75 × 107cells per ml A volume of 0 2 ml (3 5 × 106cells) tumor c

75 × 107cells per ml. A volume of 0.2 ml (3.5 × 106cells) tumor cell suspension was injected subcutaneously ventral to the right axilla of the mice (C57BL/6 for EL4, Kunming mice for S180). Mice were monitored for tumor burden by measuring the tumor size daily using a vernier calliper. Irradiation began when the tumor diameter attained 1.0 cm. Preparation of99mTc-HYNIC-Annexin V Human annexin V freeze-dried powder was purchased from Beijing Huada Protein Development Center Co. Ltd (Beijing, China). Human annexin V was conjugated with hydrazinonicotinamide (HYNIC), using methods described by Blankenberg et al. [5]. Derivatized HYNIC-annexin V was radio-labelled with a99mTc tricine precursor

complex according to literature methods see more [5,

9–11]. Ispinesib purchase After chelating with the99mTc tricine precursor complex, the radio-labeling efficiency was measured by using thin-layer chromatography Silica Gel (TLC-SG), with methyl ethyl ketone and normal saline as the developing solvent. The radiochemical purity of the tracer product was then measured with High Performance Liquid Chromatography. The radio-labelled material, prepared as described above, was diluted to have specific activities ranging from 400-800 MBq μg-1 1 ml-1 which was ready for use. Tumor irradiation The tumor-bearing mice were randomly divided into an imaging group which was irradiated and imaged using99mTc-HYNIC-Annexin V, and an selleck inhibitor observation group which was only observed for tumor regression after single-dose irradiation. The EL4 lymphoma imaging group was subdivided into 4 single-dose levels: 0, 2, 4, and 8 Gy, while the S180 sarcoma imaging group received only 2 dose levels (0 and 8 Gy),

with 4 mice each level. The observation only groups of EL4 lymphoma and S180 sarcoma both received the same dose levels of 0 Gy or 8 Gy (4 mice each level). The tumors were irradiated with the 4 ADAMTS5 MV X-rays (SSD 100 cm, 1.5 cm × 1.5 cm portal) with a 0.5 cm thick tissue-equivalent material applied to the tumor surface. The mice were anesthetized before irradiation by intraperitoneal injection of 0.15 ml of 0.7% pentobarbital and immobilized with tapes. Experiments were repeated three times. 99mTc-HYNIC-annexin V imaging of radiation-induced apoptosis At 24 hours after radiation, 0.2 ml (4-8 MBq) of the prepared99mTc-HYNIC-annexinV was injected into each mouse in the imaging groups through the tail vein. Planar images were obtained 2 hours later, using a single-head γ camera (Meridian Philips Medical Systems) equipped with a parallel-hole collimator. The energy window was centered at 140 keV with a window width of 20%, and the matrix was to 256 × 256 with a magnification factor of 3.0. The acquisition time was 1 min/image. The tumor size of mice in the observation groups was measured daily after irradiation.

No virus-specific siRNAs could be detected in mosquitoes mock-inj

No virus-specific siRNAs could be detected in mosquitoes mock-injected with cell culture medium or injected with TE/3’2J/B2, indicating that B2 protein could inhibit targeted degradation of the SINV genome in the context of infected mosquitoes (Figure 3B). Effects of B2 protein expression on SINV replication The inhibition of siRNA accumulation showed that B2 protein JQ-EZ-05 clinical trial could inhibit RNAi in mosquito cells. To determine the effects that RNAi inhibition may have on SINV replication, we first examined the ability of SINV RNA to accumulate in infected cells. Using the same total RNA samples used for siRNA detection, we examined the accumulation of viral genomic and subgenomic RNA species in Aag2

cells and mosquitoes by Northern blot analysis (Figure 4A and 4B). Starting at 24

hours post-infection, three viral RNA species were detected in cells infected with TE/3’2J, TE/3’2J/GFP, and TE/3’2J/B2 viruses. These bands represent the genomic, first subgenomic, and second subgenomic RNAs produced during virus infection. The second subgenomic RNA, expressed from the most 3′ virus promoter, is the GSK1210151A in vivo most highly transcribed RNA species for all three viruses, consistent with previous reports [22]. The observed inhibition of siRNA accumulation in TE/3’2J/B2-infected cells corresponded with a distinct increase in viral RNA accumulation. Considerably more viral RNA was detected in cells and mosquitoes infected with TE/3’2J/B2 virus beginning at 24 hours post-FAK inhibitor infection and continuing throughout all time points tested. Much less viral RNA accumulated in TE/3’2J/GFP-infected cells and mosquitoes, an expected outcome

considering the increase in genome size and accompanying decrease in Ribonucleotide reductase replication efficiency [23]. No bands were observed in RNA from mock-infected cells. Figure 4 Detection of viral RNAs in Aag2 cells (A) and Ae. aegypti mosquitoes (B). Monolayers of Aag2 cells were mock-infected or infected with TE/3’2J, TE/3’2J/GFP, or TE/3’2J/B2 virus at MOI = 0.01. Mosquitoes were intrathoracically-inoculated with cell culture medium, TE/3’2J, TE/3’2J/GFP, or TE/3’2J/B2 virus. At indicated times post infection, total RNA was isolated and an E1-specific riboprobe was used to detect virus genomic and subgenomic RNA. Ethidium bromide-stained ribosomal RNA below each blot serves as a loading control. Time post infection for each virus in (A) is 0, 24, 48, and 72 hrs, and in (B) 0, 48, and 96 hrs. G = genomic; S1 = first subgenomic; S2 = second subgenomic. Because siRNA accumulation was inhibited and viral RNA amounts increased in TE/3’2J/B2 virus-infected cells, we tested if suppression of RNAi by B2 would cause more infectious virus to be produced during infection. We performed two-step growth curve analysis in Aag2 and Vero cells to determine the effects of B2 protein expression on infectious virus production (Figure 5A).

This suspension

was subsequently dried at 100°C in a dryi

This suspension

was subsequently dried at 100°C in a drying oven and then calcined at 500°C in air for 1 h to prepare the hybrid nanocatalysts. The crystalline structure of the TiO2/MWCNTs nanocatalyst was characterised using X-ray powder diffraction (XRD) (Bruker D8 Advance, Karlsruhe, Germany) equipped with a Cu Kα radiation source operated at 40 kV and 40 mA. The powder morphology was determined by field-emission scanning electron microscopy (FE-SEM; SUPRA 55VP, Carl Zeiss, Jena, Germany) and transmission electron microscopy (TEM; Philips CM12, Amsterdam, The Netherlands; operated at 80 kV) studies. In addition, a Brunauer-Emmett-Teller RGFP966 supplier (BET) (Micromeritics, ASAP 2020, Georgia, USA) was used to determine the surface area of the nanocatalyst. The photocatalytic learn more activity of the TiO2/MWCNTs nanocatalyst was evaluated by monitoring the degradation

of methylene blue (MB) in an aqueous solution under irradiation with ultraviolet (UV) (VL-6.LC lamp) or visible light (VL) (commercial halogen tungsten lamp) using a custom-built setup. A small amount (1 mg) of the sample was suspended in 100 ml of aqueous MB solution with a concentration of 10 ppm. Prior to illumination, the solution was sonicated for 10 min and placed in a dark room for 1 h, thus permitting equilibration of the adsorption–desorption of the dye on the nanocatalyst surface. The first sample (approximately 5 mL) solution was collected immediately and was taken as the initial MB concentration APR-246 chemical structure (c 0). The solution was then Osimertinib continuously shaken at 200 rpm. Approximately 5 mL of the liquid was withdrawn every 20 min and immediately centrifuged to remove any suspended solids. To monitor the degradation of the MB, the clean solution was then analysed using a UV–Visible spectrometer (Perkin Elmer, Lambda 900 UV/Vis) in the range of 500–750 nm. Results and discussion The X-ray diffractogram of the synthesised TiO2/MWCNTs nanocatalysts showed the presence of several crystalline peaks, which are predominantly attributed to anatase TiO2 (Figure 1) [41]. The presence of this phase is due to the significantly high concentration

of TiO2 in the material as well as weak X-ray scattering by MWCNTs. Most of the TiO2 peaks were broad with the calculated crystallite size of approximately 10 nm. The presence of MWCNTs was confirmed by the existence of a peak at a 2θ angle of 42.8°, whereas two other main peaks positioned at 26.1° and 53.6° overlapped substantially with TiO2 peaks. Figure 1 X-ray diffractograms of the TiO 2 /MWCNT hybrids. Figure 2 depicts the FE-SEM images of the TiO2/MWCNTs nanocatalyst. The TiO2 nanoparticles that were produced in situ exhibit a mean particle size of approximately 10 nm. The images illustrate that the TiO2 nanoparticles were well attached to the MWCNTs. In addition, the TiO2/MWCNTs were well dispersed, although a few tangles were observed due to the length of the MWCNTs.

As a control, bacteria were grown in

As a control, bacteria were grown in Panobinostat clinical trial an equal volume of cell culturing medium. The plate was incubated at 5% CO2 and 37°C and the GW4869 absorbance was measured in a microplate reader (Multiska Ascent, Thermo labsystems, Helsingfors, Finland) at 620 nm every 30 min for 6 h. The absorbance of PMN cells only was measured and subtracted from the absorbance of the co-incubated samples (bacteria + PMN). The relative growth inhibition (delta OD620) was calculated as absorbance of bacteria-(absorbance of bacteria + PMN).

The viability of the PMN was > 80% as determined by trypan blue exclusion test 6 h after bacterial stimulation. Transwell PMN migration assay A498 cells were seeded onto a inverted 3 μm pore size transwell insert (Falcon, BD Biosciences Pharmingen, San Diego, USA) for 3 h (at 5% CO2 and 37°C) to facilitate cell settling. After 3 h the inserts were placed in 6-well plates with fresh medium and the cells were cultured on the inserts for 2 weeks at 5% CO2 and 37°C. Medium was changed every second day. The cells were pre-stimulated

with the bacteria (MOI 10) for 4 h by adding the different AMN-107 strains to the bottom wells. The PMN were prepared as described above and 106 PMN were added to the top well after the pre-stimulation. PMN cells were collected from the bottom well after 1 and 3 h and counted in a cell counter (TC10™ automated cell counter, Bio-Rad). Measurement of epithelial cytokine production An enzyme-linked immunosorbent assay (ELISA) was performed to measure the cytokine production of A498 cells stimulated with different

bacterial strains for 3 and 6 h. The cytokines IL-6 and IL-8 were measured using human IL-8 and IL-6 kits Glycogen branching enzyme (ELISA MAX™ Deluxe Sets, BioLegend, San Diego, CA, USA). Statistical analysis The variables were normally distributed and differences between groups were evaluated with the unpaired Student’s t-test or one-way ANOVA followed by Bonferroni test. Differences were considered statistically significant when p < 0.05. Data were presented as mean ± standard error of the mean (SEM), n = number of independent experiments. Results Selection and characterization of the UPEC strains The renal epithelial (A498) cells were stimulated with the different bacterial isolates for 6 h and the cell viability was assessed. Bacterial isolates that decreased the cell viability (> 20%) were not suitable for the in vitro infection study design and were excluded. Two ESBL-producing (2/8; 25%) and five non-ESBL-producing (5/11; 45%) isolates were excluded based on this criteria. Six ESBL-producing and six non-ESBL producing isolates remained for investigation. The characteristics of the different isolates included in the study are summarized in Table 1. All ESBL-producing isolates belonged to either the CTX-M-14 or CTX-M-15 enzyme type. The phylogenetic analysis showed that 50% of the susceptible strains belonged to the B2, 33% to the B1 and 17% to the D group.

PubMedCrossRef 23 Mølbak L, Johnsen K, Boye M, Jensen TK, Johans

PubMedCrossRef 23. Mølbak L, Johnsen K, Boye M, Jensen TK, Johansen M, Møller

K: The microbiota of pigs influenced PRN1371 clinical trial by diet texture and severity of lawsonia intracellularis infection. Vet Microbiol 2008, 128:96–107.PubMedCrossRef 24. Shyu C, Soule T, Bent S, Foster J, Forney L: MiCA: a Web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and 18S rRNA genes. Microb Ecol 2007, 53:562–570.PubMedCrossRef 25. Maidak BL, Cole JR, Lilburn TG, Parker CT Jr, Saxman PR, selleck inhibitor Farris RJ: The RDP-II (ribosomal database project). Nucleic Acids Res 2001, 29:173–174.PubMedCrossRef 26. Andersen AD, Mølbak L, Michaelsen KF, Lauritzen L: Molecular fingerprints of the human fecal microbiota from 9 to 18 months old and the effect of fish oil supplementation. J Pediatr Gastroenterol Nutr 2011, 53:303–309.PubMedCrossRef 27. Bacchetti De Gregoris T, Aldred

N, Clare AS, Burgess JG: Improvement of phylum- and class-specific primers for real-time PCR quantification of check details bacterial taxa. J Microbiol Methods 2011, 86:351–356.PubMedCrossRef 28. Rødgaard T, Skovgaard KSJ, Heegaard PMH: Expression of innate immune response genes in liver and three types of adipose tissue in cloned pigs. Cell Reprograming 2012, 14:407–417. 29. Hildebrandt MA, Hoffmann C, Sherrill−Mix SA, Keilbaugh SA, Hamady M, Chen YY: High-Fat diet determines the composition of Dolutegravir ic50 the murine Gut microbiome independently of obesity. Gastroenterology 2009, 137:1716–1724.PubMedCrossRef 30. Ley RE, Peterson DA, Gordon JI: Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 2006, 124:837–848.PubMedCrossRef Competing interest All authors declare no financial or any other

competing interest. Authors’ contributions MB, LM and RP designed the study experiments. RP carried out the experimental work, data and statistical analysis and wrote the manuscript. A.D.A performed the statistical analysis on T-RFLP Shannon-Weaver diversity and PCA and contributed to writing of the manuscript. JS designed and conducted the animal and the diet-intervention experiments. All authors read, corrected and approved the final manuscript.”
“Background Bacillus mycoides, a Gram positive soil rod bacillus of the B. cereus species-group [1], is characterized by hyphal colonies with cells connected at the poles in long filaments. These filaments converge into bundles that mainly curve clock- or counter-clockwise in two kinds of bacilli, both of which were attributed to B. mycoides[2]. We have previously isolated [3] examples of the two types from the environment and followed the process of colony formation on agar of two strains, i.e. DX with the right-curving colony branches and SIN with the left-curving colony branches.

No significant interface response in the S-W result has been

No significant interface response in the S-W result has been buy Thiazovivin previously observed [9], and the discrepancy may be a result of the

different annealing environments (air vs. N2). Annealing in air may lead to a thicker interface oxide (SiO x ) resulting in more evident responses in the DBRA result. The different slopes of the Al2O3 segment of the three samples indicated that the defect types or chemical environments of these samples were different. The three lines crossed one another to avoid passing through a single point of bulk sample without defects, indicating that each of the samples had more than two types of defect. As mentioned in the section ‘DBAR analysis at different annealing temperatures,’ the S Pinometostat in vivo parameter was mainly influenced by Al and neutral O vacancies. Thus, residual C during deposition and O-H bond content also possibly MLN2238 manufacturer influenced the S-W line slope. Residual C varied with the annealing temperature and may have thus influenced the environment of Al vacancies, although further investigations are needed. A thinner sample was prepared to understand the microstructure of the Al2O3/Si samples, which showed a three-layered structure in DBAR analysis. The 6-nm-thick sample was obtained using thermal ALD and observed by transmission electron microscopy (TEM). The result in Figure 6 shows three

layers, namely Si, Al2O3, and Si-Al2O3 interface layers, which have been reported for nonstoichiometric silica (SiO x ) [6, 20, 21]. Figure 6 TEM image of aluminum oxide films prepared using thermal ALD. The fitted S parameter

can be clearly analyzed in different parts of a film to gain accurate information from DBAR spectroscopy. In this study, the energy of injected positrons had a different distribution at the positron incident energy of the X-axis in the S-E plot. The positrons also reached different layers of the film. Thus, the S parameter of each point in the S-E plot contained integrated information on multiple layers. The S parameter was separated in different layers, and the density/type of vacancies was analyzed at different positions in the film. The S-E plot was fitted using the VEPFIT program to calculate the S parameter from different layers using a four-layered Terminal deoxynucleotidyl transferase mode, which corresponded to the surface/Al2O3/SiO x /Si structure observed by TEM. The obtained S parameter is shown in Figure 7. The S parameter in the Al2O3 films decreased with increased temperature, indicating that the vacancy density in the Al2O3 film decreased with increased annealing temperature. The S parameter was much lower in the SiO x layer than that in Al2O3 and the Si substrate. The S parameter also decreased with increased annealing temperature, which probably corresponded with the dominant Pb defect that decreased with increased annealing temperature [22].

Figure 1 Map of Pep3-HBcAg/pET-28a(+) prokaryotic expression plas

Figure 1 Map of Pep3-HBcAg/pET-28a(+) prokaryotic expression plasmid. The three DNA fragments were ligated and subcloned into plasmid pGEMEX-1. Then fusion gene Pep3-HBcAg was digested with restriction enzymes Eco RI and Sal I

and ligated into the equivalent sites of the pET-28a(+) vector, yielding His-tagged Pep3-HBcAg/pET-28a(+). Expression and purification of the fusion protein in Escherichia coli Recombinant plasmid Pep3-HBcAg/pET-28a(+) was introduced into Escherichia coli BL21 (DE3). Then isopropy-β-D-thiogalactoside find more (IPTG, Sigma) was added to induce fusion protein expression. The BL21 cells were harvested, supernatant and sediment were subjected for SDS-PAGE. As the fusion protein was confirmed to be present in inclusion bodies, a further lysis step was performed (8 M urea overnight). The supernatant was purified on a Ni2+-NTA affinity chromatography column (Novagen). The His-tag was removed and the concentration of purified fusion protein was measured with the Bradford assay. EGFRvIII-specific antibody (Zymed) was used to confirm the identity of the fusion protein. Immunization of mice and antibody

detection Thirty 6-8-week-old female ARRY-162 cell line BALB/c mice were purchased from Medical Experimental Animal Center, Xi’an Jiaotong University. All studies were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of Xi’an Jiaotong University. Ten mice were subcutaneously injected with fusion protein (100 μg/animal) emulsified in Freund’s complete adjuvant (Sigma) on day

0 and with the same amount of protein emulsified in Freund’s incomplete adjuvant on day 7. The third and following boosters were done only with fusion protein once a week with a total of seven immunizations. Other 20 mice were divided into two groups, and immunized with HBcAg and PBS. Immune serum samples were collected and stored at -70°C. Antibody titers ioxilan were assayed by enzyme-linked immunosorbent assay (ELISA). IFN-γ detection Enzyme-linked immunospot assay (ELISPOT) was used to evaluate CFTRinh-172 cost tumor-specific IFN-γ-secretion in splenocytes. One week after the final vaccination, spleen cells from three mice per group were harvested. Immunospot plates were coated with 100 μl anti-mouse γ-IFN monoclonal antibody (5 μg/ml, BD PharMingen). Freshly isolated splenocytes were added into plate at a density of 3 × 106 cells/well and co-cultured with 1 μg/ml EGFRvIII-specific peptide (pep-3) for 20 h at 37°C. Medium without blood-serum was added as negative control. Plates were washed and incubated with 50 μl/well of biotin-conjugated anti-mouse IFN-γ, and then stayed overnight at 4°C. Then, 10 μl/well of HRP-labelled streptavidin was added.

There are few two-phase lattice Boltzmann models that consider th

There are few two-phase lattice Boltzmann models that consider the interaction forces between nanoparticles and a base fluid for natural convection in an enclosure. Xuan et al. [26] proposed a two-phase Lattice Boltzmann model to investigate sudden-start Couette flow and convection in parallel plate channels

without researching the effect of forces on volume fraction distribution of nanoparticles. Because these forces were not investigated before our work, the effects of forces between water and nanoparticles on the fluid flow patterns were unknown. In addition, as we know, the nanoparticles in the fluid easily gather together and deposit, especially at high volume fraction. Hence, the nanoparticle distribution in the fluid flow is important for nanofluid application, which is another selleck inhibitor objective in our paper. However, the single-phase model cannot be used to investigate nanoparticle distribution. Furthermore, natural convection of a TPCA-1 ic50 square enclosure (left wall kept at a high constant temperature (T H), and top wall kept at a low constant temperature (T C)) filled with nanofluid is not investigated in the published literatures. In this paper, a two-phase Lattice Boltzmann model is proposed and applied to investigate the natural convection of a square enclosure (left wall kept at a high

constant temperature (T H), and top wall kept at a low constant temperature (T C)) filled with Al2O3-water nanofluid and the inhomogeneous distribution of nanoparticles in the square enclosure. Methods Lattice Boltzmann method The density distribution function selleck chemical for a single-phase fluid is calculated as follows: (1) (2) where is the dimensionless collision-relaxation time for the flow field, e α is the lattice velocity vector, the subscript α represents the

lattice velocity direction, is the distribution function of the nanofluid with velocity e α (along the direction α) at lattice position r and time t, is the local equilibrium distribution function, δ t is the time step, δ x is the lattice step, the order numbers α = 1,…,4 and α = 5,…,8, respectively represent Paclitaxel manufacturer the rectangular directions and the diagonal directions of the lattice, is the external force term in the direction of the lattice velocity without interparticle interaction, G = - β(T nf  - T 0)g is the effective external force, where g is the gravity acceleration, β is the thermal expansion coefficient, T nf is the temperature of the nanofluid, and T 0 is the mean value of the high and low temperature of the walls. A nanofluid is a two-phase fluid constituted by nanoparticles and a base fluid, and there are interaction forces (gravity and buoyancy force, drag force, interaction potential force, and Brownian force) between nanoparticles and the base fluid. Thus, the macroscopic density and velocity fields are simulated using the density distribution function by adding the forces term.

Salt-induced peptide formation reaction has been suggested

Salt-induced peptide formation reaction has been suggested

to be prebiotically relevant BAY 63-2521 clinical trial for the very first steps of chemical evolution (Schwendinger and Rode 1989). Based on Monte Carlo computer simulations, Rode and co-workers found that sodium chloride at concentrations above 3 M effectively acts as a dehydrating agent to overcome the thermodynamic barrier of peptide bond formation in aqueous solutions, and the first this website hydration shell of the sodium ion was assumed to no longer be saturated with water molecules (Jakschitz and Rode 2012). Furthermore, using HPLC-MS/MS analysis, a high concentration of sodium chloride was found to significantly enhance the formation of peptides from L-glutamic acid (L-Glu) in homogenous water solutions (Wang et al. 2005). All the references we have found that discuss the presence of other mono- and divalent inorganic cations in prebiotic peptide formation speculate that these

ions support the dehydrating effect of sodium chloride. However, the level of potassium exceeds that of sodium by more than an order of magnitude inside all living cells (Aronson et al. selleck inhibitor 2009), and the ion ratio is actively preserved with Na+/K+ pumps in the cell membrane, which suggests that potassium is more essential for life. The physical-chemical differences between Na+ and K+ are small (Freedman 1995), although the bio-directed activity of these ions differs dramatically; for example, K+ is required for ribosomal peptide synthesis (Spirin and Gavrilova selleck screening library 1971) and the amplification of DNA with thermostable Taq polymerase (Saiki et al. 1988), whereas Na+ attenuates these processes. The contradiction between the Na+ and K+ compositions of seawater and living cell cytoplasm led

to the hypothesis that the first protocell could have emerged in KCl solution (Natochin 2007; Natochin 2010). However, the hypothesis of the K+-driven emergence of prebiotic peptides remains to be tested. Here we investigate the relative effects of Na+ and K+ in a model peptide synthesis reaction. Methods L-glutamic acid and 1,1′-carbonyldiimidazole (CDI) were obtained from Sigma-Aldrich Co. LLC (St. Louis, USA). In total, 10 mmol KCl or 10 mmol NaCl was added to reaction mixtures containing 3 mmol L-Glu in 5 ml distilled water. The mixture was diluted to 10 ml and cooled on a crashed ice-NaCl mixture, and 6 mmol CDI was added into each mixture and incubated at room temperature for 24 h. A 10 μl sample was loaded onto a Zorbax SAX (4.6 mm × 250 mm, 5 μm) column using an autosampler. Peptide separation was performed at a flow rate of 0.5 ml/min using an NaCl gradient (2–80 % B for 80 min; buffer A: 20 % acetonitrile in 0.020 M NaH2PO4 at pH 7.0; buffer B: 2.0 M NaCl in buffer A) using an Agilent 1100 nano-HPLC system (Agilent Technologies Inc., USA). LC analysis of the peptides was performed by an established procedure (Ishihama et al.

We also thank Assoc Prof T Tsuge (Department of Innovative and

We also thank Assoc. Prof. T. Tsuge (Department of Innovative and engineered Materials, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Japan) for GC-MS analysis. This work was supported by MEXT Grant-in-Aid for Scientific Research on Priority Areas “Applied Genomics” (Grant Number 20018008) and that on Innovative Areas “”Genome Science”" (Grant

Number 221S0002). Electronic supplementary material Additional file 1: Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO 2 fixation into poly(3-hydroxybutyrate) under heterotrophic condition in CFTRinh-172 Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses (Shimizu et al.). Figure S1. DMXAA nmr Relative expression changes of phaC1 determined by qRT-PCR using three primer sets for amplification and two inner control genes for quantification. Square, amplification of the central region (primers: phaC1-5’-Cent/phaC1-3’-Cent); diamond, amplification of the N-terminal region (phaC1-5’-N/phaC1-3’-N); circle, amplification of the C-terminal

region (phaC1-5’-C/phaC1-3’-C). Open symbols, bfr2 inner control; closed symbols, 16SrRNA inner control. Materials and Methods for qRT-PCR. Figure S2. Correlation of expression ratios from RNA-seq and qRT-PCR in F26. The best-fit linear regression curve is shown with the correlation coefficient (R2). Closed circle, dapA1 (primers: dapA1-5’/dapA1-3’); closed square, phaC1 (phaC1-5’-Cent/phaC1-3’-Cent); closed triangle, cbbL (cbbL-5’/cbbL-3’); closed diamond, bfr2 (bfr2-5’/bfr2-3’). The primer sequences are listed in Table S4, and qRT-PCR was performed as described in the legend of Figure S1. Table S1. Highly transcribed genes in R. euttopha H16 during the growth on fructose.a. Table S2. Highly up-regulated genes in F26 to F16. Table S3. Highly down-regulated genes in F26 to F16. Table S4. Primers used in this study. (PDF 1 MB) References 1. Bowien B,

Kusian B: Genetics next and control of CO 2 assimilation in the chemoautotroph Ralstonia eutropha . Arch Microbiol 2002, 178:85–93.PubMedCrossRef 2. Ishizaki A, Tanaka K, Taga N: Microbial production of poly-D-3-hydroxybutyrate from CO 2 . Appl Microbiol Biotechnol 2001, 57:6–12.PubMedCrossRef 3. find more Jendrossek D: Polyhydroxyalkanoate granules are complex subcellular organelles (carbonosomes). J Bacteriol 2009, 191:3195–3202.PubMedCrossRef 4. Rehm BHA: Polyester synthases: natural catalysts for plastics. Biochem J 2003, 376:15–33.PubMedCrossRef 5. Rehm BHA: Biogenesis of microbial polyhydroxyalkanoate granules: a platform technology for the production of tailor-made bioparticles. Curr Issues Mol Biol 2007, 9:41–62.PubMed 6. Steinbüchel A, Lütke-Eversloh T: Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem Eng J 2003, 16:81–96.CrossRef 7.