Buckley J.P.,University of Chester |
Mellor D.D.,University of Nottingham |
Morris M.,University of Chester |
Occupational and Environmental Medicine | Year: 2014
Objectives The main aim of this study was to compare two days of continuous monitored capillary blood glucose (CGM) responses to sitting and standing in normally desk-based workers. Design, setting and participants This open repeated-measures study took place in a real office environment, during normal working hours and subsequent CGM overnight measures in 10 participants aged 21-61 years (8 female). Main outcomes Postprandial (lunch) measures of: CGM, accelerometer movement counts (MC) heart rate, energy expenditure (EE) and overnight CGM following one afternoon of normal sitting work compared with one afternoon of the same work performed at a standing desk. Results Area-under-the-curve analysis revealed an attenuated blood glucose excursion by 43% (p=0.022) following 185 min of standing (143, 95% CI 5.09 to 281.46 mmol/L min) compared to sitting work (326; 95% CI 228 to 425 mmol/L min). Compared to sitting, EE during an afternoon of standing work was 174 kcals greater (0.83 kcals/min; p=0.028). The accelerometer MC showed no differences between the afternoons of seated versus standing work; reported differences were thus a function of the standing work and not from additional physical movements around the office. Conclusions This is the first known 'office-based' study to provide CGM measures that add some of the needed mechanistic information to the existing evidencebase on why avoiding sedentary behaviour at work could lead to a reduced risk of cardiometabolic diseases. Source
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. The E. coli SecB gene was cloned into the pET-16b vector (Novagen) containing a His -tag and a tobacco etch virus (TEV) protease cleavage site at the N terminus. Protein samples of E. coli PhoA were produced as described before20. All E. coli MBP constructs were cloned into the pET-16b vector containing a His -tag and a TEV protease cleavage site at the N terminus. The following MBP constructs were prepared in this study (residue numbers of the boundaries are in superscript): MBP1–396, mature MBP27–396, MBP29–99, MBP67–99, MBP97–164, MBP160–201, MBP198–265, MBP260–336, MBP331–396, and the MBP variants MBPG32D/I33P, MBPY283D and MBPV8G/Y283D (MBP mutants are numbered on the basis of the amino-acid sequence of the mature form of MBP). All constructs were transformed into BL21(DE3) cells. Isotopically unlabelled protein samples were produced in cells grown in Luria-Bertani (LB) medium at 37 °C in the presence of ampicillin (100 μg ml−1) to an absorbance at 600 nm (A ) ≈ 0.8. Protein induction was induced by the addition of 0.2 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) and cells were allowed to grow for 16 h at 18 °C. Cells were harvested at A ≈ 1.5 and resuspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, pH 8 and 1 mM PMSF). Cells were disrupted by a high-pressure homogenizer and centrifuged at 50,000g. Proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by tag removal by TEV protease at 4 °C (incubation for 16 h) and gel filtration using Superdex 75 16/60 or 200 16/60 columns (GE Healthcare). Protein concentration was determined spectrophotometrically at 280 nm using the corresponding extinction coefficient. MALS was measured using DAWN HELEOS-II (Wyatt Technology Corporation) downstream of a Shimadzu liquid chromatography system connected to a Superdex 200 10/300 GL (GE Healthcare) gel filtration column. The running buffer for SecB−PhoA complexes was 20 mM KPi (pH 7.0), 100 mM KCl, 4 mM βME, and 0.5 mM EDTA, whereas for SecB−MBP complexes was 20 mM HEPES, pH 7, 150 mM KOAc and 0.05% NaN . Protein samples at a concentration of 0.05–0.2 mM were used. The flow rate was set to 0.5 ml min−1 with an injection volume of 200 μl and the light scattering signal was collected at room temperature (~23 °C). The data were analysed with ASTRA version 6.0.5 (Wyatt Technology Corporation). ITC was performed using an iTC200 microcalorimeter (GE Healthcare) at temperatures ranging from 4 °C to 25 °C. All protein samples were extensively dialysed against the ITC buffer containing 50 mM KPi (pH 7.0), 50 mM KCl, 0.05% NaN and 2 mM tris(2-carboxyethyl)phosphine (TCEP). All solutions were filtered using membrane filters (pore size, 0.45 μm) and thoroughly degassed for 20 min before the titrations. The 40-μl injection syringe was filled with ~0.05–1 mM of SecB solution and the 200-μl cell was filled with ~0.01–0.2 mM PhoA or MBP. To measure the binding affinity of MBP to SecB, the slowly folding MBPV8G/Y283D variant was used to measure the affinity of MBP for SecB. MBPV8G/Y283D was unfolded in 8 M urea, 20 mM HEPES, pH 7, 150 mM KOAc and 0.05% NaN , and diluted 20 times to give a final concentration of 2.7 μM immediately before loading into the cell. The solution containing SecB was precisely adjusted to match the urea concentration. The titrations were performed with a preliminary 0.2-μl injection, followed typically by 15 injections of 2.5 μl each with time intervals of 3 min. The solution was stirred at 1,000 r.p.m. Data for the preliminary injection, which are affected by diffusion of the solution from and into the injection syringe during the initial equilibration period, were discarded. Binding isotherms were generated by plotting heats of reaction normalized by the modes of injectant versus the ratio of total injectant to total protein per injection. The data were fitted with Origin 7.0 (OriginLab Corporation). Isotopically labelled samples for NMR studies were prepared by growing the cells in minimal (M9) medium. Cells were typically harvested at A ≈ 1.0. U-[2H,13C,15N]-labelled samples were prepared for the backbone assignment of SecB and large MBP fragments by supplementing the growing medium with 15NH Cl (1 g l−1) and 2H ,13C -glucose (2 g l−1) in 99.9% 2H O (CIL and Isotec). The 1H–13C methyl-labelled samples were prepared as described20, 29, 31. α-Ketobutyric acid (50 mg l−1) and α-ketoisovaleric acid (85 mg l−1) were added to the culture 1 h before the addition of IPTG. Met-[13CH ]- and Ala-[13CH ]-labelled samples were produced by supplementing the medium with [13CH ]-Met (50 mg l−1) and [2H ,13CH ]-Ala (50 mg l−1). For Thr labelling, a Thr-auxotrophic cell strain was used, and the medium was supplemented with [2H ,13CH ]-Thr (25 mg l−1). For Phe, Tyr, and Trp labelling, U-[1H,13C]-labelled amino acids were used. Alternative 13C-labelling of aromatic residues was performed as described32. All precursors and amino acids were added to the culture 1 h before the addition of IPTG, except Ala, which was added 30 min before induction. NMR samples were typically prepared in 50 mM KPi (pH 7.0), 50 mM KCl, 0.05% NaN , 5 mM βME and 7% D O. NMR experiments were recorded on Bruker 900, 850 and 700 MHz spectrometers. NMR spectra were typically recorded at 10 °C for the isolated PhoA and MBP fragments and at 35 °C for SecB and its complexes. Protein sample concentration ranged from 0.1 to 1.0 mM. All NMR spectra were processed using NMRPipe33 and analysed using NMRView (http://www.onemoonscientific.com). The SecB tetramer packs as a dimer of dimers and gives rise to two pairs of magnetically equivalent subunits: A and D give one set of resonances and subunits B and C give another set of resonances (Extended Data Fig. 1a). Sequential backbone assignment of SecB was achieved by the use of standard triple-resonance NMR pulse sequences. Three-dimensional (3D) 1H–15N NOESY experiments were used to confirm and extend the backbone assignment within each subunit. Side-chain assignment for methyls and aromatic residues was accomplished using the following NMR experiments: 3D (1H)–13C heteronuclear multiple-quantum coherence (HMQC)–NOESY-1H–13C HMQC, 13C-edited NOESY–HSQC, 13C-edited HSQC–NOESY, 15N-edited NOESY-HSQC, 3D (1H)–13C HSQC–NOESY-1H–15N HSQC, and 3D (1H)–15N HSQC–NOESY-1H–13C HSQC. We previously described the assignment strategy for unfolded PhoA20. We followed a similar strategy to assign MBP in the unfolded state by making use of several MBP fragments that remain soluble and unfolded when isolated (Extended Data Fig. 1c): MBP29–99, MBP67–99, MBP97–164, MBP160–201, MBP198–265, MBP260–336 and MBP331–396. Isolated MBP fragments encompassing the first 26 N-terminal residues (signal sequence) were not stable and this region could only be assigned in complex with SecB. Overlay of the spectra of the MBP fragments with the spectra of full-length MBP in 4 M urea indicated very good resonance correspondence. This is expected because all of the fragments, as well as the MBP, in 4 M urea are unfolded. Resonance assignment obtained for the various fragments was transferred to full length MBP in urea, and ambiguities were resolved by the use of 3D NMR spectra. It should be noted that although resonance dispersion in unliganded PhoA and MBP is poor, complex formation with SecB alleviates this problem (for the PhoA and MBP residues in the SecB-binding regions) with the spectra being of high resolution (Extended Data Fig. 4c). Assignment of the resonances in SecB−PhoA was accomplished by first assigning the complexes between SecB and the individual PhoA sites (SecB−PhoAa, SecB−PhoAc, SecB−PhoAd, SecB−PhoAe). We used U-12C,15N-labelled samples that contained specifically protonated methyl groups of Ala, Val, Leu, Met, Thr and Ile (δ1) and protonated aromatic residues Phe, Tyr and Trp in an otherwise deuterated background. The high sensitivity and resolution of the methyl region, combined with the high abundance of these nine amino acids in SecB (Extended Data Fig. 1a) and in the SecB-binding sites of PhoA and MBP, provided a large number of intermolecular NOEs for the SecB−PhoA and SecB−MBP complexes (Extended Data Table 1). Because PhoA in complex with SecB provided higher quality spectra than the spectra of MBP in complex with SecB, we determined first the structure of the SecB−PhoA complex (~120 kDa) by NMR. We initially characterized the structure of the each PhoA site (a–e) individually in complex with SecB (Extended Data Fig. 5). The structures of SecB−PhoAa, SecB−PhoAc, SecB−PhoAd, and SecB−PhoAe, were determined by NMR and are presented in Extended Data Fig. 5. A large number of intermolecular NOEs were collected for each one of the complexes (Extended Data Table 1). Because of the relatively short length of the polypeptides encompassing the individual PhoA sites, multiple PhoA molecules bound to SecB, as shown in Extended Data Fig. 5. We also note that we detected the presence of a small number of intermolecular NOEs that were suggestive of alternative conformations of the PhoA sites bound to SecB. However, the intensity of these sets of NOEs was much weaker, indicating that the population of such alternative complexes is low. To solve the structure of the SecB−PhoA complex, we sought to determine how each one of the PhoA sites binds to SecB in the context of the full length PhoA. To circumvent the signal overlap in this large complex, we used samples where the two proteins were isotopically labelled in different amino acids. For example, in one of these samples SecB was labelled in the methyls of Leu, Val and Met, whereas PhoA in the methyls of Ile amino acids. Because of the distinct chemical shifts of 1H and 13C resonances of the methyls and the isotope labelling scheme, it was possible to measure specific intermolecular NOEs between SecB and PhoA (Extended Data Fig. 4b). Several of these samples were used to determine as many intermolecular NOEs as possible. As expected, the NOEs were compatible with the structure of each PhoA site in complex with SecB, with the crucial difference that only one PhoA molecule could be accommodated in SecB. Owing to its short length, the isolated PhoA site b (PhoAb) binds to almost all of the exposed hydrophobic surface of SecB, as determined by NMR. In the SecB−PhoA complex with SecB, PhoA site b can only bind to the secondary binding site, as determined by NOEs. To further corroborate the structure of the SecB−PhoA complex we used PRE data (see below). The PRE-derived distances were fully compatible with the NOE data collected on SecB−PhoA. The structure of the SecB−PhoA complex was determined using the set of intermolecular NOEs collected directly in the complex and further refined using the intermolecular NOEs collected for the corresponding isolated PhoA sites in complex with SecB. It should be noted that because of the symmetry in SecB, the various PhoA sites may bind to any of the four SecB subunits. The final arrangement will be dictated by the length of the linkers tethering the SecB-recognition sites (as shown in Fig. 2), namely how far nearby recognition sites can bind from each other, and thus alternative routes of the polypeptide bound to SecB may be present. The only conceivable difference among the various conformations is the relative disposition of the PhoA sites. In all cases all of the SecB-recognition sites in PhoA are engaged by SecB in the complex and PhoA wraps around SecB. The NMR-driven structural model of the SecB−MBP complex (Extended Data Fig. 7b) was determined as follows: NMR analysis demonstrated that all seven recognition sites in MBP (labelled a–g) are bound to SecB in the SecB−MBP complex (Extended Data Fig. 7a). We have determined the high-resolution structure of MBPd and MBPe in complex with SecB (Extended Data Fig. 6). Because of their length and the short linker tethering the two sites, d and e sites most probably bind to the same side of SecB. MBP site f is the longest one, consisting of ~90 residues, and is thus entirely accommodated on the other side of SecB. With sites d, e and f occupying the primary binding sites, the other recognition sites (a, b, c and g), being much shorter, can be accommodated within the secondary client-binding sites on SecB. The structure of MBP sites d and e in complex with SecB was determined using the experimental intermolecular NOE data. The hydrophobic residues of the sites a, b, c, f, and g, showing the strongest effect upon SecB binding as determined by differential line broadening, were used to drive the docking of these sites to non-polar residues on SecB. The modelled structure shows that the entire MBP sequence can be accommodated within one SecB molecule. The structures of SecB in complex with PhoA and MBP were calculated with CYANA 3.97 (ref. 34), using NOE peak lists from 3D (1H)–13C HMQC–NOESY-1H–13C HMQC, 3D (1H)–15N HSQC–NOESY-1H–13C HSQC, 13C-edited NOESY–HSQC, and 15N-edited NOESY–-HSQC. The 13Cα, 13Cβ, 13C′, 15N and NH chemical shifts served as input for the TALOS+ program35 to extract dihedral angles (φ and ψ). The side chains of SecB residues within or nearby the PhoA and MBP binding sites were set flexible and their conformation was determined using intermolecular NOEs collected for each one of the complexes. The SecB regions remote to the binding sites were set rigid using the crystal structure coordinates for E. coli SecB26. The 20 lowest-energy structures were refined by restrained molecular dynamics in explicit water with CNS36. The percentage of residues falling in favoured and disallowed regions, respectively, of the Ramachandran plot is as follows: 99.4% and 0.6% for SecB–PhoA; 99.4% and 0.6% for SecB–PhoAa; 99.3% and 0.7% for SecB–PhoAc; 99.2% and 0.8% for SecB–PhoAd; 99.3% and 0.7% for SecB–PhoAe; 99.4% and 0.6% for SecB–MBPd; and 99.4% and 0.6% for SecB–MBPe. PRE experiments were used to confirm the position of each individual PhoA binding site in the SecB−PhoA complex. First, a ‘Cys-free’ variant of PhoA was prepared by mutating the four naturally occurring Cys residues in PhoA (Cys190, Cys200, Cys308 and Cys358) to Ser. We then introduced a Cys residue to either end of each SecB-binding site in PhoA and prepared a total of ten single-Cys mutants: T5C, T23C, K65C, M75C, G91C, G140C, Q274C, C308, N450C and C472. The protein purified from Ni-NTA column was quickly concentrated and loaded onto HiLoad 16/60 Superdex 200 gel filtration column (GE healthcare) using a buffer containing 50 mM KPi (pH 7.0), 150 mM NaCl and 0.05% NaN . Immediately after elution the purified single-Cys PhoA mutant was divided into two equal portions for parallel treatment with (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-ethanethiosulfonate (MTSL, Toronto Research Chemicals, Toronto) and a diamagnetic MTSL analogue, in a tenfold molar excess at 4 °C for 16–20 h. MTSL was prepared in a 50 mM concentrated stock in acetonitrile. Free MTSL was removed by extensive buffer exchange using Centricon Centrifugal Filter with a MWCO of 10,000 (Millipore) at 4 °C. The MTSL-labelled PhoA protein samples were then concentrated and added into the 2H-methyl-13CH -labelled SecB at a final molar ratio of PhoA:SecB = 1:1. 2D 1H,13C HMQC spectra were recorded at 28 °C. A sample of SecB in complex with PhoA cross-linked to a diamagnetic MTSL analogue was used as a reference. Residues experienced significant NMR signal intensity reduction (>50% intensity loss) were identified as sites being within 20 Å of the paramagnetic centre whereas residues experiencing more than 90% intensity loss were identified as sites being within 14 Å of the paramagnetic centre. Refolding experiments of MBP were performed as described before37 with some modifications. Briefly, MBP was first denatured in 8 M urea, 100 mM HEPES, 20 mM KOAc, 5 mM Mg(OAc) , pH 7.4, and 0.05% NaN . Refolding was initiated by rapid dilution (20 times dilution) in the urea-free buffer and the refolding process of MBP in the absence and presence of SecB or TF was monitored by the change in the intrinsic Trp fluorescence. Fluorescence intensity was measured using either a spectrofluorometer (FluoroMax-4, Horiba) or a microplate reader (Infinite 200 PRO, Tecan). The excitation and emission wavelengths were set to 295 nm and 345 nm, respectively. For measurement using the FluoroMax-4 instrument, the MBP concentration in the 1-ml cuvette was 0.4 μM, whereas for the microplate reader experiments the concentration of MBP was 4 μM in the 30 μl-plate well. All fluorescence measurements were performed at 25 °C. Data were fitted by the Prism 6 (GraphPad) software using the nonlinear regression analysis equation38. All SPR experiments were performed on a Biacore T200 system (GE Healthcare) using a NTA-coated Sensor Chip NTA (GE Healthcare) at a flow rate of 50 μl min−1. The PhoA protein sample used for SPR experiments was genetically constructed with a His -tag at the carboxy (C) terminus and a flexible (Gly-Ser) linker repeat inserted in between to avoid steric hindrance. A single-cycle kinetic procedure was used to characterize the interaction of SecB and PhoA. The His-tagged PhoA was immobilized onto a NTA sensor chip, followed by washing with the running buffer containing 50 mM phosphate, 50 mM KCl, pH 7, 0.05% NaN , and 2 mM TCEP. The reducing agent (TCEP) ensured that PhoA was in the unfolded state20. SecB (analyte) at a range of concentrations (0.1–25.6 μM) was injected, and data for a period of 30 s of association and 60 s of dissociation were collected. MBP was prepared with a His -tag at the N terminus followed by a flexible nine-residue linker to avoid steric hindrance. Multiple-cycle kinetic analysis was performed for the SPR experiments of the binding between MBP and SecB where each sample concentration was run in a separate cycle, and the surface was regenerated between each cycle using NTA regeneration buffer. His-tagged MBP was denatured in 8 M urea and immobilized onto a NTA sensor chip. Urea was quickly washed away by running buffer containing 20 mM HEPES, pH 7.4, 150 mM KOAc and 0.05% NaN . SecB was injected at concentrations ranging from 2.5 nM to 1.6 μM. The association and dissociation time for data collection was set as 90 s and 120 s, respectively. After urea was removed, MBP remained in the unfolded conformation for sufficient time to interact with SecB. This was confirmed by monitoring the refolding behaviour of MBP using an Infinite 200 PRO microplate reader (Tecan) at the temperature range of the experiments. All SPR experiments were repeated three times and highly reproducible data were obtained. The sensorgrams obtained from the assay channel were subtracted by the buffer control, and data were fitted using the Biacore T200 evaluation software (version 1.0). BLI experiments were performed using an Octet system (forteBIO) at room temperature (~23 °C). MBP was biotinylated using the biotination kit EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific). Biotin label freshly dissolved in water was added to the protein solution to a final molar ratio of 1:1 in buffer containing 50 mM KPi, pH 7, 150 mM NaCl, 0.05% NaN , and the solution was mixed at room temperature for 45 min. Unlabelled biotin label was removed by extensive buffer exchange using Centricon Centrifugal Filter with a MWCO of 10,000 (Millipore) at 4 °C using a buffer containing 20 mM HEPES (pH 7), 150 mM KoAc and 0.05% NaN . Biotin-labelled MBP (200 nM) denatured in 8 M urea was immobilized onto the streptavidin (SA) biosensor, and the biosensors were subsequently blocked with biocytin in 8 M urea solution before a quick 30 s dip into the urea-free buffer. SecB or TF previously diluted was applied in a dose-dependent manner to the biosensors immobilized with MBP. Bovine serum albumin (BSA) powder (Sigma-Aldrich) was added to a final concentration of 2% to avoid non-specific interaction. Parallel experiments were performed for reference sensors with no MBP captured and the signals were subsequently subtracted during data analysis. The association and dissociation periods were set to 2 min and 5 min, respectively.
News Article | January 19, 2016
Elm EV, an electric vehicle charging station installation company, recently launched its first crowdfunding campaign — with the aim being to raise more than £200,000 in new funding. The new funding that’s raised will reportedly be used to develop a new range of charging station products/models, and also to hire new promotional staff — with the intention being to increase public and private sector awareness of the technology/solution. Expansion into international markets is also a stated aim. “The growth of the ultra-low emission vehicle industry is phenomenal and we are in a great position,” stated Anthony Piggott, Elm EV’s technical director. “This is a one-off opportunity for members of the public, enthusiasts and investors to help build the EV charging infrastructure and to be a part of the fast-paced industry, reaping the rewards.” The crowdfunding campaign allows investors to directly purchase shares of the company — which was created in 2013, and is valued (according to the crowd-funding campaign) at £2.25 million. As Elm EV is currently one of the only companies that’s certified to install fast electric vehicle (EV) charging stations in the UK, the bet doesn’t seem to be a bad one for those so inclined — as an EV fast-charger buildout seems likely in the UK at some point in the coming decade. The company generally works with large industry players, such as Nissan GB, to provide commercial customers with charging equipment, amongst other things. The company is, notably, one of the charging station providers for the NHS commercial electric vehicle fleet — which is still growing. With the UK having seen quite a surge in EV and plug-in hybrid (PHEV) sales in 2015, I’m not going to state outright that Elm EV makes a good investment, but I am going to say that a good argument could be made that it does. Get CleanTechnica’s 1st (completely free) electric car report → “Electric Cars: What Early Adopters & First Followers Want.” Come attend CleanTechnica’s 1st “Cleantech Revolution Tour” event → in Berlin, Germany, April 9–10. Keep up to date with all the hottest cleantech news by subscribing to our (free) cleantech newsletter, or keep an eye on sector-specific news by getting our (also free) solar energy newsletter, electric vehicle newsletter, or wind energy newsletter. James Ayre 's background is predominantly in geopolitics and history, but he has an obsessive interest in pretty much everything. After an early life spent in the Imperial Free City of Dortmund, James followed the river Ruhr to Cofbuokheim, where he attended the University of Astnide. And where he also briefly considered entering the coal mining business. He currently writes for a living, on a broad variety of subjects, ranging from science, to politics, to military history, to renewable energy. You can follow his work on Google+.
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment. The Arabidopsis thaliana wild-type (Col-0), T-DNA insertion mutants mdis1-1 (GABI_463E06), mdis1-2 (GABI_090F03), mdis2 (SALK_004879) and Capsella rubella were obtained from ABRC stock centre. mik1 (SALK_095005) and mik2 (SALK_061769) were obtained from J. Zhou. The E. salsugineum seeds were obtained from Q. Xie. Plants were grown at 22 °C under long-day conditions (16-h light/8-h dark cycles). For C. rubella and E. salsugineum, the sterilized seeds were vernalized on the MS media at 4 °C for 30 days and then grown at 22 °C under long-day conditions. Pollen tubes were germinated on the germination media (1 mM CaCl , 1 mM Ca(NO ) , 1 mM MgSO , 0.01% H BO , 18% sucrose and 0.5% agarose) and cultured for 5 h at 22 °C. The germination ratio was scored under light microscopy. Mean value was calculated from three independent experiments and for each experiment, more than 300 pollen were scored. For in vivo tube growth, pollen from the wild-type and mutants were pollinated on the emasculated pistil with mature stigma as reported20. The pistils were collected at 3, 6 and 8 h after pollination and fixed for aniline blue staining. The pollen tubes in the pistil were photographed with Leica M205 microscope. The length of pollen tubes was measured with Image J software (http://rsb.info.nih.gov/ij/). Flowers at 12c stage were emasculated and left to grow for 12–24 h to achieve pistil maturation. Then about 20 pollen grains from wild-type or mutant plants, respectively, were dispersed on the stigma papillar cells with a tiny brush. After 24 h, pistils were excised and fixed in Carnoy’s fixative (75% ethanol and 25% acetic acid) as reported21, 22. The pistils were washed in 50 mM PBS buffer (NaHPO /NaH PO , pH 7.0) three times and immersed in 1 M NaOH overnight for softening. Then after three washes with PBS, the pistil was stained with 0.1% aniline blue (pH 8.0 in 0.1 M K PO ) for 6 h. The stained pistils were observed under Axio Skop2 microscope (Zeiss) equipped with an ultraviolet filter set. Ovules with micropylar guidance defect and the ratio of fertilized ovules to the number of pollen tubes in the style were calculated and the mean values from three independent experiments were compared with that of the wild type. For the dominant-negative constructions, the kinase domains were inactivated by replacing the conserved lysine residue in the intracellular ATP-binding domain with glutamic acid to generate dominant-negative constructs. For the atypical kinase, the intracellular domain was chimaerically replaced with that of BRASSINOSTEROID INSENSITIVE1 (BRI1)23 receptor kinase with an inactive kinase domain (K to E substitution). For GFP and GUS reporter expression, genomic sequences containing 2 kb native promoters and the genomic coding sequence for MDIS1 and MDIS2 were subcloned into the pCAMBIA1300-GFP binary vector. For complementation of mik mutants, full-length coding sequence driven by LAT52 promoter was cloned into pCAMBIA1300. Similarly, full-length LURE1.2 fused with a C-terminal Flag tag driven by the 35S promoter was cloned into the pCAMBIA1300. For complementation assay, the genomic fused GFP constructs were transformed into the mutant using Agrobacterium-mediated floral dip method24. To break down the reproductive isolation barrier, the full-length MDIS1 coding sequence under the LAT52 promoter was introduced into C. rubella by floral dip method. LURE1.2 and PDF2.1 lacking the putative N-terminal signal peptides (71 and 55 amino acids, respectively) were fused N-terminally with a His-tag using pET28a vector (Novagen). Similarly, the ectodomains of MDIS1, MDIS2, MIK1, MIK2 and PRK3 lacking the predicted signal peptides were fused with an N-terminal GST tag using a pGEX4T-2 vector. The fused proteins were expressed in Escherichia coli strain Rossetta DE3 (Stratagene). Cells were grown to an A value of 0.6 at 37 °C and then induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 6 h at 22 °C. The cells were lysed by sonication on ice in lysis buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, Complete Protease Inhibitor Cocktail (Roche) and 1 mg ml−1 lysozyme (Wako). After centrifugation at 12,000 g for 20 min at 4 °C, the supernatants and pellets were collected separately; the pellet was washed three times with the lysis buffer. For LURE1, the insoluble His–LURE1.2 peptides in the inclusion bodies were solved in 1 M urea supplemented with 6 M guanidine-HCl (in Tris-HCl buffer, pH 8.0) for 1 h on ice. Then the peptides were diluted at 1:10 and refolded for 3 days at 4 °C using glutathione (reduced form: oxidized form = 10:1, MERCK) and l-arginine ethyl ester dihydrochloride (Sigma-Aldrich). The folded peptides were dialysed with 3-kDa centrifugal filter (Millipore) and eluted with 50 mM Tris-HCl (pH 8.0) and then used for pull-down, co-IP, protoplasts treatment, pollen tube guidance assays and antibody generation. For purification of GST-tagged ectodomain of MDIS1, MDIS2, MIK1, MIK2 and PRK3 proteins, cells from 2 l culture were collected and lysed respectively as described above. The supernatants were used for affinity purification by glutathione agarose beads (GE, 17-0756-01) to avoid extra folding process, although more fused proteins were in the pellets than the supernatant. For GST pull-down assay, the purified proteins were mixed and incubated for 3 h and then subjected to pull-down assay with glutathione agarose beads for 3 h at 4 °C. The beads were collected by centrifugation and then washed five times with buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100 and 0.1% SDS. Finally, the proteins bound on the beads were boiled with 1× SDS sample buffer in 95–100 °C water bath and then subjected to SDS–PAGE and immunoblot with anti-GST (GE Healthcare, 27-4577-01) and anti-His (Santa Cruz) antibody. For mobility shift detection of phosphorylated proteins, phosphatase inhibitor phrostop (Roche) was added during purification and incubation. Moreover, 50 μM Phos-tag (AAL-107) and 50 μM MnCl was added to the gel according to the manufacturer’s procedure. After electrophoresis, the gel was treated with 10 mM EDTA, pH 8.0, for 10 min to remove the Mn2+ before immunoblot assay. Seedlings of LURE1.2-Flag transgenic plants were ground to fine powder in liquid nitrogen and solubilized with extraction buffer (0.05 M HEPES-KOH, pH 7.5, 150 mM KCl, 1 mM EDTA, 0.1% Triton X-100 with freshly added proteinase inhibitor cocktail (Roche)). The extracts were centrifuged at 10,000g for 10 min, and the supernatant was incubated with pre-washed anti-Flag M2 magnetic beads (Sigma-Aldrich, M8823) for 3 h at 4 °C, and then the beads was washed six times with the extraction buffer. The immunoprecipitates were eluted with 3 × Flag peptides. For co-IP in protoplasts, the transformed protoplasts expressing MDIS1–HA, MIK–HA and BAK1–HA were incubated with the purified LURE1.2–Flag or the 200 nM folded His–LURE1.2 purified from E. coli for 10 min and lysed for co-IP with pre-washed anti-HA agarose beads (Sigma-Aldrich, A2095). The precipitates were diluted with SDS sample buffer, separated on a 10% SDS–PAGE gel and subjected to immunoblot with the corresponding antibodies (anti-Flag, Sigma-Aldrich, F1804; anti-HA, Santa Cruz, sc-7392; anti-His, Santa Cruz, sc-803). Arabidopsis protoplast transformation was performed as reported previously25. For the His–LURE1-protoplast binding assay, the protoplasts incubated with 10 μm LURE1.2 for 5 min, washed three times with the culture buffer and then lysed for SDS–PAGE and immunoblot. For the enhanced interaction between MDIS1 and MIK proteins by LURE1.2, the protoplasts co-transformed with MDIS1–HA and MIK1–Flag were divided into two equal volumes. One was incubated with 0.5 nM LURE1.2 and another with equal volume of 50 mM Tris-HCl (pH 8.0) as mock control for 10 min and subjected to anti-HA immunoprecipitation. For the phosphorylation test, the transformed protoplasts were divided equally into two and incubated for 10 min with 200 nM LURE1.2 or 50 mM Tris-HCl (pH 8.0), respectively. For competition assay, protoplasts expressing MDIS1–HA, MIK1–HA and MIK2–HA were each divided equally into four centrifuge tubes and incubated with purified LURE1.2–Flag. Then active His–LURE1.2 of different concentrations was added to the protoplasts and incubated for 10 min and subsequently co-immunoprecipated with anti-HA conjugated agarose beads. For co-IP in planta, the flowers opened in the morning were collected in the afternoon at the estimated time when the pollen tubes are approaching the ovules. Total proteins were subjected to co-IP with anti-GFP conjugated agarose (ChromoTek, gta-200) or anti-LURE1.2 and protein-A-conjugated magnetic beads (Bio-Rad, 161–4013). The immunoprecipitates was subjected to SDS–PAGE and immunoblot with the corresponding antibodies (anti-GFP-HRP, Miltenyi Biotec, 130-091-833). All the co-IP experiments were repeated at least three times. For A. thaliana, the same germination media as that for in vitro germination was used. For C. rubella, a modified media (4 mM CaCl , 4 mM Ca (NO ) , 0.01% H BO , 10% sucrose and 0.5% agarose) was used. Semi-in-vitro germination and ovule-pollen attraction assay were performed as reported in A. thaliana3. Pollen tubes entered the micropyle were scored as successful breakdown of the reproductive isolation and the pollen tubes bypass outside the micropyle within 20 μm were scored as failing to enter the micropyle. For the attraction assay, gelatin (Nacalai) beads containing 40 μM LURE1.2 were made and placed beside the pollen tube tip using a micro-manipulator (Narishige) equipped with an inverted microscope (Zeiss AxioVert. A1) as described previously26. Behaviour of pollen tubes was monitored and recorded with a CCD camera. Pollen tubes growing to the beads with >30° direction change were regarded as effective pollen tube attraction. Total RNA was extracted from pollen, in vitro germinated pollen tubes (3 h after pollination) and seedlings with TRIzol reagent (Invitrogen) and then treated with DNase I (RNase-free DNase kit, Qiagen) to remove DNA. SuperScript III Reverse Transcriptase (Invitrogen) was used for the reverse transcription reactions. qPCR was performed with Power SYBR Green PCR Master Mix on the Bio-RAD C1000 Thermal Cycler using Tubulin 2 as the internal control for quantitative normalization. The specificity of the primers was examined by running the PCR products on 2.5% agarose gels and sequencing. The affinity of the purified GST, GST–MDIS1ECD, MDIS2ECD, MIK1 ECD, MIK2 ECD, ERECTAECD and PXY ECD to His–LURE1.2 was measured using the Monolith NT.115 (Nanotemper Technologies). The GST-fusion proteins were fluorescently labelled according to the manufacturer’s procedure. The solution buffer was exchanged to labelling buffer and the protein concentration was adjusted to 2 μM. Then fluorescent dye NT-647-NHS was added and mixed and incubated for 30 min at 25 °C in the dark. Finally, the labelled proteins were dialysed with column B (Nanotemper L001) and eluted with 50 mM Tris-HCl (pH 8.0) supplemented with 0.02% Tween 20. For each assay, the labelled protein (about 1 μM) was incubated with the same volume unlabelled His–LURE1.2 of 12 different serial concentrations in 50 mM Tris-HCl (pH 8.0) supplemented with 0.02% Tween 20 at room temperature for 10 min. The samples were then loaded into silica capillaries (Polymicro Technologies) and measured at 25 °C by using 20%–40% LED power and 20% MST power. Each assay was repeated three times. Data analyses were performed using Nanotemper analysis software and OriginPro 9.0 software. The constructs containing MDIS1-NE (MDIS1 fused with the N-terminal YFP), MIK1-CE and MIK2-CE (MIK1 and MIK2 fused with the C-terminal YFP, respectively) were generated as described previously8. The Agrobacterium tumefaciens EHA105 strains carrying MDIS1-NE and MIK-CE were equally mixed with and without EHA105 strain carrying LURE1.2–Flag and transformed into half of the same tobacco leaf. The transformed leaves were photographed 2 days later with a confocal laser scanning microscope (Zeiss Meta 510). Images were acquired using the same optical setting and average total pixel intensity values were calculated by sampling images of different leaves using the ImageJ software as reported27. Mean values of three experiments, each with five transformed leaves, were compared using Student’s t-test for biological significance. The E. coli cells expressing the fusion proteins were lysed and centrifuged at 4 °C. The affinity-purified fusion proteins from the supernatants were subjected to mass spectrometry. His–MDIS1KD was incubated with GST–MIK1KD in vitro in kinase assay buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl and 100 mM ATP) for 1 h at 30 °C. The proteins were separated by 10% SDS–PAGE and the gel was stained with Coomassie blue G250. The corresponding proteins band were cut into slices and subjected to alkylation/tryptic digestion followed by LC–MS/MS as reported previously28. For disulfide bonds determination, GST–MDIS1ECD, GST–MIK1ECD and GST–MIK2ECD were affinity purified from the supernatants of the bacterial lysis and eluted with 50 mM Tris-HCl, pH 8.0. Then disulfide bonds were determined by mass spectrometry as previously reported29. Alignment of protein sequences were aligned using ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Phylogenetic tree of the alignment were drawn with MEGA5 (http://www.megasoftware.net/) using the neighbour-joining method with bootstrapping based on 1,000 replicates. The leucine-rich repeat domains were predicted with LRRfinder (http://www.lrrfinder.com/) and HHPREP program. The transmembrane domains were predicted with TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The signal peptides were predicted with SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/). The coding sequences of MDIS1 or MIK1 and MIK2, respectively, were cloned into the pBT3-SUC bait or pPR3-N prey according to the manufacture’s procedure (DualsystemBiotech). Yeast strain NMY51 was co-transformed with the bait and prey constructs and grown on the selective medium lacking Trp, Leu, His and adenine. Total RNA was extracted from pollen, leaf, flower and total plant of C. rubella and E. salsugineum with TRIzol reagent (Invitrogen) and then treated with DNase I (RNase-free DNase kit, Qiagen) to remove any contaminating DNA. SuperScript III Reverse Transcriptase (Invitrogen) was used in reverse transcription reactions. ACTIN11 was used as the control for quantitative normalization. The specificity of the primers was confirmed by sequencing of the band after electrophoresis. The accession numbers for the amplified genes are as follows: CrMDIS1 (XM_006280043), EsMDIS1 (XM_006398206), CrMIK1 (XM_006285722), EsMIK1 (XM_006412864), CrMIK2 (XM_006286915), EsMIK2 (XM_006397188), CrACTIN11 (XM_006297859) and EsACTIN11 (XM_006407307). The histochemical GUS activity assay was performed in the solution containing 2 mM X-Gluc (Sigma) in 50 mM PBS (pH 7.0) and 0.5 mM potassium/ferrocyanide. GUS solution was added to the samples and incubated at 37 °C overnight. Digital images were taken with a Zeiss Axio Skop2 plus microscope. For GFP observation, images were taken with Zeiss confocal laser scanning microscope with a setting of 488 nm excitation (Carl Zeiss, Meta 510 confocal microscope). The semi-in-vitro germinated MDIS1–GFP pollen tubes were treated with 500 nM LURE1.2 and photographed by CLSM 780 (Zeiss) after different times. The anti-MIK1 and anti-MIK2 antibodies were raised in mouse with the purified His-tagged extracellular domains lacking the predicted N-terminal signal peptide. Anti-LURE1.2 antibody was raised in mouse with the folded active His–LURE1.2 fusion protein. For MIK1 and MIK2, the specificity of antibodies was tested with the fusion proteins expressed in protoplasts and the total proteins of pollen from the wild-type and corresponding mutant plants. For LURE1.2, the antibody specificity was tested with the total protein from the leaves of LURE1.2–Flag-overexpressing plants. For immunostaining, the semi-in-vitro germinated pollen tubes were fixed in 3.7% paraformaldehyde (3.7% formaldehyde, 1 mM CaCl , 1 mM MgSO , 50 mM HEPES, 5% sucrose, pH 7.4) for 30 min, washed with PME buffer (50 mM PIPES, 1 mM MgCl , 5 mM EGTA, pH 6.8) three times and then subjected to 1% Driselase and 1% cellulase for 10 min. The sample was sequentially washed with PBS buffer (pH 7.4) three times, NP40 buffer (0.5% Nonidet P-40, 1% BSA, in PBS, pH 7.4) and PBS buffer once. Antibodies diluted 1:500 (with PBS containing 3% BSA) were incubated with the sample overnight at 4 °C and then washed with PBS three times. The samples were incubated for 1 h at 4 °C with FITC-labelled goat anti-mouse secondary antibody (KBL, 202-1806) and washed with PBS three times. Anti-fade mounting medium (Invitrogen, P36934) was used for signal detection by confocal laser scanning microscopy (Zeiss Meta 510) with 488 nm excitation.
International Journal of Art Therapy: Inscape | Year: 2011
This is a retrospective analysis of individual art therapy lasting six years. Outcome measures, patient ratings and feedback, and the opinion of the therapist indicated improvement that was maintained up to three years follow-up. Process data, consisting of sessional outcome measures, indicated the severity of problems and a wide and dramatic fluctuation on a weekly basis. The examination of art work alongside data from researchers illustrated a series of phases in therapy. This material has been previously published and a summary is presented here. Given the patient's presentation, the long length of therapy was unexpected. In this paper the art therapist offers a formulation of the patient's problems and considers why art therapy was helpful when the patient had failed to improve from previous therapies. Concepts of attachment theory linked to neuroscience are used to enlighten the understanding of this case. © 2011 British Association of Art Therapists. Source