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Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.

The data presented in this article supports the rat brain sample pre- parathion procedure previous to its injection into the liquid chroma- tography–tandem mass spectrometry(LC–MS/MS) system to monitor levels of adrenaline, noradrenaline, glutamicacid, γ-aminobutyric acid, dopamine, 5-hydroxytryptamine, 5-hydroxyindoleaceticacid, and 3-methoxy-4-hydroxyphenylglycol.In addition,we describe the method validation assays (such as calibration curve, lower limit of quantification, precision and accuracy intra-and inter-day,selectivity, extraction recovery and matrix effect, stability,and carry-over effect) according to the United States Food and Drug Administration and European Medicine Agency to measure in one step different neuro- transmitters and their metabolites.The data supplied in this articleis related to the research study entitled: “Simultaneous determination of 8 neurotransmitters and their metabolite levels in rat brainusing liquid chromatography in tandem with mass spectrometry: application to the murine Nrf2 model of depression” (Wojniczetal.2016), The authors wish to thank to Instituto-Fundación Teófilo Hernando for itscontinued financial support. This dataset was also supported by “FIS No.CA12/00122” to ARN ,Minstry of Education, Culture and Sport “FPU12/02220” to AW and the Ministry of Economy and Competitiveness “SAF2012-32223” to MGL

Alternatives for the treatment of amyotrophic lateral sclerosis (ALS) are scarce and controversial. The etiology of neuronal vulnerability in ALS is being studied in motor neuron-like NSC-34 cells to determine the underlying mechanisms leading to selective loss of motor neurons. One such mechanism is associated with mitochondrial oxidative stress, Ca(2+) overload, and low expression of Ca(2+)-buffering proteins. Therefore, in order to elicit neuronal death in ALS, NSC-34 cells were exposed to the following cytotoxic agents: (1) a mixture of oligomycin 10 µM and rotenone 30 µM (O/R), or (2) phenylarsine oxide 1 µM (PAO) (to mimic excess free radical production during mitochondrial dysfunction), and (3) veratridine 100 µM (VTD) (to induce overload of Na(+) and Ca(2+) and to alter distribution of Ca(2+)-buffering proteins [parvalbumin and calbindin-D28k]). Thus, the aim of the study was to test the novel neuroprotective compound ITH33/IQM9.21 (ITH33) and to compare it with riluzole on in vitro models of neurotoxicity. Cell viability measured with MTT showed that only ITH33 protected against O/R at 3 μM and PAO at 10 μM, but not riluzole. ITH33 and riluzole were neuroprotective against VTD, blocked the maximum peak and the number of [Ca(2+)]c oscillations per cell, and restored the effect on parvalbumin. However, only riluzole reversed the effect on calbindin-D28k levels. Therefore, ITH33 was neuroprotective against oxidative stress and Na(+)/Ca(2+) overload, both of which are involved in ALS.

The mutated form of the Ca2+ channel CALHM1 (Ca2+ homeostasis modulator 1), P86L-CALHM1, has been correlated with early onset of Alzheimer’s disease (AD). P86L-CALHM1 increases production of amyloid beta (Ab) upon extracellular Ca2+ removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L-CALHM, upon Ab treatment, on the following: (i) the intracellular Ca2+ signal pathway; (ii) cell survival proteins ERK1/2 and Ca2+/cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Ab. Using aequorins to measure the effect of nuclear Ca2+ concentrations ([Ca2+]n) and cytosolic Ca2+ concentrations ([Ca2+]c) on Ca2+ entry conditions, we observed that baseline [Ca2+]n was higher in CALHM1 and P86L-CALHM1 cells than in control cells. Moreover, exposure to Ab affected [Ca2+]c levels in HeLa cells overexpressing CALHM1 and P86L-CALHM1 compared with control cells. Treatment with Ab elicited a significant decrease in the cell survival proteins p-ERK and p-CREB, an increase in the activity of caspases 3 and 7, and more frequent cell death by inducing early apoptosis in P86L-CALHM1- overexpressing cells than in CALHM1 or control cells. These results suggest that in the presence of Ab, P86L-CALHM1 shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro-cytotoxic pathways, thus potentially contributing to its deleterious effects in AD., This work was partly supported by the following grants: Ministerio de Economía y Competitividad, FPU Program, Refs. AP2009/0343 (AJMO) and AP2010/1219 (IB). ARN: FIS PI10/01426. MGL: Ministerio de Economía y Competitividad, Ref. SAF2012-23332. MFCA: Consolidación de grupos de investigación UAM-CAM 1004040047. We also thank Fundación Teófilo Hernando, Madrid, Spain, for their continued support

The mutated form of the Ca²⁺channel CALHM1 (Ca²⁺homeostasis modulator 1), P86L-CALHM1, has been correlated with early onset of Alzheimer's disease (AD). P86L-CALHM1 increases production of amyloid beta (Aβ) upon extracellular Ca²⁺removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L-CALHM, upon Aβ treatment, on the following: (i) the intracellular Ca²⁺signal pathway; (ii) cell survival proteins ERK1/2 and Ca²⁺/cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Aβ. Using aequorins to measure the effect of nuclear Ca²⁺concentrations ([Ca²⁺]n ) and cytosolic Ca²⁺concentrations ([Ca²⁺]c ) on Ca²⁺entry conditions, we observed that baseline [Ca²⁺]n was higher in CALHM1 and P86L-CALHM1 cells than in control cells. Moreover, exposure to Aβ affected [Ca²⁺]c levels in HeLa cells overexpressing CALHM1 and P86L-CALHM1 compared with control cells. Treatment with Aβ elicited a significant decrease in the cell survival proteins p-ERK and p-CREB, an increase in the activity of caspases 3 and 7, and more frequent cell death by inducing early apoptosis in P86L-CALHM1-overexpressing cells than in CALHM1 or control cells. These results suggest that in the presence of Aβ, P86L-CALHM1 shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro-cytotoxic pathways, thus potentially contributing to its deleterious effects in AD.

The vascular selectivity of PF9404C ((2′ S ),(2 S )-3-isopropylamine,1-[4-(2,3-dinitroxy)propoxymethyl]-phenoxy-2′-propanol), a new β-blocker with nitric oxide (NO)-donor and vasodilator properties, was studied in different rabbit arteries and veins. Phenylephrine (10 −6 M) or 35 mM K + were used to pre-contract the arteries and veins prior to study the relaxant effects of PF9404C and nitroglycerin. The potency of both drugs to depress the phenylephrine-induced contraction was greater than that shown in the blockade of the K + -evoked contraction in most of the vessels studied, with the exception of the central ear artery. PF9404C exhibited about three-fold higher potency than nitroglycerin to relax the majority of the vessels studied, especially when they were contracted with K + , and showed a certain selectivity of action for the renal artery. PF9404C produced autotolerance but this effect was about 20-fold less pronounced than that observed with nitroglycerin. Cross-tolerance in those preparations pre-exposed to PF9404C that were relaxed later on with nitroglycerin was much greater than autotolerance. The tolerance for nitroglycerin was practically abolished in the presence of N -acetylcysteine. However, this was not the case for PF9404C. These results indicate that, although sharing the property of being NO donors, PF9404C and nitroglycerin show a different profile in causing vasodilation; furthermore, the tolerance to this effect is lesser for PF9404C and seems to be mediated by a mechanism different to that of nitrates. This makes PF9404C a nice pharmacological tool to further develop novel NO-donor compounds with a lesser degree of vascular tolerance than those now available.

The mechanism of blockade of P/Q Ca(2+) channels by antimigraine, dotarizine, was studied in voltage-clamped bovine adrenal chromaffin cells. Inward currents through P/Q channels were pharmacologically isolated by superfusing the cells with omega-conotoxin GVIA (1 microM) plus nifedipine (3 microM). Dotarizine (10-30 microM) blocked the P/Q fraction of I(Ba) and promoted current inactivation. Thus, dotarizine caused a greater blockade of the late I(Ba), compared with blockade of the early peak I(Ba). This effect was more prominent, the longer was the duration of the depolarising pulse. The blockade of I(Ba) was also greater at more depolarising holding potentials (i.e. -60 mV), than was the blockade produced at more hyperpolarising holding potentials (i.e. -80 or -110 mV). Catecholamine secretory responses to brief pulses (2 s) of a Krebs-HEPES solution containing 100 mM K(+) and 2 mM Ca(2+) was blocked by 3 microM dotarizine. Blockade was faster and greater when dotarizine was applied on cells that were previously depolarised with Krebs-HEPES deprived of Ca(2+) and containing increasing concentrations of K(+). This voltage-dependent blockade of P/Q channels and exocytosis might be the underlying mechanism explaining the dotarizine prophylaxis of migraine attacks.

1. PF9404C is the S-S diesteroisomer of a novel blocker of beta adrenergic receptors with vasodilatory properties. It causes a concentration-dependent relaxation of rat aorta helical strips pre-contracted with 10(-6) M noradrenaline (NA; IC(50) 33 nM). It was equipotent to nitroglycerin (NTG; IC(50) 49 nM), but much more potent than isosorbide dinitrate (ISD; IC(50) 15,000 nM). 2. Oxyhaemoglobin (10 microM) shifted to the right the concentration-response curve for the relaxation induced by PF9404C (IC(50) 530 nM) or NTG (IC(50) 61 nM). 3. Either methylene blue (MB) or ODQ (1 microM each) largely prevented the vasorelaxing responses to increasing concentrations of PF9404C or NTG. 4. In rat aorta smooth muscle cells, PF9404C increased the formation of cyclic GMP from 3 pmol mg(-1) protein in basal conditions, to 53 pmol mg(-1) protein in 10 microM PF9404C. Neither metoprolol nor carvedilol enhanced cyclic GMP. 5. In the electrically driven guinea-pig left atrium, PF9404C blocked the inotropic effects of isoprenaline in a concentration-dependent manner. Its IC(50) (30 nM) was similar to that for S-propranolol (22.4 nM) and lower than the IC(50)s for metoprolol (120 nM) and atenolol (192 nM). The beta-adrenergic ligand (-)-[(3)H]-CGP12177 (0.2 nM) was displaced from its binding to rat brain membranes with K(i) of 7 nM, 17 nM, 170 nM and 1.2 microM respectively for PF9404C, S-(-)propranolol, metoprolol, and atenolol. 6. The data are consistent with the idea that the S-S diesteroisomer PF9404C, is a potent vasorelaxing agent, as well as a blocker of cardiac beta adrenergic receptors. The mechanism of its vasorelaxing effects involves the slow generation of NO. This molecule can, therefore, exhibit antihypertensive and cardioprotective actions through a double mechanism, NO donation and beta blockade.

The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca(2+) store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.

We have developed a method of liquid chromatography in tandem with mass spectrometry to monitor therapeutic levels of imatinib in plasma, a selective inhibitor of protein tyrosine kinase. After solid-phase extraction of plasma samples, imatinib and its internal standard, imatinib-D8, were eluted with Zorbax SB-C18 at 60 °C, under isocratic conditions through a mobile phase consisting of 4 mm ammonium formate, pH: 3.2 (solution A) and acetonitrile solution B. The flow rate was 0.8 mL/min with 55% solution A + 45% solution B. Imatinib was detected and quantified by mass spectrometry with electrospray ionization operating in selected-reaction monitoring mode. The calibration curve was linear in the range 10-5000 ng/mL, the lower limit of quantitation being 10 ng/mL. The method was validated according to the recommendations of the Food and Drug Administration, including tests of matrix effect (bias 10%) and recovery efficiency (80 and120%). The method is precise (coefficient of variance intra-day2% and inter-day7%), accurate (95-108%), sensitive and specific. It is a simple method with very fast recording time (1.2 min) that is applicable to clinical practice. This will permit improvement of the pharmacological treatment of patients.

Primary brain cell cultures are a useful tool for understanding the physiopathology of epilepsy and for searching new potential antiepileptic drugs. These cell types are usually prepared from murine species and few human models have been described. The main goal of this study is the establishment of experimental conditions to isolate and culture neurons and astrocytes from human brain and to test its functionality. The tissues came from antiepileptic drug-resistant epileptic patients undergoing surgery. Human neurons and astrocytes were isolated following an enzymatic and mechanical dissociation protocol. Cultures were viable for 3–6 weeks. Cytological characterization was performed by immunocytochemistry using specific antibodies against both neuron (anti-NeuN) and astrocyte (anti-GFAP) protein markers. In order to test their viability and functionality, cells were loaded with the fluorescent calcium probe fura-2 and variations in cytosolic calcium concentrations ([Ca 2+ ] c ) were measured by cell imaging. [Ca 2+ ] c increases were evoked upon cell stimulation with high K + (KCl 75 mM), glutamate (500 μM) or bicuculline (100 μM). Interestingly, spontaneous [Ca 2+ ] c transients were also observed in some neuron-like cells. A novel unreported finding in this study has been the incorporation of human serum that was critical for cell functionality. The setting of these human cultures open the opportunity to new insights on culture and calcium signalling studies on the mechanism(s) of cell resistance to antiepileptic drugs, as well as to studies on plasticity, maturation and possible neurite emission for graft studies.

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca2+]c) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca2+) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca2+ transients ([Ca2+]c) and changes of mitochondrial Ca2+ concentrations ([Ca2+]m) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca2+]c and [Ca2+]m elevations elicited by K+ pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca2+]m was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca2+]c transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca2+ channel agonist Bay K 8644 enhanced K(+)-evoked [Ca2+]m peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K+ generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca2+]c and [Ca2+]m transients elicited by K+, in PC12 cells overexpressing Bcl2, is related to the reduction of Ca2+ entry through L-type Ca2+ channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca2+ channels, the subsequent Ca2+ entry, and mitochondrial Ca2+ overload.

The hypothesis that the buffering of Ca2+by mitochondria could affect the Ca2+-dependent inhibition of voltage-activated Ca2+channels, (ICa), was tested in voltage-clamped bovine adrenal chromaffin cells. The protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the blocker of the Ca2+uniporter ruthenium red (RR), and a combination of oligomycin plus rotenone were used to interfere with mitochondrial Ca2+buffering. In cells dialyzed with an EGTA-free solution, peakICagenerated by 20 msec pulses to 0 or +10 mV, applied at 15 sec intervals, from a holding potential of −80 mV, decayed rapidly after superfusion of cells with 2 μmCCCP (τ = 16.7 ± 3 sec;n= 8). In cells dialyzed with 14 mmEGTA, CCCP did not provokeICaloss. Cell dialysis with 4 μmruthenium red or cell superfusion with oligomycin (3 μm) plus rotenone (4 μm) also accelerated the decay ofICa. After treatment with CCCP, decay of N- and P/Q-type Ca2+channel currents occurred faster than that of L-type Ca2+channel currents. These data are compatible with the idea that the elevation of the bulk cytosolic Ca2+concentration, [Ca2+]c, causes the inhibition of L- and N- as well as P/Q-type Ca2+channels expressed by bovine chromaffin cells. This [Ca2+]csignal appears to be tightly regulated by rapid Ca2+uptake into mitochondria. Thus, it is plausible that mitochondria might efficiently regulate the activity of L, N, and P/Q Ca2+channels under physiological stimulation conditions of the cell.

The novel antimigraineur, dotarizine, inhibited 5-HT (5 hydroxytryptamine)-evoked contractions of rabbit vertebral, aorta, femoral and mesenteric arteries, with IC(50)s of 1.35, 1.40, 0.52 and 1.09 microM, respectively. Flunarizine had little effect on these contractions, while ketanserin was more potent (IC(50)s of 0.17 microM for vertebral, 0.22 microM for aorta, 0.05 microM for femoral and 0.03 microM for mesenteric arteries). At 10 microM, dotarizine caused 40% blockade of K(+)-evoked contractions of rabbit aorta, and 70% inhibition of 5-HT-evoked responses; these values were 30% and 20% for 10 microM flunarizine. Contractions of rabbit aorta elicited by noradrenaline, angiotensin II or prostaglandin F(2alpha) were not affected by 10 microM dotarizine or flunarizine. Ketanserin shifted to the right, in parallel, the concentration-response curves for 5-HT in rabbit aorta; however, dotarizine caused a non-competitive type of blockade, increasing the maximum 5-HT contraction at 30 nM and decreasing it at 3 and 30 microM. K(+)-evoked contractions of rabbit aorta were halved by 3 microM dotarizine in a voltage-independent manner; flunarizine caused a delayed-type, non-reversible post-drug blockade, and exhibited some voltage-dependence. Blockade by nifedipine was voltage-dependent and fully reversible. Ca(2+)-evoked contractions of depolarised bovine middle cerebral arteries were blocked by 1--3 microM dotarizine in a non-surmountable manner. Contraction of these vessels evoked by electrical stimulation was blocked 50% and 70% by 1 and 3 microM dotarizine, respectively. Dotarizine (1--3 microM) also inhibited to a similar extent the K(+)-evoked [(3)H]noradrenaline release from cultured rat sympathetic neurones. These data suggest that the mechanism of blockade by dotarizine of cerebral vessels contractility has three components: (i) presynaptic inhibition of noradrenaline release; (ii) blockade of postsynaptic vascular 5-HT receptors; (iii) blockade of Ca(2+)entry into the vascular smooth muscle cell cytosol. The compound does not affect the vascular receptors for noradrenaline, angiotensin II or prostaglandin F(2alpha).

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología y Terapéutica. Fecha de lectura: 25 de Julio de 2001

A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of aripiprazole and its active metabolite, dehydro-aripiprazole, in human plasma. Stable isotopically labeled aripiprazole, aripiprazole-D8, has been used as the internal standard (IS) for both analytes. Only 200 μl of human plasma was needed for analyte extraction, using effective phospholipids-eliminating three-step microelution-solid-phase extraction (SPE, Oasis PRiME HLB 96-well μElution Plate). An ACE C18-PFP column was applied for chromatographic separation at 25 °C, protected by a 0.2-μm on-line filter. A combination of ammonium formate (5 mM)-acetonitrile (pH 4.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 ml/min. Run time lasted 5 min, followed by a re-equilibration time of 3 min, to give a total run time of 8 min. Five μl of the sample was injected into the chromatographic system. Aripiprazole, dehydro-aripiprazole and IS were detected using the mode multiple reaction monitoring in the positive ionization mode. The method was linear in the concentration range of 0.18–110 ng/ml and 0.35–100 ng/ml for aripiprazole and dehydro-aripiprazole, respectively. Our method has been validated according to the recommendations of regulatory agencies through tests of precision, accuracy, recovery, matrix effect, stability, sensitivity, selectivity and carry-over. Our microelution-SPE method removes more than 99% of main plasma phospholipids compared to protein precipitation and was successfully applied to several bioequivalence studies., The authors wish to thank Instituto-Fundación Teófilo Hernando for its continued financial support. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 721236. This work was also supported by FPU12/02220 from MECD to AW.

Changes in nuclear Ca(2+) homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K(+) channel interacting protein 3), is a Ca(2+)-binding protein that binds DNA and represses transcription in a Ca(2+)-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca(2+)-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory. This work was supported by grants from Spanish Ministry of Health and Science, Madrid Community, La Marató, La Caixa, Reina Sofía and Areces Foundations, the EU 6th Framework Program (NeuroNE, CureFXS), the ERA-NET Program (Neuron and E-Rare), and the Medical Research Council. S.K. has a postdoctoral contract from the Ramón y Cajal Program of the Ministry of Science and Innovation.