Discussion Ion regulation by the rectum helps mosquito larvae to survive, and adapt to, a constantly changing environment. A wealth of literature has focused on the structure and function of the larval culicine rectum and the distinction between its role in freshwater and saline tolerant species . However, the discovery that the anopheline rectum is structurally distinct from any culicine species described suggested that anophelines may utilize a unique method of ion regulation. To test this hypothesis, we compared the localization patterns of three proteins involved in ion regulation in the recta of anopheline and culicine larvae reared in fresh versus saline water. Three key points regarding the comparison of anopheline and culicine recta emerge from these data: In contrast to obligate freshwater and saline tolerant culicines which have structurally distinct recta, all anophelines examined possess a similarly structured rectum, consisting of DAR and non DAR cells .
Anopheline larvae undergo a dramatic shift in rectal Na K ATPase localization when reared in NVP-BGJ398 freshwater versus saline water. This shift is not seen in any culicine larvae examined. With the exception of Ae. aegypti, CA9 consistently localized to an anterior region of the rectum in both culicine and anopheline larvae regardless of the salinity of the rearing water. The first two key points, along with the localization patterns of Na K ATPase and V ATPase , will be used to suggest putative functions for the anopheline rectal regions. Localization of CA9 will be discussed in a separate section. Figs 4 and 5 summarize our results; Fig. 4 illustrates the differences between anopheline and culicine recta and Fig. 5 summarizes protein localization in anophelines and culicines in a schematic form. When reared in freshwater, the protein localization patterns in An. gambiae and An. albimanus recta were identical: non DAR cells contained an apical V ATPase and basal Na K ATPase whereas the DAR cells were enriched in cytoplasmic V ATPase.
The protein localization pattern of the non DAR cells in An. gambiae and An. albimanus was similar to that price Sodium valproate selleck of the freshwater culicine, Ae. aegypti, which also expressed an apical V ATPase and basal Na K ATPase in its non segmented rectum. The detection of V ATPase in Ae. aegypti larval rectum confirms the finding of Filippova et al. but conflicts with the findings of Patrick et al. who detected V ATPase mRNA in the rectum, but did not detect protein when using the same antibody against the B subunit which was used in this study. However, ultrastructural studies of the larval Ae. aegypti rectum identified a particulate coat on the apical lamellae suggesting the presence of portasomes which correspond to the V1 portion of the V ATPase .

The 50 kDa subunit is glycosylated and could be a previously undescribed P type ATPase subunit. Although the available sequence evidence is not conclusive, its N terminal sequence does not correspond to any previously reported subunit . As shown in Fig. 3, the distribution of the Na and Na K ATPase differs through distinct guinea pig kidney segments. Both enzymes are well expressed in the outer cortex, but Na ATPase expression is lower in the inner regions of the kidney and absent in the medulla. In the intestine, the Na ATPase is mainly expressed in villous and surface cells. In the crypt region , the enzyme seems to have an intracellular distribution. This particular renal and intestinal distribution probably has to do with the physiological role of this enzyme in sodium transport in these epithelia. Furthermore, IgY polyclonal antibodies raised against the purified Na and Na K ATPases differentially recognize these enzymes. Antibodies raised against the purified Na ATPase inhibit the Mg2 dependent ouabain insensitive Na stimulated ATPase activity without effect on the Na K ATPase, while antibodies raised against the purified Na K ATPase inhibit this enzyme without effect on the Na ATPase .
Na ATPase forms a phosphorylated intermediate The Na ATPase can be classified among the P type ATPases. Its Mg2 dependence, sensitivity to vanadate and capacity to Nutlin-3 selleck form a phosphorylated intermediate from ATP or Pi are the strongest pieces of evidence for this classification. It can be phosphorylated from inorganic phosphate in an ion sensitive reaction stabilized by furosemide . In that article, a phosphorylated polypeptide of about 100 kDa was identified for the first time as directly associated with the Na ATPase. In 2005, del Castillo et al. reported a phosphorylated intermediate obtained from ATP associated with the purified Na ATPase. The phosphorylation was Mg2 dependent, vanadate sensitive and stimulated by Na with two different Km values . The stimulatory effect was specific for Na and independent of anions. Phosphorylation was insensitive to ouabain but stimulated by furosemide with an EC50 of 1.8 0.54 mM. In addition, 0.
5 mM ADP partially inhibited it. Phosphorylation was also sensitive to alkaline pH and hydroxylamine, suggesting an acyl phosphate bond associated with the 100 kDa polypeptide of the enzyme. A minimum reaction cycle for the Na ATPase was proposed in which the enzyme has an E1 form that can be phosphorylated from ATP in the presence of Mg2 and Na , producing the E1.P.Na form, sensitive to ADP. Furosemide stabilizes the E1.P.Na Tenofovir form. The enzyme then changes to the E2.P.Na form, insensitive to ADP, which is susceptible to dephosphorylation. A conformational change induces Na translocation through the membrane.

In spite of their smaller cell size, the input resistance of FS interneurons is reduce than that of PYR cells as previously reported .The larger density and degree of restingNa K ATPase activity could play a function from the upkeep of a much more hyperpolarized resting membrane probable and servicing of higher frequency firing in FS cells in such ?leaky? neurons. Below ordinary resting ailments only a portion of your complete membrane bound Na K ATPase is phosphorylated and on the market to contribute for the measured adjust in membrane voltage or latest when the Na K ATPase is pharmacologically blocked. By raising inner Na , both right or indirectly , we have been capable to assess each neuron?s responsiveness to Na and their capability to activate the Na K ATPase. The outcome was greater activation of Na K ATPase dependent currents in FS interneurons and PYR1 cells than in PYR2 neurons.
During the glutamate puff experiments it had been possible to review the resting Na K ATPase exercise, measured since the change in holding latest through the first Na K ATPase blockade, mtorc2 inhibitor with all the increased Na K ATPase dependent present, measured because the component of charge induced from the glutamate puff that was sensitive to Na K ATPase block by DHO . Within this way, the romance between resting Na K ATPase exercise and complete Na K ATPase exercise activated by a Na load could be established. FS and PYR1 neurons have each higher resting Na K ATPase activity and higher ability to raise Na K ATPase exercise, making it possible for them to accommodate awider variety ofNa loadswith increases in Na K ATPase action. The subgroups of PYR neurons vary in Na K ATPase exercise but not intrinsic properties It is actually clear from the experimental data that two distinct groups of PYR neurons with varying Na K ATPase activity exist in layer V cortex; on the other hand, we now have been not able to detect any correlations in between resting Na K ATPase activity and any measured electrophysiological property.
Responses fromboth PYR groups were observed around the similar day, from your similar animal and using the similar stock of Na K ATPase antagonists. Since the Na K ATPase antagonists, especially ouabain, are difficult to wash out, we recorded from only one neuron per slice to get specific that residual Na K ATPase blockade was not contributing to our results. The presence of two distinct groups without any gradation within the distribution helped rule out probable artifacts Selumetinib solubility selleck chemicals such as depth of recording in slice, slice well being and or drug penetration. Our data strongly suggest that the differences in recorded Na K ATPase activity relate to distinctions in cell expression of Na K ATPase and never to artifacts of recording conditions, slice planning or other intrinsic properties of your recorded PYR neurons.

To our shock, 53 D, 155 D and 175 D showed stronger PP2A binding than the full Na ,K loop or 238 D and 272 D. These data suggest the likelihood there is a region in between amino acids 1 and 175 within the Na ,K loop that inhibits PP2A binding. Alternatively, the conformation within the Na ,K loop might regulate its association with PP2A and specified deletions might possibly not conserve the requisite conformation. Association in the PP2A A subunit with the Na ,K ATPase The PP2A A subunit was not pulled down together with the substantial cytoplasmic loop of your Na ,K ATPase whereas the Asubunit was immunoprecipitated together with the total length Na ,K ATPase . Its doable that there is a different website inside the Na ,K ATPase sequence that associates with PP2A A subunit, considering that many PP2A substrates are uncovered to bind to PP2A by way of the A subunit . We utilized a GST fusion protein incorporating the A domain on the Na ,K ATPase a subunit, that is composed within the N terminus plus a smaller cytoplasmic loop connecting transmembrane segments 2 and three, to check its capability to pull down the PP2A A subunit .
The Adomain includes the PKC phosphorylation web-site and in addition T0070907 selleck may well be phosphorylated by G protein coupled receptor kinases . We identified the A domain pulled down the PP2A A subunit. Consistent using the information presented in Fig. 4C, the massive cytoplasmic loop of your Na ,K ATPase didn’t precipitate the PP2A A subunit. Impact of PP2A on interaction of arrestin 2 with all the Na ,K ATPase Arrestin and GRK are leading regulators of GPCR trafficking and signaling. We now have found that Na ,K ATPase trafficking can be regulated by arrestin and GRK, with each other with spinophilin . Seeing that the association in between arrestin and GPCRs depends upon the phosphorylation of GPCRs by GRKs, PP2A might conceivably regulate Na ,K ATPase function, at the least in aspect, by means of inhibition of GRK phosphorylation and arrestin binding. To begin to test this hypothesis, we examined the result with the PP2A C subunit around the association from the Na ,K ATPase with arrestin . Fig.
six exhibits Western blot patterns of transfected COS cell lysates subjected to immunoprecipitation using the HK9 antibody after which detected using the anti flag Acetanilide antibody, which recognizes arrestin two. Arrestin 2 was co immunoprecipitated by the HK9 antibody when it was co expressed with H85N. Coexpression from the PP2A C subunit absolutely inhibited the interaction involving arrestin two as well as the H85N a subunit. Consequently, the PP2A C subunit seems to impede arrestin binding to the Na,K ATPase a subunit. This effect could be attributable to your catalytic action of your PP2A C subunit, by way of the dephosphorylation of phosphoresidues that may be very important for that arrestin interaction.

AURKA binds and phosphorylates the breast cancer associated gene products, BRAC1, in vitro and in vivo to manage its perform . It is reported that ovarian and breast epithelial carcinomas play a part during the regulation of human telomerase reverse transcriptase mRNA ranges by means of c Myc . AURKA has also been reported to override the spindle checkpoint activated by paclitaxel and nocodazole . These defects may possibly contribute to transformation. AURKA interacts with all the p53 pathway at various amounts, suggesting that these proteins type a component of an integrated practical network. AURKA interferes with p53 suppressor perform by at the least two mechanisms: it directly phosphorylates p53 at Ser315 facilitating MDM two mediated degradation of p53 in cancer cells , and furthermore, it phosphorylates p53 at Ser215 to inactivate its transcriptional activity . Moreover to these two mechanisms, our get the job done suggests regulation of p53 by means of AKT MDM two axis in gastric cancer cells . We also reported that AURKA over expression suppresses TAp73 in p53 deficient cancer cells .
TAp73, a p53 family, member has significant homology with p53 and plays an important purpose in apoptosis Vandetanib kinase inhibitor induced by cytotoxic agents . The tumor suppressor proteins p53 and p73 can activate genetic packages that halt cell proliferation transiently or completely or remove the cell altogether . Regulation of p53 and p73 by AURKA above expression can cause suppression of apoptosis of tumor cells. AKT is a major pro proliferative serine threonine kinase that promotes cell survival in the number of cell forms and prevents apoptosis induced by many apoptotic stimuli . We , and other people , have reported that AURKA up regulates AKT phosphorylation at Ser473. We reported the regulation of GSK three? and ? catenin by AURKA over expression in gastric cancer cells. A schematic overview of attainable AURKA interaction is proven in Figure 3. AURKB AURKB regulates kinetochore microtubule attachment and ensures faithful chromosome segregation .
It can be in excess of expressed in numerous human tumors like breast, colorectal, kidney, lung, and prostrate . An greater degree of AURKB correlates with advanced phases of colorectal cancer . Its above expression results in multi nucleation and polyploidy in human cells ; yet, this phenotype is exacerbated in absence of p53 . It Tofacitinib has also been reported that AURKB over expression induces chromosomes lagging in metaphase, chromosome segregation error and mistakes in cytokinesis, and so, taking part in a purpose in carcinogenesis . AURKB does not transform cells alone but is reported to induce H Ras mediated transformation . AURKB over expression is reported to correlate using the level of genomic instability inside of a tumor indicating that it contributes to the acquisition of genetic alterations essential for neoplastic transformation.

Although the cytocidal likely of autophagy stays rather controversial, mitotic catastrophe seems to become an oncosuppressive mechanism that operates upstream within the molecular machinery for cell death and cell senescence. As we’ve got mentioned over, the huge bulk of clinically made use of and experimental anticancer regimens do the job by triggering the apoptotic demise of tumor cells, programmed necrosis and mitotic catastrophe becoming a great deal significantly less employed as therapeutic targets. However, because most, if not all, cancer cells exhibit or acquire improved resistance towards pro apop totic agents, the future of anticancer therapy also relies about the exploitation of non and pre apoptotic signaling cascades. The notion of programmed necrosis has gained consensus only a number of years ago, alongside the thought of circumventing apoptosis resistance by triggering necrosis. Mitotic catastrophe can result in the activation of three distinct oncosuppressive mechanisms, i.e apoptosis, necrosis and senescence, and cancer cells appear to become intrinsically far more delicate to succumb to this kind of death than their normal counterparts.
Therefore, programmed mdv 3100 selleck necrosis and mitotic catastrophe hold fantastic promises for anticancer therapy. It will eventually be honestly interesting to view how the latest awareness that has been generated about these oncosuppressive mechanisms will be translated right into a clinical actuality. While full remissions may occur in 70?90% of individuals with Ph ALL who receive intensive chemotherapy alone, most patients relapse and die inside 12 months of treatment4 . Allogeneic HSCT substantially improves long-term survival costs, and in the huge scale trial, the 5 12 months relapse free of charge survival charge within the preimatinib era was 57% in sufferers who underwent a sibling allogeneic HSCT, 66% in patients who underwent a matched unrelated donor allogeneic HSCT, and 44% in individuals who underwent an autologous HSCT, but the survival price in patients who obtained chemotherapy alone was 10%.
Even though the allogeneic HSCT group fared worse at first due to large charges of transplantation connected mortality, the PS-341 decrease relapse risk translated to a greater five yr eventfree survival charge along with a higher five 12 months general survival price in contrast with chemotherapy alone and autologous HSCT five . A few aspects influence the outcome of patients who undergo allogeneic HSCT. Patients who underwent allogeneic HSCT in very first CR had substantially much better outcomes than people who underwent allogeneic HSCT through second or later CR. Other favorable aspects contain younger age, total body irradiation conditioning, the usage of a human leukocyte antigen identical sibling donor, and also the occurrence of acute graft versus host disorder. Not long ago, an Italian group analyzed therapy outcomes in accordance to time period.

An alternative study showed that saikosaponins have direct involvement in p53 , NF ?B and Fas Fas ligand mediated induction of apoptosis and cell cycle arrest in human hepatoma cell lines. Saikosaponins also inhibited cell survival signaling by improving the amount of I?B? while in the cytoplasm and lowering the degree and activity of NF ?B within the nucleus, and subsequently attenuated the expression of bcl xL in HepG2 and Hep3B cells. Saikosaponins hence decreased cell proliferation and induced apoptosis both in p53 favourable HepG2 and p53 negative Hep3B cells . Diosgenin, a steroidal triterpenoid acquiring two pentacyclic rings, is existing in Trigonella foenum graecum as well as other plants and continues to be shown to suppress irritation, inhibit proliferation, and induce apoptosis within a selection of tumor cells. Diosgenin inhibits osteoclastogenesis, invasion, and proliferation by the downregulation of Akt, IKK activation, and NF ?B regulated gene expression. Diosgenin suppresses NF ?B by direct DNA binding, activation of IKK, I?B? phosphorylation, I?B? degradation, p65 phosphorylation, and p65 nuclear translocation by inhibiting Akt activation.
NF ?B dependent reporter gene expression was also abrogated by diosgenin . Equivalent action High Throughput Screening was located in Withania somnifera, also known as Indian ginseng, that’s extensively made use of from the Ayurvedic procedure of medication to treat tumors, irritation, arthritis, asthma, and hypertension. Chemical investigation from the roots and leaves of this plant has yielded bioactive withanolides, a group of C28 steroidal lactone triterpenoids. Withania somnifera inhibits COX enzymes, lipid peroxidation, and proliferation of tumor cells, and it potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis as a result of the suppression of NF ?B activation and NF ?B regulated gene expression . Ursolic acid is known as a pentacyclic triterpene compound isolated from lots of types of medicinal plants and broadly existing during the human diet plan. It’s been reported to possess a broad assortment of pharmacological properties and is one particular from the most promising chemopreventive agents for cancer. It has been proven to suppress the expression of many genes linked to tumorigenesis.
It suppressed NF ?B activation induced by a variety of carcinogens like TNF, PMA, okadaic acid, hydrogen peroxide , and cigarette smoke condensate. Ursolic acid inhibited DNA binding of NF ?B. Ursolic acid inhibited I?B? degradation, I?B? phosphorylation, IKK activation, p65 phosphorylation, p65 nuclear translocation, and NF ?B dependent reporter gene expression. Ursolic acid also inhibited NF ?B dependent reporter gene Proteasome inhibitor selleck expression activated by TNF receptor , TNFR associated death domain , TNFR linked issue , NF ?B inducing kinase , IKK, and p65 . CDDO and CDDO Me, two potent oleanane triterpenoids obtaining structures similar to ursolic acid, are now in Phase I clinical trials for your treatment method of leukemia and strong tumors.

PPAR? continues to be proven to regulate IDE expression levels in rat principal neurons.54 This, taken with each other using the latest data, suggests that 1 could possibly have some result on PPAR? leading to the upregulation in IDE protein ranges. Furthermore, it remains for being witnessed if compounds 1 and two have any have an impact on on routines of one or a lot more within the A associated targets studied right here. Within this context, the computational docking scientific studies that had been carried out indicate the occurrence of favorable interaction complexes amongst the pure products one and the energetic web pages of all 4 targets indicating its potential direct effect about the ranges as well as activities of those enzymes . In summary, it’s been shown for your first time that withanolide A and asiatic acid positively modulate various targets connected by using a pathways and as a result, may perhaps be helpful in attenuating A ranges while in the AD brain by the two decreasing A production and in addition by raising A degradation .
Selumetinib kinase inhibitor Therapies determined by modulating secretases will act locally to have an effect on A manufacturing, when therapies based on increasing A degradation may perhaps show very important in acting at web pages which might be broadly separated from the A manufacturing web pages.43 This sort of multi functional and multi degree activity inside a provided therapeutic molecule could demonstrate very helpful towards AD, delivering various mechanisms to alter amyloid pathology while in the AD brain. Eventually, together with the A associated pursuits established in the present study, each one and two have been shown to induce considerable regeneration of neurites and dendrites, which could possibly support in reconstructing neuronal networks damaged in AD.56,57 Therefore, these two purely natural products may possibly serve as lead compounds to the improvement of novel therapeutic molecules with disease modifying pursuits, that are urgently desired to tackle the unwell results of the remarkably complex, multi factorial disorder like AD. Experimental Area Check Compounds Withanolide A and asiatic acid have been obtained from Chromadex Incorporation . The purity of one and 2 was 99.3% and 93.
7%, respectively. Isolation and Culture of Key Rat Cortical Neurons Major cortical neurons were isolated from 1 day outdated Sprague Dawley rat pups and cultured ZD-1839 as described by Chandler et al.58 All procedures were performed in accordance to guidelines created through the Institutional Animal Care and Use Committee at Michigan State University. The cells have been plated on poly D lysine coated, twelve effectively plates at a concentration of one 106 cells per effectively in fresh cortical medium . The experiments had been carried out on three four day previous neuronal cultures. The cells have been treated with one and two at numerous doses for 24 h.

p110 action is important for your development or maintenance of tissue web page distinct mast cell population We previously reported that genetic inactivation of p110 leads to a reduction in mast cell numbers in certain tissues, this kind of because the dermis within the ear as well as the submucosal and muscularis layers within the stomach . Mast cell numbers in other tissues, this kind of as the dermis from the back along with the mucosa layer within the abdomen, were unaffected ; Fig. 1A . We’ve now also assessed the impact of p110? deletion on mast cell numbers and uncovered comparable mast cell numbers in ?KO and WT mice at all anatomical internet sites assessed, in line with previously published information on a more constrained set of tissues . Only the dermis on the back skin showed a small reduction of toluidine blue beneficial mast cells in p110?KO mice . These information present that p110 , as opposed to p110?, has an impact on mast cell differentiation, which need to be taken into account when interpreting studies making use of D910A mice.
Inactivation of p110? or p110 will not influence vascular responsiveness to proinflammatory stimuli Not long ago, proof has been presented for your presence of p110? and p110 in endothelial cells and vascular smooth muscle cells . Given that allergic responses in p110? and p110 mutant mice are actually assessed by leakage of Evans blue from the vessels , it’s not clear to what extent altered vascular responsiveness mtorc2 inhibitor of PI3K mutant mice might possibly have contributed for the observed lowered allergic responses in these mice. To achieve insight into this question, we tested the direct result of vasoactive compounds on vascular permeability in mutant mice, once more utilizing leakage of Evans blue dye to the surrounding tissue as a read out. Injection of histamine led to a robust maximize in vascular permeability that was comparable in all genotypes . Vascular permeability responses to mast cell extracts have been also equivalent in WT, ?KO, and D910A mice . Taken with each other, these information display an intact responsiveness with the vasculature to inflammatory stimuli on systemic inactivation of p110? or p110 .
Distinct roles for p110? and p110 in adenosine signaling in mast cells In line which has a former report , we locate that adenosine stimulated Tyrphostin 9 kinase inhibitor phosphorylation of Akt, a surrogate marker of PI3K exercise, is abrogated in ?KO BMMCs . In agreement with this observation, adenosine induced Akt PKB phosphorylation was very delicate to pharmacological inhibition of p110?, with an IC50 for AS 252424 of 85 nM, as compared with three.six M for the p110 inhibitor IC87114 . We upcoming assessed the in vivo impact of PI3K deficiency on adenosine stimulated mast celldependent vascular permeability.

Whilst PI3 Kinase can bind straight to your IL 1R, it has been shown usingODNoligonucleotides also as in IRAK1 deficient cell lines that IL 1 activation of PI3 K also will depend on IRAK1 and 2 suggesting its involvement while in the signaling modules . Whereas IRAK1 seems to interact straight with all the IL 1RAcP, IRAK two preferentially associates using the IL 1R . To our know-how no protein protein interaction among PI3 K and IRAK one 2 has become reported and TRAF six mediated PI3 K perform is thought for being indirect through its association together with the tyrosine kinase Src . In IRAK1 deficient mouse embryo fibroblasts, neither IL one nor LPS induced AKT phosphorylation or IL 6 gene induction, and reintroduction of IRAK 1 rendered these cells completely responsive. 4. Part of PI3 Kinase Signaling Downstream of IL 1R, TLRs, and TCR CostimulatoryMolecules The coordinated response of innate and adaptive immune cells collectively with intestinal epithelial cells to luminal commensal and pathogenic bacteria can result in dysregulation of homeostasis resulting in inflammatory bowel disorder. This section discusses what is recognized about PI3 K signaling downstream of these receptors inside the related cell forms. 4.one.
Position of PI3 K Signaling in Innate Immune Cells 4.1.one. Dendritic Cells. Dendritic cells are crucial mediators in the innate immune response while in the intestine. Dendritic cells possess a rich supply of pattern recognition receptors and therefore are existing inside Peyers Patch and through the entire lamina propria, making purchase Vorinostat selleckchem extensions in to the gut lumen to sample luminal antigens. Human intestinal lamina propria DCs express the MHC II marker HLA DR. This lineage is largely typical CD11c myeloid DCs . A lot of the information on intestinal dendritic cells come from mouse models. Not long ago, a couple of reports have appeared on human intestinal dendritic cells . Several scientific studies have demonstrated altered DC phenotype and function in inflammatory bowel disorder . DCs are far more activated and express enhanced amounts of thematurationmarkers and TLRs at the same time as making proinflammatory cytokines . Therapy of ulcerative colitis individuals with probiotics in blend with corticosteroids induced a even more favorable phenotype with DC making much less inflammatory cytokines and reduce TLR expression .
PI3 Ks are activated in DCs by countless stimuli, as well as LPS, CpG oligodeoxynucleotide, a lot of which induce IL twelve. In DCs, PI3 K inhibits p38 Map Kinase which is important CCI-779 for transcriptional activation of IL twelve. Interleukin twelve determines the balance among Th1 cellular mediated immunity and Th2 humoral, antibody mediated immunity . Elevated IL 12 will skew in the direction of a Th1 response . Dendritic cells are integral towards the differentiation of T helper cells into T helper sort one Th1, Th2, and Th17 subsets. Interleukin six plays an essential component in regulating these 3 arms on the immune response by limiting Th1 response and marketing Th2 and Th17 responses.