BH3 domains possess 4 conserved hydrophobic residues 3 to 4 residues apart, which task into 4 hydrophobic pockets inside the ligand binding groove within the anti apoptotic proteins. On top of that, a conserved aspartic acid forms a salt bridge to a conserved arginine for the Bcl two household proteins. Mutation of any of those residues drastically lowers the affinity of BH3 proteins to their targets, compromising their pro apoptotic action. Unique models, which includes the direct activation model as well as the derepression model, are already proposed in regards to the mechanism of apoptosis regulation through the PPIs amongst the Bcl 2 members of the family. The typical attribute of those designs is that the PPIs amongst the various lessons within the Bcl two household happen via the BH3 ligand domain of professional apoptotic proteins which bind to a receptor BH3 binding groove formed by BH1 three regions over the anti apoptotic proteins. This rational was successfully made use of for development of new anti cancer therapies, during which peptidomimetics or SMIs bind in the BH3 binding groove of Bcl two proteins and behave as BH3 mimetics.
This kind of compounds hold guarantee for that advancement of new anticancer therapies. 2. Approaches for targeting the anti apoptotic Bcl 2 protein family 2. one Focusing on the protein expression level One of the methods to target the anti apoptotic proteins will be to inhibit selelck kinase inhibitor their expression degree with antisense oligonucleotides. Oblimersen sodium, a Bcl 2 particular antisense phosphorothioate oligodeoxynucleotide built to get complementary towards the 1st six codons within the human Bcl two mRNA sequence, has become evaluated being a single agent or in blend treatment in quite a few clinical scientific studies. Its clinical effect is modest and it’s not clear how correctly this kind of an antisense oligonucleotide can down regulate the Bcl two ranges in cells in vivo. 2. two Targeting the BH3 binding groove The binding profiles of different BH3 peptides and their x ray crystal structures complexed with anti apoptotic Bcl two proteins deliver valuable material for knowing the PPIs of Bcl two protein family.
For this reason, the 2nd approach that is certainly extensively studied is targeting the BH3 binding groove with BH3 mimetics therefore antagonizing the perform of Bcl 2. Peptide primarily based inhibitors have shown to attenuate Bcl two exercise, but the going here utilization of peptides being a therapeutic tactic is hindered by their lack of stability and effective delivery. To conquer this situation, a chemical strategy continues to be pursued to sustain the helical construction by introducing a chemical staple. Other approaches include things like the style and design of peptidomimetics dependant on helix mimicry towards the BH3 only proteins including compound one, a terphenyl based Bak BH3 helical peptidomimetic. SMIs, having said that, have considerable rewards over peptides and within the final decade, this kind of compounds have emerged as productive and particular inhibitors of Bcl 2 protein family.

Introduction Quite a few in vivo and in vitro scientific studies have demonstrated that human stem cells as interesting vehicles to provide the preferred gene products, building them a very promising agent in brain tumor treatment. Recently, neural stem/progenitor cells, engineered to express or carry to therapeutic agents, are recognized for his or her capability to migrate throughout the central nervous method. 1,two Cytolytic viruses and genes coding for anti tumor cytokines, professional drug converting enzymes and a variety of neurotrophic variables have all been engineered into engraftable NSCs for delivery to tumors. three Nonetheless, their clinical application is limited by ethical and logistic difficulties this kind of as their isolation and immunologic compatibility in allogenic transplantation. Human cord blood is definitely an option supply of grownup stem cells. Many research indicate that human umbilical cord blood derived stem cells are just like stem cells from bone marrow with respect to cell traits and multilineage differentiation Purification of FL HSCs E14.
5 fetal liver cells have been harvested and taken care of with red blood cell lysis buffer had been sorted applying FACS Aria cell sorter have been co cultured as previously described containing 20% FBS and penicillin streptomycin had been VX-770 molecular weight positioned on OP9 and OP9 DL1 monolayers for B and T lineage differentiation, respectively. Co culture media contained: alpha MEM, 10% FBS, 10 mM HEPES and Flt3L irradiated RAG one deficient mice. Mice had been sacrificed 5 6 weeks immediately after transplantation. The lymphoid compartments of the recipients had been analyzed by movement cytometry. The experiments shown within this research represent success from two independent experiments. likely. four,five hUCBSCs possess the capability to differentiate into a number of mesodermal tissues, endodermal tissue, and ectodermal tissue and have been proven to become much more advantageous in cell procurement, storage, and transplantation than bone marrow derived SCs.
6 Not long ago, selleck inhibitor hUCBSCs were shown to display tropism for human glioma, and the therapy of trimeric form of tumor necrosis aspect relevant apoptosis inducing ligand secreting hUCBSCs have significant anti tumor effects compared with adenoviral TRAIL gene therapy. seven Having said that, tumor unique migratory properties call for even further elucidation in relation to their likely use in therapeutic applications. Diverse components that guide the motility of stem cells are already recognized together with soluble things, cell adhesion molecules, and extracellular matrix parts. Migration by the ECM is facilitated by ECM degrading enzymes such because the matrix metal lopro teases, which free of charge bound chemokines and allow motion along the chemokine gradient in the nearby tissue.

Nevertheless, a smaller variety of non B lymphocytes that also expressed CD44 had been existing. Thus, to be able to exclude any possibility that the professional survival result of CD44 was not right produced from the tumor cells, we isolated the leukemic cells by negative selection yielding samples containing greater than 97% pure CLL cells. In these purified CLL cells, we again located that stimulation of CD44 greater the viability in all samples examined on typical by 104 49 %, which equals the average survival enhance of 103 30% in the matching PBMC samples. These results display the protective result is directly mediated by CD44 activation during the leukemic cells and independent of more cells. Contemplating that U CLL cells had larger CD44 expression than M CLL cells, we established whether or not the increased CD44 expression could translate into increased CD44 signaling and enhanced safety from apoptosis. Cell viability in PBMCs just after 3 days of culture while not CD44 stimulation was comparable in between M CLL and U CLL cells.
To estimate the number of cells particularly selleck chemical protected from apoptosis by CD44 stimulation, we subtracted the % dwell cells within the handle from your % dwell cells in the CD44 stimulated cells. Although all samples acquired a survival advantage, the result was much more prominent for U CLL than mutated CLL with 21 9% compared to 13 6% of cells, respectively, that had been rescued from apoptosis by CD44 activation. This translates right into a relative boost in viability when compared with unstimulated handle cells of 65% for U CLL cells but of only 26% for M CLL cells, indicating a alot more potent anti apoptotic result of CD44 engagement within the former subtype. Possessing shown a pro survival impact of CD44 engagement using monoclonal antibodies, we wished to test regardless of whether a physiologic ligand of CD44 would possess the same effect. To this end, we evaluated the viability of CLL cells cultured on hyaluronic acid coated plates. In these experiments, CLL cells had been incubated in wells coated with hyaluronic acid at rising concentrations.
Right after 96 hrs of culture, CLL cell viability increased in the dose dependent method. In the highest HA concentration cell viability elevated by 20% in contrast with cells cultured within the absence of HA. CD44 activates the PI3K/AKT and MAPK/ERK pathways and increases MCL 1 protein expxression We following investigated the impact of CD44 activation within the PI3K/AKT and MAPK/ERK pathways, which are already reported to be activated by CD44 in solid tumor cell lines. supplier PF-4708671 CD44 engagement on CLL cells was followed by a prompt and robust maximize of AKT phosphorylation and activation of ERK1/2. We validated AKT activation in an extended cohort of M CLL and U CLL samples. In the two subtypes, a vast majority of samples showed greater AKT phosphorylation which on common reached two.

So, combination therapies can be fair to contemplate. Indeed, synergistic effects with the combined use of statins with many medication are already reported in preclinical scientific studies each in vitro and in vivo. These medicines comprise Cox two inhibitors, tocotrienols, PPAR agonists, bisphosphonates, and numerous chemotherapeutic medication, 5 FU, gemcitabine, and paclitaxel . Also, statins can act as radiation sensitizers. Our tumor information demonstrate that statin treatment alone inhibits tumor development and this effect is even more dramatic in ACL knockdown cells. Interestingly, in contrast to the in vitro information which demonstrate that statin treatment of ACL knockdown cells doesn’t diminish cell number, in vivo, we uncovered that some tumors regressed. We repeated this in vivo experiment with A549 luc cells, focusing consideration on only two remedy arms: The ACL knockdown cells and statin remedy of those tumors. These in vivo regression data are rather striking: Several mechanisms may possibly be at play to describe why the in vivo data contrast for the modest results witnessed in vitro.
Research to assess effects about the tumor microenvironment such as angiogenesis and stromal responses are in progress. Such as, one particular could speculate that due to the fact HIFs are downstream targets within the PI3K/ AKT pathway, HIF expression could possibly be lowered by ACL knockdown and that this in flip could impact many renowned HIF targets such as VEGF, therefore affecting angiogenesis. To elucidate many of the mechanisms by which statins selleck inhibitor may be enhancing the results of ACL knockdown, we assessed the impact on PI3K/AKT and MAPK signaling. As proven in Figure 6A, B, statin therapy diminished AKT phosphorylation inside a time and dose dependent method plus the result was additional dramatic during the ACL deficient state. Yet, we observed only slight downregulation of ERK phosporylation following six h of statin treatment. We examined the effects of long term remedy with statin on MAPK signaling.
As proven in Figure 6C, a 24 h incubation with statin brought on obvious downregulation of MAPK phosphorylation inside the ACL deficient state evaluating to regulate A549 cells, suggesting that the mixture of ACL inhibition and statin remedy diminished both PI3K/AKT signaling and MAPK pathway. These data may possibly explain the sizeable anti tumor results of irreversible MEK inhibitor this blend in vivo. Without a doubt both pathways are activated in A549 cells, considering the fact that they incorporate K ras activating mutation in an LKB1 deficient background. PI3K/AKT and MAPK signaling are two of your most significant signaling cascades dysregulated in cancers. In addition, inhibition of PI3K signaling on the degree of mTORC1 has become shown to activate a suggestions loop in Ras MAPK signaling via an S6K1 and PI3K dependent course of action. Hence, dual blockade of PI3K and MAPK signaling is often expected to acquire substantial anti tumor results the two in vitro and in vivo.

There was no significant big difference in IL six, IL eight and G CSF production involving IP handled and IC taken care of groups. The two IP and IC seem to equally influence the expression of professional regenerative mediators such as IL six, IL 8 and G CSF in HLSEC. Both IP and IC appear to influence the release of IL six, IL 8 and G CSF concerning 24 to 48 hrs following H R. These results suggest that the two IP and IC could probably influence the liver regeneration signaling cascade. Therefore, the two methods demand even further evaluation employing co culture versions to find out their ultimate effect on these mediators of liver regeneration. Laparoscopic surgical ways are getting used extra frequently in the remedy of pancreatic sickness with minimal morbidity and mortality. Within this retrospective overview, we assess various variables and outcomes in the laparoscopic and open distal pancreatectomies performed at our institution more than a 4 12 months time period. We reviewed the healthcare information of 116 sufferers undergoing distal pancreatic resection from 2002 to 2006.
Data gathered incorporated demographics, pancreatic pathology, operative time, estimated blood reduction, manage of pancreas stump, pancreatic leak and other post operative complications, and length of stay. Student t check, median check, and chi square analyses have been utilised to find out sizeable differences in between laparoscopic and open groups. A p value of less than 0. 05 was regarded sizeable. Of 116 distal pancreatectomies, selleck inhibitor 26 have been performed laparoscopi cally and 90 open. Difficult dissection due to adherent tumor, bleeding, and technical limitations had been reasons for conversion. There were no considerable variations in demographics, median operative time, pancreatic leak fee or overall complication rate. Twenty 5 laparoscopic instances have been carried out for benign illness and one particular for malignant illness versus 36 for benign and 49 for malignant sickness during the open group. Median EBL was 250 mL inside the laparoscopic group versus 750 mL in the open group. A stapling device was used in 92.
3% on the laparoscopic instances to manage the pancreatic stump whereas sutures were utilized in 61. 9% with the open circumstances. Laparoscopic sufferers had a median length of stay of 5 days versus seven days while in the open group with 34. 8% with the laparoscopic group staying more than 5 days versus 67. 1% with the open group staying a lot more than selleck chemical Masitinib 5 days. Laparoscopic distal pancreatectomy yields comparable surgical benefits with drastically shorter hospital stays than open pancreatectomy. Pancreatic leak and total complication charges appear equivalent. Laparoscopic distal pancreatectomy can be a protected, productive treatment option for choose scenarios of pancreatic disease. Historically, distal pancreatectomy included removing the spleen as a result of the relations on the splenic artery and vein using the tail within the pancreas.

Considering that men and women with FAP and MAP are at elevated chance for periampullary and ampullary cancer, and because variable expression of those syndromes is well known, we hypothesized that a fraction of apparently sporadic ampullary cancer patients may have germline APC or MYH mutations as a predisposing reason behind their malignancy. A retrospective evaluation of pathology department databases and hepatobiliary/pancreatic surgeon practices on the University of Toronto identified 61 circumstances, handled amongst August 1994 and August 2005, that has a diagnosis of ampullary adenoma or ampullary cancer. A death clearance was performed by the Ontario Cancer Registry, and with all the approval on the treating surgeon, residing subjects have been approached by mail for participation within this examine. Screening for germ line APC and MYH mutations was performed making use of the protein truncation check and denaturing high effectiveness liquid chromatography evaluation, respec tively. We did not identify truncating germline APC mutations or pathogenic biallelic MYH mutations within the 33 subjects tested. One particular subject was observed to have a monoallelic G382R MYH mutation, whilst this unlikely contrib uted to her ampullary cancer, because heterozygous MYH mutations don’t seem to be a strong predisposing issue to colorectal or periampullary neoplasia.
These data propose that germline APC and MYH mutations are very likely not causative in sporadic ampullary cancer. Gallbladder carcinoma is actually a fairly uncommon malignancy with an incredibly poor prognosis. The pathologic staging of gallbladder carcinoma can be a critical determinant in the individuals prognosis and the treatment method alternatives. On the other hand, we have usually encountered sufferers in whom the course of their disease differed substantially from what can be predicted depending on their clinical selleckchem staging, which highlights the wants to think about added predictive variables. Gallbladder carcinoma takes place a lot more commonly in gals than guys, yet expression from the estrogen receptor loved ones and progesterone haven’t been studied. We utilized an immunohistochemical technique to examine the expression of ERa, ERb and PR in radically resected gallbladder carcinoma tissues and after that compared their expression standing with several clinicopathological aspects.
We immunohistochemically investigated thirty formalin fixed, paraf fin embedded specimens of gallbladder adenocarcinoma tissues using ERa, selleck ERb and PR antibodies. The expression of ERa, ERb and PR were in contrast making use of the Chi square check. Survival was analyzed employing the Kaplan Meier strategy, verified through the Log Rank test. The results indicated that adenocarcinoma of gallbladder are each negative for ERa and PR, irrespective of histologic grade, TNM stage and sex. Nevertheless, twenty two of thirty situations have been confirmed constructive for ERb, which was appreciably correlated with poor differentiated tumor grade. Overall survival costs of ERb optimistic and damaging sufferers had been 77. 3% and 37. 5%, respectively.

NOD1, which especially recognizes D glutamyl meso diaminopimelic acid, is noticed to contribute to NF kB activation and IL 8 expression through chlamydial infection. Having said that, NOD1 signals via RIP2, which has been demonstrated to activate NF kB and AP 1 transcription things, but not IRF members of the family. Past get the job done in HeLa cells demonstrated a function for NOD1 in IL eight expression while in chlamydial infection. Similarly, our final results showed that knockdown of NOD1 led to a steady decrease in chlamydial induced IFN B in HeLa cells, suggesting that NOD1 might be contributing toward optimum NF kB activation required for IFN B induction. Additionally, there needs to be an additional PRR operating in tandem with NOD1 that contributes to activation of IRF transcription variables. In this regard, it truly is important to note that NOD2, a different NLR which could activate IRF3 by way of MAVS to induce IFN B while in infection with respiratory syncytial virus, is not expressed in HeLa cells. A recent research demonstrated that the IFN B response to L.
monocytogenes was lost in STING KO mouse embryonic fibroblasts, hinting that this newly characterized protein may be a significant element in the TLR independent interferon response. In agreement with that examine, we have now shown by siRNA silencing strategies that STING can also be an aurora inhibitorAurora A inhibitor significant mediator of IFN B induced by C. muridarum in both mouse oviduct epithelial cells and human cells, suggesting that irrespective of cell style, STING plays a central part in Chlamydial induced IFN B along with the subsequent expression of interferon response genes. STING has been shown to perform downstream of your prevalent adaptor molecule MAVS. Nevertheless, knockdown of the MAVS or the upstream RLR pathway components MDA5 and RIG I did not impair the interferon response while in infection with C. muridarum. Similarly, the response to L. monocytogenes also proceeds independently of MAVS, leading to the pertinent query of what cytosolic pathway is upstream of STING.
Cytosolic delivery of dsDNA or infection with a DNA virus, such as HSV I, can lead to the type I IFN response in a STING dependent, but RIG I independent manner. Thus, it is theoretically probable that a DNA sensor may be the frequent upstream receptor protein. Known DNA sensors incorporate the host proteins DAI, absent in melanoma 2, and RNA polymerase III. However the selleck function of DAI is suggested to be unimportant for IFN B expression in lots of cell styles handled with dsDNA, absent in melanoma two has become restricted to activation of caspase one and never IFN B and RNA polymerase III recognizes only poly. Interestingly, implementing chimeric constructs it was determined that dimerization of STING was sufficient to induce signaling.

Clinical and laboratory evidence demonstrates that anti IL6 directed therapies are well tolerated in individuals, indicating their likely utility for anti cancer treatments. Humanized anti IL6 and IL6R monoclonal antibodies are already evaluated in clinical trials as well as utilization of IL6 conjugated toxins has also been proposed. These data in blend with our final results of IL6 antibody treatment options of GBM xenografts, recommend that IL6 antibody may perhaps be valuable towards GBM. When treatment method of GBMs is usually intricate by the necessity of systemic treatment options to cross the blood brain barrier, antibody based mostly therapies are actually administered intravenously and established effective for GBM. Similarly, the potential of IL6 antibody to bind and inactivate this growth component in the bloodstream may perhaps demonstrate efficacious for GBM patients. We determined that a novel molecular pathway, IL6 signaling, is linked to GSC growth and survival.
The dramatic advantage of IL6R and IL6 knockdown to the survival of mice bearing intracranial tumors along with the impact of IL6 antibody towards GBM xenografts, strongly NVP-BKM120 clinical trial recommend that focusing on IL6 signals may very well be practical as being a cancer stem cell directed therapy. Our scientific studies present proof that inhibiting IL6 pathways need to be thought about for more exploitation in therapeutic development. 1. Introduction Signal transduction proteins have enhanced value in carcinogenesis and tumor formation and signify interesting targets to the growth of novel anticancer therapeutics. The Signal Transducer and Activator of Transcription family of proteins are cytoplasmic transcription components with vital roles inside the responses to cytokines and growth things, including advertising cell development and differentiation, and inflammation and immune responses. Ordinary STATs activation is initiated from the phosphorylation of the significant tyrosine residue on the binding of cytokines or development variables to cognate receptors. STATs phosphorylation is induced by development component receptor tyrosine kinases, or cytoplasmic tyrosine kinases, including Janus kinases and Src relatives kinases.
Whilst pre existing STAT dimers happen to be detected, research present that phosphorylation induces dimerization involving two STAT monomers via a phosphotyrosine interaction with the SH2 domain. selleckchem XL184 Inside the nucleus, lively STAT dimers bind to distinct DNA response elements inside the promoters of target genes and regulate gene expression. Ordinary STAT activation is transient in accordance with physiological responses. However, the persistent activation of specific STAT members of the family, which includes Stat3 is frequently observed in lots of human tumors. It will be now very well established that aberrant activation of Stat3 contributes to malignant transformation and tumorigenesis.

Recombinant expression vectors were then purified and sequenced by an Utilized Biosystems 3730 DNA Analyzer during the DNA Sequencing and Genotyping Facility at University of Missouri Kansas City, and made use of to produce stable S2 cell lines. D. melanogaster Schneider S2 cells were maintained at 27 C in Insect Cell Culture Media, supplemented with 10% heat inactivated fetal bovine serum containing 1% penicillin streptomycin resolution. For DNA transfection, cells were seeded overnight in serum totally free medium. GenCarrier 1 transfection reagent was employed for transient transfection based on the producers guidelines. Cells in culture dishes or plates have been grown to 70% confluence prior to transfection. DES Inducible/ Secreted Kit with pCoBlast was utilised to construct secure S2 cell lines. To select steady S2 cells expressing recombinant proteins, pCoBlast was cotransfected with recombinant pMT/BiP/V5 His A vectors. Following 48h transfection, S2 cells have been centrifuged and re suspended in complete development medium containing 25 g/ml Blasticidine S hydrochloride. Resistant colonies appeared 1 week later.
For Western blot analysis, copper sulfate was additional to the steady S2 cell lines in 6 effectively plates, and protein expression was induced for 48h. Cell culture medium was collected, steady S2 cells had been homogenized in 400 l lysis buffer. The cell homogenates have been incubated on ice for 15 min and sonicated briefly several occasions, after which centrifuged at 15,000 g for 15 min at four C. The supernatants have been collected as cell extracts for Western blot analysis. The cell culture selleck media and cell extracts were separated on 10%, 12%, or 15% SDS Web page and proteins have been transferred to nitrocellulose membranes. The membrane was blocked with 5% BSA in Tris buffered saline containing 0. 05% Tween 20 at space temperature for no less than 3h then incubated overnight with principal antibody at 4 C in 5% BSA in TBS T with gentle rocking. Then, the membrane was washed four times with TBS T and incubated with secondary antibody in 5% BSA in TBS T for 2h at room temperature.
Right after washing four times with TBS T, the signal was created by utilizing ECL Chemiluminescence selleck chemicals Detection Kit or alkaline phosphatase conjugate color development Kit. Anti Flag M2 antibody and anti V5 antibody had been employed as main antibodies, horseradish peroxidase conjugate anti mouse antibody was utilized as secondary antibody for chemiluminescence, and alkaline phosphatase conjugate anti mouse antibody was made use of as secondary antibody for color growth. Immunoprecipitation assay was carried out by utilizing 300 l of cell extract, which is equivalent to around 106 cells, or equivalent cell culture medium containing recombinant proteins. The cell extracts or cell culture media had been pre cleared for 30min with 30 l Protein G Sepharose inside a complete volume of 500 l.

Past operate has connected the GSK3B isoform with NF kB16, and our final results making use of GSK3B RNAi and gene deletion support a crucial role for GSK3B in tolerance. Interestingly, in contrast to regulation of GSK3B serine phosphorylation in acutely stimulated macrophages17,18, longer tolerizing treatment method with TNF increased GSK3B tyrosine phosphorylation, therefore growing its exercise. Additionally, TNF promoted nuclear localization of GSK3B, exactly where GSK3B gains accessibility to new substrates. GSK3 mediated phosphorylation of its substrates demands past priming phosphorylation by a distinct kinase16, and TNF induces sustained signaling and phosphorylation of diverse cellular proteins in macrophages49. As a result, TNF probably alters cellular responses to GSK3 signaling a minimum of in portion by modifying substrate availability and inducing priming phosphorylation of new substrates. General, our findings assistance a model whereby longer phrase TNF exposure couples GSK3 signaling to suppression of inflammatory cytokine production by promoting suggestions inhibition of NF kB and suppressing chromatin remodeling.
The early phase of TNF induced signaling and gene induction continues to be very well characterized and linked to acute inflammatory responses. Work from our lab and other individuals characterizing later phases of TNF responses in macrophages suggests a a lot more complex role for TNF, with induction of IFN STAT1 responses and differentiation into multinucleated cells49. Within this research, we’ve extended purchase PF299804 this a lot more nuanced comprehending on the purpose of TNF in macrophages by demonstrating TNF mediated induction of a potent suggestions mechanism that suppresses inflammatory cytokine manufacturing. Suggestions inhibition via tolerization of macrophages restrains the magnitude of acute inflammatory responses, as shown by our in vitro and in vivo findings, and may possibly contribute to your protective purpose of TNF in autoimmune diseases such as numerous sclerosis and lupus14,15.
Yet, TNF is a key driver of irritation in other autoimmune diseases such as rheumatoid arthritis and inflammatory bowel sickness, indicating that in these settings TNF mediated feedback inhibition is inadequate to resolve irritation. This may perhaps selleck VX-770 arise due to the fact the homeostatic functions of TNF are compromised by counter regulatory signaling pathways such as IFN STAT133,50, or by hypomorphic TNFAIP3 alleles related with these diseases45,46. In this context, our findings supply insights that may be exploited therapeutically to enhance the suppressive and homeostatic functions of TNF by focusing on GSK3 and downstream pathways and molecules with predominantly homeostatic functions.