironment Impairs their Cytotoxic Functions Sophia Platonova 1 , Julien Cherfils-Vicini1, Liana Ghazarian1, Pierre Validire1,2, Vincent Vieillard4, Wolf Herman Fridman1, Catherine Sautès-Fridman1, Diane Damotte 1,3, Isabelle Cremer1 1 INSERM U872 Team 13, Centre de Recherche des Cordeliers, Paris, France, 2 Pathological Anatomy Service, Intitut Mutualiste Montsouris, 3-Methyladenine mw Paris, France, 3 Pathological Anatomy Service, Hôpital Hôtel Dieu, Paris, France, 4 INSERM U543, Paris, France While

NK cells were originally identified by their ability to kill tumor cells in vitro, only limited information is available on NK cells present in tumor microenvironment. Our objectives were to characterize buy VX-661 the phenotype and function of NK cells in human Non Small Cell Lung Cancers (NSCLC) patients, in tumor microenvironment, in non tumoral lung tissue, and in the blood, and to investigate the expression

of NK cell receptor ligands on tumor cells. NK cells are present both in tumoral and non tumoral lung tissues of NSCLC patients. In the tumor, they are mainly localised in the invasive margin, but outside tertiary lymphoid structures (Ti-BALT – “Tumor-induced Bronchus Associated Erastin supplier Lymphoid Tissues”) that are induced in the tumoral area. Intratumoral NK cells are not cytotoxic even after activation with IL-2,

on the contrary to NK cells from blood of the same patient, despite an activated phenotype defined by NKp44 and CD69 expression. Consistent with this observation, intratumoral NK cells display a highly significant decreased expression of activating receptors such as NKG2D, NKp30, NKp80, DNAM-1 and CD16. On the contrary, NK cells from non tumoral lung Selleck AZD1152-HQPA tissue or blood of NSCLC patients have the same phenotype than healthy donors. Analysis of NK cell receptor ligand expression revealed that inhibitory receptors ligands such as HLA-G and HLA-E are strongly expressed by tumor cells, but not by normal tissue, whereas activating receptors ligands such as MICA/B and ULBP1, 2, 3 are rarely expressed by tumor cells. Altogether these results demonstrate for the first time that the NK cells display an altered phenotype and function specifically in the tumor microenvironment and that tumor cells express high levels of inhibitory receptors ligands. This suggests a local induction of escape mechanisms established by tumor cells and directed towards NK cells. Poster No.

RPMI 1640 medium

RPMI 1640 medium see more containing 10% FBS was replaced by serum-free Opti-MEM (GIBCO, Invitrogen, USA) at 8 h later. HiPerFect Transfection Reagent and Negative control siRNA were purchased from Qiagen Technology Co. Ltd (Shanghai, China). Transfection compounds were prepared in three groups as follows: siRNA group (100 μl Opti-MEM, 6 μl HiPerFect Transfection Reagent and 5 μl JMJD2A siRNA), negative control group (100 μl Opti-MEM, 6 μl HiPerFect Transfection Reagent and 5 μl negative control siRNA) and blank control group (100 μl Opti-MEM). Transfection compounds were placed at room temperature for 10 minutes and then dropped onto 6-well plates. Bulk volume of the compounds

was 2200 μl per well. Both Opti-MEM and transfection compounds were replaced by complete medium at 24 h after transfection. FAM-siRNA was transfected to measure the efficiency of transfection simultaneously according to the manufacturer’s instructions. Quantitative real-time PCR Total RNA of three groups was extracted respectively with the RNAiso Reagent kit (TaKaRa, Dalian, China) at 48 h after transfection. cDNA was Selleck BMS202 generated by reverse transcription of 2 μg of total RNA using random primers and PrimeScript RT Master Mix Perfect Real Time (TaKaRa, Dalian, China) in a total reaction volume of 40 μl according to the manufacturer’s instructions. The sequences of forward and reverse oligonucleotide primers, specific to JMJD2A and housekeeping genes, were designed

using Primer5 software. The primers Resminostat used are: 5′-TGTGCTGTGCTCCTGTAG -3′ and 5′-GTCTCCTTCCTCTCCATCC -3′ for JMJD2A; 5′-TGACGCTGGGGCTGGCATTG -3′ and 5′-GCTCTTGCTGGGGCTGGTGG -3′ for GAPDH. Primers were synthesised by Shanghai Daweike Biotechnology Co. Ltd (Shanghai, China). Real-time quantitative PCR was performed in an ABI PRISM 7500 Real-Time System. A 10-fold dilution of each cDNA was amplified in a 20-μl volume, using the SYBR Premix Ex TaqTM Perfect Real Time (TaKaRa, Dalian, China), with 0.2 μM final concentrations of each primer.

PCR cycle conditions were 95°C for 30 s, and 40 cycles of 95°C for 5 s and 60°C for 34 s. The amplification specificity was evaluated with melting curve analysis. Threshold cycle Ct, which correlates inversely with the target mRNA levels, was calculated using the second derivative maximum algorithm provided by the iCycler software. For JMJD2A, the mRNA levels were normalized to GAPDH mRNA levels [9]. Western blot At 72 h after transfection, cells in different NF-��B inhibitor treatment groups were homogenized in Western blot analysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM PMSF, 0.28 kU/L aprotinin, 50 mg/L leupeptin, 1 mM benzamidine and 7 mg/L pepstain A. The homogenate was then centrifuged at 12, 000 rpm for 10 min at 4°C and the supernatant was retained and preserved at -80°C for later use. Protein concentration was determined using a BCA kit (Pierce).

80% to 23 74%, and the healing rates at 12 h, 24 h and 36 h (p <

80% to 23.74%, and the healing rates at 12 h, 24 h and 36 h (p < 0.001). By Sotrastaurin mouse contrast, the healing rate of NPC 5-8 F cells was not affected by treatment of lipofectamine alone and transfection of pEGFP-C3 and PinX1-FAM-siRNA (p > 0.05). Figure 6 Effect of PinX1 on wound healing ability of selleck kinase inhibitor nasopharyngeal carcinoma

5-8 F cells in scratch assay. Cells transfected with pEGFP-C3-PinX1 (a), pEGFP-C3 (b) and PinX1-FAM-siRNA(e), treated with lipofactamine alone (c), and untreated (d) were inoculated in 6-well plates pre-coated with collagen IV, cultured in media containing 10% newborn calf serum till forming monolayer, then scratched and photographed at 0 h, 12 h, 24 h and 36 h after scratching. The results show that overexpression of PinX1 by transfection of pEGFP-C3-PinX1 significantly increased the wound healing time click here of NPC 5-8 F cells, while downregulation of PinX1 by transfection of FAM-siRNA reduced has no effect on wound healing. We then examined the effect of PinX1 on hTERT mRNA level and telomerase activity. As shown in Tables 4 and 5 and Figures 7 and 8, overexpression of Pin X1 by transfection of pEGFP-C3-PinX1 significantly reduced hTERT mRNA level by 21% and decreased

telomerase activity in NPC 5-8 F cells (p = 0.000). By contrast, reduced PinX1 by transfection of PinX1-FAM-siRNA had effects on neither hTERT mRNA SPTLC1 level nor telomerase activity in NPC 5-8 F cells (p > 0.05). In addition, hTERT mRNA level and telomerase activity in NPC 5-8 F cells were not affected by transfection of pEGFP-C3 and treatment of lipofectamine alone. Table 4 hTERT

mRNA level in each group Sample hTERT mRNA F P pEGFP-C3-PinX1 0.789 ± 0.024* 117.689 0.000 pEGFP-C3 0.978 ± 0.011     Lipofectamine alone 0.987 ± 0.014     Untreated 1.000 ± 0.000     PinX1-FAM-siRNA 1.001 ± 0.085**     * vs untreated, P < 0.001, ** vs untreated, P > 0.05. hTERT mRNA level was normalized to GAPDH. Table 5 Telomerase activity in NPC cells Samples Telomerase activity F P pEGFP-C3-PinX1 36227.63 ± 2181.748* 53.816 0.000 pEGFP-C3 58346.993 ± 2181.748     Lipofectamine alone 59697.199 ± 2181.748     Untreated 62552.354 ± 2181.748     PinX1-FAM-siRNA 63600.608 ± 2181.748**     * vs untreated, P < 0.001; ** vs untreated, P > 0.05. Figure 7 Effects of PinX1 on hTERT mRNA level in NPC 5-8 F cells. PinX1 mRNA levels in NPC 5-8 F cells transfected with (a) pEGFP-C3-PinX1, (b) with pEGFP-C3, (c) treated with lipofectamine alone, (d) untreated and (e) transfected with PinX1-FAM-siRNA were measured in RT-PCR and normalized to internal control GAPDH. Data were presented as mean value of three experiments showing that overexpression of PinX1 significantly decreased hTERT mRNA level. Figure 8 Effect of PinX1 on telomerase activity in nasopharyngeal carcinoma cells.

An ankle arthrodesis had been performed at another clinic 15 mont

An ankle arthrodesis had been performed at another clinic 15 months ago, but union was not achieved, and therefore, she underwent further surgery 12 months ago to fix her ankle. This treatment also failed resulting in nonunion, and so the woman was instructed to wear a patellar tendon-bearing brace for her ankle instability and pain (Fig. 2a, b). Her laboratory data, including serum levels of alkaline phosphate, parathyroid hormone, calcium, and phosphorus, were normal, but her level of 1.25 vitamin D3 was low, and her left femoral bone density

was extremely low (0.54 mg/cm2) and 2.0 standard deviations below the normal value for her age (Table 1). Fig. 1 Radiographs of the femur. this website a 3D computed tomography images revealed an oblique fracture of the proximal shaft of

the femur. b Plain film taken 2 weeks after surgery. Callus formation is visible around the fracture site. c Plain film taken 12 weeks after surgery. Large callus and consolidation are visible Fig. 2 Radiographs of the femur. a Plain film of the ankle with Charcot arthropathy before arthrodesis. b Plain film taken before teriparatide therapy was initiated showing atrophic Necrostatin-1 nonunion of the ankle. c Plain film and sagittal CT images showing atrophic nonunion of the ankle after multiple arthrodesis operations. d Plain film and sagittal CT image taken after 12 weeks of teriparatide therapy showing complete healing of nonunion Thiamet G Table 1 Laboratory data before and after teriparatide therapy   Reference Range, Age-adjusted Pretreatment After 3 months Protein (g/dl) Total 6.7–8.3 7.5 7.2 Albumin 3.9–4.9 3.1 3.5 ALP (IU/l) 104–338 281 1033 BUN (mg/dl) 8.0–20.0 21.7 19.1 Cre (mg/dl) 0.36–1.06 0.67 0.61 Na (mEq/l) 136–147 136 134 K (mEq/l) 3.6–5.0 4.6 4.9 Cl (mEq/l) 98–109 94 99 Ca (mEq/l) 8.8–10.2

9.8 9.1 IP (mEq/l) 2.5–4.5 4.1 3.6 Mg (mEq/l) 1.8–2.4 2 1.9 HbA1c (%) 4.6–6.2 13.5 13.2 ACTH (pg/ml) 7.2–63.3 22.4   Intact-PTH (pg/ml) 10–65 31   Selleck PRI-724 Calcitonin (pg/ml) 15–86 35   1.25-Vit D3 (pg/ml) 20–60 12 72 NTx (nmol BCE/mmol・Cr) 8–70 189 327 D-Pyr (nM/mM・Cr) 2.8–7.6 31.8 21.6 We treated the femoral shaft fracture with intramedullary nail fixation 29 days after the fracture occurred, because her chronic heart failure was too poor to allow for immediate surgery. We initiated teriparatide (20-μg subcutaneous injection daily) and alfacalcidol (1-μg oral administration daily) immediately after surgery because of severe osteoporosis and in an attempt to accelerate healing of the femoral fracture. There was no immobilization of the femur, but a non-weight-bearing period of 4 weeks was implemented postoperatively. From 2 weeks after the initiation of teriparatide therapy, plain radiography began showing callus formation on the femoral shaft fracture, and after 12 weeks, almost complete healing of the fractured bone was observed (Fig. 1b, c).

For example, we observed no considerable differences in the isola

For KU-57788 manufacturer example, we observed no considerable differences in the isolation times and places

between the only human isolates (N010024, MT03) and the other strains isolated from Focus M. However, we did find a marked difference in MT. In previous studies, epidemiological investigations and traditional ecological typing studies confirmed that this case was imported from Focus C [22, 23]. In this study, N010024 was significantly different from the other strains isolated from Focus M, but had very similar MT with the strains from Focus C and gathered with them in the same subgroup. These results coincided with the conclusion of epidemiological investigations and the ecological typing, which further supported MLVA as a bacterial typing method suitable for field epidemiological investigations. There were cross-types AZD9291 molecular weight among the MTs of strains from different foci, with MT09 and MT19 being the most prominent. Foci that contained the same MT were geographically close to each other (Figure 3). For example, Foci C, D, F, and J contained MT09, and Foci C,

D, and K contained MT19, indicating that there were close relationships among the strains of adjacent foci. It is MLN2238 possible that these strains have the same source. Foci C, D, G, and K have locations adjacent to the border and even similar topography, climate conditions and hosts. The Marmota Himalayana plague focus of the Qinghai-Tibet Plateau [24] was sub-divided into four foci in recent years [11]. Cluster analysis showed that majority of the strains in the four foci were in complex 1, indicating a close relationship.

Therefore, we suggest that more accurate results will be obtained by combining the four foci in a unit when performing epidemiological and phylogenetic analysis. Foci A, B, and K are in Xinjiang province (Figure 1). The strains from Foci A and B were in the long branch of complex 1 and obviously different from other strains isolated in China. On the PLEK2 contrary, most strains from Focus K were together with the strains from foci around the Qinghai-Tibet Plateau. Foci A and B are adjacent to the Central Asia foci. Due to the lack of strains outside China in this study, it is impossible to provide a detailed and integrated relationship between the strains in Xinjiang and those of the Central Asia. However, we can confirm that there is a long genetic distance between strains of Foci A, B and other domestic strains isolated in China. To date, all the strains from Foci L and M belonged to biovar Microtus, except for one imported strain (N010024). Microtus is a newly-identified biovar that is phenotypically and genotypically different from the other three biovars [9]. Our results showed that MLVA could not only differentiate between Microtus and the other three biovars, but also divided the Microtus strains into two subclusters containing strains from foci L and M respectively.

In a similar manner, a Perl script was implemented to count the n

In a similar manner, a Perl script was implemented to count the number of bipartitions present in the whole-genome Akt inhibitor topology that were absent in the alternative topology (i.e. difference in resolution, denoted res) and to normalise the output to vary between 0 and 1. As a reference, RF distances (also known as symmetric differences) implemented in the Treedist software [78] were used. To investigate the success of the marker tree to allocate a strain to its corresponding sub-species family (according to the whole genome phylogeny), bipartition scoring in the Consense software was used and the output was compared to the pre-defined

subspecies bipartitions according to the whole-genome tree. In addition, we investigated

whether strains were assigned to the corresponding main clades of the entire Francisella genus, reporting the proportion of misidentified strains on each clade. Finally, we considered the average bootstrap support of each marker tree. It is important to consider a statistical test for topological incongruence as stochastic effects in the evolution of the sequences results in incongruence between the compared trees. To address this issue, we employed the Shimodaira-Hasegawa (SH) test [85], which is a non-parametric test for determining whether there are significant differences between conflicting topologies in specific sequence data. The null hypothesis of the SH test Ferroptosis inhibitor assumed that the compared topologies were equally probable given the data. Here, we Selleckchem Barasertib tested the marker topologies and the whole-genome topology on each respective marker sequence using the phyML software package by fixing the topologies and optimising the substitution model and crotamiton branch-length parameters. The SH test was performed within the CONSEL software package [86], which takes the output from phyML as input. Since multifurcations in topologies are strongly penalised in the phyML software, we resolved the topologies into bifurcating trees using the R package ape [84]. The substitution model

selected in the phyML analysis was based on the preferred substitution model of the jModelTest analysis. To test whether clades differed in incongruence or resolution, a Wilcoxon rank sum test with continuity correction was utilised, implemented in the R statistical package [73]. We used Spearman’s rank correlation coefficient, ρ, to quantify correlations between metrics and the average pairwise nucleotide diversity, π, of the clades. Optimisation procedure Since the number of included sequence markers in this study was moderate, we searched through all possible combinations of markers (i.e. an exhaustive search). We performed two separate analyses, one for each of the metrics used: incongruence and difference in resolution between topologies. The marker configuration(s) resulting in the lowest metric value were saved.

Sap sugars are presumably the main C and energy source for the RP

Sap sugars are presumably the main C and energy source for the RPW larvae and its microbiota, that is dominated by fermenting bacteria to obtain several metabolites including lactate and

acetate. Acknowledgements The authors thank Maria Grazia Selonsertib solubility dmso Cusimano, Rosella Maggio and Flavia Contino for technical assistance in bacterial isolation, DNA extraction and amplification, and Smad inhibitor control of DNA quality for pyrosequencing, respectively. This work was partially financed by a grant from the Italian Ministry of Education (PRIN Program 2008 prot. 200847CA28-002) and by the University of Palermo (project FFR 2012 ATE-0322 N.2785). Electronic supplementary material Additional file 1: Phoenix canariensis infested by Rhynchophorous ferrugineus (A and B); different infested palms cut in the higher part are shown. Larvae of the red palm weevil (RPW) Rhynchophorus ferrugineus, found inside the body of the infested palm (C). Female adult specimen of Rhynchophorus ferrugineus Olivier (Coleoptera, Curculionidae, Rhynchophorinae) (D). this website (PDF 171 KB) Additional file 2: Complete results of 16S pyrotag sequence clustering and taxonomic assignment by RDP of clusters and singletons at 90%, 95% and 97% ID. (XLS 93 KB) Additional file 3: Relative

abundance of bacterial families in the gut of RPW larvae as detected by pyrosequencing of the 16SrRNA gene V2 region. (JPEG 46 KB) Additional file 4: Phylogenetic tree of 16S rRNA gene amplicons clustered at 97% consensus. The tree was constructed by neighbour-joining method and Jukes Cantor distance matrix using the arb software. Bootstraps were calculated over 1000 random repetitions: values >60 and < =75 are shown as open circles, whereas values >75 are shown as filled circles. Sequences obtained in this study are indicated in bold. The scale bar represents 10% sequence divergence. (PDF 231 KB) Additional file 5: Phylogenetic tree of 16S rDNA sequences of RPW gut isolates and related sequences, as determined Selleck Rapamycin by distance

Jukes-Cantor analysis. One thousand boostrap analyses were conducted and values greater than 60% are reported. Two Archaea sequences of Methanopirus kandleri and Korarchaeum cryptophilum were used as outgroup. The scale bar represents the expected number of changes per nucleotide position. Colors indicate the isolation site or the isolation procedure described in this work and in [2]. Blue: RPW gut isolates on NA; Red: frass bacteria; Green: palm bacterial endophytes; Fuchsia: gut isolates obtained from enrichment cultures on CMC; Yellow: larval cuticle bacteria isolated from sterilization control plates. Isolate_RPWenrichAAB* was isolated from the RPW larval gut from enrichment cultures set for for Acetic Acid Bacteria isolation [42].