Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

Key resources table Reagent or resource Source Identifier Antibodies

Rabbit monoclonal anti-SPR (clone EPR9290) Abcam Cat#ab157194 Mouse monoclonal anti-β-tubulin (clone 5H1) BD Biosciences Cat#556321; RRID: AB_396360 Goat anti-Rabbit secondary antibody, HRP-conjugate Cell Signaling Technology Cat#7074; RRID: AB_2099233 Horse anti-Mouse secondary antibody, HRP-conjugate Cell Signaling Technology Cat#7076; RRID: AB_330924 Chemicals, Peptides, and Recombinant Proteins CP-TMR-SMX This paper N/A BG-TMR-SMX This paper N/A SiR-Halo This paper N/A CP-TMR Johnsson Lab N/A Sulfapyridine (≥99%) Sigma-Aldrich Cat#S6252 Sulfamethoxazole (>98%) TCI Cat#S0361 Sulfachloropyridazine Sigma-Aldrich Cat#S9882 (±)-Verapamil hydrochloride (≥99%) Sigma-Aldrich Cat#V4629 H 2 O 2 (30% (w/w), puriss. p.a.) Sigma-Aldrich Cat#31642 2-Deoxy-D-glucose (≥99%) Sigma-Aldrich Cat#D6134 6-aminonicotinamide (99%) Sigma-Aldrich Cat#A68203 Resveratrol (>99%) TCI Cat#R0071 Nicotinic acid (≥99.5%) Sigma-Aldrich Cat#72309 Nicotinamide (>98%) Sigma-Aldrich Cat#N0636 β-Nicotinamide mononucleotide (95–100%) Sigma-Aldrich Cat#N3501 Nicotinamide riboside Auwerx Lab, EPFL N/A FK866 hydrochloride hydrate (≥98%) Sigma-Aldrich Cat#F8557 Metformin (97%) Sigma-Aldrich Cat#D150959 Phenformin Sigma-Aldrich Cat#P7045 Rotenone (≥95%) Sigma-Aldrich Cat#R8875 Oligomycin A (≥95%) Sigma-Aldrich Cat#75351 NADPH tetrasodium salt (≥97%) Roche Cat#10621692001 NADP + disodium salt (≥97%) Roche Cat#10128058001 NADH disodium salt (≥95%) AppliChem Cat#A1393,0001 NAD + free acid (100%) Roche Cat#10127965001 ATP disodium salt (≥98%) AppliChem Cat#A1348,0005 ADP sodium salt (≥95%) Sigma-Aldrich Cat#A2754 GTP sodium salt hydrate (≥95%) Sigma-Aldrich Cat#G8877 L-sepiapterin Cayman Cat#81650 MitoTracker Green FM Life Technologies Cat#M7514 Hoechst 33342 Life Technologies Cat#H1399 Propidium iodide (≥94%) Sigma-Aldrich Cat#81845 Experimental Models: Cell Lines U-2 OS (Human osteosarcoma) ECACC Cat#92022711 HEK-293T (Human embryonic kidney) ATCC Cat#CRL-3216 NIH/3T3 (Mouse embroynic fibroblast) ATCC Cat#CRL-1658 HeLa (Human cervix epitheloid carcinoma) ATCC Cat#CCL-2 A549 (Human lung carcinoma) ECACC Cat#86012804 Recombinant DNA pET-51b(+) Novagen 71553 pEBTet ( Bach et al., 2007 ) N/A pET-51b(+)_NADP This paper N/A pET-51b(+)_NAD This paper N/A pEBTet_NADP-cyto This paper N/A pEBTet_NADP-nucl This paper N/A pEBTet_NADP-mito This paper N/A pEBTet_NAD-cyto This paper N/A pEBTet_NAD-nucl This paper N/A pEBTet_NAD-mito This paper N/A Software and Algorithms OriginPro 9 OriginLab Corporation http://www.originlab.com/ PyMOL Schrödinger, LLC https://www.pymol.org/ FIJI (ImageJ) ( Schindelin et al., 2012 ) https://fiji.sc/ SymPhoTime 64 PicoQuant https://www.picoquant.com/ Huygens Essential Scientific Volume Imaging https://svi.nl/HuygensEssential FlowJo v10 FlowJo, LLC https://www.flowjo.com/ R 3.4.0 R Core Team, 2017 https://www.r-project.org/ Other Leica TCS SP8 X confocal microscope - PicoHarp 300 (PicoQuant) TCSPC module Leica/PicoQuant http://www.leica-microsystems.com https://www.picoquant.com/ IN Cell Analyzer 2200 automated widefield microscope GE Healthcare Life Sciences http://www.gelifesciences.com/ Leica DMI6000B widefield microscope Leica http://www.leica-microsystems.com Chemical synthesis and sensor constructs Detailed procedures for the synthesis of the SNAP-tag substrates and plasmids construction can be found in the Appendix 1 Information. Synthesis of SiR-Halo has been described previously ( Lukinavičius et al., 2013 ). Bacterial protein expression, purification and labeling The sensor proteins were expressed in transformed Escherichia coli strain Rosetta-gami 2(DE3) (Novagen). Bacterial cultures were grown in selective (100 µg/mL ampicillin) LB medium at 37 °C to an OD 600nm of 0.8, cooled down to 16°C prior to induction with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG). After 16 hr, the cells were harvested by centrifugation, lysed by sonication in presence of a protease inhibitor cocktail (cOmplete-EDTA-free, Roche) and the resulting cell lysates were cleared by centrifugation. The proteins were purified by two successive purification steps using Ni-NTA (Qiagen) and Strep-Tactin (IBA) columns according to the supplier’s instructions. The purified proteins can be stored for several months at a concentration of 50–100 µM at −80 °C as flash frozen (N 2 liq.) small aliquots (50 µL) prepared in 50 mM HEPES, 150 mM NaCl, 1 mM DTT, 5% (v/v) glycerol, pH 7.5 or at −20 °C as stocks prepared in 50 mM HEPES, 150 mM NaCl, 1 mM DTT, 50% (v/v) glycerol, pH 7.5. For sensor labeling, the sensor protein was diluted to 5 µM in buffer (50 mM HEPES, 150 mM NaCl, pH 7.5) with 10 µM BG-TMR-SMX and 10 µM SiR-Halo and incubated at room temperature for 1 hr. The excess of SNAP-tag and Halo-tag substrates were removed by gel filtration using NAP-5 Sephadex prepacked columns (GE Healthcare). The final concentration of labeled sensor proteins was determined by measuring the absorbance at 555 nm and 650 nm in the labeling buffer supplemented with 0.1% SDS (ε(TMR) 555nm = 90,000 M −1 cm −1 , ε(SiR) 650nm = 100,000 M −1 cm −1 ). Titrations of the sensors The labeled sensors were diluted to a concentration of 20 nM in 100 µL of buffer (unless specified 50 mM HEPES, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.5) containing defined concentrations of analytes (NADP + , NAD + or NADPH/NADP + ) in black non-binding 96-well plates (Greiner Bio-One). The solutions were incubated at room temperature for at least 15 min to ensure that the sensor conformation had reached equilibrium. Fluorescence measurements were performed on an Infinite M1000 spectrofluorometer (TECAN). Both the excitation and emission bandwidth for all measurements were set to 10 nm. For the sensor constructs labeled with TMR and SiR, the emission spectra were recorded from 540 nm to 740 nm using a step size of 1 nm with an excitation at 520 nm. For the sensor constructs with EGFP and TMR, the emission spectra were measured from 480 nm to 610 nm using a step size of 1 nm with an excitation of 450 nm. The emission ratios of the FRET donor over FRET acceptor (TMR/SiR: 577 nm/667 nm; EGFP/TMR: 508 nm/577 nm) were measured as technical triplicates and were plotted as mean ± s.d. against the analyte concentration. The plots were fitted using a single binding isotherm ( Equation 3 ) to obtain the c 50 and the maximum FRET ratio change (ΔR max = R max /R min ). The c 50 values and maximum ratio changes are reported as mean ± s.d. from three independent titrations. (3) R = R m a x + R m i n − R m a x 1 + c 50 [ A n a l y t e ] (4) R = R m i n + R m a x − R m i n 1 + r 50 [ A n a l y t e ] with R being the experimental emission ratio of donor vs acceptor, [Analyte] the concentration of cofactors, R max and R min are the maximum and minimum emission ratio corresponding to the open (free) and closed (saturated) sensor, respectively. Fits were performed using OriginPro 2017 (OriginLab Corporation) with R max , R min , c 50 as free parameters. For the titrations using NADPH/NADP + , the total cofactor concentration was fixed to 100 µM while varying the ratios of NADPH vs NADP + and the plots were fitted using the single binding isotherm ( Equation 4 ) to determine r 50 defined as the NADPH/NADP + ratio corresponding to half-maximal sensor response. The prepared ratios NADPH/NADP + were corrected by measuring the percentage of NADP + present in the commercial stock of NADPH (NADPH-RO, Roche) by absorbance as described in the Supplementary Note 2. To obtain higher NADPH/NADP + ratios, the NADPH was purified by anion-exchange chromatography using a Resource Q column (GE Healthcare) and freshly used for titrations. The plots were fitted by fixing R max determined by addition of a saturating concentration of competitive free ligand (2 mM sulfamethoxazole), while setting the other parameters free. It has to be noted that the FRET donor and acceptor possess different dynamic ranges, therefore their respective emission ratio is not linearly correlated with the sensor occupancy as described previously ( Pomorski et al., 2013 ). The determination of the sensor’s K D ’ was performed by normalizing the individual fluorescence intensities of TMR or SiR by the sensor’s isosbestic point (645 nm) and fitted with the previously described Equations (3) or (4), where c 50 , r 50 are replaced by K D ’ or K 50 . K 50 is defined as the NADPH/NADP + ratios corresponding to sensor’s half-saturation with NADP + . Cell culture, transfection and cell labeling U2OS, HEK293T, NIH/3T3, HeLa cells were cultured in high-glucose DMEM with GlutaMAX-I, 1 mM pyruvate (Gibco) supplemented with 10% HyClone FetalClone II Serum (GE Healthcare) at 37 °C in a humidified incubator at 5% CO 2 . Cells were subcultured twice per week or at 90% confluency using StemPro Accutase (Gibco, Life Technologies). The cells are not known to be misidentified no cross-contaminated. The cell lines are regularly checked and not infected with mycoplasma. To generate semi-stable cell lines, the cells were transfected with the pEBTet expression vectors using Lipofectamine 3000 according to the manufacturer’s instruction. 48 hr after transfection, the cells were selected with the full growth medium supplemented with 1 µg/mL puromycin for one week. After the selection, the amplified transfected cells were continuously maintained in selective conditions and stocks were frozen in 10% DMSO at low passage numbers and stored at −80 °C for further use. Cell lines were regularly checked for mycoplasma infection (biochemical test: MycoAlert, Lonza and imaging: Hoeschst 33342 staining at 0.1 µg/mL) and used for experiments before 25 passages. Expression of the sensor proteins were induced with 100 ng/mL doxycycline for the cytosolic and nuclear sensors and 10 ng/mL doxycycline for the mitochondrial localized sensor for 24 hr, after which the cells were labelled with 1 µM fluorescent substrates (CP-TMR-SMX, SiR-Halo) in fresh pre-warmed full growth medium supplemented with 10 µM (±)-verapamil hydrochloride (Sigma-Aldrich) overnight at 37 °C, 5% CO 2 . Then, the excess of dyes was removed by washing cells three times with full growth medium followed by 2 hr incubation. The medium was exchanged one last time before imaging. The fluorescent substrates (CP-TMR-SMX, SiR-Halo) are prepared as 2 mM DMSO stock (2000x). (±)-verapamil is prepared as 10 mM stock (1000x) in cell culture grade water and sterile filtered. Live-cell quantification of NADPH/NADP + and NAD + by ratio imaging Semi-stable U2OS cell lines (NADP-Snifit: cytosol, nucleus and mitochondria and NAD-Snifit: cytosol) were passaged with StemPro Accutase (Gibco, Life Technologies) and plated (10 4 cells/well) in poly-D-Lysine coated glass-bottom 96-well plates (MatTek Corporation) and cultured in full growth medium at 37 °C, 5% CO 2 . The next day, the expression of the different constructs were induced with 100 ng/mL doxycycline for the cytosolic, nuclear sensors and 10 ng/mL doxycycline for the mitochondrial sensor. After 24 hr, the sensor proteins were labeled with 1 µM CP-TMR-SMX, 1 µM SiR-Halo and 10 µM (±)-verapamil overnight (16 hr). The excess of labeling compounds were washed three times with phenol red free full growth medium and the cells were incubated 2 hr at 37 °C, 5% CO 2 before imaging. The cells were imaged before and after being treated with 2 mM sulfapyridine (use to fully open the sensors in situ) on a IN Cell Analyzer 2200 (GE Healthcare) widefield automated microscope equipped with a sCMOS camera (2048 × 2048 pixels) using either Nikon Plan Apo 20X/0.75 CFI/60 or Plan Fluor 40X/0.60 CFI/60 air-objectives and three channels per image acquisition: Cy3/Cy3 (TMR channel), Cy3/Cy5 (FRET channel) and Cy5/Cy5 (SiR channel), with filters specification: Cy3: excitation (542/27 nm), emission (597/45 nm); Cy5: excitation (632/22 nm), emission (684/25 nm) using 200 ms exposure time at 37 °C, 5% CO 2 . Image analyses were performed in FIJI ( Schindelin et al., 2012 ). Fluorescence images in each channel were first flat-field (using flat-field reference images) and background (by subtracting the fluorescence intensity of ROIs corresponding to background region) corrected. Then, FRET images were corrected for bleed-through according to the previously determined ( Spiering et al., 2013 ) Equation (5) using single-labeled controls to determine the donor emission ratio α (i.e. bleed-through of the donor into the acceptor channel using a donor-only sample) and β (i.e. direct acceptor excitation from TMR excitation light using an acceptor-only sample). Due to the large spectral separtion between the FRET pairs, α and β are very small correction coefficients. α and β were determined to be 0.054 and 0.051 with this microscopy setup. (5) F R E T c = F R E T r a w − α ⋅ T M R − β ⋅ S i R The emission ratios (TMR/FRET c ) of 60 individual cells from three different cell preparations were tracked and measured before and 15 min after the treatment of 2 mM sulfapyridine. Sulfapyridine (SPY) treatment allows to fully open the sensors in situ and to determine the normalized FRET ratio change ΔR (ΔR = R SPY /R basal ). ΔR values were used to convert the emission ratio corresponding to the apparent sensor occupancy R of the cells at basal state (R = R max /ΔR) as the dynamic range of the sensor of the instrumental setup is similar to in vitro measurements. NADPH/NADP + ratios and NAD + are quantified using the following Equations (6 and 7) , where R max , R min , r 50 and c 50 are parameters determined by in vitro titrations at 37 °C (NADP-Snifit: R max = 4.58 ± 0.12, R min = 0.52 ± 0.02, r 50 = 30 ± 3; NAD-Snifit: R max = 4.47 ± 0.16, R min = 0.59 ± 0.03, c 50 = 130 ± 14 µM). (6) [ N A D P H ] [ N A D P + ] = r 50 R − R m i n R m a x − R (7) [ N A D + ] = c 50 R m a x − R R − R m i n Live-cell quantification of NADPH/NADP + and NAD + by FLIM Semi-stable U2OS cell lines for sensors expression in the different subcellular compartments (cytosol, nucleus, mitochondria) were passaged with StemPro Accutase (Gibco, Life Technologies), plated in poly-D-Lysinecoated glass-bottom 12-well plates (MatTek Corporation) and cultured in full growth medium at 37 °C, 5% CO 2 . Sensors expression were induced with 10 (mitochondria targeted sensors) or 100 ng/mL doxycycline. After 24 hr, the sensor constructs were labeled overnight (16 hr) either only with 1 µM CP-TMR-SMX (for donor only controls) or with 1 µM CP-TMR-SMX and 1 µM SiR-Halo each time in presence of 10 µM (±)-verapamil. The cells were washed three times in full growth medium, incubated for another 2 hr before imaging. Fluorescence lifetimes measurements were performed on a laser scanning confocal microscope (Leica TCS SP8 X) equipped with an 63x oil-immersion objective (HC PL APO 63x/1.40 CS2) and a PicoHarp 300 (PicoQuant) TCSPC module. As excitation source, the white-light laser was set 514 nm with 20 MHz pulse frequency. The FRET donor emission was measured on a hybrid photodetector for single molecule detection (Leica HyD SMD) with a detection range of 550–610 nm. The images were typically acquired using 180 × 180 µm (cytosol, nucleus) or 70 × 70 µm (mitochondria) with 512 × 512 pixels, scan speed 100 Hz, pinhole at one airy unit, a laser power adjusted to 10 5 average photon counts per second to avoid pile-up effects and a target photon counts of 500/pixel. All the measurements were performed at 37 ± 1 °C. The data acquisition and analysis were performed using SymPhoTime 64 (PicoQuant). The fluorescence decays of individual cells were extracted by ROIs (sum of the photons of all the pixels of a ROI, typically with 10 6 photon counts) and were fitted using an n-exponential reconvolution model ( Equation 8 ); (8) y ( t ) = ∑ i = 0 n − 1 I R F ⊗ | B k g r I R F | S h i f t I R F α i e x p ( − t τ i ) + B k g r D e c where the instrument response function (IRF) was calculated from the convolution integral of the model function. Bkgr IRF , Shift IRF , Bkgr Dec correspond to the corrections for the IRF background and displacement and decay background. α i and τ i correspond to the pre-exponential factors and the lifetimes. The goodness-of-fit was determined by the reduced chi-square (χ 2 < 1.2) using a nonlinear least-squares analysis and examining the weighted residuals trace. The donor-only and FRET samples were fitted according to a bi-exponential and third-order exponential fitting model. An example of fluorescence decays and fitting can be found in Appendix 1—figure 4 . The amplitude weighted average lifetimes ( Equation 9 ) were used to calculate the FRET efficiencies ( Equation 10 ) before (E, at basal cellular state) and after the treatment of cells with 2 mM sulfapyridine representing the minimal FRET efficiency (E min ). (9) ⟨ τ ⟩ = ∑ α i τ i α i (10) E = 1 − ⟨ τ D A ⟩ ⟨ τ D ⟩ and represent the amplitude weighted average lifetimes for the FRET and donor-only samples. The lifetimes measured in vitro and in U2OS cells and reported in Appendix 1—tables 5 and 6 , respectively, represent the mean ± s.d. of 10 individual cells from three independent experiments (n = 10). NADPH/NADP + ratios and NAD + are quantified using Equations (1 and 2) , where E and E min correspond to the FRET efficiency of the sensor in situ prior (basal state) and after the treatment with 2 mM sulfapyridine and E max was determined with the same setup using the purified sensor with saturating concentration of cofactor. K 50 and K D ’ are the NADPH/NADP + ratio and NAD + concentration corresponding to sensor’s half-saturation determined from in vitro titrations at 37 °C (NADP-Snifit: K 50 = 11.6 ± 3.3, NAD-Snifit: K D ’=363 ± 47 µM). Real-time monitoring of oxidative stress Semi-stable U2OS cells (cytosolic NADP-Snifit) were plated on poly-L-ornithinecoated glass coverslips (VWR 20 × 20 mm) using a 6-well plate and cultured in full growth medium at 37 °C, 5% CO 2 . Sensor expression was induced the next day by addition of 100 ng/mL doxycycline. After 24 hr, the protein construct was labeled with 1 µM CP-TMR-SMX, 1 µM SiR-Halo and 10 μM (±)-verapamil in full growth medium overnight (16 hr). The cells were washed three times with full growth medium and incubated 2 hr at 37 °C, 5% CO 2 . The medium was exchanged for HBSS (Lonza) 30 min before imaging. Glass coverslips were transferred to a Cytoo chamber (44 × 34×10 mm). Time-course experiments of sensor imaging were performed on a Leica DMI6000B wide-field microscope equipped with a Hamamatsu-C9100 EM-CCD camera and a 40x oil-immersion objective (HCX PL APO 40.0 × 1.25). Gravity fed perfusion of the chamber was performed at a flow rate of 1 mL/min. For each frame, the two channels (donor and FRET) were measured consecutively, with an interval of 10 s between individual frames. Cy3 was used as excitation filter (530/35 nm) and the emission filters were respectively Cy3 (580/40 nm) for the donor channel and Cy5 (700/72 nm) for the acceptor channel. The perfused solutions (A = 2 mM sulfapyridine, B = 10 µM H 2 O 2 , C. 100 µM H 2 O 2 , D. 200 µM H 2 O 2 ) were all prepared in HBSS (Lonza). HBSS solution was continuously perfused during the other point of the experiment. For image analysis, the 16-bit images (306 × 306 µm, 512 × 512 pixels) were background corrected and fluorescence intensity time-traces from 10 cells (defined as ROIs) were extracted for the TMR and FRET channels using FIJI ( Schindelin et al., 2012 ). For each cells and time points, the ratio (TMR/FRET) was calculated. A graph of the emission ratio (TMR/FRET) vs. time was generated as mean ± s.d (n = 10 cells). Flow cytometry measurements 10 4 semi-stable U2OS cells (NAD- and NADP-Snifit: cytosol, mitochondria) were plated in 96-well culture plates (TTP U-bottom plates) using 200 µL DMEM high glucose (GlutaMax-I, 10% FetalClone II, 1 mM sodium pyruvate) supplemented with 10 (for mitochondrial sensors) or 100 ng/mL doxycycline (for cytosolic sensors) to induce proteins expression. The constructs were labeled with 1 µM CP-TMR-SMX, 1 µM SiR-Halo and 10 μM (±)-verapamil in full growth medium overnight (16 hr). After exchanging three times the medium to remove the excess of dyes, the cells were treated for 24 hr in different conditions. The different compound were prepared in DMEM high glucose (GlutaMax-I, 10% FetalClone II, 1 mM sodium pyruvate). Then, the cells were washed with PBS and detached with 20 µL StemPro Accutase (Gibco, Life Technologies) for 5 min at 37 °C. The cells were resuspended and separated by gentle mixing with a multichannel pipette using 120 µL growth medium (in treatment condition) and 10,000 cells were analyzed on a LSR II flow cytometer (BD Biosciences) equipped with HTS module. The different lasers and filters were used to record the donor, FRET and acceptor fluorescence: 561 nm laser with 585/15 nm filter for TMR, 561 nm laser with 660/20 nm filter for FRET and 640 laser with 670/20 nm filter for SiR. Unstained cells and induced cells only labeled with either the donor or acceptor dye were used to measure fluorescence spillover. Sensor labeled with CP-TMR and SiR-Halo (forming essentially a non-functional sensor) was used as additional control to test eventual nonspecific ratio change due to the added compounds (e.g. quenching, increased fluorescence). The cell viability for the different treatment was tested by propidium iodide staining. The data were analyzed on FlowJo software. Gating strategy involved the removal of dead cells and debris (SSC-A vs FCS-A), doublets removal (SSC-A vs SSC-W) and selection of the labeled cell population (SiR vs TMR). The gated cells population in the different conditions were analyzed by determining the median of their TMR/FRET ratio. For each condition, the median was averaged from three measurements obtained from different cell preparation. The final results are represented as mean TMR/FRET ratios ± s.d from three independent experiments. For each condition, the mean ratios were normalized with the untreated cells. An example of the gating strategy and the distribution of TMR/FRET ratio of cell populations using different treatment can be found in Appendix 1—figure 7a . As we cannot experimentally determine R min , c 50 and r 50 values of our sensors on the flow cytometer and would have to use the parameters determined on a different instrument to transform FRET ratios in concentrations or ratios ( Appendix 1—table 4 ), concentrations or ratios obtained this way should only be considered as estimates.

Quantification and statistical analysis

Titrations data ( Figure 2 and Appendix 1—figure 1 ) are represented as mean ± s.d. of the emission ratio (TMR/SiR) from technical triplicates. The calculated fitting parameters (c 50 , r 50 , K D ’, K 50 , R min , R max ) used for the quantification of NAD + and NADPH/NADP + by ratio imaging, FLIM and flow cytometry (estimations) were determined as mean ± s.d. of three independent titrations (each performed in triplicates) ( Table 1 ). Flow cytometry data ( Figure 4 and Appendix 1—figure 7 ) were characterized by non-normal distributions. In essence, the sample distributions showed a positive kurtosis and skewness, and were heteroscedastic. The statistical analysis ( Appendix 1—figure 7 ) was then performed in R by a Kruskal-Wallis test with post-hoc Dunn’s test using the Benjamini-Hochberg method (FDR) for multiple comparison correction with respect to control conditions. The significance level was set to α = 0.05 and two-tailed p-values were reported (* p