(http://​www.​ncbi.​nlm.​nih.​gov/​).

Strain typing The phylogenetic group of the ESBL-producing E. coli was determined by a multiplex PCR assay [18]. Isolates belonging to phylogenetic group B2 were screened with a Selleck Doramapimod previously established PCR-based method to identify the O25b subtype [19]. Furthermore, multilocus sequence typing (MLST) using the scheme of the Institut Pasteur, Paris, France (http://​www.​pasteur.​fr/​mlst) was used to confirm that CTX-M-15-producing E. coli O25b belonged to the international clone ST131 [19]. Genetic relatedness of the ESBL-producing strains was studied by PFGE following extraction of genomic DNA and digestion with XbaI PFGE according to a standard protocol using a GenePath system (Bio-Rad). PFGE banding profiles were compared digitally using Fingerprint II software (Bio-Rad) and relatedness was calculated using the unweighted pair group method with arithmetic KPT-330 ic50 mean (UPGMA) algorithm with similarity of bands using the Dice similarity indices. Isolates were considered to belong to the same PFGE cluster if their Dice similarity

index was >80% [20]. Transfer of ESBL resistance determinants and plasmid analysis Transfer of ESBL encoding genes by conjugation was performed by matting-out assays using E. coli J53-2 RifR or E. coli HB101 StrR as recipient strains. Transconjugants were selected Fedratinib molecular weight on MH agar containing rifampin (250 μg/mL) or streptomycin (50 μg/mL) plus ceftazidime or cefotaxime (2 μg/ml). When plasmids were not transferable by conjugation, a transformation experiment was assayed. Plasmid DNA obtained using the QIAprep Spin Miniprep kit (Qiagen) were electroporated into E. coli DH10B (Invitrogen). Transformants were selected on MH agar plates supplemented with ceftazidime (2 μg/mL) or cefotaxime (2 μg/mL). Plasmids were classified according to their incompatibility group using the PCR replicon-typing scheme described previously [21]. Detection of virulence factors and plasmid addiction systems For the ESBL-producing C-X-C chemokine receptor type 7 (CXCR-7) isolates, 17 virulence-associated genes were sought as previously described: fimH (type 1 fimbriae), papG (P fimbriae adhesion) alleles I, II and III, papC, sfa/focDE (S and F1C

fimbriae), afa/draBC (Dr-binding adhesions), iha (adhesion siderophore), hra (heat(resistant agglutinin), iutA (aerobactin receptor), fyuA (yersiniabcatin receptor), cnf-1 (cytotoxic necrotizing factor type 1), hlyA (α-hemolysin), sat (secreted autoreceptor toxin), kpsMT II (group II capsule), traT (serum resistance-associated) and pheR (phenylalanine tRNA, site of insertion from PAI V) [22]. For E. coli recipient strains, seven plasmid addiction system PemK–PemI (plasmid emergency maintenance), CcdA–CcdB (coupled cell division locus) RelB–RelE (relaxed control of stable RNA synthesis), ParD–ParE (DNA replication), VagC-VagD (virulence-associated protein), Hok–Sok (host-killing) and PndA–PndC (promotion of nucleic acid) were sought by PCR as described previously [7].

New York: McGraw-Hill; 1991:563–569. 47. Boleij A, Schaeps RM, Tjalsma H: Association between Streptococcus bovis and colon cancer. J Clin Microbiol 2009, 47:516.PubMedCrossRef 48. Lee RA, Woo PC, To AP, Lau SK, Wong SS, Yuen KY: Geographical difference of Tucidinostat purchase disease association in Streptococcus bovis bacteraemia. J Med Microbiol 2003, 52:903–908.PubMedCrossRef Selonsertib chemical structure 49. Luk WK, Liu CL, Yuen KY, Wong SS, Woo PC, Fan ST: Biliary tract infection due

to bile-soluble bacteria: an intriguing paradox. Clin Infect Dis 1998, 26:1010–1012.PubMedCrossRef 50. Vaska VL, Faoagali JL: Streptococcus bovis bacteraemia: identification within organism complex and association with endocarditis and colonic malignancy. Pathology 2009, 41:183–186.PubMedCrossRef 51. Tripodi MF, Adinolfi LE, Ragone E, Durante Mangoni E, Fortunato R, Iarussi D, Ruggiero G, Utili R: Streptococcus bovis endocarditis and its association with find more chronic liver disease: an underestimated risk factor. Clin Infect Dis 2004, 38:1394–1400.PubMedCrossRef 52. Osawa R, Sasaki E: Novel observations

of genotypic and metabolic characteristics of three subspecies of Streptococcus gallolyticus. J Clin Microbiol 2004, 42:4912–4913.PubMedCrossRef 53. Hsu WH, Yu FJ, Chuang CH, Chen CF, Lee CT, Lu CY: Occult colon cancer in a patient with diabetes and recurrent Klebsiella pneumoniae liver abscess. Kaohsiung J Med Sci 2009, 25:98–103.PubMedCrossRef 54. Hiraoka A, Yamashita Y, Uesugi K, Koizumi Y, Yamamoto Y, Doi H, Hasebe A, Ichikawa S, Yano M, Miyamoto Y, et al.: Three cases of liver abscesses complicated with colon cancer without liver metastasis: importance of screening for digestive disease.

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The effect of the crystal plane orientation on the friction-induced nanofabrication was mainly attributed to the different mechanical Apoptosis inhibitor behaviors and bond structures of the various silicon crystal planes. The main conclusions can be summarized as below. (1) Friction-induced nanofabrication can be realized on Si(100), Si(110), and Si(111) surfaces, respectively. The crystal plane orientation has a significant

effect on the hillock formation on silicon surface. Under the same loading condition, the highest hillock was generated on Si(100), while the lowest hillock was formed on Si(111) either in air or in vacuum.   (2) The mechanical performance of silicon shows a strong effect on the hillock formation on various silicon crystal planes. The crystal plane with the lower elastic modulus can lead to larger pressed volume, which facilitates more deformation in silicon matrix and higher hillock.   (3) The structures of Si-Si bonds play a key role in the hillock formation on various silicon

crystal planes. High density of dangling bonds can cause much instability, facilitating the formation of more amorphous silicon and high hillock during nanoscratching.   Acknowledgment The authors are grateful for the financial support from the National Basic Research Program (2011CB707604), Natural Science PI3K Inhibitor Library in vitro Foundation of China (90923017 and 51175441). References 1. Tanaka M: An industrial and applied review of new BCKDHB MEMS selleck products devices features. Microelectron Eng 2007, 84:1341–1344.CrossRef 2. Ko WH: Trends and frontiers of MEMS. Sens Actuators A 2007, 136:62–67.CrossRef 3. Cui Z: Micro-nanofabrication

Technologies and Applications. Beijing: Higher Education Press; 2008. 4. Lin BJ: Optical lithography – present and future challenges. Comptes Rendus Physique 2006,7(8):858–874.CrossRef 5. Cerofolini G (Ed): Nanoscale Devices: Fabrication, Functionalization, and Accessibility from the Macroscopic World. Heidelberg: Springer; 2009. 6. Pires D, Hedrick JL, Silva AD, Frommer J, Gotsmann B, Wolf H, Despont M, Duerig U, Knoll AW: Nanoscale three-dimensional patterning of molecular resists by scanning probes. Science 2010, 328:732–735.CrossRef 7. Yu BJ, Dong HS, Qian LM, Chen YF, Yu JX, Zhou ZR: Friction-induced nanofabrication on monocrystalline silicon. Nanotechnology 2009, 20:465303.CrossRef 8. Ebrahimi F, Kalwani L: Fracture anisotropy in silicon single crystal. Mater Sci Eng A 1999, 268:116–126.CrossRef 9. Wang MH, Wang W, Lu ZS: Anisotropy of machined surfaces involved in the ultra-precision turning of single-crystal silicon—a simulation and experimental study. Int J Adv Manuf Technol 2012, 60:473–485.CrossRef 10. Gatzen HH, Beck M: Investigations on the friction force anisotropy of the silicon lattice. Wear 2003, 254:1122–1126.CrossRef 11. Łysko JM: Anisotropic etching of the silicon crystal-surface free energy model. Mat Sci Semicon Proc 2003, 6:235–241.CrossRef 12.

Bars represent mean values ± SEM of three independent experiments done in triplicate. For statistical analysis, samples were compared against control transfected cells by one-tailed Mann-Whitney U-test; *, p < 0.001. (C) 293 cells were transfected with constructs encoding human CEACAM1 isoform containing a short cytoplasmic domain (hCEA1), the corresponding murine

isoform (mCEA1) or an empty control vector. Cells were infected with fluorescein-labelled Opa-negative (Ngo Opa-) or OpaCEA-expressing AG-881 supplier N. gonorrhoeae (Ngo OpaCEA) at an MOI of 30 for 2 h. The uptake index was determined by flow cytometry as described in Material and Methods. Bars represent mean values ± SEM of three independent experiments. CEACAM engagement by OpaCEA-expressing N. gonorrhoeae was evaluated through functional analysis of bacterial uptake by the transfected cells. In a first set of experiments, we used an antibiotic protection assay that is based on recovery of viable intracellular bacteria after treatment of the infected cells with gentamicin, an antibiotic that kills extracellular bacteria. In the case of non-opaque gonococci, only very low numbers of bacteria were recovered from murine or human CEACAM1-4S expressing cells similar to the numbers isolated from control transfected cells (Fig. 4B). In contrast,

upon infection with OpaCEA-expressing N. gonorrhoeae, 50 – 100 times more bacteria were recovered from cells expressing human CEACAM1 (Fig. 4B). Similar to what has been observed before [18], both the short and the long isoform of human CEACAM1-4 were able to mediate efficient uptake of the pathogens (Fig. 4B). Importantly, murine CEACAM1-4S was LY3039478 not able to mediate internalization of OpaCEA-expressing N. gonorrhoeae consistent with the lack of bacterial binding to the Igv-like amino-terminal domain of murine CEACAM1 (Fig. 4B). To further confirm that full length murine CEACAM1-4S does not mediate bacterial internalization, we analysed transfected cells upon infection with fluorescein-labeled bacteria by an established flow cytometry

method [21]. Addition of trypan blue quenches the fluorescence emitted by extracellular bacteria, resulting in cell-associated fluorescence signals derived Carnitine palmitoyltransferase II exclusively from intracellular bacteria. In line with the results of the antibiotic protection assay, non-opaque N. gonorrhoeae was not internalized, whereas OpaCEA-expressing bacteria were taken up by cells transfected with human CEACAM1-4S (Fig. 4C). Moreover, cells expressing murine CEACAM1-4S did not harbor intracellular bacteria, further corroborating the notion that OpaCEA proteins of N. gonorrhoeae do not functionally engage CEACAM1 orthologues of other mammalian species (Fig. 4C). Microscopic Epoxomicin nmr determination of Neisseria gonorrhoeae internalization via CEACAM1 To finally demonstrate the selective binding and internalization of OpaCEA-expressing N. gonorrhoeae by human, but not murine CEACAM1, we analysed infected samples with confocal fluorescence microscopy.

In SA treatments, PPO response with or without stress conditions was irregular. Although, PPO activity

was comparatively lesser in SA+EA plants, it followed the same trend as we observed in EA plants. P. resedanum association and SA-dependent responses under CDK inhibitor abiotic stress We also assessed the effect of endophytic elicitation with or without the treatment of SA on endogenous SA level. The results showed that SA was significantly Angiogenesis inhibitor low in non-stressed control. However, the stress periods has increased the endogenous SA levels (Figure 7). Similarly, in endophyte-associated plants, the endogenous SA was significantly higher than control under normal growth conditions. While after 2 days stress, its level in-significantly increased. The 4 and 8 days stress significantly increased SA contents in EA plants. This level was significantly higher than that of control and SA treated plants. In sole SA treatments, the plant synthesized find more low level of SA without any stress. However, upon 2 and 4 days stress, the SA level increased significantly while after 8 days, it decreased. In case of SA+EA plants, the endogenous SA followed the

same trend as we noticed in sole SA treatments, however, the quantity of SA synthesized was significantly higher during similar conditions (Figure 7). The overall SA biosynthesis pathway activation in sole SA was lower than EA and SA+EA plants. The EA and SA+EA plants have significantly activated endogenous SA biosynthesis 4-Aminobutyrate aminotransferase with or without stress conditions. Figure 7 Endogenous salicylic acid (SA) synthesis of pepper plants inoculated with or without P. resedanum under osmotic stress and normal growth conditions.

EA = infected with P. resedanum; SA = treated with SA; SA+EA = endophytic fungal associated plants treated with SA. NST, 2-DT, 4-DT and 8-DT represent non-stressed, 2, 4 and 8 days drought stressed plants respectively. The different letter (s) in each stress period showed significant difference (P<0.05) as evaluated by DMRT. Discussion Endophyte-association helps in biomass recovery The results of the present study support and give additional information on the mechanism of endophyte’s ameliorative potential during abiotic stress to crop plant. The results revealed that endophyte-association rescued growth of pepper plants during stress by increasing shoot length. Plant-fungus relationship has been proclaimed a pivotal source for plant growth and development [30, 31]. Endophytic fungi have been regarded as plant protectant and growth regulator during normal and extreme environmental conditions [15–20, 31–33]. Various novel endophytic fungal species like Piriformospora indica, Neotyphodium sp., Curvularia protuberate, and Colletotrichum sp. etc [19, 20, 31, 32, 34] have been known to improve plant growth during abiotic stress conditions. Penicillium species have been known as a vital source for bioactive secondary metabolites [35].

Regardless of the mechanism, higher bacterial MP under MRG conditions may contribute towards increased survival under the conditions examined. Another important cellular property examined in this study is membrane integrity (MI). Like MP, higher MI is strongly correlated with bacterial viability [61]. Higher MI was found under MRG conditions for both E. coli and S. aureus

grown in LB, but not in M9 minimal media and diluted LB, respectively. Dramatically https://www.selleckchem.com/products/Trichostatin-A.html higher percentages of dead cells were found under normal gravity conditions in rich media. Interestingly, in congruence with earlier E. coli gene expression studies [33], MP and MI observations are consistent with the observation that E. coli grown under MRG conditions exhibits enhanced ability to survive

sub-lethal doses of antimicrobial agents [13, 22]. As these stress- survival assays require growth Selonsertib nmr of E. coli in culture, it is possible that differences in MP and MI account for bacterial phenotypes observed under MRG conditions. Conclusions Documented responses to MRG or microgravity conditions include large scale changes in gene expression as well as more basic responses, such as higher cell numbers. Our study demonstrates that such changes are accompanied by increased membrane potential and lower percentages of dead cells both of which are critical to bacterial population growth. The two species examined, generally, exhibited similar responses. However, responses observed varied with growth phase and were medium-dependent revealing that nutrient availability is a modulator of responses to these conditions. Overall, our data provides novel information about E. coli and S. aureus MP and MI under MRG conditions and suggest that bacteria are physiologically more active and a larger percentage

are viable under MRG as compared to NG conditions. Future studies are needed to elucidate the mechanism leading to increased MP and MI and to determine if these differences are consistently observed regardless of bacterial species and growth conditions. Finally, our findings have LCZ696 mw implications for fundamental biological next responses, namely the ability for living cells to detect and respond to mechanical stimuli [19]. Further study is needed to examine the inter-play between responses to mechanical conditions and other aspects of the environment and to explore potential mechanisms by which such conditions are sensed or detected to determine if they are conserved across taxa. Methods Bacterial strains Escherichia coli K-12 MG1655 (ATCC 700926), Staphylococcus aureus (ATCC 25923) Growth media Full strength Luria broth (LB) and M9 Minimal media (+ 0.4% glucose and 1 μg/ml thiamine) were used to cultivate E. coli. Full strength LB and diluted LB (1:50) were used to cultivate S. aureus. In this case, diluted LB was used instead of M9 minimal media because M9 did not support the growth of S. aureus (data not shown).

Ideally, the oxide surface should be covered with a monolayer of dye molecules to achieve efficient electron injection. When dye molecules undergo aggregation, electron injection becomes less efficient, and overall conversion efficiency declines. However, Yan et al. [39], on the other hand, observe the surface etching of ZnO nanoflowers after a long sensitization

time. Surface etching also leads to a significant loss in overall conversion efficiency. For ZnO-based cells, it is essential to optimize the dye adsorption time to minimize the formation of dye Epigenetics inhibitor aggregates and the damage to ZnO surfaces. Because the dye molecules must penetrate the mesoporous oxide film before they attach to the interfacial surface, the optimal dye adsorption time likely depends on the thickness of the ZnO film. Thus, this study investigates both the film thickness and the dye adsorption time. Although these two factors have been learn more individually investigated before and certain studies have reported the influences of dye concentration and adsorption time on DSSC performance [32, 36], a detailed and systemic study of the effects of film thickness PLX-4720 mouse and dye adsorption time for ZnO-based DSSCs is lacking. This study reports the preparation of DSSC photoelectrodes using

commercially available ZnO nanoparticles sensitized with the acidic N719 dye. This study also systematically investigates the influences of ZnO film thickness and dye adsorption time on the performance of the resulting DSSCs. To further understand the effect of dye adsorption time,

electrochemical impedance spectroscopy (EIS) was used to investigate the electron transport characteristics of the fabricated cells. This study shows the correlation Liothyronine Sodium between J SC and dye loading as a function of the dye adsorption time and reports the at-rest stability of the best-performing cell. Methods Fabrication of solar cells ZnO films (active area 0.28 cm2) of various thicknesses (14 to 35 μm) were deposited on fluorine-doped tin oxide (FTO) substrates (8 to 10 Ω/□, 3 mm in thickness, Nippon Sheet Glass Co. Ltd, Tokyo, Japan) by screen printing. Screen-printable ZnO paste was prepared by dispersing commercially available ZnO nanoparticles (UniRegion Bio-Tech, Taiwan) in an equal proportion of α-terpineol (Fluka, Sigma-Aldrich, St. Louis, MO, USA) and ethyl cellulose. Before dye adsorption, the ZnO films were sintered at 400°C for 1 h to remove any organic material in the paste. This thermal treatment sintered the nanoparticles together to form an interconnecting network. Dye sensitization was achieved by immersing the sintered ZnO films in a 0.5 mM solution of cis-diisothiocyanato-bis(2,2′-bipyridyl-4,4′-dicarboxylato)-ruthenium(II) bis(tetrabutylammonium) (N719, Solaronix; Solaronix SA, Aubonne, Switzerland). The solvent used to prepare the dye solution consisted of equal parts of acetonitrile and tert-butanol.

PubMedCrossRef 31. Nanagara R, Li F, Beutler A, Hudson A, Schumacher

HR: Alteration of Chlamydia trachomatis biologic behavior in synovial membranes. Suppression of surface antigen production in reactive arthritis and Reiter’s syndrome. Arthritis Rheum 1995,38(10):1410–1417.PubMedCrossRef 32. Patton DL, Askienazy-Elbhar M, Henry-Suchet J, Campbell LA, Cappuccio A, Tannous W, Wang SP, Kuo CC: Detection of Chlamydia trachomatis in fallopian tube tissue in women with postinfectious tubal infertility. Am J Obstet Gynecol 1994,171(1):95–101.PubMed 33. Batteiger BE, Tu W, Ofner S, Van Der Pol B, Stothard DR, Orr DP, Katz BP, Fortenberry JD: Repeated Chlamydia trachomatis genital infections in adolescent women. J Infect Dis 2010,201(1):42–51.PubMedCrossRef 34. Golden MR, Whittington WL, Handsfield HH, Hughes buy Pexidartinib JP, Stamm FK228 in vivo WE, Hogben M, Clark A, Malinski C, Helmers JR, Thomas KK, et al.: Effect of expedited treatment of sex partners on recurrent or persistent gonorrhea or chlamydial infection. N Engl J Med 2005,352(7):676–685.PubMedCrossRef 35. Elman M, Slatkine M, Harth Y: The effective treatment of acne vulgaris by a high-intensity, narrow band 405–420 nm light source. J Cosmet Laser Ther 2003,5(2):111–117.PubMedCrossRef 36. Lembo AJ, Ganz RA, Sheth S, Cave D, Kelly C, Levin P, Kazlas PT, Baldwin PC, Lindmark WR, McGrath JR, et al.: Treatment of Helicobacter pylori infection with Selleck Thiazovivin intra-gastric violet

light phototherapy: a pilot clinical trial. Lasers Surg Med 2009,41(5):337–344.PubMedCrossRef 37. Murdoch

LE, Maclean M, MacGregor SJ, Anderson else JG: Inactivation of Campylobacter jejuni by exposure to high-intensity 405-nm visible light. Foodborne Pathog Dis 2010,7(10):1211–1216.PubMedCrossRef 38. Maclean M, Macgregor SJ, Anderson JG, Woolsey GA: The role of oxygen in the visible-light inactivation of Staphylococcus aureus. J Photochem Photobiol B 2008,92(3):180–184.PubMedCrossRef 39. Ashkenazi H, Malik Z, Harth Y, Nitzan Y: Eradication of Propionibacterium acnes by its endogenic porphyrins after illumination with high intensity blue light. FEMS Immunol Med Microbiol 2003,35(1):17–24.PubMedCrossRef 40. Boncompain G, Schneider B, Delevoye C, Kellermann O, Dautry-Varsat A, Subtil A: Production of reactive oxygen species is turned on and rapidly shut down in epithelial cells infected with Chlamydia trachomatis. Infect Immun 2010,78(1):80–87.PubMedCrossRef 41. Dong F, Su H, Huang Y, Zhong Y, Zhong G: Cleavage of host keratin 8 by a Chlamydia-secreted protease. Infect Immun 2004,72(7):3863–3868.PubMedCrossRef 42. Zhong G, Fan P, Ji H, Dong F, Huang Y: Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med 2001,193(8):935–942.PubMedCrossRef 43. Sun J, Schoborg RV: The host adherens junction molecule nectin-1 is degraded by chlamydial protease-like activity factor (CPAF) in Chlamydia trachomatis-infected genital epithelial cells.

The purpose of this study was to assess (1) the energy, macro- and micronutrient intakes as well as (2) the eating Batimastat attitudes of a group of elite adolescent female figure skaters to assess the potential nutritional risks among them. The results of this study will identify potential nutrient inadequacies and disordered eating attitudes and behaviors to inform the nutrition education and counseling needs of elite adolescent female skaters. Methods Participants Participants were 36 nationally-ranked elite adolescent female figure skaters who had a mean

age of 16 ± 2.5 SD years (range 13–22 years) and who attended an elite training camp at the US Olympic Training Center in Colorado Springs, CO between 1998 and 1999. Written

informed consent was obtained from all participants and, where necessary, by their legal guardians prior to participation in the study. The Sports Medicine Advisory Board of the US Figure Skating Association and the US Olympic Committee Human Subject Review Board approved this study. The Human Investigation Review Committee at Tufts Medical Center in Boston, MA approved the secondary analysis of the data. Height and weight Participants were weighed and measured (in light clothing and without shoes) in the morning prior to engaging in physical activity. Body weight was measured using a beam balance scale to the nearest 50 g and height was measured Ganetespib chemical structure with a stadiometer to the nearest 0.5 cm. Body mass index (BMI) was then calculated as the ratio of weight (kg) to height (m) Erastin mw squared (kg/m2); BMI-for-age percentiles were calculated for all participants ≤19y using growth charts from the Centers for Disease Control

and Prevention CDC; [19]. Dietary intake Dietary intake was assessed to determine the chief sources of energy and key micronutrients in skaters’ diets. After participants were provided detailed instructions, three-day food records (2 nonconsecutive weekdays and 1 weekend day) were recorded during a period of active training two months prior to attendance at the training camp. During the first week of training camp, participants met with a study staff member to review their food records and clarify missing or ambiguous data. The skaters then received a brief individualized nutrition education session with recommendations to help them improve their intakes if they exhibited problems. Diet records were then verified, coded, entered and analyzed by a registered dietitian on the study staff using Nutritionist IV (version 4.1, 1997, First Data Bank, Inc., San Bruno, CA). Estimated intakes of calories, macronutrients, and micronutrients were compared to age and gender appropriate https://www.selleckchem.com/products/Cyt387.html normative data from the National Health and Nutrition Examination Survey of 1999–2000 NHANES; [20–23]. Eating attitudes The Eating Attitudes Test EAT-40; [24] served as a measure of disordered attitudes and behaviors towards eating and body weight control.

Under generally applied experimental conditions, the endogenous oxidizing and reducing agents are not present. In absence of selleck compound electron donors and acceptors, charge recombination occurs on the μs to ms time-scale, (e.g., Brettel 1997; Vassiliev et al. 1997). However, electrons can also escape from the Fe4S4 selleck chemicals cluster to other electron acceptors, such as oxygen (Rousseau et al. 1993). Therefore, in absence of electron donors and presence of light all P700s are soon blocked in their oxidized (closed/P700+) state (Savikhin 2006). To study the kinetics of PSI with open RCs, reducing agents are added to the buffer. Most often phenazine

methosulfate (PMS) reduced by sodium ascorbate (NaAsc) is used for this purpose. PMS is supplied at different concentrations: 10 μM (e.g., Gobets et al. 2001; Ihalainen et al. 2005; Turconi et al. 1993), 20 μM (Engelmann et al. 2006; Giera et al. 2010; Karapetyan et al. 1997; Nuijs et

al. 1986), 60 μM (Slavov et al. 2008) or 150 μM (Byrdin et al. 2000). In this work, we study how fast PMS re-reduces P700+ to its neutral state, and use these rates to estimate the fraction of closed RCs under different light intensities. We show that PMS itself is quenching fluorescence of light harvesting complexes. And we show see more that closing the RC of higher plant PSI increased the fluorescence quantum yield by only 4%. Materials and methods Purification until of photosynthetic complexes Thylakoids were isolated from Arabidopsis thaliana plants as described previously (Bassi and Simpson 1987). The major light

harvesting complex of PSII (LHCII) and the PSI complex were obtained by mild solubilization of the thylakoids followed by the sucrose gradient density centrifugation, as described in (Caffarri et al. 2001). For all the fluorescence measurements, the obtained PSI complexes were run over a second sucrose gradient to improve the purity. Indeed, the low temperature emission shows that the sample is very pure (Wientjes et al. 2009). Photosystem II membranes were obtained as described in Berthold et al. (1981). The PSI light-harvesting antenna Lhca1/4 was obtained as described in Wientjes and Croce (2011). Absorption and fluorescence spectroscopy Absorption spectra were recorded on a Cary 4000 UV–Vis spectrophotometer (Varian, Palo Alto, CA). Fluorescence spectra were recorded on a Fluorolog 3.22 spectrofluorimeter (HORIBA Jobin-Yvon, Longjumeau, France); samples were diluted to an optical density of 0.05/cm at the Q y maximum, unless stated otherwise. P700 and fluorescence kinetics The P700 oxidative state and fluorescence kinetics were measured using the Dual-PAM-100 (Heinz Walz, Effeltrich, Germany). For P700+ detection, the 830 minus 875 nm absorption difference signal was used.