The results refuted

The results refuted Ku0059436 the initial hypothesis that low DO is one of the main pre-requisite conditions for the transcription of nirK and norB genes in N. europaea. On the other hand, these results indeed supported our other hypothesis that higher NO2 – concentrations constitute the principal trigger for increased relative transcription related to autotrophic denitrification reactions. The distinct responses

observed during the exponential and stationary phase to both DO limitation and nitrite toxicity highlight the need to understand the specific regulatory mechanisms employed by N. europaea to jointly counter substrate starvation and stress. Methods Cultivation of batch N. europaea cultures N. europaea (ATCC 19718, Manassas, VA) batch cultures

were cultivated in the dark in batch Navitoclax in vitro bioreactors (Bellco Glass, Vineland, NJ, working volume = 4 L, agitation speed = 200 rpm) in a growth medium containing 280 mg-N/L and in addition (per liter): 0.2 g of MgSO47H2O, 0.02 g of CaCl22H2O, 0.087 g of K2HPO4, 2.52 g EPPS (3- [4-(2-Hydroxyethyl)-1-piperazine] propanesulfonic acid), 1 mL of 13% EDTA-Fe3+, 1 mL of trace elements solution (10 mg of Na2MoO42H2O, 172 mg of MnCl24H2O, 10 mg of ZnSO47H2O, 0.4 mg of CoCl26H2O, and 100 mL of distilled water), 0.5 mL of 0.5% phenol red, and 0.5 mL of 2 mM CuSO45H2O. Reactor pH was controlled in the range 6.8-7.4 by manual addition of pre-sterilized 40% potassium bicarbonate solution. Batch growth experiments were conducted at three DO concentrations, 0.5 ± 0.05, 1.5 ± 0.05 and 3.0 ± 0.05 mg O2/L. Batch reactor DO was measured and controlled with a fermentation DO probe and benchtop dissolved oxygen meter and controller

system (Cole-Parmer, Vernon Hills, IL) using a combination of filter sterilized (0.2 μm pore size, Millipore®, Ann Arbor, MI) nitrogen gas or air. In select experiments conducted at DO = 1.5 ± 0.05 mg O2/L, the feed medium additionally contained 280, or 560 mg NO2 –N/L before N. europaea inoculation, which enabled the determination of batch growth in the presence of these high NO2 –N concentrations. NH3 (gas-sensing electrode, Corning, Corning, NY), NH2OH [30], NO2 – (diazotization, [31], cell concentration (direct counting) and gaseous NO (chemiluminescence, CLD-64, Ecophysics, Ann Arbor, MI) were measured once a day during the batch growth profile. All batch growth experiments were conducted in duplicate. Detection of intracellular and extracellular nitric oxide Intracellular NO presence was determined by staining with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (Molecular Probes, Eugene, OR) for 30 min in the absence of light. Stained cells were washed twice with sterile NH3-free medium and quantified immediately with epifluorescence microscopy (Nikon ECLIPSE 80 i) using a minimum of 10 randomly-chosen microscopic fields (each 0.30 × 0.22 mm2).

Therefore, PNI and postoperative recurrence rate are closely rela

Therefore, PNI and postoperative recurrence rate are closely related. Consequently, if the mechanism of CCA PNI could be understood and interrupted in early-stage CCA, the prognosis of CCA patients could be greatly improved. Anatomic

Foundation of Cholangiocarcinoma PNI In the human hepatoduodenal ligament, the pampiniform nerve plexus can be clearly seen, and it can be classified into hepatic anteplex and hepatic metaplex. The hepatic anteplex is composed of the left and right celiac ganglia and left vagus nervous ramification, which includes the cystic duct, gallbladder and cholo-pancreatic common bile duct ramification. The scabbard is formed around the hepatic artery, and leads, via the hepatic artery, into the liver. The hepatic

metaplex is composed PCI-32765 chemical structure of the right celiac ganglia and right vagus nerve ramification, which are mainly distributed along the extrahepatic bile duct and portal vein; some of its ramification links with the anteplex nervous ramification. The sensory fibers of the right phrenic nerve are distributed in the coronary ligament, the falciform ligament of the liver, and the vicinal liver capsule[11], while part of the fibers combined with the liver ante- and metaplex, along with the fibers of the hepatic plexus, and distributes into the exterior and interior biliary CHIR99021 system of the liver. The whole liver is controlled by the sympathetic and parasympathetic nerves. They are distributed

in the hepatic artery, vena portae hepatic, liver interior and extrahepatic bile duct; the sympathetic nerve originates from celiac ganglia, while the parasympathetic nerve comes from the vagus nerve[12]. Therefore, the biliary system is typical of organs with extremely fundamental autonomic nerves, which could be controlled by an extensive neural system. The nerve terminal is partially removed through the porta hepatic hemal tube structure, surrounded by the bile duct and blood vessel. The bile duct is one of IMP dehydrogenase the most important components of the liver, which is also the channel of choleresis and excretion. As the nerve terminal acts on the liver hemal tube system, the patho- and physiological functions of bile duct epithelium are inevitably affected, providing the anatomic foundation for CCA metastasis via PNI. Cholangiocarcinoma PNI as independent metastasis pathway Among gastrointestinal malignancies, PNI is often seen in pancreatic and biliary system cancers, and occasionally in rectal cancer. It is a local diffusion mode for tumors, and it plays a critical role in prognosis. Current study shows tumor perineural invasion to be uncorrelated with patient’s age or sex as well as whether or not tumor metastasis in distant (including liver metastasis or abdominal cavity, peritoneum metastasis). However, it is highly correlated with tumor volume, location, depth of invasiveness, angiogenesis and lymph node involvement[13].

Then, we will demonstrate that such wires can be used as a templa



deposited at high temperature using trimethylaluminium (TMAl) and NH3 precursors. Under such growth conditions, the polarity of the AlN layer is Al-polar [15], and its thickness has no significant influence on the later GaN wire growth. According to our previous work [11], a thin SiN x layer is first deposited on the AlN surface to prevent GaN planar growth. Self-assembled catalyst-free GaN wires are then grown for 500 s using TMGa and NH3 precursors with a low V/III ratio (approximately 20) and silane injection to favour the vertical growth [16]. Results and discussion Figure 1 shows a typical 45° tilted SEM image of the resulting vertically aligned GaN wires. They exhibit an irregular Selleck Talazoparib Neratinib datasheet hexagonal cross section and a quite large dispersion in length and diameter. Due to the very low wire density (approximately 106 wires/cm2), specular X-ray reflectivity (not shown in this paper) allows measurement of the total layer thickness on top of silicon. Well-contrasted interference fringes corresponding to a thickness of 25 ± 0.5 nm are measured close to the target value for the AlN layer. HRTEM cross sections have shown no significant planar growth

on the surface. This is in agreement with the deposition of the SiN x passivation layer on top of AlN, as already observed for the growth of GaN wires on sapphire [11]. Figure 1 SEM picture of GaN wires. 45° tilted view of GaN wires grown by MOVPE on Si (111) with an intermediate AlN layer. The structural properties of the wires were first investigated by laboratory XRD using symmetric (Θ-2Θ) and rocking (ω) scans. Figure 2a shows the Θ-2Θ diffraction pattern of the as-grown samples with a cobalt radiation source. The GaN (0001), AlN (0001) and Si (111) Bragg peaks are indexed, indicating a GaN wire growth orientation along the c-axis. The disorientation of the GaN wires was investigated by the Δω rocking curves of the GaN (0002) and GaN (0004) Bragg peaks. As shown in Figure 2b, the 1.



pairs CT-MSR-01/CT-MSR-02 and 2144-01/2144-02 were used to investigate transcription of NMB0044 and NMB2144 respectively. RT-PCR of RmpM (NMB0382) using RG7204 supplier primerset CT-class4-1/CT-class4-2, was used as loading control. Sequence analysis was carried out to confirm the identity of the generated RT-PCR products. Cell fractionation Meningococci were


MALDI-TOF mass spectrometry was carried out as described previously [64]. Acknowledgements Melanie Nguyen is acknowledged for her technical assistance. This research was partly funded by the Sixth Framework Programme of the European Commission, Proposal/Contract no.: 512061 Galactosylceramidase (Network of Excellence ‘European Virtual Institute for Functional Genomics of Bacterial Pathogens’, http://​www.​noe-epg.​uni-wuerzburg.​de References 1. Ebright RH: RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II. J Mol Biol 2000, 304:687–698.PubMedCrossRef 2. Gross CA, Chan CL, Lonetto MA: A structure/function analysis of Escherichia coli RNA polymerase. Philos Trans R Soc Lond B Biol Sci 1996, 351:475–482.PubMedCrossRef 3. Gross CA, Chan C, Dombroski A, Gruber T, Sharp M, Tupy J, Young B: The functional and regulatory roles of sigma factors in transcription. Cold Spring Harb Symp Quant Biol 1998, 63:141–155.PubMedCrossRef 4. Murakami KS, Darst SA: Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 2003, 13:31–39.PubMedCrossRef 5. Sweetser D, Nonet M, Young RA: Prokaryotic and eukaryotic RNA polymerases have homologous core subunits. Proc Natl Acad Sci USA 1987, 84:1192–1196.PubMedCrossRef 6.


Mol PS-341 research buy Microbiol 2005, 57:196–211.CrossRefPubMed 13. Morgan E, Campbell JD, Rowe SC, Bispham J, Stevens MP, Bowen AJ, Barrow PA, Maskell DJ, Wallis TS: Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium. Mol Microbiol 2004, 54:994–1010.CrossRefPubMed 14. Knodler LA, Celli J, Hardt WD, Vallance BA, Yip C, Finlay BB:Salmonella effectors within a single pathogeniCity island are differentially expressed and translocated by separate type III secretion systems. Mol Microbiol 2002, 43:1089–1103.CrossRefPubMed 15. Jones MA, Hulme SD, Barrow PA, Wigley P: The Salmonella pathogeniCity island 1 and Salmonella pathogeniCity island 2 type III secretion

systems play a major role in pathogenesis of systemic disease and gastrointestinal tract colonization of Salmonella enterica serovar Typhimurium in the chicken. Avian

Pathol 2007, 36:199–203.CrossRefPubMed 16. Bohez L, Gantois I, Ducatelle R, Pasmans F, Dewulf J, Haesebrouck F, Van Immerseel F: The Salmonella PathogeniCity Island 2 regulator ssrA promotes reproductive tract but not intestinal colonization in chickens. Vet Microbiol 2008, 126:216–224.CrossRefPubMed 17. Dieye Y, Ameiss Selleckchem Silmitasertib K, Mellata M, Curtiss R III: The Salmonella PathogeniCity Island (SPI) 1 contributes more than SPI2 to the colonization of the chicken by Salmonella enterica serovar Typhimurium. BMC Microbiol 2009, 9:3.CrossRefPubMed 18. Bohez L, Ducatelle R, Pasmans F, Botteldoorn N, Haesebrouck F, Van Immerseel F:Salmonella enterica serovar Enteritidis colonization of the chicken caecum requires the HilA regulatory protein. Vet Microbiol 2006, 116:202–210.CrossRefPubMed 19. Desin TS, Lam PK, Koch B, Mickael C, Berberov E, Wisner AL, Townsend HG, Potter AA, Koster W:Salmonella enterica serovar enteritidis pathogeniCity island 1 is not essential for but facilitates rapid systemic spread in chickens. Infect Immun 2009, 77:2866–2875.CrossRefPubMed

20. Galyov EE, Wood MW, Rosqvist R, Mullan PB, Watson PR, Carnitine palmitoyltransferase II Hedges S, Wallis TS: A secreted effector protein of Salmonella dublin is translocated into eukaryotic cells and mediates inflammation and fluid secretion in infected ileal mucosa. Mol Microbiol 1997, 25:903–912.CrossRefPubMed 21. Shea JE, Hensel M, Gleeson C, Holden DW: Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci USA 1996, 93:2593–2597.CrossRefPubMed 22. Karasova D, Sebkova A, Vrbas V, Havlickova H, Sisak F, Rychlik I: Comparative analysis of Salmonella enterica serovar Enteritidis mutants with a vaccine potential. Vaccine 2009, 27:5265–5270.CrossRefPubMed 23. Hapfelmeier S, Stecher B, Barthel M, Kremer M, Muller AJ, Heikenwalder M, Stallmach T, Hensel M, Pfeffer K, Akira S, et al.: The Salmonella pathogeniCity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms. J Immunol 2005, 174:1675–1685.PubMed 24.

The relative humidity was stable at 43% during the race The ‘Bik

The relative humidity was stable at 43% during the race. The ‘Bike Race Marathon MTB Rohozec’ in Liberec took place from 9th June to 10th June 2012. The course comprised a 12.6 km track with an elevation of 250 m. The track surface consisted of paved and unpaved roads and paths. There was one aid station located at the start and finish area with food and beverages similar to those mentioned above. The temperature was +19˚C at the start, rose to a maximum of +23˚C, dropped to +6˚C during the night and changed to +11˚C until

the end of the race. Weather conditions varied from sunny to cloudy with a short PLX4032 shower in the afternoon and relative humidity increased from 44% to 98%. Procedures, measurements and calculations Participants were instructed to keep a training diary until the start of the race. The training three months before the race (i.e. training

units in hours, cycling units in hours, training distances in kilometers, cycling speed, heart rate during training units, volume of kilometers in the year 2011, and the years of active cycling) was recorded. Participant recruitment and pre-race testing took place during event registration in the morning before the race between 07:00 a.m. and 11:00 a.m. in a private room adjacent to the registration area. The athletes were informed of the procedures and gave their informed written consent. Post-race measurements were taken between 12:00 and 1:00 p.m. immediately upon completion of the out race in the same place. No measurements were made during the race. Between the pre- and the post-race measurements, all athletes recorded their fluid intake using a written record. Anthropometric measurements and plethysmography of the foot Anthropometric measurements were recorded in all forty-nine ultra-MTBers (37 males and 12 females) (Table  2, also Figure  1) to estimate skeletal muscle mass and fat mass. Body mass, total body water, extracellular fluid and intracellular fluid were measured using a multiple-frequency bioelectrical impedance analyser (InBody 720, Biospace, Seoul, South Korea). Inbody 720 has a tetra polar

8-point tactile electrode system performing at each session 30 impedance measurements by using six different frequencies (i.e. 1 kHz, 5 kHz, 50 kHz, 250 kHz, 500 kHz, and 1,000 kHz) at each five segments (i.e. right arm, left arm, trunk, right leg, and left leg). Subjects were barefoot and generally clothed in cycling attire for both the pre- and post-race measurements and participants were advised to void their urinary bladder prior to the anthropometric measurements. Body height was determined using a stadiometer (TANITA HR 001, Tanita Europe B.V., Amsterdam, The Netherland) to the nearest 0.01 m. Body mass index was calculated using body mass and body height. The circumferences of mid-upper arm, mid-thigh and mid-calf were measured on the right side of the body to the nearest 0.

F Kaeppeli, Zurich, Switzerland)

F. Kaeppeli, Zurich, Switzerland). SCH772984 price These blood samples were analyzed for hemoglobin concentration and hematocrit, which were used to calculate changes in PV according to Dill and Costill [26]. Body composition measurement A densitometer (Lunar iDXA™, GE Healthcare, Madison, WI, USA) was used for the determination of total lean body mass and lean soft tissue mass of the legs. Dual-energy X-ray absorptiometry (DXA) measurements were performed just before the constant-load trials every second day throughout the intervention periods to assess leg lean mass as an indicator of glycogen content. According to the DXA two-component soft tissue model, lean soft tissue mainly



of sphericity RXDX-106 ic50 Thiamet G was violated, the degrees of freedom were corrected using the Greenhouse-Geisser estimates of sphericity. When F ratios were significant, post hoc comparisons of main effects were performed using a Student’s paired t-test with Bonferroni correction. PV data were not normally distributed and thus log-transformed before using the described analysis. All data are presented as means ± SD. The effect size is denoted as ηp 2 (partial eta-squared). The level of significance was set at P < 0.05. The statistical analyses were conducted using the software SPSS Statistics 20.0 (SPSS, Chicago, IL, USA). Results As judged by the leftover pill count, average compliance with NaHCO3 and placebo supplementation was 100%. T lim increased by 23.5% following NaHCO3 ingestion (F (1,7) = 35.45, P = 0.001, ηp 2 = 0.84; Figure 2a). However, there was neither an effect of time (F (4,28) = 1.1, P = 0.375, ηp 2 = 0.14) nor an intervention x time interaction (F (4,28) = 0.74, P = 0.464, ηp 2 = 0.01; Figure 2b). No differences in CP, as measured before the first and second supplementation period, could be found (306.8 ± 21.4 W vs. 309.0 ± 30.4 W; F (1,7) = 0.15, P = 0.708, ηp 2 = 0.02). Also, no difference could be found between CP as determined before the NaHCO3 and placebo intervention (304.3 ± 25.6 W vs. 311.5 ± 26.5 W; F (1,7) = 1.99, P = 0.202, ηp 2 = 0.22). Figure 2 Time-to-exhaustion with NaHCO 3 and placebo supplementation.

J Am Coll Surg 2008, 206:685–693 PubMed 32 Gavant ML, Schurr M,

J Am Coll Surg 2008, 206:685–693.PubMed 32. Gavant ML, Schurr M, Flick PA, et al.: Predicting clinical outcome of nonsurgical management of blunt splenic injury: using CT to reveal abnormalities of splenic vasculature. AJR 1997, 168:207–212.PubMed 33. Thompson BE, Munera F, Cohn SM, et al.: Novel computed tomography scan scoring system predicts the need for intervention after splenic injury. J Trauma 2006, 60:1083–1086.CrossRefPubMed

34. Rhodes CA, Dinan D, Jafri SZ, et al.: Clinical outcome of active extravasation in splenic trauma. Emerg Radiol 2005, 11:348–352.CrossRefPubMed 35. Marmery H, Shanmuganathan K, Alexander M, et al.: Optimization of Selection for Nonoperative Management of Blunt Splenic Injury: Comparison of MDCT Grading Systems. AJR 2007, 189:1421–1427.CrossRefPubMed 36. Norrman G, Tingstedt B, Ekelund M, et al.: Nonoperative Management of Blunt Splenic Trauma: Also Feasible and Safe in Centers with Low Trauma Incidence and in the Presence of Established Risk Factors. Eur J Trauma Emerg Surg 2009, 35:102–107.CrossRef 37. Dent D, Alsabrook G, Erikson BA, et al.: Blunt splenic injuries: high nonoperative management rate can be achieved with selective embolisation. J Trauma 2004, 56:1063–1067.CrossRefPubMed 38. Wasvery H, Howells G, Villalba M, et al.: Nonoperative ZD1839 purchase management of adult blunt splenic trauma: a 15 year experience. Am Surg 1997, 63:694–699. 39. Schurr MJ, Fabian

TC, Gavant M, et al.: Management of Blunt Splenic Trauma: Computed Tomographic Contrast Blush Predicts Failure of Nonoperative Management. J Trauma

1995, 39:507–512.CrossRefPubMed 40. Becker CD, Poletti P-A: The trauma concept: the role of MDCT in the diagnosis and management of visceral injuries. Eur Radiol Suppl 2005,15(Suppl 4):D104-D109. 41. Bessoud B, Denys A, Calmes JM, et al.: Nonoperative Management of Traumatic Splenic Injuries: Is There a Role for Proximal Splenic Artery Embolisation? AJR 2006, 186:779–785.CrossRefPubMed 42. Sclafani SJ, Shaftan GW, Scalea TM, et al.: Non-operative salvage of computer-tomography diagnosed splenic injuries: utilisation of angiography for triage and embolisation for haemostasis. J Trauma 1995, 39:818–825.CrossRefPubMed 43. Sclafani SA, Weisberg A, Scalea T: Blunt splenic injuries: nonsurgical treatment with CT, arteriography, and transcatheter arterial Erastin supplier embolisation of the splenic artery. Radiology 1991, 181:189–196.PubMed 44. Hagiwara A, Yukloka T, Ohat S, et al.: Nonsurgical management of patients with blunt splenic injury: efficacy of transcatheter arterial embolisation. AJR 1996, 167:156–166. 45. Ekeh AP, McCarthy MC, Woods RJ, et al.: Complications arising from splenic embolisation after blunt splenic trauma. Am J Surg 2005, 189:335–339.CrossRefPubMed 46. Gaarder C, Dormagen JB, Eken T, et al.: Nonoperative Management of Splenic Injuries: Improved Results with Angioembolisation. J Trauma 2006, 61:192–198.CrossRefPubMed 47. van der Hul RL, van Overhagen H, Dallinga RJ, et al.

coelicolor also showed defective chromosome segregation during sp

coelicolor also showed defective chromosome segregation during sporulation. In prokaryotes, motor proteins such as FtsK and SpoIIIE containing a conserved RecA domain are often associated with DNA translocation during processes of cell division, conjugation and sporulation [25]. In S. coelicolor, FtsK and ParA/ParB are required for proper chromosome

segregation during sporulation [15, 16]. However, despite detectable levels of errors in chromosome segregation in FtsK or ParAB mutants, the majority of chromosomes still appear to segregate properly, suggesting that other proteins are also involved in chromosome partition or segregation. According to analysis using the Protein Homology/analogY Recognition Engine PHYRE http://​www.​sbg.​bio.​ic.​ac.​uk/​phyre/​html/​index.​html, CmdB protein was predicted containing a RecA domain (from positions 77 to 407, expectation value 1.7 × 10-21) or E. coli-FtsK motor domain this website (3.3 × 10-12), suggesting that it might be an ATP/GTP-dependent motor protein. CmdB displays homology with VirB4-like proteins from Frankia, Brevibacterium, Geobacillus and Thermoanaerobacter (expectation values 3 × 10-42, 1 × 10-39, 7 × 10-9 and BIBW2992 2 × 10-9, respectively) etc. The VirB4, an essential component of the bacterial type IV system, interacts with other membrane proteins in the vir operon to assemble a pore for transfer of a DNA-protein complex [26, 27]. Since CmdB is also located on the cell membrane, it is

likely that CmdB along with other five membrane proteins from the same gene cluster might form a complex on the cell membrane. Further study will be needed to explore the existence of such a complex and to investigate

whether it could form a type IV-like channel on cell membrane for chromosome and/or plasmid translocation in Streptomyces. About 836 and 69 genes of S. coelicolor genome are predicted to encode membrane and ATP/GTP-binding proteins, respectively ([28]; http://​www.​sanger.​ac.​uk/​Projects/​S_​coelicolor/​classwise.​html#class4.​1.​0). Among these, SCO6878, SCO6880 and SCO6881, located Tenofovir order in a cluster of 14 probably co-transcribed genes SCO6871-6884, highly resemble cmdB, cmdC and cmdD, respectively. However, null mutants of SCO6878 or SCO6881 did not display defective sporulation or over-production of blue pigment on MS medium (our unpublished data). Thus, either these genes are not involved in sporulation and antibiotic production, or their role may be masked by functional overlap with other genes, or the phenotype might be manifested only under particular conditions. Conclusion This study describes the identification of six co-transcribed genes cmdABCDEF, deletions of which displayed over-expression of blue-pigmented Act, defective sporulation and especially abnormalities in chromosome segregation, indicating that cmdABCDEF are new genes involved in antibiotic production and differentiation of S. coelicolor. Methods Bacterial strains, plasmids and general Methods S.

elongatus and cobalt resin prepared by charging chelating Sepharo

elongatus and cobalt resin prepared by charging chelating Sepharose fast flow resin according to the manufacturer’s instructions (GE Healthcare Life Sciences). Crude thylakoid membranes were prepared from T. elongatus by glass bead breakage and differential centrifugation as described by Boehm et al. (2009) and re-suspended in buffer A (50 mM MES–NaOH pH 6.0, 10 mM MgCl2, 5 mM CaCl2, 10 % (w/v) glycerol) as used by Kashino et al. (2002). Thylakoids were solubilised with 1 % (w/v) β-DDM at a Chl concentration of 0.2 mg/ml for 10 min on ice in a final volume of 0.5 ml. After pelleting insoluble material

by centrifuging in a microfuge, 0.45 ml of the supernatant was removed and diluted by addition of 0.45 ml of buffer A to which was added 0.1 ml of cobalt resin (50 µl of resin resuspended to final volume of 100 µl by addition of buffer A). Samples were then incubated on a rotating wheel at 4 °C for 2 h. After removal of the membrane extract, the cobalt resin was washed four times with 500 µl of buffer A, with the final wash kept for analysis. Bound proteins were eluted with

100 µl of buffer A containing 100-mM imidazole followed by 100 µl of 1× SDS sample buffer used for electrophoresis. Chelating selleckchem Sepharose lacking bound metal ions was used as a control. Salt washes of purified PSII complexes and thylakoid membranes PSII complexes in buffer A2 (20 mM MES–NaOH pH 6.5, 1 mM MgCl2, 1 mM CaCl2, 10 % (w/v) glycerol, 0.03 % (w/v) β-DDM) purified either by two-step anion-exchange or by

nickel-affinity chromatography were incubated with buffer A2 supplemented with 1 M CaCl2 on ice for 30 min in the dark. Immediately after incubation samples were concentrated on 100,000 Erastin mw MWCO Vivaspin 500 centrifugal concentrators (Sartorius AG). Green retentate and flow-through containing removed extrinsic proteins were desalted by two buffer exchanges using Vivaspin 500 centrifugal concentrators, with MWCO of 100,000 and 3,000, respectively. Chlorophyll concentration was adjusted to 1 mg/ml and the volume of the filtrate was adjusted to match the volume of the green retentate. In the case of thylakoid membranes, proteins were extracted by high salt or high pH using the Freeze–Thaw approach described by Boehm et al. (2009). Protein analysis, isolation of protein and immunoblotting Thermosynechococcus elongatus CyanoP and Psb27 were over-expressed in E. coli and purified as described previously (Michoux et al. 2010, 2012). These proteins plus CyanoQ isolated here were used to raise antibodies in rabbit. Protein samples were separated on 18 % (w/v) polyacrylamide gels containing 6 M urea as described by Boehm et al. (2009). Immunoblotting analyses were performed as described by Boehm et al. (2009) using the following antibodies and dilutions: αD1 (1:5000), αPsbO (1:1000), αCyanoP (1:2500), αCyanoQ (1:5000) and αPsb27 (1:2500).