Combined Metabolic Activators with Different NAD+ Precursors Improve Metabolic Functions in the Animal Models of Neurodegenerative Diseases.

BACKGROUND: Mitochondrial dysfunction and metabolic abnormalities are acknowledged as significant factors in the onset of neurodegenerative disorders such as Parkinson's disease (PD) and Alzheimer's disease (AD). Our research has demonstrated that the use of combined metabolic activators (CMA) may alleviate metabolic dysfunctions and stimulate mitochondrial metabolism. Therefore, the use of CMA could potentially be an effective therapeutic strategy to slow down or halt the progression of PD and AD. CMAs include substances such as the glutathione precursors (L-serine and N-acetyl cysteine), the NAD+ precursor (nicotinamide riboside), and L-carnitine tartrate. METHODS: Here, we tested the effect of two different formulations, including CMA1 (nicotinamide riboside, L-serine, N-acetyl cysteine, L-carnitine tartrate), and CMA2 (nicotinamide, L-serine, N-acetyl cysteine, L-carnitine tartrate), as well as their individual components, on the animal models of AD and PD. We assessed the brain and liver tissues for pathological changes and immunohistochemical markers. Additionally, in the case of PD, we performed behavioral tests and measured responses to apomorphine-induced rotations. FINDINGS: Histological analysis showed that the administration of both CMA1 and CMA2 formulations led to improvements in hyperemia, degeneration, and necrosis in neurons for both AD and PD models. Moreover, the administration of CMA2 showed a superior effect compared to CMA1. This was further corroborated by immunohistochemical data, which indicated a reduction in immunoreactivity in the neurons. Additionally, notable metabolic enhancements in liver tissues were observed using both formulations. In PD rat models, the administration of both formulations positively influenced the behavioral functions of the animals. INTERPRETATION: Our findings suggest that the administration of both CMA1 and CMA2 markedly enhanced metabolic and behavioral outcomes, aligning with neuro-histological observations. These findings underscore the promise of CMA2 administration as an effective therapeutic strategy for enhancing metabolic parameters and cognitive function in AD and PD patients.

Anticancer Potential of Novel Cinnamoyl Derivatives against U87MG and SHSY-5Y Cell Lines.

BACKGROUND: Glioblastoma multiforme (GBM) is probably the most malignant and aggressive brain tumor belonging to the class of astrocytomas. The considerable aggressiveness and high malignancy of GBM make it a tumor that is difficult to treat. Here, we report the synthesis and biological evaluation of eighteen novel cinnamoyl derivatives (3a-i and 4a-i) to obtain more effective antitumor agents against GBM. METHODS: The chemical structures of novel cinnamoyl derivatives (3a-i and 4a-i) were confirmed by NMR and MS analyses. The physicochemical properties and evaluation of the ADME profile of 3a-i and 4a-i were performed by the preADMETlab2.0 web program. Cinnamoyl derivatives 3a-i and 4a-i were tested in vitro for their cytotoxicity against the human healthy fibroblast (HDFa) cells using an MTT cell viability assay. Derivatives with no toxicity on HDFa cells were tested both on human glioblastoma (U87MG) and neuroblastoma (SHSY- 5Y) cells, chosen as an experimental model of brain tumors. Cell death mechanisms were analyzed by performing flow cytometry analyses. RESULTS: Cinnamoyl derivatives 3a-i and 4a-i showed good physicochemical and ADME properties suggesting that these compounds could be developed as oral drugs endowed with a high capability to cross the blood-brain barrier. Compounds (E)-1-methoxy-4-(2-(phenylsulfonyl)vinyl)benzene (2c) and (E)-N-benzyl-N-(2- (cyclohexylamino)-2-oxoethyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (3e) did not show cytotoxicity on healthy human fibroblast cells up to 100 µg/mL. The most anticarcinogenic molecule, compound 3e, emerged as the most potent anticancer candidate in this study. Flow cytometry results showed that compound 3e (25 µg/mL) application resulted in nearly 86% and 84% cytotoxicity in the U87MG and the SHSY-5Y cell lines, respectively. Compound 2c (25 µg/mL) resulted in 81% and 82% cytotoxicity in the U87MG and the SHSY-5Y cell lines, respectively. CONCLUSION: Cinnamoyl derivative 3e inhibits the proliferation of cultured U87MG and SHSY-5Y cells by inducing apoptosis. Further detailed research will be conducted to confirm these data in in vivo experimental animal models.

Costunolide and Parthenolide Ameliorate MPP+ Induced Apoptosis in the Cellular Parkinson's Disease Model.

Monoamine oxidase B (MAO-B) is an enzyme that metabolizes several chemicals, including dopamine. MAO-B inhibitors are used in the treatment of Parkinson's Disease (PD), and the inhibition of this enzyme reduces dopamine turnover and oxidative stress. The absence of dopamine results in PD pathogenesis originating from decreased Acetylcholinesterase (AChE) activity and elevated oxidative stress. Here, we performed a molecular docking analysis for the potential use of costunolide and parthenolide terpenoids as potential MAO-B inhibitors in the treatment of PD. Neuroprotective properties of plant-originated costunolide and parthenolide terpenoids were investigated in a cellular PD model that was developed by using MPP+ toxicity. We investigated neuroprotection mechanisms through the analysis of oxidative stress parameters, acetylcholinesterase activity and apoptotic cell death ratios. Our results showed that 100 µg/mL and 50 µg/mL of costunolide, and 50 µg/mL of parthenolide applied to the cellular disease model ameliorated the cytotoxicity caused by MPP+ exposure. We found that acetylcholinesterase activity assays exhibited that terpenoids could ameliorate and restore the enzyme activity as in negative control levels. The oxidative stress parameter analyses revealed that terpenoid application could enhance antioxidant levels and decrease oxidative stress in the cultures. In conclusion, we reported that these two terpenoid molecules could be used in the development of efficient treatment strategies for PD patients.

Combined metabolic activators improve cognitive functions in Alzheimer's disease patients: a randomised, double-blinded, placebo-controlled phase-II trial.

BACKGROUND: Alzheimer's disease (AD) is associated with metabolic abnormalities linked to critical elements of neurodegeneration. We recently administered combined metabolic activators (CMA) to the AD rat model and observed that CMA improves the AD-associated histological parameters in the animals. CMA promotes mitochondrial fatty acid uptake from the cytosol, facilitates fatty acid oxidation in the mitochondria, and alleviates oxidative stress. METHODS: Here, we designed a randomised, double-blinded, placebo-controlled phase-II clinical trial and studied the effect of CMA administration on the global metabolism of AD patients. One-dose CMA included 12.35 g L-serine (61.75%), 1 g nicotinamide riboside (5%), 2.55 g N-acetyl-L-cysteine (12.75%), and 3.73 g L-carnitine tartrate (18.65%). AD patients received one dose of CMA or placebo daily during the first 28 days and twice daily between day 28 and day 84. The primary endpoint was the difference in the cognitive function and daily living activity scores between the placebo and the treatment arms. The secondary aim of this study was to evaluate the safety and tolerability of CMA. A comprehensive plasma metabolome and proteome analysis was also performed to evaluate the efficacy of the CMA in AD patients. RESULTS: We showed a significant decrease of AD Assessment Scale-cognitive subscale (ADAS-Cog) score on day 84 vs day 0 (P = 0.00001, 29% improvement) in the CMA group. Moreover, there was a significant decline (P = 0.0073) in ADAS-Cog scores (improvement of cognitive functions) in the CMA compared to the placebo group in patients with higher ADAS-Cog scores. Improved cognitive functions in AD patients were supported by the relevant alterations in the hippocampal volumes and cortical thickness based on imaging analysis. Moreover, the plasma levels of proteins and metabolites associated with NAD + and glutathione metabolism were significantly improved after CMA treatment. CONCLUSION: Our results indicate that treatment of AD patients with CMA can lead to enhanced cognitive functions and improved clinical parameters associated with phenomics, metabolomics, proteomics and imaging analysis. Trial registration ClinicalTrials.gov NCT04044131 Registered 17 July 2019, https://clinicaltrials.gov/ct2/show/NCT04044131.

Combined metabolic activators improve metabolic functions in the animal models of neurodegenerative diseases.

BACKGROUND: Neurodegenerative diseases (NDDs), including Alzheimer's disease (AD) and Parkinson's disease (PD), are associated with metabolic abnormalities. Integrative analysis of human clinical data and animal studies have contributed to a better understanding of the molecular and cellular pathways involved in the progression of NDDs. Previously, we have reported that the combined metabolic activators (CMA), which include the precursors of nicotinamide adenine dinucleotide and glutathione can be utilized to alleviate metabolic disorders by activating mitochondrial metabolism. METHODS: We first analysed the brain transcriptomics data from AD patients and controls using a brain-specific genome-scale metabolic model (GEM). Then, we investigated the effect of CMA administration in animal models of AD and PD. We evaluated pathological and immunohistochemical findings of brain and liver tissues. Moreover, PD rats were tested for locomotor activity and apomorphine-induced rotation. FINDINGS: Analysis of transcriptomics data with GEM revealed that mitochondrial dysfunction is involved in the underlying molecular pathways of AD. In animal models of AD and PD, we showed significant damage in the high-fat diet groups' brain and liver tissues compared to the chow diet. The histological analyses revealed that hyperemia, degeneration and necrosis in neurons were improved by CMA administration in both AD and PD animal models. These findings were supported by immunohistochemical evidence of decreased immunoreactivity in neurons. In parallel to the improvement in the brain, we also observed dramatic metabolic improvement in the liver tissue. CMA administration also showed a beneficial effect on behavioural functions in PD rats. INTERPRETATION: Overall, we showed that CMA administration significantly improved behavioural scores in parallel with the neurohistological outcomes in the AD and PD animal models and is a promising treatment for improving the metabolic parameters and brain functions in NDDs.

Drug Synergism of Anticancer Action in Combination with Favipiravir and Paclitaxel on Neuroblastoma Cells.

Background and Objectives: Favipiravir (FPV) is an antiviral medication and has an inhibitory effect on Cytochrome P450 (CYP2C8) protein, which is mainly involved in drug metabolism in the liver, and the expression of this gene is known to be enhanced in neuronal cells. The metabolization of Paclitaxel (PTX), a chemotherapeutic drug used in cancer patients, was analyzed for the first time in the human SH-SY5Y neuroblastoma cell line for monitoring possible synergistic effects when administered with FPV. Materials and Methods: Further, in vitro cytotoxic and genotoxic evaluations of FPV and PTX were also performed using wide concentration ranges in a human fibroblast cell culture (HDFa). Nuclear abnormalities were examined under a fluorescent microscope using the Hoechst 33258 fluorescent staining technique. In addition, the synergistic effects of these two drugs on cultured SH-SY5Y cells were determined by MTT cell viability assay. In addition, the death mechanisms that can occur in SHSY-5Y were revealed by using the flow cytometry technique. Results: Cell viability analyses on the HDFa healthy cell culture showed that both FPV and PTX have inhibitory effects at higher concentrations. On the other hand, there were no significant differences in nuclear abnormality numbers when both of the compounds were applied together. Cell viability analyses showed that FPV and PTX applications have higher cytotoxicity, which indicated synergistic toxicity against the SHSY-5Y cell line. Also, PTX exhibited higher anticancer properties against the neuroblastoma cell line when applied with FPV, as shown in both cytotoxicity and flow cytometry analyses. Conclusions: In light of our findings, the anticancer properties of PTX can be enhanced when the drug application is coupled with FPV exposure. Moreover, these results put forth that the anticancer drug dosage should be evaluated carefully in cancer patients who take COVID-19 treatment with FPV.

Do neurosteroids have impact on depression and cognitive functions in cases with acromegaly?

OBJECTIVE: Neurosteroids (NSs) are a distinct hormone group and, they are known for their contribution into the status of mood and cognitive functions. Whether they are also involved in the mood disturbances and cognition in acromegaly is not known. Herein we aimed to evaluate the relation of mood status and cognitive functions with the NS levels in cases with acromegaly. DESIGN: A total of 33 cases with acromegaly composed the acromegaly group (AG) and, 30 age and gender-matched cases without acromegaly composed the control group (CG). The levels of Allopregnanolone (AP), pregnenolone (PRG), 24S-hydroxycholesterol (24OHC), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androsterone (ADT), GH and IGF-1 were measured in each group. Beck Depression Inventory (BDI) was used to assess depressive symptoms, whereas an extensive neuropsychological assessment with several neurocognitive tests were carried out for each subject by an experienced psychologist. RESULTS: Cases with acromegaly had lower 24OHC and DHEA levels (p = 0.002 and p = 0.007, respectively) in comparison to CG. Of the cognitive functions time to complete 1 s Series was significantly higher and, the scores on Switching Verbal Fluency Test, Boston Naming Test (BNT)-semantic and BNT-phonological, the highest learning point of Oktem Verbal Memory Processes Test (VMPT) were significantly lower in cases with acromegaly in comparison to those in controls (p = 0.004, p = 0.01, p < 0.001, p = 0.02 and p = 0.05, respectively). KAS-perseveration errors were higher in CG (p = 0.03). In AG the levels of AP were negatively correlated with the scores on Months backward Test (MBT), Animal Naming Test, Construction, BNT-spontaneous and positively correlated with BNT-incorrect answers; PRG was positively correlated with VMPT-retention scores, ADT was negatively correlated with MBT and 3 s Series scores, DHEAS was positively correlated with VMPT-the highest learning point whereas it was negatively correlated with MBT scores. Additionally, the scores on BDI were positively correlated with DHEA levels in AG. CONCLUSION: Cognitive changes may be encountered in acromegaly and, neurosteroids may contribute to the changes in certain cognitive functions.

Boron Nitride Nanoparticles Loaded with a Boron-Based Hybrid as a Promising Drug Carrier System for Alzheimer's Disease Treatment.

The search for an innovative and effective drug delivery system that can carry and release targeted drugs with enhanced activity to treat Alzheimer's disease has received much attention in the last decade. In this study, we first designed a boron-based drug delivery system for effective treatment of AD by integrating the folic acid (FA) functional group into hexagonal boron nitride (hBN) nanoparticles (NPs) through an esterification reaction. The hBN-FA drug carrier system was assembled with a new drug candidate and a novel boron-based hybrid containing an antioxidant as BLA, to constitute a self-assembled AD nano transport system. We performed molecular characterization analyses by using UV-vis spectroscopy, Fourier transform infrared spectrophotometer (FTIR), scanning electron microscope (SEM), Energy-dispersive X-ray spectroscopy (EDS) and Zeta potential investigations. Second, we tested the anti-Alzheimer properties of the carrier system on a differentiated neuroblastoma (SHSY5-Y) cell line, which was exposed to beta-amyloid (1-42) peptides to stimulate an experimental in vitro AD model. Next, we performed cytotoxicity analyses of synthesized molecules on the human dermal fibroblast cell line (HDFa) and the experimental AD model. Cytotoxicity analyses showed that even higher concentrations of the carrier system did not enhance the toxicological outcome in HDFa cells. Drug loading analyses reported that uncoated hBN nano conjugate could not load the BLA, whereas the memantine loading capacity of hBN was 84.3%. On the other hand, memantine and the BLA loading capacity of the hBN-FA construct was found to be 95% and 97.5%, respectively. Finally, we investigated the neuroprotective properties of the nano carrier systems in the experimental AD model. According to the results, 25 µg/mL concentrations of hBN-FA+memantine (94% cell viability) and hBN-FA+BLA (99% cell viability) showed ameliorative properties against beta-amyloid (1-42) peptide toxicity (50% cell viability). These results were generated through the use of flow cytometry, acetylcholinesterase (AChE) and antioxidant assays. In conclusion, the developed drug carrier system for AD treatment showed promising potential for further investigations and enlightened neuroprotective capabilities of boron molecules to treat AD and other neurodegenerative diseases. On the other hand, enzyme activity, systematic toxicity analyses, and animal studies should be performed to understand neuroprotective properties of the designed carrier system comprehensively.

In vitro Evaluation of Selective Cytotoxic Activity of Chaerophyllum macropodum Boiss. on Cultured Human SH-SY5Y Neuroblastoma Cells.

Neuroblastoma is the most common solid tumor in children. New treatment approaches are needed because of the harmful side effects and costs of the methods used in the treatment of neuroblastoma. Medicinal and aromatic plants are important for new treatment approaches due to their minimal side effects and economic advantages. Therefore, the present study was carried out to examine the cytotoxic effect of Chaerophyllum macropodum extract on human neuroblastoma (SH-SY5Y) and fibroblast (HDFa) cell lines. 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release (LDH) assays were used to determine the cytotoxic effect of C. macropodum. The extracts were analyzed for their phenolic content by HPLC-PDA. Major components were determined as 63.600% o-coumaric acid, 15.606% catechine hydrate, 8.713% rosmarinic acid, 4.376% clorogenic acid, and 3.972% salicylic acid. The obtained results from cytotoxicity testing revealed that C. macropodum exerted a significant cytotoxic effect on human neuroblastoma cells at all tested concentrations (p < 0.05). But it did not lead to any cytotoxic potential on human fibroblasts. As a result, the obtained data clearly revealed C. macropodum exerted a selective cytotoxic action on neuroblastoma cells for the first time.

Borax relieved the acrylamide-induced hematotoxic, hepatotoxic, immunotoxic and genotoxic damages in rainbow trout by regulating apoptosis and Nrf2 signaling pathway.

Acrylamide(AA) is a compound with wide usage areas including paper, dyes, and plastics industries. Due to its broad spectrum and water solubility suggest that this vinyl compound may cause serious environmental problems. AA was shown to exhibit neurotoxic, immunotoxic, reproductive toxicant as well as carcinogenic potency on animals. Especially in recent years, the therapeutic effects of boron and boron containing compounds like borax(BX), ulexite(ULX) and colemanite(COL) had been reported. However, the ameliorative potential by boron compounds against AA-induced toxicities had not been investigated yet. Therefore, in this investigation rainbow trout were exposed acutely to AA in the presence and absence of BX. The hematological indices and genotoxic end-points were examined in the fish blood tissue. In addition to oxidative stress response, the levels of DNA damage, CASP3, TNF-α, Nrf-2 as well as IL-6 amounts were determined in both blood and liver tissues of fish. The obtained results executed that AA induced toxic conditions in both tissues. In fact, an increase in the amount of oxidative stress and ROS, and a decrease in GSH levels were observed. AA exposure led to an increase in CASP3levels and 8-OHdG formation. It was also found that Nrf-2 pathway contributed to the initiation of oxidative stress that associated with AA-induced toxicity. On the contrary, our findings indicated that co-exposure of BX with AA elicited oxidative stress and cell death. In a conclusion BX was suggested as a useful and effective natural agent for the prevention and early treatment of AA toxicity in fish.

Structural, biocompatibility, and antibacterial properties of Ge-DLC nanocomposite for biomedical applications.

Integrative production of new nanocomposites has been used to enhance favorable features of biomaterials for unlocking ultimate potential of different molecules. In the present study, advantageous properties of diamond like carbons (DLC) and germanium (Ge) like greater biocompatibility and antibacterial attributes were aimed to combined into a thin film. For this purpose, 400 nm DLC-Ge nanocomposite was coated on the borosilicate glasses via the magnetron sputtering and surface characteristics was analyzed by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and The Raman spectrum. Biocompatibility analysis were performed by 3-(4,5-Dimethylthiazol-2-yl) (MTT) cell viability assay and Hoechst 33258 fluorescent staining genotoxicity assessments on the human fibroblast cell line (HDFa). Finally, antibacterial properties of DLC-Ge nanocomposite coatings were investigated by Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 25923) bacterial attachment analysis. As a result of magnetron sputtering coating, nearly 400 nm thick DLC-Ge nanocomposite film showed a smooth, a non-porous, and a dense characteristic. Cell viability analysis showed that Ge-DLC coatings permits %95 cell surface growth of fibroblast cells. Also, there were no significant difference in aspect of nuclear abnormalities compared to the (-) control which showed nonmutagenic features of the thin film. Finally, antibacterial attachment analysis put forth that Ge-DLC coatings inhibits bacterial adhesion as %40 and %25 rates for P. aeruginosa and S. aureus bacterial strains, respectively. From these results, DLC-Ge nanocomposites could be proposed as a potential new biomaterial for various biomedical applications.

Lipoic Acid Conjugated Boron Hybrids Enhance Wound Healing and Antimicrobial Processes.

Complications of chronic non-healing wounds led to the emergence of nanotechnology-based therapies to enhance healing, facilitate tissue repair, and prevent wound-related complications like infections. Here, we design alpha lipoic acid (ALA) conjugated hexagonal boron nitride (hBN) and boron carbide (B4C) nanoparticles (NPs) to enhance wound healing in human dermal fibroblast (HDFa) cell culture and characterize its antimicrobial properties against Staphylococcus aureus (S. aureus, gram positive) and Escherichia coli (E. coli, gram negative) bacterial strains. ALA molecules are integrated onto hBN and C4B NPs through esterification procedure, and molecular characterizations are performed by using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and UV-vis spectroscopy. Wound healing and antimicrobial properties are investigated via the use of cell viability assays, scratch test, oxidative stress, and antimicrobial activity assays. Based on our analysis, we observe that ALA-conjugated hBN NPs have the highest wound-healing feature and antimicrobial activity compared to ALA-B4C. On the other hand, hBN, ALA-B4C, and ALA compounds showed promising regenerative and antimicrobial properties. Also, we find that ALA conjugation enhances wound healing and antimicrobial potency of hBN and B4C NPs. We conclude that the ALA-hBN conjugate is a potential candidate to stimulate regeneration process for injuries.

Oxidant and anti-oxidant status in common brain tumors: Correlation to TP53 and human biliverdin reductase.

OBJECTIVE: To assess oxidant and antioxidant status in patients with common brain tumors; namely meningiomas, low-grade gliomas (LGG) and high-grade gliomas (HGG) and to compare with normal brain tissues. PATIENTS AND METHODS: Almost nine biomarkers were measured in 59 brain tumors obtained during surgery and 15 normal brain tissues that were collected during autopsy. Results were compared between two groups. RESULTS: In general, protein oxidation and lipid peroxidation increased while antioxidant capacity decreased significantly in tumors compared to the controls (p<0.05) and higher the grade of the tumor, higher the levels of oxidation and lower the anti-oxidation. CONCLUSIONS: Reactive oxygen species may play a crucial role in the pathogenesis of these common brain tumors. As the processes at the molecular level understood, targeted-treatment adjunct to surgical removal will be possible to cope with these devastating brain tumors.

The efficacy of donepezil administration on acetylcholinesterase activity and altered redox homeostasis in Alzheimer's disease.

Alzheimer's disease (AD) is a serious multifactorial disorder with progressive neurodegenerative outcomes related with impaired redox homeostasis. Inhibition of the enzyme acetylcholinesterase (AChE), as one of the major therapeutic strategies, is considered to be offering only symptomatic relief and moderate disease modifying effect. We intended to investigate the effects of acetylcholinesterase inhibition via donepezil on protein carbonyl (PCO), advanced protein oxidation products (AOPP) and ischemia modified albumin (IMA) as protein oxidation markers and ferric reducing antioxidant power (FRAP), prooxidant-antioxidant balance (PAB), total thiol (T-SH), protein thiol (P-SH) as antioxidant status markers and also kynurenine (KYN), N-formyl kynurenine (N-FKYN) and protein bound dityrosine (DT) levels all in one demonstrating the redox homeostasis in Alzheimer patients also correlated with AChE activity. The AChE activity and PCO, KYN, N-FKYN and DT levels were found to be significantly higher in the AD group than the control group. The FRAP, T-SH and P-SH levels were significantly lower in the AD group than in the control group. The AChE activity was significantly higher both in donepezil treated and untreated groups when compared with the control group. PCO levels were significantly higher in Alzheimer's untreated group than the healthy control and donepezil treated groups. AChE activity was positively correlated with PCO, IMA, PAB, KYN and N-FKYN levels and negatively correlated with FRAP, T-SH and P-SH levels in all participants. Our data showed that treatment with donepezil had ameliorating effects on redox homeostasis in Alzheimer patients. AChE inhibition seems to be exhibiting a potent antioxidant role and may inhibit protein oxidation by decreasing AChE activity in AD, thus medicinal natural substances exhibiting the similar mechanism of action with their antioxidant behaviours can be recommended for the emphasis on new drug new drug development. Further clinical and experimental studies are needed to support our current findings and conclusions.