To promote their pathology CD4 T-cells from patients with rheumatoid arthritis (RA) have to clonally expand LY341495 and differentiate into cytokine-producing effector cells. inflammation. Rebalancing glucose utilization and repairing oxidant signaling may provide a book therapeutic technique to prevent autoimmunity in RA. Intro The autoimmune disease ARTHRITIS RHEUMATOID (RA) problems tendons cartilage and bone tissue and shortens life span through acceleration of coronary disease (1 2 Compact disc4 T-cells in RA individuals maintain synovitis promote autoantibody development facilitate osteoclast differentiation and impose endothelial dysfunction (3). When triggered RA Compact disc4 T-cells insufficiently upregulate the glycolytic enzyme PFKFB3 and generate much less ATP and lactate (4). It really is currently unknown whether and exactly how metabolic abnormalities are linked to their pro-inflammatory features mechanistically. The cardinal feature of na?ve Compact disc4 T-cells may be the capability to proliferate when encountering antigen massively. When transitioning from na?ve to effector position T-cells expand 40-100 fold within times (5) building them highly reliant on energy and biosynthetic precursors (6). Relaxing lymphocytes depend on oxidative phosphorylation and fatty acidity break down but upon activation change to aerobic glycolysis and tricarboxylic acidity flux designating blood sugar as the principal resource for ATP era in triggered lymphocyte. Anabolic rate of metabolism of blood sugar not merely provides energy but also macromolecular blocks for the exponentially growing biomass typically by shunting blood sugar in to the pentose phosphate pathway (PPP) (7). In the 1st rate-limiting step from the PPP G6PD oxidizes G6P to 6-phosphogluconolactone to create 5-carbon sugar (pentoses) ribose 5-phosphate a precursor for nucleotide synthesis and NADPH among the cell’s primary reductants. As an electron carrier NADPH provides Rabbit Polyclonal to GRP94. reducing equivalents for biosynthetic reactions and by regenerating decreased glutathione protects against reactive air varieties (ROS) toxicity. Cytoplasmic NADPH can be an total necessity to convert oxidized glutathione (GSSG) to its decreased type (GSH) which can be converted when hydrogen peroxide can be reduced to drinking water. Oxidative stress outcomes from the actions of ROS short-lived oxygen-containing substances with high chemical substance reactivity towards lipids proteins and nucleic acids. Until lately ROS LY341495 were thought to be merely damaging real estate agents but are actually named second messengers that regulate mobile function through oxidant signaling (8 9 Cells can make ROS in a number of of their organelles and still have specialized enzymes like the category of NADPH oxidases (NOX) to provide fast and managed gain access to. Quantitatively mitochondria stick out as continual ROS suppliers using the respiratory string complexes I and LY341495 III liberating superoxide in to the mitochondrial matrix as well as the intermembrane space (9 10 It really is incompletely realized how redox signaling impacts T-cell proliferation and differentiation and exactly how cell-internal ROS relate with pathogenic T-cell features. The current research has investigated practical implications of metabolic and redox dysregulation in RA T-cells. We discover that RA T-cells neglect to correctly stability mitochondrial ROS creation as well as the mobile anti-oxidant equipment. Molecular studies place excessive activity of G6PD at the pinnacle of abnormal T-cell regulation in RA and provide a new paradigm for the connection between metabolic activities abnormal proliferative behavior and pro-inflammatory effector functions. Mechanistically PPP hyperactivity oversupplies RA T-cells with reducing equivalents increasing NADPH and depleting ROS. This insufficient oxidative signaling prevents sufficient activation of the cell cycle kinase ATM and allows RA T-cells to bypass the G2/M cell cycle checkpoint. ATM deficiency shifts differentiation of na?ve CD4 T-cells towards the Th1 and Th17 lineage creating an inflammation-prone T-cell pool. Several metabolic interventions are able to rebalance glucose utilization away from the PPP towards glycolytic breakdown easing reductive stress and preventing hyperproliferation and maldifferentiation of RA T-cells. Such interventions represent possible drug candidates for a novel LY341495 strategy in anti-inflammatory therapy. Results.
Background Liver cell injury in alcoholic hepatitis (AH) is in Rabbit Polyclonal to OR2A5/2A14. part due to macrophage generated proinflammatory cytokines i. antibody-labeling we profiled the receptors cytokines and chemokines seen in M1 M2a M2b and M2c macrophages in liver organ biopsies from sufferers with AH. Outcomes The increased Compact disc 163 expression within previous research was confirmed aswell yet another macrophage phenotypic marker Compact disc206 recommending that AH pathogenesis at least partly consists of M2a and M2c macrophages. TGF-β was present to become more than expressed by liver organ sinusoidal macrophages robustly. Macrophage expression from the phenotypic markers TLR-2 TLR-4 and TLR-8 – within both M1 and M2 macrophages – aswell as the chemokines CCL-1 and CCL-18 was discovered. Nevertheless IRF-4 which relates to IL-4 creation and M2a polarization aswell as the cytokines CCL-1 and Il-1β as well as the chemokine CXCL-1 had been also observed recommending that M2a and M2b also are likely involved in AH pathogenesis. Bottom line Livers with AH display solid macrophage over appearance of TGF-β a rise factor additionally connected with M2 type macrophages and mainly known because of its fibrogenetic properties. Nevertheless our immunoprofiling of macrophage over appearance also implies that AH is powered by receptors interferons and cytokines that are generally associated not only with M2 macrophages but with M1 aswell. Thus a complicated interplay between various kinds of macrophages expressing a different array of substances and receptors is certainly involved with AH. Keywords: Alcoholic hepatitis Macrophages Compact disc163 TLR-4 Launch Liver cell damage in AH is certainly in part because of macrophage generated proinflammatory cytokines and sinusoidal blockage. The response of some phenotypic subtypes of macrophages (Kupffer cells) causes problems for hepatocytes by method of innate immune system damage in response to endotoxin. This is within rodent types of early alcoholic liver organ disease and perhaps in AH in human beings (Miller et al. 2011 These noticeable changes are increased in response to severe alcoholic beverages ingestion. They are replies that are reversible when ethanol ingestion is certainly ended in experimental alcoholic beverages fed rodent versions. The question is certainly: what macrophages get excited about chronic alcohol mistreatment in humans who’ve AH? Plasticity and useful polarization are hallmarks of various kinds of macrophages i.e. M1 M2a M2c and M2b that will be involved with AH. This differential modulation of the sort of macrophage-chemokine program integrates polarized macrophages in pathways of level of resistance to or advertising of immune-regulation tissues repair and redecorating (Mellins et al. 2011 The T cell response to cytokines and chemokines differs when M1 and M2 macrophages are compared. M1 includes a Th1 response to LPS and IFNα. M2a b and c result in a Th2 response of immune-regulation matrix remodeling and deposition. M2a is a reply to IL-4 and 13 M2b is certainly a reply to TLR/IL-1R agonists and M2c responds to 1L-10 and suppresses immune system responses to tissues remodeling (Bleesing et al. 2007 Mellins et al. 2011 The type of macrophages located in the liver sinusoids determines the type of the inflammatory process in AH. The question remains as to the type of macrophage response that exists in AH. Monocytes derived from blood have provided the basis of studies for 17 alpha-propionate LPS-sensitive cellular response to induce TNF-α expression (Hill et 17 alpha-propionate al. 1992 using isolated Kupffer cells and cell-cultured natural 264.7 cell-line (Gobejishvili et al. 2006 In this statement we used immunofluorescence antibodies against macrophage markers to more fully classify the type of macrophages involved in alcoholic hepatitis. Using immunofluorescent antibody-labeling we profiled the proinflammatory markers and chemokines observed in M1 M2a M2b and M2c macrophages in liver biopsies from patients with AH. Methods Eight archived liver biopsies diagnosed as alcoholic hepatitis and 2 archived control livers were used 17 17 alpha-propionate alpha-propionate in order to study the type of molecules expressed by macrophages in liver sinusoids. Immunohistochemistry Liver tissue was fixed in 10% buffered zinc formalin. These sections were either single or double stained using antibodies raised in rabbit mouse or goats (observe Table 1 for list of antibodies and their origin). A Nikon 400 fluorescent microscope.
Latent infection of B lymphocytes by Epstein-Barr trojan (EBV) results in their immortalization into lymphoblastoid cell lines (LCLs); this latency system is controlled from the EBNA2 viral transcriptional activator which focuses on promoters via RBPJ a DNA binding protein in the Notch signaling pathway. binding at sites near EBNA3A- or EBNA3C-regulated genes is definitely specifically regulated from the respective EBNA3. To investigate EBNA3 binding specificity we recognized sequences and transcription factors enriched at EBNA3A- EBNA3B- and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is definitely enriched at EBNA3A- and EBNA3C-bound sites and exposed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-bad BJAB cells we demonstrate that IRF4 is essential for EBNA3C but not EBNA3A or EBNA3B binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for unique subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with additional cell transcription factors. IMPORTANCE Epstein-Barr computer virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines test. Using this approach we confirmed 10 of 12 EBNA3A- 9 of 10 EBNA3B- and 9 of 12 EBNA3C-bound sites recognized by ChIP-seq (Fig. 2). In addition we found evidence of EBNA3B binding at 3 sites (EIF2AK3 QSK and ALOXE3) by ChIP-qPCR that were not observed by ChIP-seq. Based on these results we estimated the overall level of sensitivity and specificity of our EBNA3 ChIP-seq experiments relative to ChIP-qPCR to be 92% and 83% respectively. FIG 2 ChIP-qPCR validation of EBNA3 binding sites in LCLs recognized by ChIP-seq. The Scoparone pub plots display enrichment of genomic DNA from ChIP of EBNA3A EBNA3B or EBNA3C relative to input. Each EBNA3 was specifically ChIPed using HA antibody with either the EBNA3A-F-HA … EBNA3-bound sites are overrepresented at promoter and enhancer elements. We constructed average histone profile Scoparone plots for ±2-kb areas centered on Rabbit polyclonal to ITPK1. EBNA3 maximum summits for the activation marks H3K9Ac H3K27Ac H3K4me1 H3K4me2 and H3K4me3; the repressive marks H3K27me3 and H3K9me3; and the transcribed-region-associated mark H3K36me3 using ENCODE histone ChIP-seq data units (48). For each EBNA3 protein the signals from acetylation marks (H3K9Ac and H3K27Ac) were strong as were mono- di- and trimethylation of H3K4 (Fig. 3A). For those EBNA3s levels of repressive marks characteristic of facultative (H3K27me3) and constitutive (H3K9me3) heterochromatin were very low despite the prior observation that H3K27me3 levels are improved at EBNA3A- and EBNA3C-repressed genes such as CDKN2A and BCL2L11 (BIM). We also annotated EBNA3-bound peaks according to their locations within the epigenetic scenery (49). The results for EBNA3B were standard: 8% of EBNA3B sites reside within active promoters defined by high H3K4me3 and H3K9Ac 12 within poor and poised promoters characterized by high H3K4me3 and low H3K27Ac or high H3K27me3 33 within strong enhancers with high H3K4me1 and high H3K27Ac and 25% within poor enhancers with intermediate H3K4me1 and little H3K27Ac and 22% were found in heterochromatin regions characterized by the absence of these histone marks (Fig. 3B). Therefore the EBNA3 proteins despite their functions in the repression of multiple cell genes (19 -25) bind mainly at genomic sites bearing marks of transcriptionally active chromatin. FIG 3 Characterization of EBNA3-bound sites. (A) Average histone profile Scoparone plots for EBNA3A- EBNA3B- and EBNA3C-bound sites. The average densities of ChIP-seq reads for the indicated histone modifications are plotted for ±2-kb windows round the Scoparone summits … Overlap among EBNA3A EBNA3B EBNA3C EBNA2 and RBPJ sites in LCLs. Because the EBNA3 proteins cooperatively regulate many cell genes and because EBNA2 EBNA3A and EBNA3C must interact with the RBPJ transcription element to transform B lymphocytes we wanted to examine Scoparone the degree of overlap among EBNA3A- EBNA3B- EBNA3C- and EBNA2-bound sites. Significant overlap between EBNA3A and EBNA3C has been mentioned previously (35). Our experiments estimate that this overlap is about 26% of EBNA3A peaks that are Scoparone EBNA3C cobound and reveal the overlap extends to EBNA3B-bound sites. For EBNA3B 21 are shared with EBNA3A 22 with EBNA3C and 37% with EBNA2. McClellan et al. found that 80% of the genes closest to an EBNA3-bound site were also the closest genes to an EBNA2-bound site (33). Using their approach we found only 36% of EBNA3-bound genes to be EBNA2 cobound though this proportion increases to 50% for EBNA3B-bound genes (observe Table S4 in the supplemental material). However in LCLs there is considerable overlap among EBNA2-.
(CS) is a unicellular green alga. with TLR4 receptor. These CS-activated signaling pathways could further activate NF-have been suggested as potential remedies for improving human being health insurance and wildly utilized as botanical foods. For instance extracts were utilized as nutrition health supplements in relieving hypertension and remedies for modulating human being immune reactions [1-4]. It had been reported that components can elicit different beneficial pharmacological results against malignancies  bacterial infections  and viral replication [7 8 From an earlier study extracts Epirubicin were shown to strongly increase the production of IFN-and IL-2 and activate Th1 cells to strengthen immune system and host defense . Hasegawa et al. demonstrated the roles of extracts in inducing IFN-and IL-2 mRNA expression and activating cell-mediated immunity . DCs are professional antigen-presenting cells (APCs) and have unique ability in linking innate and adaptive immunity [11 12 Immature DCs are able to ingest antigens. Once activated DCs go through a series of maturation processes that include migration to lymphoid tissues downregulation of antigen uptake upregulation of major histocompatibility complex (MHC) class II costimulatory molecules (CD40 CD80 and CD86) and a specific maturation marker CD83 [13-15] and finally presenting antigenic peptides to T lymphocytes . The MAPK families (p38 ERK and JNK) were activated in response to a variety of cellular stress or stimuli including oxidative stress LPS and TNF-LPS (L8274 (CS) is a commercially available product (International Epirubicin Cryptomonadales Biotechnology Taiwan). Fifty grams of powders Epirubicin were refluxed with 150?mL distilled water for 1?h. The extracts (polysaccharide fraction) were filtered through no. 5 filter paper (Toyo Roshi Toyo Japan) and vacuum concentrated at 60°C. The presence of LPS was detected by the chromogenic amebocyte lysate assay (Charles River Laboratories Inc. Wilmington MA USA). The cytotoxicity of CS against normal cells (PBMC) was assessed by Alamar Blue assay (AbD Serotec Oxford UK) because of its low toxicity to normal cells [19 20 ISGF-3 CS was not toxic to PBMC at the highest concentration tested (>100?secreted from DCs or T cells were assayed with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Minneapolis MN Epirubicin USA). The absorbance of the plate was detected by a SpectraMax M5 microplate reader (Molecular Devices Sunnyvale CA USA) with input wavelength at 450-540?nm. The detecting limits of these ELISAs were 31.3?pg/mL for IL-12 and 15.6?pg/mL for IFN-was quantified by ELISA. T-cell proliferation was detected by Alamar Blue assay after being cocultured with DC for 5 days. The number of viable cells correlated with the magnitude of dye reduction and was quantified as percentage of alamarblue reduction. The percentage of alamarblue reduction (% reduction) is calculated according to the following formula: < 0.05. All data were mean ± SEM of three independent experiments unless indicated otherwise. 3 Results 3.1 CS Induces Phenotypic Maturation and IL-12 Production of Human Monocyte-Derived DC by Activating NF-by T cells (Figures 2(a) and 2(b)). Figure 2 Allogeneic T-cell responses induced by CS-treated DCs. Immature DCs were stimulated with CS Epirubicin (30?were reported to induce immune responses in human or mice [35-37]. With this scholarly research we studied the result of polysaccharides components Epirubicin on human being immune system reactions. It had been reported that polysaccharides extracted from many origins can stimulate DC maturation and immune system reactions . As additional polysaccharide components polysaccharide fraction components may also maturate DC and activate immune system response by inducing IL-12 secretion in DCs. IL-12 can be very important to activating organic killer cells (NKs) and causing the differentiation of T helper cells toward Th1 cells. Th1 response can skew the disease fighting capability toward cellular immune system response which increase the killing effectiveness of macrophage boost Compact disc8+ T-cell proliferation and activate organic killer cells . Furthermore Th1 response is essential in fighting with each other against disease tumor and disease . Since NF-. Nevertheless ERK pathway was proven to differentiate DC towards tolerogenic DC . One feasible explanation would be that the strength and length of signaling activation could be different predicated on stimuli and various activation patterns of signaling pathways may bring about various consequences..
Withaferin A (WFA) is a steroidal lactone with antitumor results manifested in multiple levels that are mechanistically obscure. and CHOP. Collectively our results present mechanistic understanding into how WFA inhibits breasts tumor 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 development. (23) breast tumor cells had been treated with WFA as indicated for 10-times; colonies had been counted. of breasts tumor cells in the current presence of WFA was assayed by colony development in smooth agar (24). was performed utilizing a commercially obtainable XTT assay package (Roche Applied Technology Indianapolis IN). Apo2 Breasts tumorigenesis assay MDA-MB-231 MDA-MB-231-pLKO.1 MDA-MB-231-DR5shRNA1 and MDA-MB-231-DR5shRNA2 xenografts had been generated as previously referred to (24) grouped in 2 experimental organizations (8 mice/group) and treated with intraperitoneal injections of either vehicle (10% DMSO 40 cremophor-EL and 50% PBS) or vehicle containing 4 mg Withaferin A (ChromaDex Inc. Irvine CA)/kg bodyweight 5days/week 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 for 5 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 weeks. The dosage and path of WFA administration had been selected from earlier research documenting effectiveness of WFA (8). Tumors had 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 been collected after four weeks of treatment; assessed subjected and weighed to help expand analysis by immunohistochemistry RT-PCR and traditional western blotting. At least four arbitrary nonoverlapping representative pictures from each tumor section from eight tumors of every group had been captured using ImagePro software program for quantitation of benefit pRSK CHOP pElk1 and DR5 manifestation. MMTV-neu mice model- Mammary tumor cells from our previously released prevention research in MMTV-neu mice (11) had been also used to look for the expression of the proteins by traditional western blotting. With this research WFA administration led to a statistically significant reduction in macroscopic mammary tumor size microscopic mammary tumor region (11). All animal research were relative to the rules of Johns Hopkins University University and IACUC of Pittsburgh IACUC. Phospho-Antibody Array Evaluation Breast tumor cells had been treated with WFA as well as the phospho-antibody array evaluation was performed using the Proteome Profiler Human being Phospho-Kinase Array Package ARY003 from R&D Systems based on the manufacturer’s guidelines. Array images had been analyzed using the GeneTools picture evaluation software program (Syngene). Subcellular fractions Immunoblotting transfection RNA disturbance Immunofluorescence and confocal imaging had been prepared pursuing previously published process (25). was completed as referred to (26). The blots are representative of multiple independent bar and experiments diagrams are included showing quantitation of western blot signals. Breast tumor cells had been with ERK CHOP Elk1-WT and 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 Elk1-S383A-mutant vectors using Lipofectamine-2000 (Invitrogen) and treated with WFA as indicated. as referred to (24). Chromatin immunoprecipitation (ChIP) and RNA isolation RT-PCR ChIP analyses had been performed using our released treatment (27). Total mobile RNA was extracted using the TRIZOL Reagent package (Life Systems Inc. Rockville MD). RT-PCR was performed using particular antisense and feeling PCR primers. Steady knockdown using Lentiviral brief hairpin RNA Five-six pre-made lentiviral DR5 CHOP and RSK brief hairpin RNA (shRNA) constructs and a poor control construct developed in the same vector program (pLKO.1) were purchased from Open up Biosystems (Huntville AL). Constructs were useful for transient transfections using Lipofectamine or Fugene. Paired steady knockdown cells had been generated pursuing our previously founded process (25). Statistical Evaluation All experiments had been performed thrice in triplicates. Statistical evaluation was performed using Microsoft Excel software program. Significant differences had been analyzed using student’s physiological relevance of our results by analyzing whether WFA got inhibitory effects for the advancement of breasts carcinoma in nude mouse versions. Tumor development was considerably inhibited in WFA-treated experimental group compared to the control group (Shape 1C). Ki-67 a nuclear nonhistone protein is among the main markers of tumor proliferation (31) utilized like a decision-making device for adjuvant therapy (32). The immunohistochemical evaluation of tumor.
Previous studies showed that a new word that is similar to many known words will be learned better than a new word that is similar to few known words (Storkel et al. words. The results of these experiments show that new words influence the recognition of previously known words. In addition to newly learned words influencing the processing of previously known words work by Storkel et al. (2006) suggests that characteristics of known words can influence how easily a new word is learned. Storkel et al. had adults learn novel words that were either similar to few known words (i.e. the new words had a sparse phonological neighborhood) or to many known words (i.e. the new words had a dense phonological neighborhood). At the end of training Storkel et al. found that participants learned a higher proportion of novel nonwords that were similar to many known words (i.e. they had dense phonological neighborhoods) than novel nonwords that were similar to few known words (i.e. they had sparse phonological neighborhoods). Whereas Gaskell and Dumay (2003) CCNG2 found that newly XL647 learned words influence how known words are processed Storkel et al. (2006) found that the number of known words that resemble a new word influences the acquisition of a new word. In the work of Gaskell and Dumay (2003) Storkel et al. (2006; Vitevitch & Storkel 2013 and many others there is an assumption-at least implicitly-that the phonological representations that are known and acquired are abstract stripped of indexical information and other types of acoustic-phonetic variability. However there is an increasing amount of evidence that suggests the lexicon may contain not only abstract word-forms but also exemplar representations of words (e.g. Goldinger 1998 Johnson 1997 McLennan & Luce 2005 Vitevitch & Donoso 2011 XL647 Vitevitch et al. 2013 (The idea of acoustic-phonetic exemplars in the lexicon should not be confused with the idea of experiencing multiple and variable examples of an object with the same name as examined in Perry et al. 2010 If such exemplar representations of words exist in the lexicon how do these exemplars influence the process of learning a new word? From the exemplar perspective a word that occurs often in the language will have many exemplars of that word stored XL647 in the lexicon whereas a word that occurs less often in the language will have fewer exemplars of that word stored in the lexicon. With that in mind consider the word-learning model proposed by Storkel et al. (2006) to account for the influence XL647 on acquisition of the number of known words that are neighbors with a novel word. If the number of exemplar representations of a known-word influences word-learning in the same way that the number of abstract representations of known phonological neighbors influences word-learning then a high frequency word-which has more exemplars to resonant with than a low frequency word-would have an advantage in “attracting” a novel neighbor to the lexicon. To test this prediction about the number of exemplars of a known-word (as a function of word frequency) influencing the acquisition of a phonological neighbor of that known word we identified words in the lexicon that had no phonological neighbors-so called lexical hermits (Vitevitch 2008 which varied in their frequency of occurrence in the language. We then created novel words that were phonological neighbors of these high and low frequency known words. For example participants were asked to learn the novel word /depaim/ which is a phonological neighbor of the low frequency hermit = 433.69 occurrences per million; = 242.04) and the remaining five hermit words are used rarely in the language (= 17.75 occurrences per million; =16.42; (8) = 3.83 < .01). There was no difference in the Age-of-Acquisition (AoA; Kuperman Stadthagen-Gonzalez & Brysbaert 2012 of the frequent words (= 4.31 rated AoA; =.77) and the infrequent words (= 4.64 rated AoA; =.79; (8) = .67 = .52). There was no difference in phonotactic probability of the frequent words and the infrequent words either. The mean for the frequent hermits = .2205 (= .06) and for the infrequent hermits = .2044 (= .03) (8) = .52 = .61. The mean sfor the frequent hermits = .0105 (= .005) and for the infrequent hermits = .0112 (= .006); (8) = .19 = .85; Vitevitch & Luce 2004 Finally there was no difference in the phonotactic probability of the novel words that were neighbors of the hermits. The novel neighbor of the frequent hermits had a mean = .1973 (= .04) and the novel neighbor of the infrequent hermits XL647 had a mean.