The prevention of falls in patients with Parkinson's disease with in-home monitoring using a wearable system: a pilot study protocol.

BACKGROUND: Parkinson's disease (PD) is a chronic, progressive neurodegenerative condition that gradually worsens motor function and leads to postural instability and, eventually, falls. Several factors may influence the frequency of future falls, such as slowness, freezing of gait, loss of balance, and mobility problems, cognitive impairments, and the number of previous falls. The TED bracelet is an advanced technological wearable device able to predict falls. AIMS: This principal aim is to investigate the feasibility of a full-scale research project that uses the TED bracelet to identify whether individuals with PD are at risk of falling. METHODS: This study will involve a pilot prospective observational study design; the subjects will include 26 patients suffering from mild PD and 26 others with no PD and no gait problems. Data will be collected from the TED bracelet and then compared to a paper-based fall diary. The enrolled participants will have a scheduled outpatient evaluation to collect both clinical and instrumental data as well as biological samples. DISCUSSION: This pilot study could then be implemented in a larger form to further evaluate the effectiveness of the TED device. Finally, it will help further develop gait monitoring systems for people with Parkinson's disease and other neurodegenerative diseases that can affect physical function and mobility, such as dementia and Alzheimer's. CONCLUSIONS: Preventing falls and their complications could lead to major advancements in the quality of home care for patients with PD, which would significantly impact the quality of life of both these patients and their caregivers.

Structure activity relationship studies on Amb639752: toward the identification of a common pharmacophoric structure for DGKα inhibitors.

A series of analogues of Amb639752, a novel diacylglycerol kinase (DGK) inhibitor recently discovered by us via virtual screening, have been tested. The compounds were evaluated as DGK inhibitors on α, θ, and ζ isoforms, and as antagonists on serotonin receptors. From these assays emerged two novel compounds, namely 11 and 20, which with an IC50 respectively of 1.6 and 1.8 µM are the most potent inhibitors of DGKα discovered to date. Both compounds demonstrated the ability to restore apoptosis in a cellular model of X-linked lymphoproliferative disease as well as the capacity to reduce the migration of cancer cells, suggesting their potential utility in preventing metastasis. Finally, relying on experimental biological data, molecular modelling studies allow us to set a three-point pharmacophore model for DGK inhibitors.

Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.

Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc.

Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia.

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

Alteration of BIRC3 and multiple other NF-κB pathway genes in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

The host genetic background of DNA repair mechanisms is an independent predictor of survival in diffuse large B-cell lymphoma.

Several drugs used for diffuse large B-cell lymphoma (DLBCL) treatment rely on DNA damage for tumor cell killing. We verified the prognostic impact of the host DNA repair genotype in 2 independent cohorts of DLBCL treated with R-CHOP21 (training cohort, 163 cases; validation cohort, 145 cases). Among 35 single nucleotide polymorphisms analyzed in the training series, MLH1 rs1799977 was the sole predicting overall survival. DLBCL carrying the MLH1 AG/GG genotype displayed an increased death risk (hazard ratio [HR] = 3.23; P < .001; q =0 .009) compared with patients carrying the AA genotype. Multivariate analysis adjusted for International Prognostic Index identified MLH1 AG/GG as an independent OS predictor (P < .001). The poor prognosis of MLH1 AG/GG was the result of an increased risk of failing both R-CHOP21 (HR = 2.02; P = .007) and platinum-based second-line (HR = 2.26; P = .044) treatment. Survival analysis in the validation series confirmed all outcomes predicted by MLH1 rs1799977. The effect on OS of MLH1, a component of the DNA mismatch repair system, is consistent with its role in regulating the genotoxic effects of doxorubicin and platinum compounds, which are a mainstay of DLBCL first- and second-line treatment.

Alterations of negative regulators of cytokine signalling in immunodeficiency-related non-Hodgkin lymphoma.

We investigated immunodeficiency-related non-Hodgkin lymphoma for the presence of molecular alterations affecting negative regulators of the Janus family protein tyrosine kinase/signal transducer and activator of transcription pathway. Protein tyrosine phosphatase, non-receptor type 6/Src homology 2-containing tyrosine phosphatase-1 epigenetic silencing was recurrent in primary effusion lymphoma (100%), and diffuse large B-cell lymphoma (63%), with a higher prevalence in the non-germinal centre subtype, and was associated with the activation of the Janus family protein tyrosine kinase/signal transducer and activator of transcription 3 pathway. Suppressor of cytokine signalling (SOCS)1 and SOCS3 epigenetic silencing were occasionally detected, whereas SOCS1 was frequently mutated in diffuse large B-cell lymphoma and polymorphic post-transplant lymphoproliferative disorders, possibly as a cause of aberrant somatic hypermutation. However, the mutation profile of the coding region of the gene was different from that expected from the aberrant somatic hypermutation process, suggesting that, at least in some cases, SOCS1 mutations may have been selected for their functional activity.

UPO Biobank: The Challenge of Integrating Biobanking into the Academic Environment to Support Translational Research.

Biobanks are driving motors of precision and personalized medicine by providing high-quality biological material/data through the standardization and harmonization of their collection, preservation, and distribution. UPO Biobank was established in 2020 as an institutional, disease, and population biobank within the University of Piemonte Orientale (UPO) for the promotion and support of high-quality, multidisciplinary studies. UPO Biobank collaborates with UPO researchers, sustaining academic translational research, and supports the Novara Cohort Study, a longitudinal cohort study involving the population in the Novara area that will collect data and biological specimens that will be available for epidemiological, public health, and biological studies on aging. UPO Biobank has been developed by implementing the quality standards for the field and the ethical and legal issues and normative about privacy protection, data collection, and sharing. As a member of the "Biobanking and Biomolecular Resources Research Infrastructure" (BBMRI) network, UPO Biobank aims to expand its activity worldwide and launch cooperation with new national and international partners and researchers. The objective of this manuscript is to report an institutional and operational experience through the description of the technical and procedural solutions and ethical and scientific implications associated with the establishment of this university research biobank.

Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility, and differentiation.

Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse.

Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques.

Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery.

Molecular history of Richter syndrome: origin from a cell already present at the time of chronic lymphocytic leukemia diagnosis.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia to aggressive lymphoma. We explored intraclonal diversification (ID) of immunoglobulin genes in order to (i) follow the evolutionary history of the RS clone (ii) compare the role of ID in clonally related RS vs. clonally unrelated cases. Most (10/11, 90.9%) clonally related RS stem from the predominant clone observed at CLL diagnosis. One single RS had a transformation pattern compatible with sequential evolution from a secondary CLL subclone. Once RS transformation had occurred, all secondary CLL subclones disappeared and were substituted by the dominant RS clone with its own descendants. These observations suggest that genetic lesions associated with RS transformation are acquired by a cell belonging to the original CLL clone, rather than being progressively accumulated by later CLL subclones. Accordingly, most (9/11, 81.1%) clonally related RS harbored a genetic lesion disrupting TP53 that was already present, though at subclonal levels, in 5/11 (45.5%) samples of the paired CLL phase. A fraction of clonally related RS switched off ID (4/11, 36.4%) or reduced the levels of ID (5/11, 45.4%) at transformation. Conversely, all clonally unrelated RS harbored ID and were characterized by a significantly higher mutation frequency compared to clonally related RS (median: 1.18 × 10(-3) vs. 0.13 × 10(-3); p =0.002). These data indicate that (i) clonally related RS stems from a cell that is already present within the initial CLL clone and (ii) clonally unrelated and clonally related RS are biologically distinct disorders also in terms of antigen affinity maturation.

Integrated DNA copy number and methylation profiling of lymphoid neoplasms using a single array.

Changes in DNA copy number (CN) and DNA methylation represent important aberrations for lymphomas and other cancers. Here, for the first time, we show that the Illumina Infinium Methylation (IIM) assay, although not originally designed for CN profiling, is able to estimate CN changes. We compared the IIM CN profiles to those obtained with a standard technique in a series of diffuse large B-cell lymphomas: the profiles showed a high degree of consensus. The demonstration of CN profiling as an additional function of the IIM assay may impact the choice of platform for methylation profiling of haematological and solid tumours.

Multiple myeloma shows no intra-disease clustering of immunoglobulin heavy chain genes.

BACKGROUND: Characterization of the immunoglobulin gene repertoire has improved our understanding of the immunopathogenesis of lymphoid tumors. Early B-lymphocyte precursors of multiple myeloma are known to exist and might be susceptible to antigenic drive. DESIGN AND METHODS: To verify this hypothesis, we collected a database of 345 fully readable multiple myeloma immunoglobulin sequences. We characterized the immunoglobulin repertoire, analyzed the somatic hypermutation load, and investigated for stereotyped receptor clusters. RESULTS: Compared to the normal immunoglobulin repertoire, multiple myeloma displayed only modest differences involving only a few genes, showing that the myeloma immunoglobulin repertoire is the least skewed among mature B-cell tumors. Median somatic hypermutation load was 7.8%; median length of complementarity determining-region 3 was 15.5 amino acids. Clustering analysis showed the absence of myeloma specific clusters and no similarity with published chronic lymphocytic leukemia or lymphoma subsets. CONCLUSIONS: Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor.

Public Attitude towards Biobanking: An Italian University Survey.

Biobanks have established a critical role in biomedical research by collecting, preserving, organizing, and disseminating biospecimens and related health data, contributing to precision medicine development. Participation in biobanks is influenced by several factors, such as trust in institutions and scientists, knowledge about biobanking, and the consideration of benefit sharing. Understanding public attitudes, fears, and concerns toward biobanking is fundamental to designing targeted interventions to increase trust towards biobanks. The aim of our study was to investigate the level of knowledge and perception of biobanks in students and personnel of the University of Piemonte Orientale. An online questionnaire was designed and administered via e-mail. A total of 17,758 UPO personnel and students were invited to participate in the survey, and 1521 (9.3%) subjects completed the survey. The results showed that 65.0% of the participants were aware of the term "biobank" and knew what the activity of a biobank was, and 76.3% of subjects were willing to provide biospecimens to a research biobank, whereas 67.3% of the respondents were willing to contribute, in addition to biospecimens, their health and lifestyle data. Concerns were raised about the confidentiality of the information (25.6%) and the commercial use of the samples (25.0%). In conclusion, participants were aware of the role that biobanks play in research and were eager to participate for the sake of furthering scientific research. Still, several concerns need to be addressed regarding the confidentiality of the data along with the commercial use of the samples and associated data.

Dissecting the Mechanism of Action of Spiperone-A Candidate for Drug Repurposing for Colorectal Cancer.

Approximately 50% of colorectal cancer (CRC) patients still die from recurrence and metastatic disease, highlighting the need for novel therapeutic strategies. Drug repurposing is attracting increasing attention because, compared to traditional de novo drug discovery processes, it may reduce drug development periods and costs. Epidemiological and preclinical evidence support the antitumor activity of antipsychotic drugs. Herein, we dissect the mechanism of action of the typical antipsychotic spiperone in CRC. Spiperone can reduce the clonogenic potential of stem-like CRC cells (CRC-SCs) and induce cell cycle arrest and apoptosis, in both differentiated and CRC-SCs, at clinically relevant concentrations whose toxicity is negligible for non-neoplastic cells. Analysis of intracellular Ca2+ kinetics upon spiperone treatment revealed a massive phospholipase C (PLC)-dependent endoplasmic reticulum (ER) Ca2+ release, resulting in ER Ca2+ homeostasis disruption. RNA sequencing revealed unfolded protein response (UPR) activation, ER stress, and induction of apoptosis, along with IRE1-dependent decay of mRNA (RIDD) activation. Lipidomic analysis showed a significant alteration of lipid profile and, in particular, of sphingolipids. Damage to the Golgi apparatus was also observed. Our data suggest that spiperone can represent an effective drug in the treatment of CRC, and that ER stress induction, along with lipid metabolism alteration, represents effective druggable pathways in CRC.

B-cell receptor, clinical course and prognosis in chronic lymphocytic leukaemia: the growing saga of the IGHV3 subgroup gene usage.

The immunoglobulin heavy chain variable gene (IGHV) mutational status has been recognized as an important predictor of prognosis in chronic lymphocytic leukaemia (CLL) since 1999. More recently, other features of the B-cell receptor, such as stereotypy, have been identified as capable of refining the prognostic potential of IGHV status in the clinical assessment of CLL patients. In this context, different genes belonging to the IGHV3 subgroup, the most frequently used subgroup in CLL, have been shown to denote disease subsets that either display a bad prognosis (i.e. IGHV3-21, IGHV3-23) or are associated with particularly good clinical outcomes, including a highly stable/indolent clinical course, even prone to spontaneous regression (i.e. IGHV3-72, IGHV3-30). The present review focuses on the molecular and biological features of CLL-expressing specific genes belonging to the IGHV3 subgroup that are known to mark disease subsets with completely different clinical courses, and may be possibly related to CLL pathogenesis via antigen and/or superantigen involvement.

Identification of Key Phospholipids That Bind and Activate Atypical PKCs.

PKCζ and PKCι/λ form the atypical protein kinase C subgroup, characterised by a lack of regulation by calcium and the neutral lipid diacylglycerol. To better understand the regulation of these kinases, we systematically explored their interactions with various purified phospholipids using the lipid overlay assays, followed by kinase activity assays to evaluate the lipid effects on their enzymatic activity. We observed that both PKCζ and PKCι interact with phosphatidic acid and phosphatidylserine. Conversely, PKCι is unique in binding also to phosphatidylinositol-monophosphates (e.g., phosphatidylinositol 3-phosphate, 4-phosphate, and 5-phosphate). Moreover, we observed that phosphatidylinositol 4-phosphate specifically activates PKCι, while both isoforms are responsive to phosphatidic acid and phosphatidylserine. Overall, our results suggest that atypical Protein kinase C (PKC) localisation and activity are regulated by membrane lipids distinct from those involved in conventional PKCs and unveil a specific regulation of PKCι by phosphatidylinositol-monophosphates.

Persistence of Neutralizing Antibodies to SARS-CoV-2 in First Wave Infected Individuals at Ten Months Post-Infection: The UnIRSA Cohort Study.

Longitudinal mapping of antibody-based SARS-CoV-2 immunity is critical for public health control of the pandemic and vaccine development. We performed a longitudinal analysis of the antibody-based immune response in a cohort of 100 COVID-19 individuals who were infected during the first wave of infection in northern Italy. The SARS-CoV-2 humoral response was tested using the COVID-SeroIndex, Kantaro Quantitative SARS-CoV-2 IgG Antibody RUO Kit (R&D Systems, Bio-Techne, Minneapolis, USA) and pseudotype-based neutralizing antibody assay. Using sequential serum samples collected from 100 COVID-19 recovered individuals from northern Italy-mostly with mild disease-at 2 and 10 months after their first positive PCR test, we show that 93% of them seroconverted at 2 months, with a geometric mean (GeoMean) half-maximal neutralization titer (NT50) of 387.9. Among the 35 unvaccinated subjects retested at 10 months, 7 resulted seronegative, with an 80% drop in seropositivity, while 28 showed decreased anti-receptor binding domain (RBD) and anti-spike (S) IgG titers, with a GeoMean NT50 neutralization titer dropping to 163.5. As an NT50 > 100 is known to confer protection from SARS-CoV-2 re-infection, our data show that the neutralizing activity elicited by the natural infection has lasted for at least 10 months in a large fraction of subjects.

Single nucleotide polymorphism-arrays provide new insights in the pathogenesis of post-transplant diffuse large B-cell lymphoma.

Post-transplant lymphoproliferative disorders (PTLD) are complications of solid organ transplantation associated with severe morbidity and mortality. Diffuse large B-cell lymphoma (DLBCL) represents the most common form of monomorphic PTLD. We studied 44 cases of post-transplant DLBCL (PT-DLBCL) with high-density genome wide single nucleotide polymorphism-based arrays, and compared them with 105 cases of immunocompetent DLBCL (IC-DLBCL) and 28 cases of Human Immunodeficiency Virus-associated DLBCL (HIV-DLBCL). PT-DLBCL showed a genomic profile with specific features, although their genomic complexity was overall similar to that observed in IC- and HIV-DLBCL. Among the loci more frequently deleted in PT-DLBCL there were small interstitial deletions targeting known fragile sites, such as FRA1B, FRA2E and FRA3B. Deletions at 2p16.1 (FRA2E) were the most common lesions in PT-DLBCL, occurring at a frequency that was significantly higher than in IC-DLBCL. Genetic lesions that characterized post-germinal center IC-DLBCL were under-represented in our series of PT-DLBCL. Two other differences between IC-DLBCL and PT-DLBCL were the lack of del(13q14.3) (MIR15/MIR16) and of copy neutral LOH affecting 6p [major histocompatibility complex (MHC) locus] in the latter group. In conclusion, PT-DLBCL presented unique features when compared with IC-DLBCL. Changes in PT-DLBCL were partially different to those in HIV-DLBCL, suggesting different pathogenetic mechanisms in the two conditions linked to immunodeficiency.

Genome wide DNA-profiling of HIV-related B-cell lymphomas.

Non-Hodgkin lymphomas (NHL) represent a frequent complication of human immunodeficiency virus (HIV) infection. To elucidate HIV-NHL pathogenesis, we performed a genome-wide DNA profiling based on a single nucleotide polymorphism-based microarray comparative genomic hybridization in 57 HIV-lymphomas and, for comparison, in 105 immunocompetent diffuse large B-cell lymphomas (IC-DLBCL). Genomic complexity varied across HIV-NHL subtypes. HIV-Burkitt lymphoma showed a significantly lower number of lesions than HIV-DLBCL (P = 0.032), whereas the median number of copy number changes was significantly higher in Epstein-Barr virus negative (EBV-) HIV-DLBCL (42.5, range 8-153) compared to EBV+ cases (22; range 3-41; P = 0.029). Compared to IC-DLBCL, HIV-DLBCL displayed a distinct genomic profile with no gains of 18q and specific genetic lesions. Fragile sites-associated genes, including FHIT (FRA3B), WWOX (FRA16D), DCC (FRA18B) and PARK2 (FRA6E) were frequently inactivated in HIV-NHL by interstitial deletions, and a significantly higher prevalence of FHIT alterations was observed in HIV-DLBCL compared to IC-DLBCL. The same genes involved by fragile site deletions were also frequently affected by aberrant methylation of regulative regions.