Even so, analyses of BMP stimulated hypertrophy suggested that ALP activity steadily elevated more than a three day period, when Col X mRNA reached maximal levels inside 24 h. Experi ments in which pre hypertrophic chick chondrocytes were transfected with luciferase constructs regulated by sequences in the avian kind X collagen gene demon strated that a b2 area two. six 2. 0 kilobases upstream of your ColX transcription start off web page, when joined to 640 base pair region with the proximal promoter, was transcrip tionally activated by BMP two, four, and 7. Northern blot analyses following cyclohexamide remedy showed that new protein synthesis isn’t necessary for BMP induced Col X expression.
Added studies indicated that the mechanism for variety X collagen promoter regulation prob ably entails BMP activated Smads interacting having a Runx2 Cbfa1 transcription factor, and that retinoic acid stimulation of Col X expression is by means of precisely the same 316 bp area. While long-term therapy of chondrocytes selleck mTOR inhibitors with ascorbate benefits in elevated levels of kind X collagen mRNA, there is no information regarding the potential of ascorbate to regulate the kind X collagen promoter. In osteoblastic cells, BMPs and ascorbate have been shown to operate through mechanisms that at the least partly involve mitogen activated protein kinases. As an example, ascorbate promotes extracellular matrix pro duction which, in turn, activates the extracellular signal regulated kinases, in an osteoblas tic cell line. MAP kinases including ERK1 2, p38 and PI3 kinase have also been reported to become needed for BMP rely ent induction of osteoblast differentiation.
Nonetheless, these pathways can act oppositely in certain BMP induced processes which include osteocalcin synthesis by osteoblasts. Generally, MAP kinase pathways involving ERK1 2 stim selleck inhibitor ulate proliferation, growth and differentiation, whereas those that stimulate p38 kinase lead to differentiation and apoptosis. In early stages of chondrocyte differentia tion, an increase in p38 and decrease in ERK1 two activity is required for the progression to cartilage nodule formation in chick limb buds. In hypertrophying chondrocytes p38 has been shown to be needed for Col X mRNA syn thesis. In apoptosis of articular chondrocytes as well as other cell varieties, ERK1 2 inhibits and p38 stimu lates the apoptotic pathway.
Chick sternal chondrocytes are a well known model for the study of chondrocyte maturation because beneath typical development chondrocytes from the cephalic portion with the sternum undergo hypertrophy followed by minerali zation and bone formation, whereas the caudal portion remains as cartilage. In this study we investigate the roles of ERK1 2, p38 and two upstream pathways, protein kinase C and PI3 kinase, within the maturation of chick prehypertrophic sternal chondrocytes induced by BMP two and ascorbate.
Nonetheless, at the present time there’s insufficient proof for the viral hypothesis in Hashimotos thyroiditis. Microorganisms causing chronic inflammation have grow to be increasingly investigated as you possibly can cancer initiators promoters. There has been very little consideration on the prospective role of infectious method inside the pathogenesis of thyroid cancer, though inflammation has been implicated in the development of thyroid cancer. Herpes simplex virus variety 2 was discovered to be substantially connected with papillary thyroid cancer plus the presence of lymph node metastases. In addition, human parvovirus B19 has been regularly present in thyroid tissues of Hashimotos thyroiditis and papillary thyroid cancer. While the thyroid gland is amongst the CMV reservoirs, no previous study has examined the presence of CMV in thyroid cancer.
The findings of this selleckchem P22077 study suggest that CMV infection is unlikely to be linked with papillary thyroid cancer. CMV infection normally exhibits an altered pattern of IE protein expression. Such proteins act through very sophisticated mechanisms to facilitate viral production and to avoid detection and elimination with the virus by the immune system. Interestingly, BRAF activation is involved within the expression of CMV IE antigen. Sorafenib is often a tyrosine kinase inhibitor being utilized in advanced iodine refractory thyroid cancer and is identified to inhibit BRAF kinase phosphorylation within the MAPK pathway, of note would be the reality that sorafenib also inhibits CMV replication. In our study, about 78% of papillary thyroid cancer harbored the BRAF mutation.
Bigger tumor size, extrathyroidal invasion, lymph node metastasis, and more advanced TNM stage had been linked with all the BRAF selelck kinase inhibitor mutation. This is in maintaining with all the knowledge of other individuals. The prevalence of your BRAF mutation in papillary thyroid cancer varies from 32% to 90% within the literature, depending on detection procedures and histopathological subtypes. Provided that the majority of our individuals had classic subtype of papillary thyroid cancer, our constructive price of BRAF mutation was compatible to that reported by other endocrine surgery centers. We recognize a few of the limitations of our study. The amount of patients studied was smaller, and final results obtained from our selected population may not be extrapolated to other populations. Moreover, we did not investigate CMV serological status among our individuals.
A prior study has shown that CMV DNA may be extensively distributed in organs of both seropositive and seronegative healthier folks. For that reason, we straight assayed the presence of your CMV DNA and protein inside the thyroid gland with out serological tests. Conclusions CMV DNA and protein weren’t detected in fresh frozen tissues of follicular adenoma and papillary thyroid cancer, irrespective of the presence or absence of BRAF mutation.
The antibiotic concentration in serum was then applied to assess numerous PK and PD parameters. PK parameters assessed had been Cmax, defined as the peak plasma concentration of a drug following adminis tration of a dose, Cmin, defined as the lowest concentration that a drug reaches ahead of the following dose is administered, location below the concentration curve 0 six, an integral of the concentration time curve measured in ug. ml?1. h?1, t1 two, defined because the biological half life, which can be the time necessary for the concentration of your drug to attain half of its original value measured in hours, and ke, defined as the elimination rate constant which is the rate at which drugs are removed in the physique measured in per hour.
Among the PD parameters assessed were the AUC MIC ratio, which takes each the antimicrobial concentration and time into account for predicting outcomes of concentration independent anti biotics, T MIC, defined because the time period during which the serum antibiotic selleck chemicals concentration remains above the MIC level measured in hours, Cmax MIC may be the ratio of maximum achievable concentration of the drug in serum to MIC. Protein binding in serum We have assumed that unbound or cost-free drug equili brates with all the extravascular space and that the total concentration of antibiotic in any offered space is a com bination of your free and protein bound drug has been thought of for binding of protein in serum. Moreover the actual levels of absolutely free drug adjustments incredibly small with al terations in binding to serum proteins of as a great deal as 80% or 90%. Hence the total concentration of antibiotic in serum has been estimated for studying the in vivo ef ficacy of your therapy.
Survival price study Determination of the efficacy of mixture antibiotic therapy against pneumococcal pneumonia was initial established in survival price research. Groups of 12 mice were inoculated intranasaly with S. pneumoniae as de scribed above. Treatments with AMP at 200 mg kg physique weight and AZM at 50 mg selleckchem kg body weight either alone or in combination by intravenous route have been initiated 18 hours post infection. Control mice received sterile saline. Survival rate was recorded every 24 hour until day three p. i. Therapy regimens 18 hours after bacterial inoculation, groups of mice had been treated using a single intravenous dose of either AMP or AZM only as monotherapy or administered both as mixture therapy within a 0. 1 mL volume, and sacrificed for sample collection in the previously stated time point, beginning at 18th hours and continuing till 24th h with an interval of 1 h in in between two successive sam pling point.
The PI3K AKT pathway, the PLC? 1 pathway plus the MAPK cascades are downstream targets of the CaSR. In our study, calcium treatment resulted inside a clearly enhanced activity of AKT PKB and PLC? 1 in bone metastasizing cells but not in non metastasizing cells. Also, in bone me tastasizing cells, calcium had an activating impact on the MAP kinases p38 and JNK. The focal adhesion adapter protein paxillin also as c Jun, each downstream targets of JNK, showed comparable activity patterns. Inhi biting CaSR with NPS 2143 these enhancements had been pre vented as well as the phosphorylation in the signal mediator together with the highest calcium sensitivity, AKT, was decreased. The more reduction of AKT activity soon after inhibition of CaSR indicates a basement activity of CaSR even without the need of adding calcium.
The culture medium includes a low amount of calcium not specified by the company. Presumably this low calcium concentration leads to a slightly activation of CaSR and consequently also of AKT phosphorylation. This impact appears to become inhibited selelck kinase inhibitor by NPS 2143. The reduced AKT activity induced by NPS 2143 therapy confirms the responsibility of CaSR for the calcium dependent effects. In contrast, calcium had no activating impact on ERK. This suggests AKT, PLC? 1, p38 and JNK paxillin signaling path ways, that are described as downstream targets of CaSR, getting the crucial pathways in the CaSR signaling in RCC cells promoting bone distinct metastasis. Even so, ERK as a downstream target of CaSR is discussed controversially and a few research hypothesize the ERK pathway getting in volved in extracellular calcium induced cell migration, once more confirming a cell sort distinct function of CaSR as currently described.
The key regulator in the AKT pathway could be the tumor selleckchem PD-183805 suppressor PTEN. As an antagonist on the PI3Kinase, PTEN inhibits the activa tion of AKT and thereby down regulates cell prolifera tion and migration. Moreover, in our former investigations we established a correlation among low PTEN expression in specimens of RCC sufferers and poor prognosis brought on by metastasis. In bone me tastasizing RCC cells, PTEN expression was approxi mately 50% lower than in non metastasizing cells. The expression of PTEN correlated inversely with the activ ity of AKT. Moreover, the expression of PTEN was very calcium sensitive. Calcium remedy resulted in an just about full decline inside the expression of PTEN. This implicates that the per se low PTEN expression in bone metastasizing RCC cells is further lowered by the bone microenvironment, consequently activating the AKT signaling pathway and advertising bone metastasis. Our study indicates that bone metastasis of RCC is promoted by an enhanced expression of CaSR.
RNA extraction and quantification Total RNA was extracted working with Trizol based on the manufac turers directions. Reverse transcription was performed employing One particular Step PrimeScriptmiRNA cDNA Synthesis Kit. True time PCR was performed applying SYBRPremix Ex TaqTM II with an iCyclerthermal cycler. U6 RNA was applied as a miRNA internal control. The primers of miR 92b was as follows, Colony formation assay U251 and U87 cells had been transfected with miR 92b mimics, a manage oligonucleotide and a miR 92b inhibitor. Immediately after the transfection, the U251 and U87 cells have been counted and seeded in 12 well plates at a density of 50 and 60 cells per properly, respectively. The culture medium was replaced just about every 3 days. The number of colonies was counted around the sixth day just after seeding.
The price of colony formation extra resources was calculated using the following equation, colony formation price ? 100%. MTT assays The MTT assay was employed to decide cell viability. Each of the cells had been seeded into 96 properly culture plates in standard development medium. The cells transfected with miR 92b mimics. control and inhibitors have been grown for four days. One particular plate was developed immediately soon after the medium transform as well as other plates were developed each and every 24 hours for four days. Assays were initiated by adding 20 L of MTT substrate to every well and incubating the cells for an further three hours. Ultimately, the medium was removed and 200 L DMSO was added to every nicely. The absorbance was measured at 492 nm applying an Automated Microplate Reader. RT PCR Analysis was employed to establish the relative expression levels of miRNAs.
Total RNA was isolated making use of TRI ZOLTM reagent according to the suppliers instruction. Reverse transcrip tion was carried out utilizing One Step PrimeScript miRNA cDNA Synthesis Kit. Genuine time PCR was performed working with SYBR Green Supermix with an iCyclerthermal cycler. Primers of all genes have been in Supple mentary. The find more information data had been collected and analyzed applying the comparative Ct system using GADPH as the reference gene. MicroRNA target prediction The target genes of miR 92b were predicted by the fol lowing computer aided algorithms, TargetScan Human Release six. two. Luciferase assay The three UTR of human DKK3, containing the putative target web sites for miR 92b, was amplified by PCR. The wild type and mutant inserts were transfected in to the PGL3 promoter vector. Dual Luciferase reporter assays were performed according to the companies directions as previously described.
Flow cytometric analysis of apoptosis Cells were plated in six properly plates in antibiotic totally free medium and transfected with manage oligonucleotide or inhibitor working with Lipofectamine 2000 based on the makers recommendation. Luciferase and renilla signals were measured 48 h immediately after transfection using the Annexin V FITC apoptosis detection kit as described by the manufac turers directions.
Of them, the TYST mainly occurs in neo nates and infants, unique from adolescences or adults who composed of various germ cells and obtaining own biological characters. The TYST is still a extremely malignant neoplasm with poor prognosis, elevated resistance to chemotherapy, recurrence right after initial chemotherapy or surgery, and the unwanted effects of chemotherapeutics, even though the survival price of patients with TYST was enhanced following surgical resection or platinum based combination che motherapy, e. g. cisplatin, etoposide and bleomycin. The regulation of cell differentiation from immature malignant tumor cells to mature was suggested as a possible therapy for tumors. Conventional radio therapy and or chemotherapy have been discovered to suppress the bone marrow and immune function via influen cing cell phenotypes.
The cell apoptosis is closely related with all the tumorigeness, tumor development and insensitivity of chemotherapy radiation therapy. You will find limited MP-470 research on human TYSTs, even though YST has been studies in cells from male murine embry onal carcinoma in vitro and ovarian YST cell lines. The present studies aimed at establishing the animal model of TYST plus the human TYST cell line and eval uating the traits with the disease and bio function of human TYST cells. The present study evaluated the role of ATRA as an inducer of differentiation within a vari ety of tumor cells in the growth TYST cell lines in vitro and explored the molecular mechanism of TYST cell proliferation. Effects of cisplatin on TYST cell apoptosis and the expression of P53 and Bcl two genes had been further much more investigated.
Supplies selleck and strategies TYST and sampling TYST tissues have been sampled kids with TYST, aging about two three years old, through the testicular surgery, with out any radiotherapy or chemotherapy. The study proto col and informed consent of the sampling for scientific research had been authorized by The Ethical Committee of Clinical Investigation of Second Affiliated Hospital of Wenz hou Medical College. Informed written consents were approved from guardians around the behalf of the kids participants involved inside the study. Tumors with dia meters about 50 70 mm and without the encapsulation were severely adhered together with the surrounding tissues. Plasma levels of alpha fetoprotein had been above 1200 ng ml, corresponded with all the typical reference worth of 0 7 ng ml. TYST samples have been good in immunohisto chemical staining against cytokeratins and alpha fetoprotein. A xenograft tumor model Male BALB C mice using the autosomal recessive nude gene in homozygous, aging four weeks, have been purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences.
Even though infection with both of these strains appears to possess related clinical manifesta tions in humans along with other mammals, many reviews recommend that influenza brought about by H3N2 viruses is usually much more significant than that brought on by H1N1 subtype, The IVA genomes consist of eight single stranded RNA segments of negative polarity that encode as much as 11 pro teins, These RNA segments are associated with all the NP along with the RNA dependent RNA polymerase, which comprises 3 polymerase subunits to type viral ribonucleoprotein complexes, repre senting the minimum set of infectious viral structures. Influenza viruses pursue a nuclear replication system.
so, the RNPs must be exported from the nucleus for the cytoplasm to get enveloped Tosedostat ic50 with other viral proteins with the cell membrane, The cellular response to growth elements, inflammatory cytokines, and other mitogens is usually mediated by recep tors that are either G protein linked or intrinsic protein tyrosine kinases, The binding of ligand to receptor transmits a signal to one particular or more cascades of serine thre onine kinases that utilize sequential phosphorylation to transmit and amplify the signal, These kinase cas cades are collectively often known as mitogen activated protein kinase signaling cascades, The Raf MEK ERK pathway represents among the top characterized MAPK signaling pathways. MAPK cascades are essential regula tors of cellular responses for example proliferation, differenti ation, and apoptosis, Quite a few unfavorable strand RNA viruses induce cellular signaling via MAPK cascades, Infection with IVAs or IVBs upregulates the Raf MEK ERK cascade to assistance virus replication inside of the infected host cells, This signal cascade, that is activated late for the duration of influenza infection, is important for effective export of nuclear RNPs.
MEK inhibition continues to be proven to impair the nuclear RNP export and minimizes virus yields, Recently, we demonstrated that HA accumulation with the cell membrane and its tight association with lipid raft domains trigger virus induced ERK activation, present ing a significant part of HA like a viral inducer of MAPK signaling. While GSK1838705A HA appears to become significant, we are able to not exclude the involvement of other viral proteins or processes in activating MAPK signaling. In this examine, we examined the activation levels of MAPK signaling induced by two currently circulating human strains. A Hong Kong 218847 06 plus a Hong Kong 218449 06, These viruses were isolated from two diverse individuals in Hong Kong in 2006. We observed that the H3N2 strain replicates extra efficiently in tissue culture than does the H1N1 as well as induced larger amounts of ERK phosphorylation.
Phosphorylated Akt and ERK1 2 can be detected in RV contaminated cells from 48 hours p. i, and band intensity increased from 48 96 hrs p. i. compared to total ranges, Phosphorylated Akt and ERK2 had been detected from the mock contaminated cells at 96 hours p. i. but not just before, whereas complete amounts of Akt and ERK one two had been detectable in any respect time points, Therapy of RV contaminated cells with PI3K inhibitor LY294002 and MEK1 two inhibitor U0126 entirely inhibited activation of Akt and ERK1 2 respectively, The phosphorylation of Akt and ERK and their down stream targets p70S6K, GSK 3, c myc and Poor were also examined by Western blotting among 12 96 hrs p. i, Phosphorylated Akt and ERK1 2 had been detectable in RV contaminated cells at 48 and 36 hrs p. i. respectively.
p70S6K is phosphorylated by FRAP mTOR downstream of Akt at Thr389 and at Thr421 Ser42, downstream from the Ras Raf MEK ERK pathway. Phosphorylation p53 inhibitor at Thr389 was observed at 12, 24, 60, 84 and 96 hours p. i, Phosphorylation of the Thr421 Ser42 internet site was observed in any way time factors, though increases in band intensity could be seen at twelve, 24, 60, 84 and 96 hours p. i, mirroring the phosphorylation at Thr389. Phosphorylation of Thr421 Ser424 but not Thr389 was observed within the mock infected cells, albeit at a reduced level than in RV infected cells. The phosphorylation of GSK 3, downstream of Akt, greater from 12 and 96 hrs p. i. and was related to that of Akt. Phosphorylation of Poor, a further substrate for Akt, nevertheless, could not be detected in RV infected or mock contaminated cells.
The phosphorylation of c myc, a tran scription factor activated by ERK1 2 phosphorylation, decreased in between twelve and 96 hours p. i, in contrast to your phosphorylation profile of ERK1 2. GSK three and c myc had been also detectable selleck chemical while in the mock contaminated cells at 96 hours p. i. The effects of LY294002 and U0126 on cell viability in RV contaminated cells RV induces apoptosis in RK13 cells with characteristic morphological and biochemical characteristics, The XTT assay was applied to examine the effect of RV infection and LY29002 and U0126 treatment on cellular metabolic process above time. XTT is actually a tetrazolium salt, that’s cleaved through the succinate dehydrogenase system of mitochondria in To assess the part of PI3K dependent signaling for the duration of RV infection, the results of PI3K inhibitor LY294002 about the advancement of RV induced apoptosis were exam ined, 12 96 hrs p.
i, by caspase activity assay, trypan blue exclusion staining, DNA fragmentation and light microscopy, RV induced apoptotic signaling has been reported to take place amongst twelve 24 hours p. i, with peak caspase exercise occurring around 72 hrs p. i. at a multiplicity of infection of 3 PFU cell, Fig. 3A exhibits that which has a MOI of 4 PFU cell the peak of RV induced caspase action takes place earlier at 60 hrs p.
BEAS 2B cells have been exposed to AgNPs for 4 and 24 h. No considerable toxicity was observed following 4 h for almost any of your AgNPs, However, major tox icity was observed after 24 h to the 10 nm citrate coated as well as the ten nm PVP coated AgNPs in the highest dose, None in the lar ger sized AgNPs altered the cell viability, Some AgNPs happen to be proven to interact with all the LDH assay via enzyme inhibition or binding, To investigate this challenge we incubated AgNPs with cell ly sates and detected the LDH exercise after 0, four and 24 h, The reduction in enzyme activity was most pronounced to the ten nm AgNPs, primarily for that citrate coated particles, and occurred in a time and dose dependent method.
The the original source enzyme inhibition is most likely correlated together with the Ag release since Ag ions are proven to inhibit the catalytic activity of LDH enzyme, As a result, LDH outcomes really should be interpreted with caution plus the likelihood of false nega tive effects be viewed as, specially for particles with reduced stability that release Ag ions. AgNPs induce DNA harm in human lung cells The potential of AgNPs to induce DNA harm was in vestigated with two distinct assays. alkaline comet assay that provides indication to the total DNA harm and H2AX foci for mation, which is mostly a marker of DNA double strand breaks. The alkaline comet assay was utilized to find out the DNA injury connected with exposure to non cytotoxic concentrations of AgNPs in BEAS 2B cells.
No sizeable enhance during the percentage of DNA in the comet tail was observed immediately after four h publicity to any from the AgNPs, On the other hand, a statistically substantial boost in total DNA harm was observed straight from the source immediately after 24 h for all AgNPs, independent of size and coating, The H2AX foci formation was assessed by immuno cytochemistry in BEAS 2B cells beneath the same condi tions as for that comet assay, i. e. four and 24 h publicity to ten ug mL AgNPs. All fluorescent stainings have been unfavorable for H2AX the two following 4 h and 24 h. Fluorescence pictures are proven in Figure 3C for two on the investigated particles, ten nm and 75 nm citrate coated AgNPs. Etoposide, a topoisomerase inhibitor, was utilized as being a optimistic handle. The outcomes show that none in the AgNPs induced DNA double strand breaks in BEAS 2B cells underneath given check situations. No cellular ROS improve on exposure to AgNPs The kinetics of intracellular ROS formation after expos ure of BEAS 2B cells to AgNPs was measured using the dichlorodihydrofluorescein diacetate assay. The DCFH DA probe can detect cytosolic radicals such as hydroxyl, peroxyl, alkoxyl, nitrate and carbonate, but will not be capable to pass organelle membranes, None with the AgNPs induced any considerable ROS enhance soon after 24 h, at doses as much as 20 ug mL, The positive management, tert butyl hydroperoxide, induced a 2.
On the other hand, the web page of action of KCNQ channel openers just isn’t absolutely clarified hence far. On this research, i. c. v. injection of XE 991 didn’t impact the analgesic impact of retigabine, regardless of precisely the same dose stopping retigabine mediated inhibition of electro convulsion, sup pression of exploratory habits, and rotarod efficiency. These effects propose that KCNQ channel opening from the brain may perhaps play a negligible part from the growth of retigabine induced analgesia in inflammatory soreness. More much more, we demonstrated that intraplantar administration of retigabine or ICA 27243 suppresses formalin induced lick ing habits. Whereas the contribution of KCNQ channels during the spinal cord can not be ruled out, these findings sug gest opening of KCNQ channels in only the peripheral nerves creates sufficient analgesia.
Additionally, its re ported that intraplantar injection of retigabine selleck chemicals NVP-BKM120 attenuated bradykinin induced nociceptive habits and that topical injection on the KCNQ channel opener, flupirtine, in to the sciatic nerve substantially reversed thermal hyper algesia inside a rat neuropathic ache model induced by partial sciatic nerve ligation, These reports recommend that per ipheral KCNQ channels contribute to ache pathway. Extra more than, retigabine continues to be reported to cut back sensitivity to noxious heat of nociceptive A delta fibers making use of isolated skin nerve planning as well as the excitability of periph eral human C fibers, On top of that, its reported that intraplantar injection of XE 991 induced nociceptive behav ior and improved the responses of a delta fibers to noxious heat during the electrophysiological examine, These re ports assistance our hypothesis that KCNQ channel opening inside of the peripheral nerves controls hyperalgesia.
In con trast, Xu et al. reported that i. c. v. injection of retigabine alleviated CFA induced pan JAK inhibitor inflammatory ache inside the temporo mandibular joint, suggesting the involvement of brain KCNQ channels in retigabine mediated analgesia. The dis crepancies involving their and our soreness designs are unclear. The examine of Xu et al. evaluated mechanical allodynia utilizing von Frey filaments whereas our research evaluated thermal hyperalgesia.
The main difference in pain modality might make clear the discrepancy because thermal hyperalgesia through in flammation is regarded as to end result from up regulation and or sensitization with the heat activated channels, transient re ceptor prospective channel, subfamily vanilloid mem ber one and TRPV2, in peripheral nerves, Intraplantar injection of formalin in rats can also be acknowledged to induce two distinct phases of discharges from spinal dorsal horn neurons, which modulate nociceptive habits and are inhibited by sciatic nerve block, From the CFA induced inflammatory pain model, inflammatory mediators directly and or indirectly activate non selective cation channels in a fiber and or C fibers of main sensory nerves and as a result depolarize neurons, The depolarization induces action potentials and triggers pain.