If this is not the

case, as for intracellular Ca2 + in RBCs of patients with sickle cell disease,59 cytosolic free Ca2 + cannot be estimated from the 45Ca2 + distribution between the cells and the medium. Most of the Ca2 + in that case is accumulated in the intracellular inside-out vesicles that are most likely enriched with Ca2 + pumps,60 and an increase in the intracellular45Ca2 + levels is not always followed by the activation of Ca2 +-sensitive K+ (Gardos) channels. Measurements of ion fluxes bear a common limitation: flux measurements are performed in suspension, and the considerations discussed above in “Obtaining pure cell preparations” (Section 3.1) apply. So far, studies Cabozantinib solubility dmso to assess the role of WBC and platelet contamination in possible artefact generation when measuring ion fluxes using radioactive tracers are lacking. Another point that is seldom taken into account is the effect of the electro-neutrality of compartments on ion movements. Cation movements, such as those mediated by Gardos channel activity, that lead to cell dehydration are known to be rate limited by anion movements. In many cell suspension experiments, thiocyanate (SCN−) is used to bypass this limitation of anion movements. Ten millimolar is usually sufficient to saturate this

effect,61 avoiding important changes in the isoelectric point of impermeant anions and RBC hydration that are observed at higher SCN− concentrations.62 Apart from ion flux experiments, this could also apply to patch-clamp experiments aiming to investigate cation channel activity. Even if this consideration does Palbociclib ic50 not apply

in the whole-cell configuration because the anion supply is provided by the pipette content, it can impair the movement of cations in the cell-attached configuration. In this case, run-down of channel activity might be observed and conclusions can be drawn erroneously. During the past three decades, electrophysiological studies have revealed that the human RBC membrane is endowed with a large variety of ion channels.[63], [64], [65], [66] and [67] However, their physiological role remains widely unclear; they barely participate Oxalosuccinic acid in the RBC homeostasis, which is based on an almost total absence of cationic permeability and minute anionic conductance.68 Nevertheless, due to the pioneering work of Hamill on human and frog RBCs,[61] and [69] the patch-clamp technique applied to RBCs has proven to be a powerful method to decipher the involvement of ionic conductances mainly in pathophysiological scenarios.[32], [42], [62], [65], [70], [71] and [72] The main problem when attempting to perform patch-clamp on RBCs lies in the small size of the cells, which especially holds true for mammalian RBCs (2.1–9.4 μm) (cp. Section (3.3) “Interspecies studies”). This small size imposes major challenges. The opto-mechanical properties of the hardware require high-quality microscopes and at least 20 × objectives.

All suffers responded positively to local injections of BoNT/A that resulted in less headaches and precranial muscle tenderness (Dolly and O’Connell, 2012) (Relja and Telarovic, 2004). Furthermore, www.selleckchem.com/products/BKM-120.html Elza compared BoNT/A

with other currently available drugs for the treatment of migraines. Their results suggested that the BoNT/A was more effective for the group of patients with frequent episodic migraines. However, considering the clinical benefits and the lack of undesirable side effects such as weight gain and constipation, they argued that BoNT/A should be considered for use in the patients with chronic headaches as an alternative therapy or in patients with contraindications for the use of other classes of drugs. They also reminded that further investigation is needed to define patient subgroups that might benefit from BoNT/A (Magalhães et al., 2010). Arthritis is an important and growing public health problem (Lawrence et al., 2008), There is a growing need for novel treatments of refractory arthritis joint pain as aging

population is expanding with many sufferers who cannot receive the joint replacement surgery. In 2008, Jasvinder et al. reported the use of intra-articular BoNT/A in two rheumatoid arthritis (RA) patients with persistent painful monoarthritis in ankle/feet joints. Both patients had monaticular BIBF 1120 ic50 pain despite a good response of all other joints to a combination therapy that also included anti-tumor necrosis factor therapy. All intra-articular corticosteroid injections and declined surgical options were failed in both patients. They began with a single “off-label” intra-articular injection of BoNT/A into the right ankle (100 units) and left first metatarsophalangeal joint (25 units). As a result, their GNA12 pain and function improved significantly

(>40%) in both patients and the function lasted 15–18 months. They concluded that the intra-articular BoNT/A may provide an additional therapeutic option in RA patients with persistent monoarthritis (Singh and Mahowald, 2009). In 2009, Maren et al. conducted several small open label studies in which they injected BoNT/A into the joints with arthritis. They found that two third of the patients had more than 50% reduction in the joint pain severity that was associated with a significant improvement in function. Importantly, no serious adverse effects of BoNT/A were reported. They continued their studies using the same method in shoulders and knees. The results showed that BoNT/A produced a significant decrease in shoulder pain severity in one month (6.8–4.4 on VAS, p = 0.22). Furthermore, BoNT/A produced a significant 48% decrease in McGill Total Pain Score in the knees in one month (p = 0.11). This was still significant three months after the injection (p = 0.02).

, 2008), and Cd was also shown to cause cell death in a large number of different cell types (Templeton and Liu, 2010). Cell death induction by Cd was ascribed to the causation of ER-stress

(Wang et al., 2009), mitochondrial depolarization (Messner et al., 2009), increase in ceramides and calpain-activation (Lee and Thevenod, 2008), ROS-production (Yang et al., 2007), and DNA-damage (Liu and Jan, 2000). Intriguingly, the reported final outcome of Cd-induced cell death is highly diverse, ranging from classical apoptosis (Jung et al., Torin 1 supplier 2008) and necrosis (Kaji et al., 1992 and Kishimoto et al., 1991) to programmed necrosis (Messner et al., 2009) and autophagy (Dong et al., 2009). This study was conducted to precisely define the final outcome of Cd-induced cell death in endothelial cells, and to study the cellular processes involved therein with a “bottom up” research strategy. Many previous studies on cadmium-induced cell death focussed primarily on upstream signalling analyses, lacking a hard fact characterization of the ultimate outcome. As the endpoint of cell death defines whether an agent (Cd) causes inflammation (necrosis) or not (apoptosis), the clear definition of the mode of cell death is crucial Carfilzomib for the pathophysiological understanding of Cd-caused diseases. All reagents used were of purissimum or analytical grade quality and were purchased from

Sigma–Aldrich (Sigma–Aldrich, Vienna, Austria) unless specified otherwise. The isolation and culture of human umbilical vein endothelial cells (HUVECs) has been described previously (Bernhard et al., 2003). The isolation and analysis of HUVECs were approved by the Ethics Committee of the Medical University of Innsbruck (No.: UN2979) and the Ethics Committee of the Medical University of Vienna (EK Nr. 1183/2012). Cells were routinely passaged in 0.2% gelatine-coated (Sigma, Steinheim, Germany) polysterene culture flasks (TPP, Switzerland) in endothelial growth medium (EGM, Lonza) in a humidified see more atmosphere containing 5% CO2. For cell death analyses, 3 × 105 HUVECs per well were

seeded onto gelatine-coated 6-well plates (TPP, Switzerland). Prior to each experiment, medium was replaced by fresh medium. BCL-XL viruses: For constitutive over-expression of human BCL-XL in HUVECs, BCL-XL encoding cDNA was PCR amplified and recombined into pDONR-207 (Invitrogen) using Invitrogen’s B/P recombination kit. A sequence verified clone was used for L/R recombination with pHR-SFFV-dest-IRES-Puro thereby generating the lentiviral expression plasmid pHR-SFFV-BCLXL-IRES-Puro (Sigl et al., 2009). For lentiviral transduction, human HEK 293T cells were transiently transfected with lentiviral plasmids containing cDNAs coding for human BCL-XL or eGFP, along with the packaging plasmids pCMV 8.91 and pVSV-G (kindly provided by Didier Trono). Forty eight and 72 h after transfection lentiviral supernatant was sterile filtered (0.

The formation of lipid droplets in the cytoplasm, mineral nodules and cartilage extracellular matrix in the mDPSC culture after chemical defined conditions confirmed

the adipogenic, osteogenic and chondrogenic differentiation potential, respectively. Not all the cells in mDPSC cultures had the differentiation capability and, in fact, a uniform induced differentiation free of non-responsive cells is very difficult to achieve in mesenchymal stem cell cultures.35 Interestingly, some elongated cells spontaneously acquired a contractile capacity. In addition of the induced differentiation described in this study, in one isolate it was observed spontaneous differentiation in adipocyte lineage (data not shown). These data indicate the high Epacadostat manufacturer plasticity of the mDPSC even in the absence of specific stimuli. Stem cells obtained from human or rat dental pulp also exhibit extensive capability of osteogenic, chondrogenic and adipogenic differentiation.6, 7 and 11 However, Balic and Mina34 demonstrated that cultures derived from pulps of unerupted and erupted mouse incisors were incapable of differentiating into adipocytes and chondrocytes. The authors

suggest that the differentiation in these cell types may be masked by the significant number of osteo/progenitor cells present in the culture which should be investigated in experiments aiming to evaluate the differentiation potential as in vivo transplantation assays. The time of culture, the cell Seliciclib passage or medium used are other factors that may have hampered the differentiation of the cell isolates obtained

by Balic and Mina. This study provides the description of stem cells obtained from mouse dental pulp, generating cell lines positive for GFP that can be used to track the fate of these cells when injected into different mouse models of disease. The data presented herein demonstrate that mDPSC comprise a morphologically heterogeneous population of cells that exhibit some phenotypic and functional features of both embryonic and mesenchymal stem cells, such as observed in the human dental pulp. The ability to expand and differentiate opens the futures possibilities Aspartate in the study of the cell therapies in animal models. Funding: This work was supported by CNPq, FAPESB, FINEP, and FIOCRUZ. Competing interests: There are no conflicts of interest. Ethical approval: All of the experimental were approved by the Animal Ethics Committee of the Gonçalo Moniz Research Center-FIOCRUZ, Salvador, Bahia. “
“Despite numerous investigations1, 2, 3, 4 and 5 the precise mechanisms involved in the formation and enlargement of jaw cysts have not been completely established. Cyst formation is believed to be related to the proliferation of epithelial remnants that are activated by the release of cytokines and growth factors.

(2006), who found M. leucophaeata in the Gulf of Finland (northern Baltic Sea), the mussel larvae had been transported there in ship ballast waters from the North Sea. The occurrence of this species in the Gulf of Finland could have depended on cooling water discharged from power plants. But there are no such ‘hot spots’ near the part of the Gulf of Gdańsk where I found these mussels. One question that still awaits

an answer is whether young M. leucophaeata will be able to develop successfully and reproduce in the Gulf of Gdańsk, as adult specimens have not yet been found in this area. I am very grateful to Prof. Anna Szaniawska for her constructive comments on the manuscript, to Dr Ari Laine for his help with identifying the species, and to Dr Urszula Janas and Halina 3-MA purchase Rzemykowska MSc. for their support in obtaining the necessary information on M. leucophaeata. “
“Figure options Download www.selleckchem.com/products/RO4929097.html full-size image Download as PowerPoint slide It is with deep sadness that we announce the passing of Professor Halina Piekarek-Jankowska on 26th May 2011. She was a distinguished scientist at the University of Gdańsk, a lecturer to and educator of students, an outstanding expert in the field of marine geology, contributing generously to the life of the scientific community. She was also a long-standing member of the Editorial Board of ‘Oceanologia’. Halina Piekarek-Jankowska was born at Rawa Mazowiecka on 8th March 1948. In 1965 she

began her studies of geology at the University of Warsaw. During her final student years she gained her first scientific experiences in the hydrogeology of post-lacustrine Liothyronine Sodium regions, when she participated in research in the Suwałki Lake District under the tutorship of Professor Zdzisław Pazdro. She incorporated some of the materials from this research into her M. Sc. thesis. Already then, as her fellow students recall, she displayed an outstanding intelligence and personality. In 1970 she graduated from the Faculty of Geology, University of Warsaw, with

distinction. On the recommendation of Professor Pazdro, she joined the team of geologists in the Department of Geology and Cartography at the newly founded University of Gdańsk (UG), and from 1975 onwards she assisted Doc. Leonard Bohdziewicz in organizing a course in marine geology at the Institute of Oceanography UG. It was in this institute that she climbed the ladder of her scientific career: from 1971 to 1970 she was employed as assistant lecturer, from 1979 to 1996 as reader, from 1996 to 2002 as associate professor, and since 2008 as full professor. She was awarded her professorship in 2002. At the start of her career she conducted research in the Kashubian Lake District into the hydrodynamic links between quaternary aquifers and the waters of Lakes Radunia and Ostrzyce. These were pioneering studies in this part of Poland and were of a distinctly utilitarian nature. She used the materials from this research in her Ph.D.

Such precipitates can also affect the HTS resulting in poor liquid dispenses on the automation equipment. Tris buffer contains a free amine group which can react with enzymes and/or substrates, altering the equilibrium of the system. Tris is also able to chelate metal ions which could have

deleterious effects on the activity of enzymes requiring metals for catalysis or structure (Desmarais et al., 2002). There are many subtleties to consider when choosing a detection method for following an enzymatic check details reaction in HTS, including throughput, sensitivity, cost and assay robustness, as well as the nature of the reaction under investigation and that of the products and/or substrates to be measured. No detection method is perfect – they are all utilized with some caveats – but for most enzyme classes, it is possible to strike a balance between these requirements to develop a useful assay. Many of the methods that are introduced here will be discussed with respect to specific enzyme classes and technologies later in this review. Directly monitoring a reaction as it is happening is referred to as a continuous read. Continuous reading typically requires a spectrophotometer/fluorometer capable of rapidly collecting data Selleckchem SB203580 from multiple time points and the ability of the

molecules being monitored to absorb or emit light in a reaction dependent way. Some examples of suitable systems used

in continuous detection are observing the change in either absorbance or fluorescence upon the interconversion of NAD and NADH, the production of fluorescent labels such as amino methyl coumarin (AMC) by proteolysis of AMC-labeled peptides, and the ability to observe changes in light scattering upon large protein complex formation. Continuous detection provides the advantage of observing an entire reaction time course Dapagliflozin from a single mixture of substrate and enzyme, which minimizes the error in data by minimizing the need for multiple transfers and excess handling of the reaction components. However timing is a key variable that must be controlled particularly if a single time point is chosen for the assay as it can be difficult to stop a continuous reaction without disrupting the system or interfering with detection. In the specific case of fluorescence detection for enzyme assays one method to address “overriding” of the assay signal by compound fluorescence is to measure the reaction progress in a kinetic mode. Unless the reaction under study is slow, on the order of tens of minutes, only fast-scanning readers or whole-plate imagers (such as the PerkinElmer ViewLux™) allow for unbiased and speedy repeated measurements of microtiter plates. However, often a simple method where two-time points are collected allows one to estimate the reaction rate by simple subtraction of the two data points.

APs can affect a number of reproductive

parameters in fish, including gonadal development (Meier et al., 2007b), induction of plasma vitellogenin (Vtg) in male and juvenile fish GSI-IX (Jobling and Sumpter, 1993 and White et al., 1994), inhibition of spermatogenesis (Gimeno et al., 1998, Jobling and Sumpter, 1993, Miles-Richardson et al., 1999 and Weber et al., 2002), and oogenesis (Tanaka and Grizzle, 2002 and Weber et al., 2003). Tollefsen et al. (2007) and Tollefsen and Nilsen (2008) found that APs were able to bind to plasma sex steroid-binding proteins (rtSBP) in rainbow trout (Oncorhynchus mykiss). The highest affinity was seen for mono-substituted APs with 4–8 carbon chain length, but this was still 104–106 times lower than the affinity for the natural sex steroid 17β-estradiol (E2). The results suggested that endocrine disruption may occur after exposure to realistic concentrations of APs and a variety of other PW compounds. Tollefsen et al. (2006) further showed that chemicals in solid phase extracts of PW were able to displace E2 from the rtSBP and induce estrogenic effects. The bioactive chemicals were not identified. Tollefsen et al. (2011) demonstrated that complex mixtures

of oil-related compounds could modulate the endocrine physiology APO866 concentration of Atlantic cod. Fish were exposed to either diluted PW (0.5% and 0.1%), dispersed oil (0.2 mg L−1), or artificial PW water mixed with nine low to medium molecular weight APs and PAHs. The total sex-steroid binding capacity was up-regulated in the blood of female cod, indicating interference with blood steroid transport. Induction of plasma Vtg was not found, Glutamate dehydrogenase although the number of males and females with elevated Vtg was higher in certain exposure groups than in the control group. General health parameters such as gonadosomatic, hepatosomatic or fish condition index were not affected, which

suggests that the endocrine disrupting effect was too low to elicit clear physiological or growth effects. When exposing late larvae and juveniles of Atlantic cod to PW Meier et al. (2010) found that individuals exposed to 1% PW had significantly higher levels of Vtg and CYP1A in plasma and liver, respectively. No similar effects were seen at exposure to 0.1% and 0.01% PW. Serious reproductive disturbance was demonstrated by Meier et al. (2007b) in first-time spawning Atlantic cod that were force fed a paste containing C4–C7 APs. Total AP doses during 1 and 5 weeks were 0.02–80 mg kg−1 body weight. Treatment impaired oocyte development, reduced estrogen levels, and delayed spawning by 17–28 days in female fish.

, 2004). For a variety of animal species and for different modalities it has been demonstrated BTK inhibitor solubility dmso that single neurons respond in a temporally sparse manner (Reinagel, 2001, Jadhav et al., 2009, Olshausen et al., 2004 and Hromádka et al., 2008) when stimulated

with natural time-varying input. In the mammal this is intensely studied in the visual (Dan et al., 1996, Vinje and Gallant, 2000, Reinagel and Reid, 2002, Yen et al., 2007, Maldonado et al., 2008, Haider et al., 2010 and Martin and Schröder, 2013) and the auditory (Hromádka et al., 2008, Chen et al., 2012 and Carlson et al., 2012) pathway as well as in the rodent whisker system (Jadhav et al., 2009 and Wolfe et al., 2010). Sparseness increases across sensory

processing levels and is particularly high in the neocortex. Individual neurons emit only a few spikes positioned at specific instances during the presentation of a time-varying input. Repeated identical stimulations yield a high reliability and temporal precision of responses (Herikstad et al., 2011 and Haider et al., 2010). Thus, single Rucaparib price neurons focus only on a highly specific spatio-temporal feature from a complex input scenario. Theoretical studies addressing the efficient coding of natural images in the mammalian visual system have been very successful. In a ground breaking study, Olshausen et al. (1996) learned a dictionary of features for reconstructing a large set of natural still images under Reverse transcriptase the constraint of a sparse code to obtain receptive fields (RFs), which closely resembled the physiologically measured RFs of simple cells in the mammalian visual

cortex. This approach was later extended to the temporal domain by van Hateren and Ruderman (1998), learning rich spatio-temporal receptive fields directly from movie patches. In recent years, it has been shown that a number of unsupervised learning algorithms, including the denoising Autoencoder (dAE) (Vincent et al., 2010) and the Restricted Boltzmann Machine (RBM) (Hinton and Salakhutdinov, 2006, Hinton et al., 2012 and Mohamed et al., 2011), are able to learn structure from natural stimuli and that the types of structure learnt can again be related to cortical RFs as measured in the mammalian brain (Saxe et al., 2011, Lee et al., 2008 and Lee et al., 2009). Considering that sensory experience is per se dynamic and under the constraint of a temporally sparse stimulus representation at the level of single neurons, how could the static RF model, i.e. the learned spatial feature, extend into the time domain? Here we address this question with an unsupervised learning approach using RBMs as a model class.

Cerebral bleeding after treatment also occurred on the opposite MDV3100 cost side of the brain infarction, suggesting a causal link to the substantially higher energy and lower frequency of the “sonothrombolysis probe” compared with the energy of diagnostic US probes. In vivo experiments evaluating the therapeutic efficacy and safety of using highly energetic, low-frequency (20 kHz) US in treating rats with an embolic MCA occlusion showed

an increased incidence of cerebral edema [24] and [25], thus indicating the unsuitability of this kind of US for clinical use. So far, “diagnostic” transcranial US remains the only form of US appropriate for sonothrombolysis. Skoloudik et al. [7] performed a

pilot study on 9 patients who had suffered an AIS with acute MCA or basilar artery occlusion and undergone endovascular sonothrombolysis within an 8-h time window from symptom onset. For this purpose, a 3F microcatheter with a US probe of 2.05–2.35 MHz was used. Complete recanalization at the end of treatment was achieved in one third of patients, and partial recanalization occurred in an additional 44% of patients at the end of the procedure. At admission, the National Institutes of Health Stroke Scale (NIHSS) scores were in the range of 10–33 (median, 19.0). At 3 months, 4 (44%) patients were functionally independent (modified ZD1839 supplier Rankin Scale [mRS] score, 0–3; median mRS score, 4). No sICHs occurred for 24 h after endovascular sonothrombolysis

until a control computed tomography (CT) scan at 24 h. These researchers concluded that this endovascular system might serve as a new treatment option for patients suffering from acute stroke. The thrombolytic effect of US has generally been regarded as a tool for improving recanalization. However, as several US follow-up studies have shown, reocclusion of a vessel after recanalization can occur in 20% or more (up to 29%) of patients after rtPA treatment [1] and [26]. Sawaguchi et al. [27] recently 4��8C reported interesting results from a novel use of US treatment in AIS. They found that continuous US (500 kHz, 0.72–0.28 W/cm2) significantly suppressed thrombus growth in vitro. Based on their findings, these researchers suggested low-intensity, low-energy US as a possible simple and safe tool to prevent reocclusion of intracranial vessels after rtPA treatment. Determining the most efficient US settings for sonothrombolysis is complicated by the fact that there is a tremendous number of possible combinations of its parameters. Wang et al. [28] presented results from an in vitro experiment for the systematic and rapid evaluation of the thrombolytic effect of 500-kHz US as the ultrasonic spatial intensity increased from 0.1 to 0.7 mW/cm2.

DNA concentration was measured with GeneQuant pro spectrophotometer (Amersham Biosciences, Cambridge, England) at λ = 260 nm from 10 μl of total DNA volume. Purity of DNA was

assessed using the ratio of OD260 nm/280 nm. Samples were amplified using ABI 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). STR genotyping was performed using PowerPlex16™ System PCR Amplification Kit (nDNA; Promega Corporation, Madison, WI, USA) version 3.1. Y-STR genotyping was conducted using AmpFlSTR® Yfiler™ Amplification Kit (Applied Biosystems, Foster City, CA, USA). Polymerase chain reaction (PCR) was carried out according to the manufacturer’s instructions. Fluorescence detection of genotypes was performed with ABI Prism® 3100-Avant Genetic Analyzer and by using Data Collection v.2.0, and Gene Mapper ID v.3.2 analysis software (Applied Biosystems, Foster City, USA). For mtDNA analysis we amplified click here HV1 (Primer L15996: 5′-CTC CAC CAT TAG

CAC CCA AAG C-3′; Primer H16401: 5′-TGA TTT CAC GGA GGA TGG TG-3′) and HV2 (Primer L29: 5′-GGT CTA TCA CCC TAT TAA CCA C-3′; Primer H389: Sirolimus 5′-CTG GTT AGG CTG GTG TTA GG-3′) regions. Considering that nucleotide positions within the mtDNA are numbered from 1 to 16,569 using the L-strand from control region, HV1 region spans positions 16,024 to 16,365 (342pb) and HV2 covers positions 73 to 340 (268pb). PCR products of mtDNA were purified from residual primers with Exonuclease I (EXO I; Amersham Biosciences – E70073Z; GE HealthCare©) and Shrimp Alkaline Phosphatase (SAP; Amersham Biosciences – E7009; GE HealthCare©), and sequenced directly by cycle sequencing. Hyper variable segments were sequenced with BigDye Terminator Cycle Sequencing kit from Applied Biosystems on ABI PRISM™ 3100-Avant DNA sequencer. ABI PRISM™ 3100-Avant Genetic Analyzer

was used for separation and detection of the fluorescence-labelled 4��8C chain termination products. The sequences of mtDNA were manually checked using CHROMAS18 and aligned with CLUSTAL-X.19 DNA profiles obtained from the teeth of the deceased were compared to the DNA profiles of reference samples obtained from close relatives. Relatives’ genomic DNA was extracted from leucocytes by a standard method.20 DNA analyses of relatives were performed as described above. This project was approved by the Research Ethics Committee of the Pontifical Catholic University of Rio Grande do Sul (Tel. +55 51 33203345; protocol #1107/05) and the consent or assent to take part in this study was obtained from Forensic Laboratory of Instituto-Geral de Perícias of Rio Grande do Sul, Brazil. Allele identification was carried out with Gene Mapper ID software version 3.2 using ABI Prism 3100 from Applied Biosystems. Comparisons between the results obtained from the different protocols were examined using ANOVA or ANOVA followed by Student Newman’s Keul Post Hoc and Pearson’s correlation (SPSS software package for Windows version 13.0; SPSS, Inc.). A P value of <0.