Controls consisted of PBS/0 25 μM H2O2 in the absence (control) o

Controls consisted of PBS/0.25 μM H2O2 in the absence (control) or presence of diluted plasma. Data were calculated Temsirolimus supplier as a change in fluorescence over time (900 s) minus the fluorescence observed at time zero (ΔFI). Results are calculated as ΔFI after 300 sec and shown as % change from pre-LY2603618 cost damage values. Plasma interleukin (IL)-6. Plasma (100 μL), collected pre and 12, 36 and 60 hours post damage was measured for IL-6 using a sandwich ELISA, purchased from R&D Systems, (Minneapolis, MN, USA). Plasma antioxidant capacity. The capacity to reduce ferric ions was determined using the ferric reducing antioxidant power (FRAP) assay as described by Benzie and Strain [27]. Briefly,

an aliquot of 8.5 μL of normal (non- deproteinized) serum was added to 275 μL of diluted FRAP reagent (pre-warmed to 37°C) using a microplate and the plates were incubated at 37°C for 30 mins before measuring

the absorbance at 595 nm using a plate reader (ELX 808 Ultra Microplate Reader (Bio-tek Instruments. Inc, USA)). The working FRAP reagent was prepared by mixing 10 volumes of 300 mmol/L acetate buffer, pH 3.6, with 1 volume of 10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol/L hydrochloric acid and with 1 volume of 20 mmol/L ferric chloride. A standard curve was prepared using different concentrations (200–2000 μmol/L) of FeSO4.7H2O. FRAP was calculated and expressed as either μmol/L or % of pre-treatment values. Statistical analyses Data were analyzed using Statistical Analysis Software (SAS) 9.1 for Windows (version 5.1.2600). Using a repeated measures analysis of variance (ANOVA), comparison between conditions (blueberries MK-0457 research buy and DCLK1 control) over time for each

measure (independent variable) were determined, providing levels of significance for Trial effect, Treatment effect, and interaction effect between Treatment and Trial. Where significance permitted, post-hoc tests were performed to identify significant differences at each time point. Represented values are means ± standard deviation (or standard error) for n = 10 at a 95% significance level (p = 0.05). Paired t-tests were used to determine order effects for performance measures and effort during the 300 maximal eccentric contractions of the quadriceps. Pearson’s Product Moment Correlation Coefficient’s were determined using SPSS 15.0 for Windows. This allowed us to investigate any relationships between certain variables (i.e. antioxidant activity with muscle performance measures) by giving an r-value between 0.0 and 1.00 (or −0.0 and −1.00). Results Intervention diet All subjects completed the study and there were no reported adverse effects from the dietary intervention. Performance (muscle function) Overall changes in volunteer’s physical performance following a strenuous exercise designed to cause muscle damage were evaluated by measuring the torque generated during a series of isometric, eccentric and concentric exercises over a 60 hour recovery period (Table 2).

6 × 107 to 1 7 × 108 CFU over 24 hours, (n = 3, Figure 1) This i

6 × 107 to 1.7 × 108 CFU over 24 hours, (n = 3, Figure 1). This indicates that Bdellovibrio effectively suppressed the population growth of P. tolaasii, most likely due to killing by predation. Figure 1 Reduction in P. tolaasii OD600 nm over 24 hours, in vitro , in the presence of Bdellovibrio bacteriovorus . Mean OD600nm of P. tolaasii

2192T samples in the absence AZD5363 manufacturer or presence of live B bacteriovorus HD100 added at 4 × 106 or 1.6 × 107 Plaque Forming Units (PFU) (n = 4). The MI-503 nmr increase in OD600nm in the absence of Bdellovibrio indicates P. tolaasii 2192T growth, while no increase in the presence of 4 × 106 or 1.6 × 107 B. bacteriovorus HD100 indicates inhibition of P. tolaasii 2192T growth. Error bars indicate 95% Confidence Intervals for each OD600nm value. Brown blotch lesion intensity was reduced by Bdellovibrioapplication onto mushrooms Given B. bacteriovorus HD100 was observed to suppress P. tolaasii 2192T growth in vitro, we reasoned that Nutlin-3 in vitro this effect might be replicated in a more natural environment. We first aimed to determine whether symptoms of P. tolaasii infection, a function of bacterial metabolism and growth, were reduced with Bdellovibrio treatment in a natural context. The intensity of lesions formed

by P. tolaasii 2192T on the post-harvest pileus surface of the cultivated button mushroom Agaricus bisporus was measured in the presence and absence of B. bacteriovorus HD100, as shown in Figure 2 . Mushroom pilei inoculated with P. tolaasii

2192T alone, in the absence of any treatment with B. bacteriovorus HD100, formed dark, wet surface lesions, the primary symptom of brown blotch disease, after 48 hours at 29°C (mean intensity MTMR9 = 0.019 1/PV ± 0.0005, n = 30). In contrast, pilei treated with a King’s Medium B control (the preferred growth medium of P. tolaasii) did not form these dark lesions (mean intensity = 0.012 1/PV ± 0.0005, n = 30); similarly, those treated with B. bacteriovorus HD100 alone, and not inoculated with P. tolaasii 2192T, also did not form dark lesions (mean intensity = 0.010 1/PV ± 0.0005, n = 30), so Bdellovibrio application itself did not have a significant adverse effect on the appearance of mushroom pilei. Figure 2 Lesion intensity on P. tolaasii -inoculated mushrooms in the presence and absence of Bdellovibrio . Lesion intensities on mushroom pilei under 5 different treatment conditions, detailed to the right of the graph. Each P tolaasii 2192T inoculation contained 1.7 × 106 CFU, and each B. bacteriovorus HD100 inoculation contained 2.9 × 106 PFU. Higher lesion intensity indicates a greater level of brown blotch disease symptoms and therefore a higher level of P. tolaasii infection. Horizontal black bars indicate the mean lesion intensity value for each treatment group. Student’s t-test of significance between B. bacteriovorus HD100 treated and non-treated mushrooms inoculated with P. tolaasii 2192T: **p < 0.01, ***p <0.001. Post-harvest mushrooms treated with B.

At the recruitment visit, the transdermal buprenorphine patch in

At the recruitment visit, the transdermal buprenorphine patch in these patients was replaced by a 25 μg/h transdermal fentanyl patch, positioned at different skin LY2606368 research buy site on the thorax, arm or back. The BTDS group were patients at the screening visit who had taken fentanyl TTS 75 μg/h and suffered side-effects and refractory pain and had taken this dose continuously during the pre-recruitment week. The transdermal fentanyl patch in these patients was replaced by a 52.5 μg/h transdermal buprenorphine patch, positioned at different skin site on the thorax, arm

or back. Rescue medication with 20 mg of immediate-release oral morphine was prescribed to each patient up to three times a day. At the end of the recruitment visit (V1) all the patients were asked to return after one week for selleck the first control visit (V2), and to continue keeping their daily diaries. Assessment of analgesic efficacy Mean weekly pain on the basis of the VAS scores in diaries (VAS 0 = no pain to VAS 100 = intolerable pain) was recorded throughout the 4 week period. The Present Pain Intensity (PPI, 0 = no pain, 1 = mild, 2 = discomforting, 3 = distressing, 4 = horrible, 5 = excruciating) and Pain Rating Index (PRI) were assessed during each visit from V1 to V4. The PRI was taken from the Short-Form Mc Gill Pain Questionnaire and comprised 15 items investigating both the sensorial (11 items) and the emotional sphere

of pain (4 items) with a score from 0 to 3 for each item (0–45). In all cases the necessity of rescue medication was registered as milligrams

of oral morphine per day. Another parameter taken into consideration was the patients’ satisfaction with the new therapy. It was evaluated by means of the simple question: “”Are you satisfied with your analgesic treatment?”" The patients could answer only “”Yes/No”". The primary efficacy measure was pain reduction as recorded by patients both in a daily dairy using VAS and during the visits by PPI and PRI. The secondary efficacy measure was the reduction of rescue mediation consumption as milligrams of IR oral morphine per day. Assessment of adverse events In all patients, the presence (Yes) or absence (No) of AEs was evaluated and recorded in response to questions posed for nausea and/or vomiting, constipation, and dysphoria. The level of sedation was evaluated by a 4-point scale (0 = no sedation, Branched chain aminotransferase 1 = slight sedation, 2 = moderate sedation, 3 = severe sedation). Statistical analysis For each of the two treatment groups, a paired Student t test was used to compare the mean values of the primary efficacy parameters (VAS, PPI, and PRI) and rescue medication consumption for the same patients measured at Visits 2, 3, and 4 compared to baseline values (Visit 1). A Student t test for independent variables was used to compare the two independent treatment groups. Results In total, 40 Caucasian patients were screened and 32 were enrolled. All the enrolled patients completed the study.

cruzi ubiquitin intergenic region (TcUIR – 278 bp) and the casset

cruzi ubiquitin intergenic region (TcUIR – 278 bp) and the cassette containing the T. cruzi Dm28c pol

I promoter (617 bp) followed by a TcUIR and a hexahistidine tag were synthesized in vitro (GenScript, Piscataway, USA) (Figure 6). The third DNA segment, represented by the RfA cassette (Invitrogen) (1711 bp), was PCR-amplified from pCR-Blunt and was inserted into pBluescript(r) II KS+. Restriction sites were placed in specific positions of the sequence, to insert the various cassettes or remove some segments of DNA, such that new segments could be inserted for the construction of new vectors. Figure LY3023414 order 6 Schematic drawing showing the vector construction steps. The elements shown are the neomycin (NEO) and hygromycin (HYGRO) resistance genes, the T. cruzi intergenic region from ubiquitin locus (TcUIR), the attachment sites for Gateway(r) recombination (attB1, attB2, attR1 and attR2), the chloramphenicol resistance gene (CmR), the gene for negative selection during cloning (ccdB), the fusion tags (6xhis, GFP, YFP, CFP, TAP and c-myc) and the ribosomal promoter (PR). In A, the steps for vectors construction are represented. In B, the

vector reading frame with start and stop codons are shown. The plasmid containing the three cassettes was named pTc6HN. We constructed some derivative vectors from pTc6HN, by replacing the polyhistidine tag with a TAP tag, the sequence of the c-myc epitope or with genes coding CHIR-99021 purchase for fluorescent proteins (EGFP, CFP and YFP). All tags were amplified from plasmid vectors with the exception of c-myc, which was synthesized as two single-strand oligonucleotides (Additional file 5 – Table S2). For c-myc strands hybridization, 1.3 μg of each strand was used. The single strands

were incubated in 10 mM NaCl Palmatine buffer at 95°C for 10 min. The temperature was then slowly lowered to allow hybridization. After N-terminal tag insertion, the original vectors were identified as pTcTAPN, pTcGFPN, pTcCFPN, pTcYFPN, pTcMYCN and pTcGFPH (neomycin resistance was replaced with hygromycin resistance in pTcGFPN). All of the constructs were sequenced by the commercial Macrogen facility (Macrogen, Seoul, Korea). The analysis of ab1 files was performed on SeqMan software (DNASTAR, Inc., Madison, USA). The sequences are available in GenBank under accession numbers HM162840 (pTcYFPN), HM162841 (pTcMYCN), HM162842 (pTcTAPN), HM162843 (pTcGFPN), HM162844 (pTcGFPH), HM162845 (pTcCFPN) and HM162846 (pTc6HN). Oligonucleotides used for the construction and sequencing of vectors are listed in Additional file 5 – Table S2 and Additional file 6 – Table S3, respectively. Validation of vectors Five T. cruzi genes were used in the validation process: TcRab7 (Tc00.1047053508461.270), PAR 2 (Tc00.1047053511215.119), a putative centrin (Tc00.1047053506559.380), Tcpr29A (Tc00.1047053506167.40), and TcrL27 (Tc00.1047053506817.30).

J Mater Chem 2011, 21:6020 CrossRef 14 Zhang SB, Wei SH, Zunger

J Mater Chem 2011, 21:6020.CrossRef 14. Zhang SB, Wei SH, Zunger A: Intrinsic n -type versus p -type doping asymmetry and the defect physics of ZnO. Phys Rev B 2001, 63:075205.CrossRef 15. Zhang Y, Wang LW, Mascarenhas A: “”Quantum coaxial cables”" for solar energy harvesting. Nano Lett 2007, 7:1264.CrossRef 16. Aranovich JA, Golmayo D, Fahrenbruchn AL, Bube RH: Photovoltaic properties of ZnO/CdTe heterojunctions prepared by spray pyrolysis. J Appl Phys 1980, 51:4260–4268.CrossRef 17. Jasieniak J, MacDonald BI, Watkins SE, Mulvaney P: Solution-processed sintered nanocrystal solar cells via layer-by-layer assembly. Nano Lett 2011, 11:2856–2864.CrossRef 18. Panthani MG, Kurley JM, Crisp RW, Dietz TC, Ezzyat T, Luther JM, Talapin

DV: High efficiency solution processed sintered CdTe nanocrystal solar cells: the role of interfaces. Nano Lett 2014, 14:670–675.CrossRef 19. Lee SH, Zhang XG, Parish CM, Lee HN, Smith DB, He Y, Xu J: Nanocone

tip-film GANT61 clinical trial solar cells with efficient charge transport. Adv Mater 2011, 23:4381–4385.CrossRef 20. Michallon J, Zanuccoli M, Kaminski A, Consonni V, Morand A, Bucci D, Emieux F, Szambolics H, Perraud S, Semenikhin I: Comparison of optical properties of Si and ZnO/CdTe core/shell nanowire arrays. Mater Sci Eng B 2013, 178:665.CrossRef 21. Lévy-Clément C, Katty A, Bastide S, Zenia F, Mora I, Munoz-Sanjose V: A new CdTe/ZnO columnar composite film for Eta -solar cells. Phys E 2002, 14:229.CrossRef 22. Tena-Zaera R, Katty A, Bastide S, Lévy-Clément C, O’Regan B, Muñoz-Sanjosé V: ZnO/CdTe/CuSCN: a promising heterostructure to act as inorganic eta -solar cell. Thin Solid Films 2005, 483:372–377.CrossRef 23. Aga RS, Jowhar D, Blebbistatin Ueda A, Pan Z, Collins WE, Mu R, Singer KD, Shen J: Enhanced photoresponse in ZnO nanowires decorated with CdTe quantum dot. Appl Phys Lett 2007, 91:232108.CrossRef 24. Cao X, Chen P, Guo Y: Decoration of textured ZnO nanowires array with CdTe quantum dots: enhanced light-trapping effect and photogenerated

charge separation. J Phys Chem C 2008, second 112:20560–20566.CrossRef 25. Aga RS, Gunther D, Ueda A, Pan Z, Collins WE, Mu R, Singer KD: Increased short circuit current in organic photovoltaic using high-surface area electrode based on ZnO nanowires decorated with CdTe quantum dots. Nanotechnol 2009, 20:465204.CrossRef 26. Chen HM, Chen CK, Chang YC, Tsai CW, Liu RS, Hu SF, Chang WS, Chen KH: Quantum dot monolayer sensitized ZnO nanowire-array photoelectrodes: true efficiency for water splitting. Angew Chem 2010, 122:6102–6105.CrossRef 27. Wang X, Zhu H, Xu Y, Wang H, Tao Y, Hark S, Xiao X, Li Q: Aligned ZnO/CdTe core-shell nanocable arrays on indium tin oxide: synthesis and photoelectrochemical properties. ACS Nano 2010, 4:3302.CrossRef 28. Chen ZH, Liu CP, Wang HE, Tang YB, Liu ZT, Zhang WJ, Lee ST, Zapien JA, Bello I: Electronic structure at the interfaces of vertically aligned zinc oxide nanowires and sensitizing layers in photochemical solar cells. J Phys D Appl Phys 2011, 44:325108.CrossRef 29.

Conclusion The potential for contracting a microbial pathogen is

Conclusion The potential for contracting a microbial pathogen is highest within a hospital environment and hospital acquired infections are significant contributors to morbidity and mortality. Despite the lack of direct evidence to prove that environmental contaminants are responsible for hospital acquired infections, there is an increasing evidence suggesting that the environment may act as a reservoir for at least some of the pathogens causing nosocomial infections. This

work showed that many different bacterial species can persist on surfaces for months and years. The level of bacterial contamination was related with the this website presence of humidity on selleck inhibitor the surface, and tap water (biofilm) was a point of dispersion of bacterial species, usually involved in nosocomial infections as Pseudomonas

aeruginosa, Stenotrophomonas maltophilia and Enterococcus feacalis. Their presence in the environment, as it seems to be pointed by the analysis of the diversity, increases the risk of transmission to the different materials during hand manipulation. Methods Sampling (ICU, Medicine I, Medicine II and Urology) The study was carried out at the Hospital de Faro, Portugal, which serves a resident population of about 253 thousand people and this value may double or triple the population seasonally (in http://​www.​hdfaro.​min-saude.​pt/​site/​index.​php). Between January 2010 and

September, 2011, the hospital was evaluated 12 times (sampled each two months) and four different wards were investigated for environmental contamination of the following surfaces and equipment: sink, tap (biofilm), surface countertop and workbench of the nurses area, shower (and handrail), bedside table, handrail bed (including bed), serum support, oxygen flask, stethoscope, equipment at bedside, other medical equipment, tray used by nurses, hand gel/soap, table (meal and work). The equipment considered in this study is included in the category of noncritical hospital objects and surfaces. These items have been Tau-protein kinase said to pose no risk to patients, nevertheless, these surfaces and equipment are frequently touched by hand can contribute to the spread of healthcare-associated pathogens as Pseudomonas aeruginosa, Staphilococus aureus, or Acinetobacter baumanii. The evaluation was performed in wards of the Medical Unit I and II, Urology and Intensive Care Unit. Samples were collected in the wards, always in the same period of the day, at the end of the morning and during lunch time, after the medical visits and treatments, and also sometime after a ward cleaning. Swabs were used for collecting the organisms present in an area of 10X10 cm of each surface. Taps were sampled by removing the biofilm.

We thank Mari Nyyssönen for help with the microarray experiments,

We thank Mari Nyyssönen for help with the microarray experiments, and thank Jizhong Zhou and Liyou Wu for providing the microarrays. The work was supported by a grant from U.S Department of Energy, Office of Science, DE-FG02-04ER63923 and by the WCU (World Class University) program through the National Research Foundation

of Korea funded by the Ministry of Education, Science and Technology (R33-10076). References 1. Villemur R, Lanthier M, Beaudet R, Lépine F: The Desulfitobacterium genus. FEMS Microbiology Reviews 2006, 30:706–733.PubMedCrossRef 2. Kunapuli U, Jahn MK, Lueders T, Geyer R, Heipieper HJ, Meckenstock RU: Desulfitobacterium aromaticivorans sp. nov. and Geobacter toluenoxydans sp. nov., iron-reducing bacteria capable of anaerobic degradation of monoaromatic hydrocarbons. PI3K inhibitor Int J Syst Evol Microbiol 2010,60(3):686–695.PubMedCrossRef 3. Maymo-Gatell GSK458 in vitro X, Chien Y, Gossett JM, Zinder SH: Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 1997, 276:1568–1571.PubMedCrossRef 4. Madsen T, Licht D: Isolation and characterization of an

anaerobic chlorophenol-transforming bacterium. Appl Environ Microbiol 1992, 58:2874–2878.PubMed 5. Christiansen N, Ahring BK: Desulfitobacterium hafniense sp. nov., an anaerobic, reductively dechlorinating bacterium. Int J Syst Bacteriol 1996, 46:442–448.CrossRef 6. Niggemyer A, Spring S, Stackebrandt E, Rosenzweig RF: Isolation and characterization of a novel As(V)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacterium . Appl Environ Microbiol 2001, 67:5568–5580.PubMedCrossRef 7. Lie TJ, Godchaux W, Leadbetter ER: Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria ( Desulfitobacterium spp.) and sulfate-reducing bacteria: effects of inhibitors of sulfidogenesis. Pazopanib purchase Appl Environ Microbiol 1999,65(10):4611–4617.PubMed 8. Suyama A, Iwakiri R, Kai K, Tokunaga T, Sera

N, Furukawa K: Isolation and characterization of Desulfitobacterium sp. strain Y51 capable of efficient dechlorination of tetrachloroethene and polychloroethanes. Biosci Biotechnol Biochem 2001, 65:1474–1481.PubMedCrossRef 9. Nonaka H, Keresztes G, Shinoda Y, Ikenaga Y, Abe M, Naito K, Inatomi K, Furukawa K, Inui M, Yukawa H: Complete genome sequence of the dehalorespiring bacterium Desulfitobacterium hafniense Y51 and comparison with Dehalococcoides ethenogenes 195. J Bacteriol 2006,188(6):2262–2274.PubMedCrossRef 10. Suyama A, Yamashita M, Yoshino S, Furukawa K: Molecular characterization of the PceA reductive dehalogenase of Desulfitobacterium sp. Strain Y51. J Bacteriol 2002,184(13):3419–3425.PubMedCrossRef 11. Juhala RJ, Ford ME, Duda RL, Youlton A, Hatfull GF, Hendrix RW: Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages. Journal of Molecular Biology 2000,299(1):27–51.PubMedCrossRef 12.

Free PHB granules, i e PHB granules that were not in contact

Free PHB granules, i.e. PHB granules that were not in contact AZD1390 nmr to the nucleoid region were not observed. Apparently, constitutive over-expression of phaM resulted in formation of an increased number of small and nucleoid-attached PHB granules. If PhaM is responsible

for the formation of small granules and for the close contact to the nucleoid region, deletion of phaM should have a phenotype. In fact, R. eutropha ∆phaM cells accumulated only very few (0–2) PHB granules that were significantly larger in diameter than those of the phaM over-expressing mutant or of the wild type (Figure 5). Since the diameters of PHB granules of the ∆phaM strain were considerably larger even at early time points a precise analysis whether or not the granules were attached to the nucleoid region was difficult. In most ∆phaM cells the PHB granules were still located close to the nucleoid; however, BLZ945 ic50 at least in some cells a detachment of PHB granules from the nucleoid region could not be excluded for the wild type or for the phaM over-expressing strain. A clear decision whether the absence of PhaM resulted in detachment from the nucleoid can, however, not be made. Since

R. eutropha expresses at least one other protein with DNA-binding and PHB-binding property (PhaR) [30, 31] it might be that PhaR also contributes to association of PHB with DNA. In summary, our data on mutants with altered expression of PhaM clearly show that number, diameter and subcellular localization of PHB granules depends on the presence and concentration of PhaM. Time course of formation and localization of PHB granules in R. eutropha over-expressing PhaP5 PhaP5 had previously been identified as a phasin in R. eutropha by its in vivo interaction with PhaP2 and other phasins [22]. Remarkably, PhaP5 also interacted

with PhaM. To investigate the influence of PhaP5 on RANTES PHB granule formation the phaP5 gene was cloned in a broad host range plasmid (pBBR1MCS-2) under control of the strong and constitutive phaC1 promotor (PphaC), transferred to R. eutropha H16 and HF39 via conjugation and investigated for PHB granules formation and localization under PHB permissive conditions (Figure 6). In case of strain HF39 a eypf-phaP5 fusion was cloned and used to confirm localization of PhaP5 on the PHB granules by fluorescence microscopy. Controls showed that free eYfp is a soluble protein in R. eutropha (Figure 7). Figure 7 Fluorescence microscopical (FM) investigation of R. eutropha H16 (pBBR1MCS-2-P phaC – eyfp -c1) with over-expression of eYfp (a); R. eutropha H16 (pBBR1MCS-2-P phaC – phaP5 ) with over-expression of PhaP5 (b), and R. eutropha H16 (pBBR1MCS-2-P phaC -eyfp- phaP5 ) with over-expression of eYfp-PhaP5 fusion (c) at various stages of PHB formation.

pneumoniae putative surface protein Orf50 53176-54000 E S pneumo

pneumoniae putative surface protein Orf50 53176-54000 E S. pneumoniae DNA replication protein Orf72 79231-80088 E S. pneumoniae putative bacteriocin Orf51 53993-54478 E S. pneumoniae DUF 3801 Orf73 80162-80773 E S. pneumoniae Predicted transcriptional regulator Orf52 54475-55209 E S. pneumoniae phage antirepressor protein Orf74 80766-81749 E S. pneumoniae Protein with unknown function Orf53 55202-56890 E S. pneumoniae TraG/TraD family protein Orf75 82268-82621 E S. pneumoniae transcriptional regulator, ArsR family Orf54 57454-58486 E – DUF Entospletinib 318 Predicted Permease (HHPred) Orf76 82696-83940 E S. pneumoniae major facilitator superfamily MFS_1 Orf55 59048-59398 D C. fetus glyoxalase

family protein Orf77 83927-84403 E S. pneumoniae toxin-antitoxin system, toxin component, GNAT domain protein Orf56 59411-59938 D C. fetus transcriptional regulator Orf78 84758-86491 E S. pneumoniae DNA topoisomerase III Orf57 59988-61910 D C. fetus tetracycline resistance R406 clinical trial protein Orf79 86484-87449 E S. pneumoniae possible DNA (cytosine-5-)-methyltransferase Orf58

62225-63082 D C. fetus aminoglycoside 6-adenylyltransferase (AAD(6) Orf80 87436-95079 E S. pneumoniae superfamily II DNA and RNA helicase Orf59 63575-64348 E S. pneumoniae replication initiator/phage Orf81 95123-95779 E S. pneumoniae putative single-stranded DNA binding protein Orf60 64345-65172 E S. pneumoniae replicative DNA helicase Orf82 95939-96841 E S. pneumoniae transcriptional regulator, XRE family Orf61 65314-65814 E S. pneumoniae Cyclooxygenase (COX) TnpX site-specific recombinase family protein Orf83 97071-98282 E S. pneumoniae transporter, major facilitator family/multidrug resistance protein 2 Orf62 65938-66399

E S. pneumoniae flavodoxin Orf84 C 99739-98462 E S. pneumoniae relaxase/type IV secretory pathway protein VirD2 Orf63 66817-67302 E S. pneumoniae putative conjugative transposon protein Orf85 C 101169-99795 E S. pneumoniae conjugal transfer relaxosome component TraJ Orf64 67299-68033 E S. pneumoniae phage antirepressor protein Orf86 C 101403-100321 E S. pneumoniae toxin-antitoxin system, toxin component, Fic family Orf65 68026-69816 E S. pneumoniae TraG/TraD family protein/putative conjugal transfer protein Orf87 C 101878-101396 E S. pneumoniae putative membrane protein Orf66 70395-70706 E S. pneumoniae putative single-strand binding protein Orf88 C 102435-101887 E S. pneumoniae putative toxin-antitoxin system, toxin component Orf67 70934-71797 E S. pneumoniae conjugative transposon membrane protein Orf89 C 102845-102444 E S. pneumoniae regulator/toxin-antitoxin system, antitoxin component Orf68 72099-72509 E S. pneumoniae conjugative transposon membrane protein Orf90 103034-103555 E S. pneumoniae conserved hypothetical protein Orf69 72580-74823 E S. pneumoniae type IV conjugative transfer system protein Orf91 103825-104235 E S. pneumoniae sigma-70, region 4 Orf70 74831-77410 E S. pneumoniae conjugative transposon cell wall hydrolase/NlpC/P60 family Orf92 104966-106712 E S.

Conventional photolithography and photoresist stripping processes

Conventional photolithography and photoresist stripping processes were employed to construct channels with

the desired depth. A silicon (Si) wafer was cleaned in H2SO4:H2O2 solution LY3009104 (volume ratio of 10:1) at 120°C for 10 min, followed by deionized water (DI) for 4 cycles, then HF:H2O solution (1:50) at 22°C for 1 min and DI water for 4 cycles, and finally spin-dried in hot N2 gas for 15 min. Then, the Si wafer was processed by hexamethyldisiloxane (HMDS) coating and positive photoresist HPR 504 spin-coated at 4,000 rpm for 30 s. The wafer was soft-baked on a hot plate at 110°C for 60 s before exposing to UV via the Mask Aligner (SUSS Microtec MA6-2, Garching Germany) for 5 s. The photoresist was developed using FHD-5 for 60 s and post-baked on a hot plate at 120°C for 60 s. The micropatterns were successfully defined at this stage. The Si wafer was then RG7112 research buy etched by a DRIE machine (Surface Technology Systems, Newport, UK) and followed by photoresist stripping in PS210 Photoresist Asher (PVA Tepla AG, Kirchheim, Germany) for 25 min. After constructing the microchannels, 10 nm of thermal oxide was grown using a diffusion furnace to form silica on the channel wall. After drilling the inlets and outlets on the Si chip by a mechanical driller, the chip has to be sealed to form a closed channel. A thin film of polydimethylsiloxane (PDMS) was applied for such purpose due to the good adhesion between PDMS and the Si chip. PDMS was formulated

from Sylgard 184 silicone elastomer mixture (Dow Corning Corporation, Midland, MI, USA) at a weight ratio of base:curing agent = 10:1. Then, it was poured onto a Si wafer with saline coating on the surface and pressed against a cleaned glass slide. After curing PDMS in an oven at 60°C for 2 h, the microchip was constructed by pressing the Si chip against the glass slide Selleckchem Nutlin-3 with the thin layer of PDMS on its surface. The fabricated microchip is shown in Figure  2a. The microreactor is comprised of two microchannels: channels A and B with a width of 300 μm and a depth of 12 μm and an array (20 channels) of 1D nanochannels that connected the two microchannels

to demonstrate the injection process. It is not necessary to adopt 20 nanochannels. One can increase or decrease the number according to their applications. Fewer nanochannels will result in higher precision, and more nanochannels will give a higher throughput. The inset (a1) in Figure  2a illustrates the multilayer structure showing the PDMS, the silicon chip, and the glass slide. Another inset (a2) shows the structure of the two microchannels connected by the nanochannel array that is highlighted by the green dashed square. When the electric field across channel A and channel B was applied, fluid flowed from channel A to channel B through the nanochannel array as indicated by the green arrow in the same figure. The enlarged scanning electron microscopy (SEM) image of the nanochannel array is shown in Figure  2b. The channel width observed was 10 μm.