Naked DNA, usually in plasmid form, is the simplest form of non-v

Naked DNA, usually in plasmid form, is the simplest form of non-viral transferring of a gene into a target cell [13–16]. Because of low transferring efficiency of a bare plasmid, several physical (electroporation, ultrasound, gas-filled micro-bubbles) and chemical (liposomes) approaches have been exploited to enhance their transformation efficiency [17]. In another type of classification, non-viral delivery vectors can be categorized as organic (lipid complexes, conjugated

polymers, cationic polymers, etc.) and inorganic (magnetic nanoparticles, quantum dots, carbon nanotubes, gold nanoparticles, etc.) systems [18]. Among the materials used to design non-viral vectors, attention has recently increased on the natural GSK2126458 in vivo biomaterials due to their unique properties such as biodegradability, biocompatibility, and controlled release. The delivery carriers necessitate being small enough to be internalized into the cells

and enter the nucleus passing through the cytoplasm and escaping the endosome/lysosome process following endocytosis (Figure 1). The use of nanoparticles in gene delivery can provide both the targeted and sustained gene delivery by protecting the gene against nuclease degradation and improving its stability [19–22]. Figure 1 Internalization of non-viral vectors into cell and passage to nucleus through cytoplasm following endocytosis. Nanoparticles in gene delivery In the field of nanomedicine, selleck kinase inhibitor nanotechnology methods focus on formulating therapeutic biocompatible agents such as nanoparticles, nanocapsules, micellar systems, and conjugates [22, 23]. Nanoparticles are solid and spherical structures ranging to around 100 nm in size and prepared from from natural or synthetic polymers [24]. To reach the large-size nucleic acid molecule, the cytoplasm, or even the

nucleus, a suitable carrier system is required to deliver genes to cells which enhance cell internalization and protect the DNA molecule from nuclease enzymatic degradation (e.g., virosomes, cationic liposomes, and nanoparticles). To achieve the suitable carrier system, the nanoparticles can be considered as a good candidate for therapeutic applications because of several following reasons: (1) They exist in the same size domain as proteins,(2) they have large surface areas and ability to bind to a large number of surface functional groups, and (3) they possess controllable absorption and release properties and particle size and surface characteristics [25]. Nanoparticles can also be coated with molecules to produce a hydrophilic layer at the surface (PEGylation) to increases their blood circulation half-life. Poloxamer, poloxamines, and chitosan have also been studied for surface modifications.

Some of these findings have been supported by mechanistic studies

Some of these findings have been supported by mechanistic studies in various muscle cell cultures, where IGF-1 [10], myogenesis [11] and protein synthesis [10, 12, 13] were increased, and also a more explorative approach using microarrays on muscle biopsies from creatine supplemented individuals revealed cytoskeleton remodelling, protein and glycogen synthesis regulation, as well as cell proliferation and differentiation [8]. Other techniques such as proteomics and metabonomics may reveal additional insight into some of the biochemical effects of creatine supplementation at the protein and metabolite level. see more High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy is

JNK inhibitors high throughput screening a well-established analytical technique for metabolic fingerprinting of biofluids and various tissues and has also been used for elucidating the metabolic effects of dietary factors in both humans [14–17], animals [18–20], and also in cell cultures [21]. These studies have demonstrated that NMR-based metabonomics is extremely efficient in detecting endogenous and exogeneous metabolic perturbations. However, while being capable of identifying biomarkers and

metabolic perturbations, the metabolic network responsible for the perturbations can only be hypothesised. Proteomics displays protein products as a result of gene expression and efficiency of translation, and has been used to separate and identify differentially regulated proteins from in response to various treatments of cultured cells [22, 23] and muscles [24]. Linking information obtained from metabolic fingerprinting with proteomics would pave the way for obtaining a better understanding of the primary pathways

involved in perturbations associated with CMH supplementation. In this study we have for the first time examined and integrated the NMR metabolite profile and the proteomic profile of myotubes in the presence and absence of creatine supplementation in a systems biology approach. Methods Muscle Cell Culture Myotube cultures were established from a mouse myoblast line (C2C12) originally derived from a thigh muscle [25] (American Type Culture Collection, Manassas, VA). A clone from this cell line, which effectively fused and formed myotubes, was isolated [26]. The clone was grown in 80 cm2 culture flask in 10 mL of medium consisting of Dulbecco’s modified Eagle’s medium (DMEM), 10% (vol/vol) fetal calf serum (FCS), and supplemented with 1% antibiotics giving 100 IU/mL penicillin, 100 μg/mL streptomycin sulfate, 3 μg/mL amphotericin B, and 20 μg/mL gentamycin (growth medium). Cells were maintained in an atmosphere of 95% air and 5% CO2 at 37°C. Prior to confluence, cells were harvested in 0.25% trypsin and sub-cultured into 80 cm2 culture flasks or 96 well plates.

athalia P argus Temperature (T; in °C) Low T ≤ 19 5

athalia P. argus Temperature (T; in °C) Low T ≤ 19.5 Selleck AUY-922 T ≤ 20 T ≤ 14 T ≤ 22 Intermediate 19.5 < T ≤ 25.5 20 < T ≤ 31 14 < T ≤ 25 22 < T ≤ 28 High T > 25.5 T > 31 T > 25 T > 28 Radiation (R; in °C) Low R ≤ 12 R ≤ 10 R ≤ 14 R ≤ 17 Intermediate 12 < R ≤ 28 10 < R ≤ 20 14 < R ≤ 31 17 < R ≤ 20 High R > 28 R > 20 R > 31 R > 20 Cloudiness (C; in %) Low C ≤ 15 C ≤ 15 C ≤ 25 C = 0 Intermediate 15 < C ≤ 60 15 < C ≤ 70 25 < C ≤ 70 0 < C ≤ 20 High C > 60 C > 70 C > 70 C > 20 Wind speed (W; in Bft) Low W ≤ 1 W ≤ 2 W ≤ 3 W ≤ 2 Intermediate 1 < W ≤ 2 2 < W ≤ 4 3 < W ≤ 4 2 < W ≤ 3 High

W > 2 W > 4 W > 4 W > 3 Time budget analysis For each tracked individual, we calculated the proportion of time devoted to a certain behaviour. We tested for differences between weather categories in proportion of time spent flying as opposed to non-flight behaviour, using Wilcoxon rank sum test (W) in R 2.7.0. Ten individuals devoting their total tracked time to flight behaviour, were excluded from the analysis, because these individuals were lost from Tideglusib order sight within the first recorded bout. Time budget analysis (Miron

et al. 1992) is complementary to survival analysis, since possible changes in bout duration are compensated by changes in occurrence of these bouts. Spatial analysis Spatial coordinates were recorded at a constant time interval (2006: 10 s; 2007: 1 s) by the GPS device. Coordinates derived from the Garmin eTrexVenture™ were transformed into.shp files using GPS2Shape software (Jochem 2006). Successive points were connected with straight lines and are further referred to as steps. For each individual, we analysed PIK3C2G the total pathway, determining tortuosity as the standard

deviation in turning angle in proportion to a full circle (in radians divided by 2π) and the net displacement of the pathway (i.e. the distance between the track starting and ending points; in metres). The effects of weather variables on tortuosity and net displacement were tested using regression analysis with generalized linear models in R 2.7.0. In addition, we compared the tortuosity and net displacement of the pathways of released individuals of M. jurtina with pathway characteristics of individuals tracked within their habitat using Wilcoxon rank sum test (W) in R 2.7.0. The effects of weather variables and presence of habitat on tortuosity and net displacement were tested using regression analysis with generalized linear models in R 2.7.0 and Akaike’s information criterion for model selection (Burnham and Anderson 2002). Colonization frequency Data on colonization frequency were obtained from the Dutch Butterfly Monitoring Scheme monitoring (Van Swaay et al. 2008), with standardized transect counts over the period 1990–2008. The total number of transects where the study species were sighted strongly differed between species: 452 for C. pamphilus, 737 for M. jurtina, 22 for M. athalia, and 155 for P. argus. Because of the small sample size, we excluded M. athalia from this analysis.

Protein Sci 2006,15(6):1550–1556 CrossRefPubMed 38 Joshi B, Jand

Protein Sci 2006,15(6):1550–1556.CrossRefPubMed 38. Joshi B, Janda L, Stoytcheva Z, Tichy P: PkwA, a WD-repeat protein, is expressed in spore-derived mycelium of Thermomonospora curvata and phosphorylation of its WD domain could act as a molecular switch. Microbiology 2000,146(Pt 12):3259–3267.PubMed 39. Ackerley DF, Barak Y, Lynch SV, Curtin J, Matin A: Effect of chromate stress on Escherichia coli K-12. J Bacteriol 2006,188(9):3371–3381.CrossRefPubMed 40. Hu P, Brodie EL, Suzuki Y, McAdams HH, Andersen

GL: Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus. J Bacteriol 2005,187(24):8437–8449.CrossRefPubMed 41. Silver S, Phung LT: Bacterial heavy metal resistance: new surprises. Annu Rev Microbiol 1996, 50:753–789.CrossRefPubMed 42. Munkelt D, Grass G, Nies DH: The chromosomally encoded cation diffusion RG-7388 chemical structure facilitator proteins DmeF and FieF from Wautersia metallidurans CH34 are transporters of broad metal specificity. J Bacteriol 2004,186(23):8036–8043.CrossRefPubMed

43. Henne KL, Turse JE, Nicora CD, Lipton MS, Tollaksen S, Lindberg C, Babnigg G, Giometti CS, Nakatsu CH, Thompson DK, et al.: Global Proteomic Analysis of the Chromate Response in Arthrobacter sp. Strain FB24. J Proteome Res 2009,8(4):1704–1716.CrossRefPubMed 44. Cervantes C: Bacterial interactions with chromate. Antonie Van Leeuwenhoek 1991,59(4):229–233.CrossRefPubMed 45. Coleman NV, Mattes TE, Gossett JM, Spain JC: Phylogenetic and kinetic diversity of aerobic vinyl chloride-assimilating bacteria from contaminated sites. Appl Environ Microbiol 2002,68(12):6162–6171.CrossRefPubMed 46. Mattes TE, Adavosertib Coleman NV, Spain JC, Gossett JM: Physiological and molecular genetic analyses of vinyl chloride and ethene biodegradation in Nocardioides sp. strain JS614. Arch Microbiol 2005,183(2):95–106.CrossRefPubMed 47. McLeod MP,

Warren RL, Hsiao WW, Araki N, Myhre M, Fernandes C, Miyazawa D, Wong W, Lillquist AL, Wang D, et al.: The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proc Natl Acad Sci USA 2006,103(42):15582–15587.CrossRefPubMed 48. Jerke KH: Physiological and Genetic Analysis of Plasmid-Mediated Metal Resistance in Arthrobacter sp strain AK-1. West Lafayette: Purdue University 2006. 49. Biebl H, Pfenning N: Isolation of members new of the family Rhodospirillaceae. The Prokaryotes (Edited by: Starr MP, Stolp H, Truber HG, Balows A, Schlegel HG). Berlin: Springer-Verlag KG 1981. 50. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: a laboratory manual. 2 Edition Cold Spring Laboratory, Cold Spring Harbor, NY 1989. 51. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al.: Clustal W and Clustal X version 2.0. Bioinformatics 2007,23(21):2947–2948.CrossRefPubMed 52. The Universal Protein Resource (UniProt) 2009 Nucleic Acids Res 2009, (37 Database):D169–174. 53.

For several rats, one trabecula was selected and analyzed as it d

For several rats, one trabecula was selected and analyzed as it developed this website over time after PTH

treatment. Figure 7 shows how PTH in this particular trabecula first led to filling and overfilling of cavities, while later, more bone was added to the surface of the trabecula resulting in a thicker trabecula. Also, resorption still appeared to take place in this trabecula. Another trabecula after segmentation of the image appeared cleaved due to OVX-induced increased resorption. PTH treatment led to bone formation, which took place where it was most beneficial, i.e., at the cleaved site, restoring the trabecula. This indicates that there see more probably was still a thin line of bone left in the center, which was unaccounted for after segmentation, but large enough for bone formation to take place. It was found that for all rats, the maximum trabecular thickness continued to increase over time. Therefore, no maximum limit for trabecular thickness appeared to be present. Fig. 7 A trabecula in two PTH-treated ovariectomized rats was tracked over time

to determine the development of bone formation (1 and 2). On the left of 1 and 2, you see three-dimensional segmented images of a trabecula, after PTH treatment is started at week 8, taken at weeks 8 (a), 10 (b), 12 (c), and ADP ribosylation factor 14 (d). On the right, you see overlaid two-dimensional segmented sections comparing weeks 8 and 10 (e), 10 and 12 (f), and 12 and 14 (g). Yellow indicates resorbed bone, green newly formed bone, and red unchanged bone. Bone formation is clearly seen over time in both trabeculae. In trabecula 1, bone is mostly deposited in the cavities in the first 2 weeks, while later on bone is added to the surface. In trabecula 2, the trabecula appears cleaved after segmentation, although most likely

there was still a thin line of bone present. PTH treatment leads to bone formation at the cleaved site, where it is most needed hereby restoring the trabecula Prediction of gain in bone mass after PTH treatment The linear correlations between several structural parameters and the gains in bone mass, gain in bone volume fraction, final bone mass, and final bone volume fraction after PTH treatment varied between the specific parameters as well as bone regions (Table 1). More significant predictions were found for the metaphysis than the epiphysis. Best correlations were found between BV and BV/TV at week 0 and BV and BV/TV at week 14, respectively, in both the meta- and epiphysis. Paradoxically, the loss of bone after OVX did not predict the gain of bone after PTH treatment well. From structural parameters evaluated at week 8, bone surface (BS) was the best predictor of the gain in bone after PTH.

The optical system was configured with a 75 W Xe lamp, circular l

The optical system was configured with a 75 W Xe lamp, circular light polarizer and end-mounted photomultiplier. The instrument had previously been calibrated with (D)-camphorsulfonic acid. Temperature was regulated using a Neslab RTE-300 circulating programmable water bath (Neslab Inc). CD spectra were recorded at 298 K in a 10 mm path length cell over a wavelength range of 215–345 nm in steps of either 1 0r 2 nm, with

3 nm entrance/exit slit widths: the number of counts was set to 10,000 with adaptive sampling APO866 price set to 500,000. The spectra were corrected by subtracting the spectrum of the same buffer solution of 100 mM potassium chloride and 10 mM potassium phosphate at pH 7.0. Annealing and melting profiles were recorded using a thermoelectric temperature

controller (Melcor) on 4 μM DNA samples with and without 3.5 mol.equiv. of ligands using 0.5 K temperature increments and a cooling or heating rate of 0.2 K/min over the temperature range 298-368 K. Cells and culture conditions BJ fibroblasts expressing DAPT ic50 hTERT (BJ-hTERT) or hTERT and SV40 early region (BJ-EHLT), were obtained as previously reported [15]. Cells were grown in Dulbecco Modified Eagle Medium (D-MEM, Invitrogen Carlsbad, CA, USA) supplemented with 10% fetal calf serum, 2 mM L-glutamin and antibiotics. Proliferation assay 5 × 104 cells were seeded in 60-mm Petri plates (Nunc, MasciaBrunelli, Milano, Italy) and 24 h after plating, 0.5 μM of freshly dissolved compound was added to the culture medium. Cell counts (Coulter Counter, Kontron Instruments, Milano, Italy) and viability (trypan blue dye exclusion) were determined daily, from day 2 to day 8 of culture. Immunofluorescence Cells were fixed in 2% formaldehyde and permeabilized in 0.25% Triton X100 in PBS for 5 min at BCKDHA room temperature. For immunolabeling, cells were incubated with primary antibody, then washed in PBS and incubated with the secondary antibodies. The following primary antibodies were used: pAb and mAb anti-TRF1 (Abcam Ltd.; Cambridge UK); mAb (Upstate, Lake Placid, NY) and pAb anti-γH2AX (Abcam). The following secondary antibody were

used: TRITC conjugated Goat anti Rabbit, FITC conjugated Goat anti Mouse (Jackson ImmunoResearch Europe Ltd., Suffolk, UK). Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by a Leica FW4000 deconvolution software (Leica, Solms, Germany). This system permits to focus single planes inside the cell generating 3D high-resolution images. For quantitative analysis of γH2AX positivity, 200 cells on triplicate slices were scored. For TIF’s analysis, in each nucleus a single plane was analyzed and at least 50 nuclei per sample were scored. Fluorescence in situ hybridization (FISH) For metaphase chromosome preparation cells were treated with demecolcine (Sigma, Milan, Italy) 0.

A) The relationship

A) The relationship YM155 research buy between the cell elongation rate and the interval between two divisions during YgjD depletion (Movie 2, additional files), and B) for MG1655 (Movie 3, additional files). For YgjD depletion, cell elongation rate starts to decrease from generation 3 on. However, this decrease in cell elongation rate is initially not compensated for by an increase in the interval between two divisions. Points below the contour line correspond to cells that divide before they double in size, and whose size thus steadily declines. The inset lists the result of a non-parametric correlation analysis between ‘cell elongation

rate’ and ‘time to division’, performed separately for every generation. A negative correlation indicates coupling of the interval between division and the cell elongation rate. For MG1655, the majority of cells cluster around the contour line. C) and D) show the result of the independent contrast correlation analysis for YgjD depletion in TB80, and MG1655 growth. Each point depicts the difference (residual) between two sister cells in the

cell elongation rate (horizontal axis) and in the interval between cell divisions (vertical axis). Cells that have a higher elongation rate than their sister tend to have a shorter interval between divisions. The inset lists the result of a non-parametric correlation analysis between ‘difference in cell elongation rate’ and ‘difference EVP4593 nmr in interval between two divisions’, performed separately for every generation. Again, Florfenicol negative correlation indicates coupling of the interval between division and the cell elongation rate. The phenotype

induced by YgjD depletion was specific, and depletions of other essential genes lead to different cellular morphologies. We analyzed time-lapse images of the depletion of three other essential genes (dnaT, fldA and ffh). Depletion of each protein resulted in cellular phenotypes that were different from each other and from YgjD when depleted (Additional file 6 – Figure S3; also see Additional Files 7, 8 and 9 – movies 4, 5 and 6). Also, the effects of YgjD depletion were different from the consequences of exposure to two antibiotics that we tested: we followed wildtype E. coli cells exposed to the translational inhibitors kanamycin and chloramphenicol at minimum inhibitory concentration (2.5 μg/ml for chloramphenicol, 5 μg/ml for kanamycin), and observed no decrease in cell size (Additional file 10 – Figure S4, and Additional Files 11 and 12 – movies 7 and 8). For reference, we also analyzed images of growing microcolonies of wildtype E. coli MG1655 cells on LB medium supplemented with glucose.

Nano Lett 2008,8(12):4469–4476 CrossRef 7 Hu W, Peng C, Luo W, L

Nano Lett 2008,8(12):4469–4476.CrossRef 7. Hu W, Peng C, Luo W, Lv M, Li X, Li D, Huang Q, Fan C: Graphene-based antibacterial

paper. ACS Nano 2010,4(7):4317–4323.CrossRef 8. Akhavan O, Ghaderi E: Photolytic reduction of graphene oxide nanosheets on TiO 2 thin film for photo inactivation of bacteria in solar light irradiation. J. Phy. Chem. C 2009, 113:20214–20220.CrossRef 9. Akhavan O, Ghaderi E: Toxicity of graphene and graphene oxide nanowalls against bacteria. ACS Nano 2010,4(10):5731–5736.CrossRef 10. Ma J, Zhang J, Xiong Z, Yong Y, Zhao XS: Preparation, characterization and find more antibacterial properties of silver-modified graphene oxide. J Mater Chem 2011, 21:3350–3352.CrossRef 11. Gurunathan S, Han JW, Dayem AA, Eppakayala V, Kim JH: Oxidative stress-mediated antibacterial activity of graphene oxide and reduced graphene oxide in Pseudomonas aeruginosa . Int J Nanomedicine 2012, 7:5901–5914.CrossRef 12. Akhavan O, Choobtashani M, Ghaderi E: Protein degradation and RNA efflux of viruses photocatalyzed by graphene−tungsten oxide composite under visible light irradiation. J. Phy. Chem.

C 2012, 116:9653–9659.CrossRef 13. Yang K, Zhang S, Zhang G, Sun X, Lee ST, Liu Z: Graphene in mice: ultrahigh in vivo tumor uptake and efficient photothermal therapy. Nano Lett 2010,10(9):3318–3323.CrossRef 14. Yang K, Wan J, Zhang S, Tian B, Zhang Y, Liu Z: The influence of surface chemistry and size of nanoscale

graphene oxide on photothermal therapy of cancer using ultra-low laser power. Biomaterials 2012,33(7):2206–2214.CrossRef 15. Robinson JT, Tabakman SM, Liang Y, Wang H, Casalongue SH, Rebamipide Vinh D, Dai HJ: Ultrasmall reduced graphene oxide with high near-infrared absorbance for photothermal therapy. Am Chem Soc 2011,133(17):6825–6831.CrossRef 16. Liu Z, Robinson JT, Sun X, Dai H: PEGylated nanographene oxide for delivery of water-insoluble cancer drugs. J Am Chem Soc 2008,130(33):10876–10877.CrossRef 17. Zhang L, Xia J, Zhao Q, Liu L, Zhang Z: Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs. Small 2010,6(4):537–544.CrossRef 18. Zhang W, Guo Z, Huang D, Liu Z, Guo X, Zhong H: Synergistic effect of chemo-photothermal therapy using PEGylated graphene oxide. Biomaterials 2011, 32:8555–8561.CrossRef 19. Agarwal S, Zhou X, Ye F, He Q, Chen GCK, Soo J, Boey F, Zhang H, Chen P: Interfacing live cells with nanocarbon substrates. Langmuir 2010,26(4):2244–2247.CrossRef 20. Heo C, Yoo J, Lee S, Jo A, Jung S, Yoo H, Lee YH, Suh M: The control of neural cell-to-cell interactions through non-contact electrical field stimulation using graphene electrodes. Biomaterials 2011,32(1):19–27.CrossRef 21. Wu J, Agrawal M, Becerril HA, Bao Z, Liu Z, Chen Y, Peumans P: Organic light-emitting diodes on solution-processed graphene transparent electrodes. ACS Nano 2010,4(1):43–48.CrossRef 22.

In this study, we first identified three effective MDR1 siRNAs fr

In this study, we first identified three effective MDR1 siRNAs from four candidate siRNA sites by qRT-PCR. The three siRNA plasmids were pooled at an equal molar concentrations

and transfected into L2-RYC cells. All three siRNAs were specific for MDR1 target gene but at different mRNA degradation sites, so increased the target gene knock-down efficiency of random-designed siRNAs. The decreased concentration of individual siRNAs could reduce potential off-target effects. Our result confirmed that the pooled siRNAs have higher inhibition efficacy than that of potent individual siRNAs. Effective siRNA DNA delivery into cells and in vivo has been a great challenge for the broad use of RNAi therapeutics. The most commonly used JQ-EZ-05 in vivo carriers for delivering nucleic acids into mammalian cells are non-viral and viral vectors. Liposome-mediated Luminespib transfection is simple and powerful, but has cytotoxic side effects [26]. Calcium phosphate co-precipitation has rigorous conditions of transfection and a small range of target cells [42, 43]. Virus-mediated transfection is high efficient and available to achieve sustainable transgene expression. However the

biosafety for in vivo use remains a concern [44]. Recently, ultrasound contrast agents (in a form of microbubble) have been used to deliver gene and drug in vitro and in vivo, providing a new and efficient therapeutic technique [22–25]. Ultrasound microbubble-mediated destruction has been shown to enhance cell membrane permeability and improve gene and drug delivery. It has been shown that ultrasound microbubble-mediated destruction can transfect DNA into a variety of mammalian cells [22, 24, 26, 45]. The change of cell membrane permeability is recoverable when ultrasound energy and exposure time are within a suitable range. Thus ultrasound exposure will not cause permanent damage to cells [45, 46]. We first determined the optimal ultrasound parameters of acoustic intensity and exposure time for L2-RYC cell transfection. When cultured L2-RYC cells

were exposed to ultrasound with intensity Unoprostone of 0.75 W/cm2 and 1 W/cm2, the survival rates was too low to be used in the study. Although ultrasound with intensity of 0.25 W/cm2 did not affect cell viability, plasmids DNA delivery into cells was poor. Fortunately, we found out ultrasound with intensity of 0.5 W/cm2 for 30 s could effectively transfect plasmids into cells without causing significant amount of cell death. Our previous study on bone marrow mononuclear cells also reported gene delivery by ultrasound with intensity of 0.5 W/cm2 did not reduce cell viability and not destroy membrane of treated cells [45]. Under the chosen condition, we found that 30% GFP-positive cells can be achieved by gene transfection using ultrasound microbubble-mediated delivery.

The column was equilibrated with 4% acetonitrile containing 0 1%

The column was equilibrated with 4% acetonitrile containing 0.1% formic acid at 0.5 μL min-1 and the samples eluted with an acetonitrile gradient

(4%-31% in 32 min). MS/MS spectra of ionisable species were acquired in a data-dependant fashion as follows: Ionisable species (300 < m/z < 1200) were trapped and the two most intense ions in the scan were independently fragmented by collision-induced dissociation. Post acquisition, MS and MS/MS spectra were subjected to peak detection using Bruker’s DataAnalysis software (version 3.4). Data were imported into BioTools. MS/MS data were searched as described above, but with an MS mass tolerance and MS/MS tol of 0.3 and 0.4 Da, respectively, and EPZ015666 a peptide charge of 1+, 2+ and 3 + . Western

blotting analysis The intracellular concentrations of heat shock protein (HSP) GroEL and a recombination protein RecA were analysed by Western blotting. Aliquots of cell lysates from both planktonic and biofilm cultures equivalent to 15 μg of protein, were separated by electrophoresis on 12%T 3.3% C polyacrylamide gels (100 V, 1.5 h) [33]. The proteins were then electro-transferred to an Immuno-Blot PVDF membrane SBI-0206965 (Bio-Rad Laboratories, CA, USA) using Mini Trans-Blot Cell (250 mA, 2 h) (Bio-Rad Laboratories, CA, USA) followed by blocking (1 h, room temperature) using 5% (w/v) ECL Blocking Agent (GE Healthcare, Buckinghamshire, UK). The washed membrane was then treated with either mouse anti-human Hsp60 monoclonal antibody (SPA-087, Stressgen before Biotechnologies, British Columbia, Canada) diluted 1:1000 or mouse anti-E. coli RecA monoclonal antibody (MD-02 + 3, MBL International,

IF, USA) diluted 1:1000 for 24 h at 4°C. The washed membrane was then probed for 1 h at room temperature with anti-mouse alkaline phosphatase conjugate secondary antibody (1 mAB: 5000 BSA- tris-buffered saline-tween 20 (TBS-T)). The target protein was detected using ECF substrate and scanned using a Typhoon Scanner. The expression of the protein was analysed using ImageQuant TL software. EFC substrate, Typhoon Scanner and ImageQuant TL software were purchased from GE Healthcare (Buckinghamshire, UK). Quantitative real-time PCR (qRTPCR) Gene sequences of groEL, dnaK and recA and 16S rRNA were retrieved from the Oralgen Databases (http://​www.​oralgen.​lanl.​gov) and primers were designed using the web-based tool Primer 3-PCR (Additional file 2: Table S2). 16S rRNA was used as reference gene. Bacterial samples from each culture type (4 mL) were harvested and incubated in 4 mL of RNAlater (Ambion, Austin, TX, USA) overnight at 4°C. RNAlater was then removed by centrifugation (5,000 × g, 4°C, 15 min). Cell pellets were resuspended in 1 mL of fresh RNAlater and stored at −80°C until required. Total RNA was extracted from the bacterial pellets using the RiboPure-Bacteria Kit (Ambion, TX, USA) following the manufacturer’s instructions.